Systems for transgene expression

Abstract

The present disclosure provides engineered transcriptional modulators relevant to control of gene transcription, e.g. for use in gene therapy systems.

Description

SYSTEMS FOR TRANSGENE EXPRESSIONBACKGROUND

[0001] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0002] Gene therapy has the potential to treat and cure disease by delivering therapeutic genes in vivo. Most lead applications employ adeno-associated virus (AAV)-based delivery vectors. The present disclosure identifies the source of various problems in many available gene therapy systems, provides solutions to such problems, and, moreover, provides a variety of technologies useful, among other things, in regulating gene expression including specifically expression of transgenes as may be utilized in gene therapy applications.SUMMARY

[0003] The present disclosure provides important technologies relevant to control of gene expression, and in particular to gene transcription.

[0004] Among other things, the present disclosure provides engineered transcriptional modulators, for example including (a) a DNA binding element; and (b) a transcription modulation element (e.g., a transcription activation element). Such engineered transcriptional modulators provided by and / or utilized in accordance with the present disclosure, in many embodiments, further include (c) a regulatory element that, for example, renders the engineered transcriptional modulator responsive to an exogenous signal such as, for example, a small molecule agent (e.g., presence, level, or form thereof). In many embodiments, an engineered transcriptional modulator provided by and / or utilized in accordance with the present disclosure further includes one or more linkers between adjacent element(s).

[0005] Remarkably, the present disclosure demonstrates feasibility of achieving effective transcriptional modulation with engineered transcriptional modulators of unusually compact size (e.g., in some embodiments below about 1.8 kb, typically not more than 3.5kb). As described herein, in many embodiments, one particularly desirable feature of provided compact engineeredtranscriptional modulators is their amenability to be delivered into cells, and, in many embodiments organisms (e.g., humans) through use of a gene therapy vector system, such as a viral vector system, and in particular an AAV-based system, notwithstanding size constraints of such system. Indeed, the present disclosure provides engineered transcriptional modulators sufficiently compact that they can be encoded by a construct that also includes a gene of interest and regulatory sequences sufficient to render such gene of interest responsive to such encoded engineered transcriptional modulator while remaining small enough to be delivered by AAV-based technologies. In particular embodiments, as described further herein below, a compact engineered transcriptional modulator provided and / or utilized in accordance with the present disclosure is so compact that a gene encoding it can be included on the same construct as an expressible gene of interest and appropriate regulatory sequences that render such gene of interest under control of the compact engineered transcriptional modulator, and the whole construct is still of a size amenable to AAV-based delivery.

[0006] Still further, the present disclosure documents effective exogenous regulation, e.g., by a small molecule regulator which, in many embodiments, may be an approved drug, of compact engineered transcriptional modulators provided and / or utilized in accordance with the present disclosure. The present disclosure documents effective design and use of engineered transcriptional modulators including a regulatory element that renders the compact engineered transcriptional modulator responsive to exogenous regulation, e.g., by a small molecule regulator including, in some embodiments, small molecule regulators that are approved drugs. Indeed, the present disclosure remarkably achieves such exogenous regulation with a variety of different strategies, including strategies that had previously only been described as useful in the context of much larger polypeptides (see, for example, Khalil et al., US Patent Number 11,530,246 to Boston University, issued Dec. 20, 2022 with a prior publication on Dec. 3, 2020 as US2020 / 0377564), thus precluding AAV delivery, particularly on a construct that also includes a an expressible gene of interest and regulatory sequences as described herein. The present disclosure surprisingly documents successful adaptation of such exogenous control systems to a compact environment as described herein.

[0007] Thus, the present disclosure, among other things, provides and demonstrates effectiveness of exogenously-regulated (specifically including drug-regulated) engineered transcription factor systems for the control of AAV transgene expression.

[0008] The present disclosure further provides nucleic acid constructs encoding engineered transcriptional modulators as described herein (e.g., compact engineered transcriptional modulators). The present disclosure also provides nucleic acid constructs including an expressible gene of interest and appropriate regulatory sequences to render such gene of interest responsive to an engineered transcriptional modulator as described herein (e.g., a compact engineered transcriptional modulator).

[0009] As noted above, in some embodiments, a single nucleic acid construct provided by and / or utilized in accordance with the present disclosure includes both a gene encoding an engineered transcriptional modulator as described herein (e.g., a compact engineered transcriptional modulator), and an expressible gene of interest associated with appropriate regulatory sequences to render it responsive to the encoded engineered transcriptional modulator (e.g., the encoded compact engineered transcriptional modulators). The present disclosure provides certain insights relating to particularly useful and / or effective design of such constructs (e.g., relating to arrangement of included sequence elements).

[0010] For example, among other things, the present disclosure demonstrates that a particular arrangement of elements, referred to herein as an “upstream divergent” configuration, in many embodiments, is particularly useful. Alternatively or additionally, in some embodiments, inclusion of one or more insulator sequences can desirably reduce system leakiness while, surprisingly, without materially reducing expression of a relevant gene of interest (e.g., in an “on” state as described herein).

[0011] Those skilled in the art, reading the present disclosure will appreciate that embodiments that utilize an engineered transcriptional modulator that is responsive to exogenous regulation (e.g., that includes a regulatory element) together with a construct in which an expressible gene of interest in associated with appropriate regulatory sequences can transition between “on” and “off’ states (referring to expression of the gene of interest, which in turn reflects activity status of the engineered transcriptional modulator), and, in some embodiments, can achieve different level(s) of “off’ or “on”, depending on the exogeneous signal (e g., presence or level of a small molecule - e.g., a particular form thereof). The present disclosure documents effective transitioning between such “on” and “off’ states in response to an exogenous signal (e.g.,presence or level of a small molecule which, in many embodiments, may be an approved drug as described herein).

[0012] In some embodiments, provided systems are characterized by significant differences in expression and / or activity of a regulated gene of interest (a regulated transgene) as described herein between an “off-” state as compared with an “on-” state. For example, in some embodiments, detected expression and / or activity is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 90-fold, at least 95-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold or more that detected in the “off’ state.

[0013] In some embodiments, expression and / or activity of a regulated gene of interest (e.g., a transgene, such as may have been delivered by AAV) is undetectable in the off state; in some such embodiments, an insulator sequence is utilized.

[0014] Among other things, described herein are novel genetically-inducible AAV-driven transgene expression systems that can be useful in gene therapy applications. The disclosed systems for conditional activation of a transgene, e.g., a therapeutic transgene, use genetically compact control modalities.

[0015] In some embodiments, the present disclosure provides expression systems comprising (i) a transcription factor binding site and a minimal promoter operably linked to a gene of interest, and (ii) a synthetic transcription factor (synTF) comprising an activation domain and a zinc finger domain, wherein a synTF binds to a transcription factor binding site. In some embodiments, a synTF as used herein is also referred to as an engineered transcriptional modulator.

[0016] In some embodiments, a zinc finger domain of a synTF comprises a minimal zinc-finger protein architecture.

[0017] In some embodiments, a zinc finger domain is or comprises ZF1 or ZF6.

[0018] In some embodiments, an activation domain is or comprises VP64, p65.1, p65.2, or RTA.2, or fragments, or variants of any of the foregoing. In some embodiments, an activation domain of a synTF is activated in the presence of a drug.

[0019] In some embodiments, a synTF further comprises a protease configured to cleave an activation domain from a zinc finger domain, and wherein a protease is inhibited in the presence of a drug.

[0020] In some embodiments, a synTF further comprises a protease and a nuclear export sequence (NES) or degradation domain, wherein a protease is configured to cleave off a NES or degradation domain from a synTF, and wherein a protease is inhibited in the presence of a drug. In some embodiments, a degradation domain is an ODC-1 sequence incorporated as 1, 2, 3, or 4 tandem repeats, and wherein a protease is NS3p.

[0021] In some embodiments, a synTF further comprises a nuclear translocation domain activated in the presence of a drug. In some embodiments, a nuclear translocation domain is ERT2.

[0022] In some embodiments, a synTF further comprises a linker comprising about 5 to about 15 amino acids between a protease and an activation domain and / or zinc finger domain.

[0023] In some embodiments, a drug is an FDA-approved drug. For example, in some embodiments, a drug is grazoprevir (GZV) or 4-hydroxytamoxifen / tamoxifen (4OHT). In some embodiments, a drug can cross a blood-brain-barrier (BBB).

[0024] In some embodiments, an expression system comprises more than one transcription factor binding site. In some embodiments, transcription factor binding sites are configured in a compact or spaced architecture.

[0025] In some embodiments, a gene of interest is a gene therapy.

[0026] In some embodiments, the present disclosure provides expression systems comprising a compact, e.g., miniature, synthetic transcription factor disclosed herein.

[0027] In some embodiments, the present disclosure provides vectors comprising an expression system according to any one of the embodiments herein. In some embodiments, a vector is a viral vector. In some embodiments, a viral vector is an adeno-associated virus (AAV) vector or a lentiviral vector.

[0028] In some embodiments, the present disclosure provides vectors comprising an expression system disclosed herein. In some embodiments, a vector is an adeno-associated virus (AAV) vector.

[0029] In some embodiments, the present disclosure provides method of delivering a gene therapy, comprising contacting a cell with a vector disclosed herein (e.g., the foregoing embodiments).

[0030] The present disclosure provides, among other things, engineered transcriptional modulator polypeptides. In some embodiments, an engineered transcriptional modulator polypeptide comprises: (a) one or more DNA binding elements; (b) one or more transcription modulation elements comprising one or more transcription activation elements or one or more transcription repression elements; (c) one or more regulatory elements that confer responsiveness to a small molecule regulator; and (d) optionally one or more linker elements.

[0031] In some embodiments, an engineered transcriptional modulator polypeptide is characterized in that it has a sufficiently compact length such that it can be encoded by a nucleic acid construct having a total length of not more than 4.8 kb. In some embodiments, a nucleic acid construct having a total length of not more than 4.8 kb comprises: (i) a nucleic acid sequence that encodes an engineered transcriptional modulator polypeptide, operatively linked with a first promoter and, optionally, a first set of transcription regulatory sequences; and (ii) a nucleic acid sequence encoding one or more genes of interest operatively linked with a second promoter and transcription regulatory sequences including at least one binding site recognized by the DNA binding element.

[0032] In some embodiments, one or more DNA binding elements comprise a helix-turn-helix domain, a homeodomain, a basic region leucine zipper domain, a nuclear hormone receptor domain, a zinc finger domain, or any variant, fragment or combination thereof. The terms domain and elements are used interchangeably herein in reference to polypeptides that can be useful in an engineered transcriptional modulator disclosed herein.

[0033] In some embodiments, one or more DNA binding elements comprise one or more zinc finger DNA binding element.

[0034] In some embodiments, one or more DNA binding elements do not comprise a zinc finger DNA binding element.

[0035] In some embodiments, one or more regulatory elements comprise a protease element, a localization element, a pair of proximity elements, or fragments, or variants or any combination thereof.

[0036] In some embodiments, one or more transcription modulation elements comprise a transcription activation element.

[0037] In some embodiments, one or more transcription modulation elements comprise a transcription repression element.

[0038] In some embodiments, one or more transcription modulation elements comprise a VP64 domain, a p65.1 domain, a p65.2 domain, a RTA.2 domain, a VP16 domain, a VPR domain, a p300 domain, a CBP domain, a Hsfl domain, a Swi / Snf domain, a MED15 domain, a E1A domain, a Gal4-AD domain, a B42 domain, a p65 domain, or any biological equivalent thereof, or any variant thereof or any fragment thereof.

[0039] In some embodiments, a zinc finger DNA binding element comprises one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, and / or ZF19.

[0040] In some embodiments, a zinc finger DNA binding element is or comprises ZF6.

[0041] In some embodiments, a zinc finger DNA binding element is or comprises ZF1.

[0042] In some embodiments, an engineered transcriptional modulator is characterized in that, when present in a cell that also includes a nucleic acid construct in which one or more genes of interest is operatively linked with at least one binding site recognized by one or more DNA binding elements, expression of one or more genes of interest is modulated responsive to the small molecule regulator.

[0043] In some embodiments, a nucleic acid construct comprises: (i) a nucleic acid sequence encoding an engineered transcriptional modulator polypeptide operatively linked with an engineered promoter, and optionally with one or more regulatory sequences, and (ii) one or more genes of interest operatively linked with at least one binding site recognized by a DNA binding element.

[0044] In some embodiments, one or more transcriptional modulation elements are a human element, or a humanized element, or a fragment of either of the foregoing.

[0045] In some embodiments, one or more regulatory elements are or comprise a protease element.

[0046] In some embodiments, a protease element is or comprises an NS3p protease. In some embodiments, an NS3p has an amino acid sequence of SEQ ID NO: 85 or a sequence having at least 85% identity thereto. In some embodiments, when a protease element is or comprises NS3p, a small molecule regulator comprises grazoprevir.

[0047] In some embodiments, one or more regulatory elements are or comprise a localization element. In some embodiments, a localization element comprises a nuclear localization element.

[0048] In some embodiments, one or more localization elements are or comprise ERT2. In some embodiments, ERT2 has an amino acid sequence of SEQ ID NO: 86 or a sequence having at least 85% identity thereto. In some embodiments, when a localization element is or comprises ERT2, the small molecule regulator comprises 4-hydroxytamoxifen.

[0049] In some embodiments, one or more regulatory elements further comprise one or more destabilization elements, e g., one or more degradation domains (e.g., degradation tag (degron)) or a nuclear export signal.

[0050] In some embodiments, one or more regulatory elements are situated between one or more DNA binding elements and one or more transcription modulation elements.

[0051] In some embodiments, a linker element is present between one or more DNA binding elements, one or more transcription modulation elements, and / or one or more regulatory elements.

[0052] In some embodiments, one or more DNA binding elements are situated at the N terminus of one or more transcription modulation elements, or one or more DNA binding elements are situated at the C terminus of one or more transcription modulation elements.

[0053] In some embodiments, an engineered transcriptional modulator polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1 - 37, or a sequence with at least 85%, at least 90%, at least 95% or 100% identity thereto.

[0054] In certain embodiments, the present disclosure provides expression systems comprising an engineered transcriptional modulator polypeptide as described herein or a plurality of engineered transcriptional modulator polypeptides as described herein.

[0055] In certain embodiments, the present disclosure provides delivery systems comprising an engineered transcriptional modulator polypeptide as described herein or a plurality of engineered transcriptional modulator polypeptides as described herein.

[0056] In certain embodiments, the present disclosure provides nucleic acid constructs encoding an engineered transcriptional modulator polypeptide as described herein, or a plurality of engineered transcriptional modulator polypeptide polypeptides as described herein.

[0057] In certain embodiments, the present disclosure provides nucleic acid constructs. In some embodiments, a nucleic acid construct comprises: (a) a first sequence encoding an engineered transcriptional modulator polypeptide, operatively linked with a promoter and, optionally with one or more transcriptional regulatory sequences; and (b) a second sequence encoding one or more payloads operatively linked with a promoter and at least one transcriptional regulatory sequence that includes a binding site for a DNA binding element in the engineered transcriptional modulator.

[0058] In some embodiments, a nucleic acid construct comprises one or more additional elements. In some embodiments, an additional element comprises an insulator element. In some embodiments, an insulator element is situated between a first sequence and a second sequence. In some embodiments, an insulator element comprises a sequence of SEQ ID NO: 100 or a fragment or a variant thereof.

[0059] In some embodiments, a payload is a polypeptide payload. In some embodiments, a payload is a nucleic acid payload (e.g., an RNA payload).

[0060] In some embodiments, a first sequence is positioned 5’ of a second sequence in a nucleic acid construct. In some embodiments, a first sequence is positioned 3’ of a second sequence in a nucleic acid construct.

[0061] In some embodiments, a first sequence and a second sequence are positioned in opposite directions to each other in a nucleic acid construct.

[0062] In some embodiments, a first sequence and a second sequence are in a divergent configuration in a nucleic acid construct.

[0063] In some embodiments, a first sequence is 5’ to a second sequence.

[0064] In some embodiments, a first sequence is 3’ to a second sequence.

[0065] In some embodiments, a first sequence and a second sequence are separate transcription units.

[0066] In some embodiments, a nucleic acid construct is no more than 4.8 kb in length.

[0067] In some embodiments, a nucleic acid construct has a sequence of any one of SEQ ID NOs: 39 to 70, or a sequence with at least 85% identity thereto.

[0068] In certain embodiments, the present disclosure provides expression systems. In some embodiments, an expression system comprises a nucleic acid construct as described herein, or a plurality of nucleic acid constructs as described herein.

[0069] In certain embodiments, the present disclosure provides delivery systems. In some embodiments, a delivery system comprises a nucleic acid construct as described herein, or a plurality of nucleic acid constructs as described herein.

[0070] In some embodiments, a delivery system comprises a vector (e g., a viral vector), a lipid-based system and / or a polymer-based system.

[0071] In certain embodiments, the present disclosure provides recombinant adeno-associated virus (rAAV) particles. In some embodiments, a recombinant adeno-associated virus (rAAV) particle comprises: (i) an AAV capsid protein; and (ii) a nucleic acid construct as described herein.

[0072] In some embodiments, a nucleic acid construct comprises one or more ITR sequences. In some embodiments, a nucleic acid construct comprises a pair of AAV ITRs flanking: (i) a nucleic acid sequence encoding an engineered transcriptional modulator polypeptide, and (ii) a nucleic acid sequence encoding one or more genes of interest.

[0073] In some embodiments, an AAV capsid protein comprises a capsid protein from: AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 (rh10), AAV11, AAV12,AAV13, AAV-DJ, or AAV-PHP.eB. In some embodiments, an AAV capsid protein comprises an AAV2 capsid protein.

[0074] In certain embodiments, the present disclosure provides cells. In some embodiments, a cell comprises one or more nucleic acid constructs as described herein.

[0075] In some embodiments, a cell is or has been engineered to express one or more engineered transcriptional modulator polypeptides as described herein.

[0076] In some embodiments, a cell is or has been engineered to express a payload from a nucleic acid construct that comprises a sequence encoding a payload operatively linked with a promoter and at least one transcriptional regulatory sequence. In some embodiments, a nucleic acid construct comprises a binding site for a DNA binding element. In some embodiments, a nucleic acid construct further comprises a transcriptional modulator polypeptide.

[0077] In some embodiments, a cell is or has been engineered to express a transcriptional modulator polypeptide as described herein, or a plurality of transcriptional modulator polypeptides as described herein.

[0078] In some embodiments, a cell further comprises at least one payload sequence operatively linked with a promoter and at least one transcriptional regulatory sequence that includes a binding site for a DNA binding element.

[0079] In some embodiments, a cell is contacted with a small molecule regulator. In some embodiments, contacting a cell with a small molecule regulator results in binding of the small molecule regulator to a regulatory element.

[0080] In some embodiments, binding of a small molecule regulator to a regulatory element results in stabilization, a change in localization, a change in conformation and / or a change in activity of an engineered transcriptional modulator polypeptide.

[0081] In some embodiments, expression of a payload is modulated responsive to a small molecule regulator.

[0082] In some embodiments, a cell is in vitro, ex vivo or in vivo.

[0083] In some embodiments, a cell is a mammalian cell. In some embodiments, a cell is a human cell.

[0084] In some embodiments, a cell is a cell from a central nervous system. In some embodiments, a central nervous system cell is or comprises a CNS epithelial cell, a nerve cell, a CNS connective tissue cell, a stem cell, a progenitor cell, a CNS immune cell, a spinal cord cell, a cell that lines one or more brain ventricles, a nerve support cell, a glial cell, a fat cell, a meninges cell, or a combination thereof.

[0085] In some embodiments, a cell is in a subject.

[0086] In certain embodiments, the present disclosure provides compositions. In some embodiments, a composition comprises one or more engineered transcriptional modulator polypeptides as described herein. In some embodiments, a composition comprises one or more nucleic acid constructs as described herein.

[0087] In some embodiments, a composition further comprises an expression system.

[0088] In some embodiments, a composition further comprises a delivery system.

[0089] In certain embodiments, the present disclosure provides pharmaceutical compositions comprising a composition as described herein and one or more pharmaceutically acceptable excipients.

[0090] In certain embodiments, the present disclosure provides methods. In some embodiments, a method comprises delivering one or more engineered transcriptional modulator polypeptides as described herein to a cell tissue or subject. In some embodiments, a method comprises delivering one or more nucleic acid constructs as described herein to a cell tissue or subject.

[0091] In some embodiments, a method comprises introducing into a cell, tissue or subject a nucleic acid construct encoding an engineered transcriptional modulator polypeptide.

[0092] In some embodiments, delivering comprises administering.

[0093] In some embodiments, a method is a treatment method.

[0094] In some embodiments, a method is a prevention method.

[0095] In some embodiments, a method delivers a payload to a cell, tissue or subject.

[0096] In some embodiments, a payload is a polypeptide payload.

[0097] In some embodiments, a payload is a polynucleotide payload (e.g., an RNA payload).

[0098] In some embodiments, a payload is a therapeutic payload or a diagnostic payload.

[0099] In some embodiments, a method further comprises delivering (e.g., administering) a small molecule regulator to a cell, tissue or subject.

[0100] In some embodiments, a method delivers a payload and expression of the payload is modulated responsive to a small molecule regulator.

[0101] In some embodiments, the present disclosure provides methods of expressing a payload in a cell, comprising introducing into a cell, a nucleic acid construct comprising a sequence encoding a payload operatively linked with a promoter and at least one transcriptional regulatory sequence.

[0102] In some embodiments, a nucleic acid construct comprises a binding site for a DNA binding element. In some embodiments, an engineered transcriptional modulator polypeptide comprising a DNA binding element that recognizes a binding site is present in a cell, tissue or subject. In some embodiments, payload expression level is controlled by modulating exposure to a small molecule regulator. In some embodiments, contacting a cell, tissue or a subject with a small molecule regulator induces expression of a payload.

[0103] In certain embodiments, the present disclosure provides methods of treating a disease, disorder or condition responsive to a payload polypeptide.

[0104] In some embodiments, a method of treating a disease, disorder or condition responsive to a payload polypeptide, comprises administering to a subject suffering from or susceptible to such disease disorder or condition a pharmaceutical composition provided herein that comprises or delivers the payload polypeptide.

[0105] In some embodiments, a method of treating a disease, disorder or condition responsive to expression of a gene under control of at least one transcriptional regulatory sequence that is recognized by a DNA binding element, comprising administering to a subject suffering from or susceptible to such disease, disorder or condition, a pharmaceutical composition provided herein that comprises or delivers an engineered transcriptional modulator.

[0106] In some embodiments, an engineered transcriptional modulator is characterized in that exposure to a small molecule regulator modulates stabilization, localization and / or conformation of the engineered transcriptional modulator, as compared to stabilization, localization and / orconformation of an engineered transcriptional modulator in the absence of, or before exposure to the small molecule regulator.

[0107] In some embodiments, exposure to a small molecule regulator increases expression and / or activity of a payload, as compared to expression and / or activity of a payload in the absence of, or before exposure to the small molecule regulator.

[0108] In certain embodiments, the present disclosure provides kits. In some embodiments, a kit comprises an expression system described herein. In some embodiments, a kit comprises a delivery system described herein.

[0109] In certain embodiments, the present disclosure provides engineered organisms. In some embodiments, an organism is or has been engineered to express a cell described herein, one or more nucleic acid constructs described herein, or one or more engineered transcriptional modulator polypeptides described herein.

[0110] In some embodiments, an organism is a non-human animal.

[0111] In some embodiments, a non-human animal is a rodent. In some embodiments, a rodent is a rat or a mouse.

[0112] In some embodiments, a delivery system comprises an AAV, a lentiviral vector, an all-DNA viral vector, a transposon, or a DNA template (e g., for recombinase or HDR mediated genomic insertion).

[0113] The foregoing general description and following detailed description are exemplary and explanatory and are intended to provide further explanation of the disclosure as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0114] FIGs. 1A- IK are schematics and bar graphs showing design, construction and validation of exemplary engineered transcriptional modulators disclosed herein (e.g., as described in Example 2). Constructs were tested via transient transfection of COMET synTF32and exemplary engineered transcriptional modulator (also referred to as “mini-synTFs”) andreporter plasmids in HEK293FT cells, and fluorescent reporter expression was measured via flow cytometry. For FIGs. 1A- 1J, mini-synTF plasmids: pAC285, pAC286, pAC287, pAC288, pAC289, pAC290, pAC291, pAC292; reporter plasmids: pHIE467, pAC317; transfection control: pPD1033; and fdler DNA: pHIE298 were used. For FIG. IK, mini-synTF plasmids: pAC286, pAC289, pAC291, pHIE282; reporter plasmid: pHIE467; transfection control: pPD1033; and filler DNA: pHIE298 were used. See, Tables 1-7 for more information. Bars show mean absolute fluorescence of three biological replicates; error bars indicate S. E. M. Fold induction (reporter expression with ligand treatment divided by reporter expression without ligand treatment) values are indicated above each bar for that condition pair. Unpaired Welch’s t-test with Holm-Sidak method was used: n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** / ? < 0.001.

[0115] FIGs. 1A- 1C are schematics showing exemplary engineered transcriptional modulator architectures. FIG 1A is a schematic showing structures of two exemplary grazoprevir-(GZV-) induced stabilization (“GZV-on”) engineered transcriptional modulators, with distinct topologies 1 A and IB, as disclosed herein. FIG. IB is a schematic showing structures of two exemplary GZV-induced destabilization (“GZV-off’) engineered transcriptional modulators, with distinct topologies 2A and 2B, as disclosed herein. FIG. 1C is a schematic showing structures of two exemplary 4-hydroxytamoxifen / tamoxifen-(4OHT-) induced nuclear localization (“4OHT-on”) engineered transcriptional modulators, with distinct topologies 3A and 3B, as disclosed herein. Abbreviations: NS3p, protease from the non- structural 3 protein of the Hepatitis C virus; AD, transcriptional activation domain; DBD, DNA-binding domain; NES, nuclear export sequence; ERT2, mutated estrogen receptor 2.

[0116] FIGs. ID - IF are bar graphs showing expression of an exemplary fluorescent reporter transgene (in absolute units of Molecules of Equivalent PE-Texas Red, “MEPTRs”) in HEK293FT cells following transient transfection with plasmids encoding exemplary engineered transcriptional modulators, as assessed with flow cytometry. FIG. ID shows expression from exemplary GZV-induced stabilization (“GZV-on”) engineered transcriptional modulators with topologies 1A and IB shown in FIG. 1A, in the presence of DMSO vehicle control or IpM GZV FIG. IE shows expression from exemplary GZV-induced destabilization (“GZV-off’) engineered transcriptional modulators, with distinct topologies 2A and 2B (with either NES or degron regulatory element) as shown in FIG. IB, in the presence of DMSO vehicle control or 1 pM GZV. FIG. IF shows expression from exemplary 4OHT-induced nuclear localization(“4OHT-on”) engineered transcriptional modulators, with distinct topologies 3 A and 3B as shown in FIG. 1C, in the presence of EtOH control or IpM 40HT.

[0117] FIG. 1G is a pair of schematics showing designs of exemplary synthetic promoter architectures, one with a compact arrangement of engineered transcriptional modulator binding sites (top) and one with a spaced arrangement of engineered transcriptional modulator binding sites (bottom).

[0118] FIGs. 1H- 1J are bar graphs showing expression of an exemplary fluorescent reporter transgene (in absolute units of MEPTRs) in HEK293FT cells following transient transfection with plasmids encoding exemplary engineered transcriptional modulators and synthetic promoters, as assessed with flow cytometry. FIG 1H shows results from an exemplary “GZV-on” engineered transcriptional modulator with topology IB and synthetic promoters with compact or spaced architectures, as shown in FIG. 1G, in the presence of DMSO vehicle control or IpM GZV. FIG II shows results from an exemplary “GZV-off (degron)” engineered transcriptional modulator with topology 2A and synthetic promoters with compact or spaced architectures, as shown in FIG. 1G, in the presence of DMSO vehicle control or IpM GZV. FIG 1J shows results from an exemplary “4OHT-on” engineered transcriptional modulator with topology 3 A and synthetic promoters with compact or spaced architectures, as shown in FIG. 1G, in the presence of DMSO vehicle control or I uM GZV.

[0119] FIG. IK is a bar graph showing expression of an exemplary fluorescent reporter transgene (in absolute units of MEPTRs) in HEK293FT cells following transient transfection with plasmids encoding exemplary engineered transcriptional modulators (mini-synTF plasmids: pAC286, pAC289, pAC291, pHIE282; reporter plasmid: pHIE467; transfection control: pPD1033; and filler DNA: pHIE298; see Tables 1-7 for more information) to a constitutively active COMET synTF32employing the same activation domain (VP64) and zinc-finger DNA-binding domain (ZF6), as assessed with flow cytometry.

[0120] FIGs. 2A-2L are plots showing expression of an exemplary fluorescent reporter transgene (in absolute units of MEPTRs) in HEK293FT cells for exemplary engineered transcriptional modulators (depicted schematically) used for sensitivity analysis (see, Example 3). All constructs were tested via transient transfection of COMET- and exemplary engineered transcriptional modulator (“mini-synTF”) and reporter plasmids in HEK293FT cells, andfluorescent reporter expression was measured via flow cytometry. Bars show mean absolute fluorescence of three biological replicates; error bars indicate S. E. M. Fold induction (reporter expression with ligand treatment divided by reporter expression without ligand treatment) values are indicated above each bar for that condition pair. Unpaired Welch’s t-test with Holm-Sidak method was used: n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0121] FIGs. 2A- 2C show expression for exemplary engineered transcriptional modulators (mini-synTF plasmids: pAC286, pAC289, pAC291, pAC294, pAC297, pAC299; reporter plasmids: pHIE467, pHIE542; transfection control: pPD1033; and fdler DNA: pHIE298 described herein) comprising a ZF 1 DNA binding element or a ZF6 DNA binding element. FIG.2A shows expression from exemplary “GZV-on” engineered transcriptional modulators, in the presence of DMSO vehicle control or IpM GZV. FIG.2B shows expression from exemplary “GZV-off’ engineered transcriptional modulators comprising a degron, in the presence of DMSO vehicle control or 1 pM GZV. FIG. 2C shows expression from exemplary “4OHT-on” engineered transcriptional modulators, in the presence of EtOH control or 1 pM 4OHT.

[0122] FIG. 2D and FIG. 2E show expression for exemplary engineered transcriptional modulators comprising certain transcription modulation elements, including a VP64 transcription activation domain, a p65.1 transcription activation domain, a p65.2 transcription activation domain, or a RTA.2 transcription activation domain (mini-synTF plasmids: pAC286, pAWOOl, pAW002, pAW003, pAC291, pAC337, pAC338, pAC339; reporter plasmid: pHIE467; transfection control: pPD1033; and filler DNA: pHIE298 described herein). FIG. 2D shows expression from exemplary “GZV-on” engineered transcriptional modulators, in the presence of DMSO vehicle control or 1 pM GZV. FIG. 2E shows expression from exemplary “4OHT-on” engineered transcriptional modulators, in the presence of EtOH control or IpM 4OHT.

[0123] FIG. 2F and FIG.2G show expression for exemplary “GZV-on” engineered transcriptional modulators comprising exemplary linkers (mini-synTF plasmids: pAC286, pAW005, pAW008, pAW023, pAW002, pAW024, pAW025, pAW007, pAW009, pAW045; reporter plasmid: pHIE467; transfection control: pPD1033; and filler DNA: pHIE298) in the presence of DMSO vehicle control or IpM GZV. FIG. 2F shows expression for exemplary engineered transcriptional modulators comprising linkers between DNA binding element (ZF6) and regulatory element (NS3p) (“A”), between regulatory element and transcription modulationelement (VP64 or p65.2) (“B”), or at both locations (“A & B”), compared to the same modulator without linker(s). FIG. 2G shows expression for exemplary engineered transcriptional modulators comprising linkers of different lengths (5 amino acids (GGGGS) or 15 amino acids (GGGGSXJ or GGSx5)) and stiffness (more stiff: GGSx5; less stiff GGGGSx3 or GGGGS)), compared to the same modulator without a linker.

[0124] FIG. 2H shows expression for exemplary “GZV-oflf’ engineered transcriptional modulators comprising exemplary degron regulatory elements (a single degradation tag from the murine or human ornithine decarboxylase enzyme (mODC-1 or hODC-1) or a multimer of 2 or 3 of same), in the presence of DMSO vehicle or IpM GZV Mini-synTF plasmids: pAC335, pAW016, pAW017, pAW020, pAW021, pAW022; reporter plasmid: pHIE467; transfection control: pPD1033; and filler DNA: pHIE298 were used.

[0125] FIG. 21 shows expression for exemplary constructs comprising an exemplary “4OHT-on” engineered transcriptional modulator and engineered promoters comprising different numbers of engineered transcriptional modulator binding sites (depicted schematically at left), in the presence of EtOH control or IpM 4OHT. Mini-synTF plasmid: pAC338; reporter plasmids: pAC340, pAC341, pHIE467, pAC342, pAC343, pHIE324; transfection control: pPD1033; and filler DNA: pHIE298 were used.

[0126] FIG. 2J shows expression for exemplary “GZV-oflf” engineered transcriptional modulators comprising a NES or degron and linkers of different lengths (5 amino acids (GGGGS) or 15 amino acids (GGGGSx3 or GGSx5)) and stiffness (more stiff / higher serine content: GGSx5; less stiff / lower serine content GGGGSx3 or GGGGS)), compared to the same modulator without a linker, in the presence of DMSO vehicle or IpM GZV. mini-synTF plasmids: pAC287, pAC289, pAC331, pAC332, pAC333, pAC334, pAC335, pAC336; reporter plasmid: pHIE467; transfection control: pPD1033; and filler DNA: pHIE298 were used.

[0127] FIG. 2K shows expression for exemplary “GZV-oflf’ engineered transcriptional modulators comprising a mODC-1 degron regulatory element comprising different residues from MODC-1, in the presence of DMSO vehicle or IpM GZV. mini-synTF plasmids: pAC335, pAWOlO; reporter plasmid: pHIE467; transfection control: pPD1033; and filler DNA: pHIE298 were used.

[0128] FIG. 2L shows expression for constitutively active COMET synTFs for conditions with engineered promoters comprising different numbers of synTF binding sites. COMET synTF plasmid: pHIE282; reporter plasmids: pAC340, pAC341, pHIE467, pAC342, pAC343, pHIE324; transfection control: pPD1033; and fdler DNA: pHIE298 were used.

[0129] FIGS. 3A-3M show designs and validation of AAV vectors comprising exemplary transcriptional modulators as disclosed herein (see, e.g. Example 3). Bars show mean absolute fluorescence or mean % reporter+ cells of three biological replicates; error bars indicate S. E. M. In FIGS.3C, 3E, 3G-3I, calculated mean values of fold induction (reporter expression with ligand treatment divided by reporter expression without ligand treatment) are shown above each pair of bars. In FIGS.3D, 3F, 3L, calculated mean values of the percentage of live cells expressing the reporter are shown above each bar. Unpaired Welch’s t-test with Holm- Sidak method was used: n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. For FIGS. 3A-3F and FIGS. 3K-3M, AAV2 transfer vector plasmids: pAC360, pAC361, pAC362, pAC363, pAC364, pAC365, pAC366, pAC367, pAC368, pAC369, pAC370, pAC371, pAC372, pAC373, pAC374, pAC375, pAC377, pAC378; rep / cap plasmid: pLIO; and helper plasmid: pLl l were used. For FIG.3G - FIG. 3J, AAV2 transfer vector plasmids: pAC360, pAC361, pAC362, pAC363, pAC364, pAC365, pAC366, pAC367, pAC368, pAC369, pAC370, pAC371, pAC372, pAC373, pAC374, pAC375, pAC377, pAC378; transfection control: pPD1033; and fdler DNA: pHIE298 were used. See Tables 1 -7 for more information.

[0130] FIG.3A is a schematic showing two exemplary designs of transcriptional units for AAVs, including an exemplary transcriptional unit comprising an exemplary constitutive chicken P-actin promoter (CBA) promoter (“pCBA”), engineered transcriptional modulator (“mini-synTF”), and an exemplary bovine growth hormone polyadenylation signal (“bGH polyA”) (top) and an exemplary transcriptional unit comprising an engineered inducible promoter (“pSyn”), an exemplary reporter transgene, a hybrid intron31, and an exemplary simian virus 40 polyadenylation signal (“SV40 polyA tail”).

[0131] FIG.3B is a schematic showing three exemplary AAV vector configurations of transcriptional units such as those shown in FIG. 3 A (Upstream tandem “UT”, upstream divergent “UD”, and downstream divergent “DD”), wherein transcriptional units are flanked byinverted terminal repeats (“ITRs”) and, optionally, are separated by a chicken hypersensitive site 4 insulator core (“cHS4”).

[0132] FIGs. 3C - 3D are bar graphs showing transduction efficiency for and expression of an exemplary fluorescent reporter transgene (in absolute units of MEPTRs) in HEK293FT cells transduced with AAVs comprising exemplary “4OHT-on” engineered transcriptional modulators comprising an ERT2 regulatory element, a p65.2 transcription activation domain, and a ZF6 DNA binding element (in presence of EtOH control or 1 pM 4OHT) for designs with or without an insulator sequence (as shown in FIG. 3A) and for different configurations of transcription units (as shown in FIG. 3B). FIG. 3C shows reporter expression in MEPTRs. FIG. 3D shows transduction efficiency expressed as a percentage of live cells expressing the reporter. For both FIG. 3C and 3D, HEK293FT cells were transduced with a viral dose of 1×106vg / cell; cells received ligand treatment at time of transduction as well as 72 h later. Cells were analyzed 5 d post-transduction. AAV-mediated reporter expression levels (dsRedExpress2) were quantified via flow cytometry. Bars show mean absolute fluorescence or transduction efficiency of three biological replicates; error bars indicate S. E. M. Fold induction (reporter expression with ligand treatment divided by reporter expression without ligand treatment) or transduction efficiency (percentage of live cells expressing the reporter) values are indicated above each bar for that condition pair.

[0133] FIGs. 3E - 3F are bar graphs showing transduction efficiency and expression of an exemplary fluorescent reporter transgene (in absolute units of MEPTRs) in HEK293FT cells transduced with AAVs comprising exemplary “GZV-on” engineered transcriptional modulators comprising an NS3p regulatory element, a p65.2 transcription activation domain, and a ZF6 DNA binding element (in presence of DMSO vehicle control or IpM GZV) for designs with or without an insulator sequence (as shown in FIG. 3A) and for different configurations of transcription units (as shown in FIG. 3B). FIG.3E shows reporter expression in MEPTRs. FIG.3F shows transduction efficiency expressed as a percentage of live cells expressing the reporter. For both FIG. 3E and 3F, HEK293FT cells were transduced with a viral dose of 1×106vg / cell; cells received ligand treatment at time of transduction as well as 72 h later. Cells were analyzed 5 d post-transduction. AAV-mediated reporter expression levels (dsRedExpress2, in this case) were quantified via flow cytometry. Bars show mean absolute fluorescence or transduction efficiency of three biological replicates; error bars indicate S. E. M. Fold induction (reporter expression withligand treatment divided by reporter expression without ligand treatment) or transduction efficiency (percentage of live cells expressing the reporter) values are indicated above each bar for that condition pair.

[0134] FIGs. 3G -3M show validation of AAV transfer vectors and vector configurations using transient transfection into HEK293FT cells with plasmids encoding ITR-containing AAV transfer vectors comprising exemplary engineered transcriptional modulators. Three different vector configurations (UT, UD, DD)were tested for each type of engineered transcriptional modulator (see schematic in FIG. 3A), as well as inclusion of a cHS4 insulator between the constitutive and inducible transcriptional units (see schematic in FIG. 3A). FIG.3G is a bar graph showing expression of an exemplary fluorescent reporter transgene (in absolute units of MEPTRs) for a “4OHT-on” engineered transcriptional modulator in presence of EtOH control or IpM 4OHT. FIG. 3H is a bar graph showing expression of an exemplary fluorescent reporter transgene (in absolute units of MEPTRs) with a “GZV-on” engineered transcriptional modulator in presence of DMSO vehicle control or IpM GZV FIG. 31 is a bar graph showing expression of an exemplary fluorescent reporter transgene (in absolute units of MEPTRs) with a “GZV-off’ engineered transcriptional modulator in presence of DMSO vehicle control or IpM GZV. FIG. 3J is a bar graph showing expression with a control vector CMV-driven reporter. FIG. 3K is a bar graph showing expression of an exemplary fluorescent reporter transgene (in absolute units of MEPTRs) with “GZV-off’ engineered transcriptional modulators in presence of DMSO vehicle control or IpM GZV. FIG. 3L is a bar graph showing transduction efficiency (expressed as a percentage of live cells expressing the reporter) of an exemplary fluorescent reporter transgene with “GZV-off’ engineered transcriptional modulators in presence of DMSO vehicle control or IpM GZV. FIG.3M is a pair of bar graphs showing expression (left) and transduction efficiency (right) with a control vector CMV-driven reporter used as a benchmark for FIGs. 3K and 3L.

[0135] FIGs. 4A- 4F are schematics and plots showing expression of an exemplary fluorescent reporter transgene (in absolute units of MEPTRs) via AAV2 transduction of HEK293FT cells with exemplary engineered transcriptional modulators (depicted schematically) as discussed in Example 3. All constructs were tested using a viral dose of 1×106vg / cell as determined via qPCR. Bars show mean absolute fluorescence of three biological replicates; error bars indicate S. E. M. Fold induction (reporter expression with ligand treatment divided by reporter expression without ligand treatment) values are indicated above each bar for that condition pair. UnpairedWelch’s t-test with Holm-Sidak method: n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. AAV2 transfer vector plasmids: pAC366, pAC371, pAC373, pAW026, pAW027, pAW028, pAW029, pAW030, pAW031, pAW032, pAW033, pAW034, pAWO35, pAW036, pAW037, pAW038; rep / cap plasmid: pLI0; and helper plasmid: pLI1 were used. See Tables 1 -7 for more information.

[0136] FIG 4Ais a bar graph showing expression for exemplary “4OHT-on” engineered transcriptional modulators comprising certain transcription modulation elements, including a VP64 transcription activation domain, a p65.1 transcription activation domain, a p65.2 transcription activation domain, or a RTA.2 transcription activation domain, in the presence of EtOH control or 1 pM 4OHT.

[0137] FIG 4B is a bar graph showing expression for exemplary “GZV-on” engineered transcriptional modulators comprising certain transcription modulation elements, including a VP64 transcription activation domain, a p65.1 transcription activation domain, a p65.2 transcription activation domain, or a RTA.2 transcription activation domain, in the presence of DMSO vehicle control or 1 pM GZV.

[0138] FIG. 4C is a schematic depicting an exemplary “4OHT-on” engineered transcriptional modulator (left) and an exemplary engineered (“synthetic”) promoter.

[0139] FIG. 4D is a bar graph showing expression from engineered systems depicted schematically in FIG. 4C, comprising “4OHT-on” engineered transcriptional modulators (in the presence of EtOH control or IpM 4OHT) comprising ZF6 DNA binding elements and engineered promoters with different numbers of binding sites to which the ZF6 binding elements bind.

[0140] FIG. 4E shows expression for exemplary “GZV-off ’ engineered transcriptional modulators comprising different regulatory elements and / or linkers, including: two human ODC-1 degrons (with 1 or 3 multimers), and NES (with a GGGGSx3 or GGSx5 linker, in the presence of DMSO vehicle control or IpM GZV.

[0141] FIG. 4F shows expression for a CMV-driven constitutive reporter control reference.

[0142] FIGs. 5A- 5C are bar plots showing expression of an exemplary fluorescent reporter transgene (in absolute units of MEPTRs) in SH-SY5Y cells following transduction by AAVdelivery of exemplary “4OHT-on” engineered transcriptional modulators or CMV control, following incubation for 96h with 1 p 40HT or EtOH control, as assessed with flow cytometry. All constructs were tested via AAV2 transduction of SH-SY5Y cells using a viral dose of 1×105vg / cell as determined via qPCR. AAV2 transfer vector plasmids: pAC366, pAC363, pAC364, pAC365; rep / cap plasmid: pLI0; and helper plasmid: pLI1 were used. See Tables for more information. Bars show mean absolute fluorescence of three biological replicates; error bars indicate S. E. M. Fold induction (reporter expression with ligand treatment divided by reporter expression without ligand treatment) values are indicated above each bar for that condition pair. Unpaired Welch’s t-test with Holm-Sidak method was used: n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. FIG. 5A shows expression for all single cells analyzed. FIG. 5B shows expression for reporter-positive cells transduced with 4OHT-on system AAV vectors, quantified via post-hoc analysis. FIG. 5C shows expression following AAV delivery of pCMV, used as a benchmark for on-state reporter expression.

[0143] FIGs. 6A-6M are plots showing gating strategy and beads conversion from MFI to absolute units for flow cytometry analysis.

[0144] FIGs. 6A- 6F are plots showing gating strategy for transfected HEK293FT cells. FIG.6A is a scatter plot showing FSC-A and SSC-A for all events, with the gate for putative cells shown on the plot and the arrow above the plot indicating that all putative cells were carried through to the next step of the analysis shown in FIG. 6B. FIG. 6B is a scatter plot showing FSC-A and FSC-H for all cells from FIG. 6A, with single cells identified with the quadrilateral gate drawn approximately around the x=y line in the plot, and the arrows above the plot representing transition from all cells (left) in FIG. 6Ato mock transfection plotted in FIG. 6C.FIG 6C is a histogram of Pacific Blue channel fluorescence, using a mock transfection control to set the transfection gate for single cells determined as shown FIG. 6B (as indicated by arrow above plot), where the top 0.1 of negative control cells were included in the transfection gate. FIG. 6D is a histogram showing Pacific Blue MFI for single cells from FIG. 6B, gated as shown in FIG. 6C, with the arrow above the plot indicating that transfected cells as determined in this step were carried through to the next stage of analysis shown in FIG. 6E. FIG. 6E is a histogram of PE-TexasRed fluorescence using a transfection control to set the gate for transduced (reporter-i-) single cells, where the top 0.1% of negative control cells were included in thetransduced gate. FIG. 6F is a histogram showing PE-Texas Red MFI for all transfected cells, where 58.2% were determined to be transduced (“reporter-ON”).

[0145] FIGs. 6G - 6K show gating strategy for AAV transduction experiments (all cell lines).FIG. 6G is a scatter plot showing FSC-A and SSC-A for all events, with the gate for putative cells shown on the plot and the arrow above the plot indicating that all putative cells were carried through to the next step of the analysis shown in FIG. 6H. FIG. 6H is a scatter plot showing FSC-A and FSC-H for all cells from FIG. 6G, with single cells identified with the quadrilateral gate drawn approximately around the x=y line in the plot, and the arrows above the plot representing transition from all cells (left) in FIG. 6G to live cells as assessed as shown in FIG.61 FIG 61 is a scatter plot showing FSC-A and DAPI MFI, with the box in the figure showing cells that were determined to be live cells and arrows above the plot indicating the transition from single cells (left) in FIG. 6H to further analysis of live cells as shown in FIG. 6J. FIG. 6J is a histogram of PE-TexasRed fluorescence using a mock transduction control to set the gate for live transduced (reporter+) cells, where the top 0.1% of negative control cells were included in the transduced gate. FIG. 6K is a histogram showing PE-Texas Red MFI for all live cells, where 72.1% were determined to be transduced (“reporter+”).

[0146] FIG. 6L and FIG. 6M show beads conversion from MFI to absolute units. FIG. 6L is a scatter plot showing FSC-A and SSC-A, used to identify and gate beads, and a gate was drawn tightly around them to exclude any doublets or debris. FIG. 6M is a scatter plot showing bead MFI for PE and Pacific-Blue), from which 9 peaks were identified and gated upon (black boxes in figure). Fluorescence in the PE-TexasRed channel was extracted from each peak and used to create a standard curve using manufacturer provided fluorophore values used to convert MFI to absolute units.

[0147] FIGs. 7A- 7C are bar graphs showing expression of fluorescent reporter (in absolute units of MEPTRs) in HEK293FT cells following delivery of exemplary engineered transcriptional modulators comprising mechanosensitive transcription modulation elements, as assessed with flow cytometry. FIG. 7A shows expression for exemplary “GZV-on” engineered transcriptional modulators comprising mechanosensitive transcription modulation elements (MRTF-A, eNFR2, 3x9aa, and combinations; see Mahata 2023) compared to a p65.2 transcription modulation element delivered by transient transfection. FIG. 7B shows expressionfor exemplary “GZV-on” engineered transcriptional modulators comprising an MRTF-A (left) or a p65.2 (right) transcription modulation element, delivered by AAV transduction, in the presence of DMSO vehicle control or IpM GZV FIG. 7C shows expression for exemplary “4OHT-on” engineered transcriptional modulators comprising an MRTF-A (left) or a p65.2 (right) transcription modulation element, delivered by AAV transduction, in the presence of EtOH control or IpM 40HT. In FIGs. 7B and 7C, AAV transfer vectors were engineered in an upstream tandem (UT), and upstream divergent (UD) configuration with (+ins) or without (-ins) an insulator. All constructs were tested using a viral dose of 1×106vg / cell as determined via qPCR. Bars show mean absolute fluorescence of three biological replicates; error bars indicate S. E. M. Fold induction (reporter expression with ligand treatment divided by reporter expression without ligand treatment) values are indicated above each bar for that condition pair. Unpaired Welch’s t-test with Holm-Sidak method: n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0148] FIGs. 8A- 8C show exemplary engineered transcriptional modulator amino acid sequences with annotation of various elements. FIG. 8A shows the amino acid sequence (SEQ ID NO: 19) of an exemplary 4OHT-on engineered transcriptional modulator comprising from 5’ to 3’: an ERT2 regulatory element, a p65.2 transcription activation element, a GGGGSx3 linker, and a ZF6 DNA binding element. FIG. 8B shows the amino acid sequence (SEQ ID NO: 22) of an exemplary GZV-on engineered transcriptional modulator comprising from 5’ to 3’: a ZF6 DNA binding element, an NS3p regulatory element, a p65.2 transcription activation element, and an SV40 nuclear localization sequence. FIG. 8C shows the amino acid sequence (SEQ ID NO: 33) of an exemplary GZV-off engineered transcriptional modulator comprising from 5’ to 3’: three copies of a hODC-1 regulatory element, a GGGGSx3 linker, an NS3p regulatory element, an SV40 nuclear localization sequence, a VP64 transcription activation domain, a GGGGSx3 linker, and a ZF6 DNA binding element.DETAILED DESCRIPTION

[0149] Gene therapies can be useful in delivering a payload (also referred to herein as a gene of interest or a transgene) to a particular tissue and / or organ. However, several limitations exist in currently available gene therapies including the inability to fine-tune payload expression, e.g.,based on a subject’s response to a therapy. While there are existing systems that can be used to modulate payload expression, such systems require additional elements (e.g., regulatable elements) to be present in the delivered gene therapy. This is particularly challenging due to the constraints on packaging size of DNAthat can be encapsidated in viral particles, e.g., AAV particles. For example, due to packaging requirements, the maximum length of DNAthat can be delivered with an AAV based system is about 4.7kb. Currently available systems to modulate payload expression include inducible transcription factor-based systems that are relatively large and include many elements all of which are required to ensure tunability of such systems.Accordingly, use of such systems in a nucleic acid construct (e.g., for use in an AAV particle) that can only be 4.7kb in length at most restricts the size of a payload. This imposed limit on payload length can preclude the use of many therapeutic transgenes in gene therapy delivering such payloads or reduce the efficacy of such gene therapies, making such expression control systems unsuitable for addressing many needs in gene therapy.

[0150] This disclosure recognizes these challenges and provides technologies that can address such challenges. Technologies provided herein include engineered transcriptional modulators, e.g. comprising one or more promoters that are sufficiently compact (e.g., encoded by a nucleic acid sequence less than 1.8 kb in length) such that they can be encoded by a nucleic acid construct that also includes a gene of interest and a transcriptional regulatory sequence sufficient to render such gene of interest responsive to such encoded engineered transcriptional modulator while remaining small enough to be delivered by AAV-based technologies. In particular embodiments, as described further herein below, a compact engineered transcriptional modulator provided and / or utilized in accordance with the present disclosure is so compact that a gene encoding it can be included on the same construct as an expressible gene of interest and appropriate regulatory sequences that render such gene of interest under control of the compact engineered transcriptional modulator, and the whole construct is still of a size amenable to AAV-based delivery.

[0151] Accordingly, disclosed herein are novel synthetic regulators of transgene expression (also referred to herein as engineered transcriptional modulators). This disclosure enables, among other things, the construction of gene and cell therapies in which transgene expression can be controlled by administration of known drugs, such as FDA-approved drugs. The disclosed systems are unique due, at least, to their relative genetic compactness, which render themcompatible with numerous vectors, including size-limited vectors such as those based upon adeno-associated virus (AAV).

[0152] Unlike natural promoters, genetic elements disclosed herein can signal via transcription factors of the Composable Mammalian Elements of Transcription (COMET) platform, which utilizes synthetic zinc finger-based transcription factors. For example, a typical COMET platform comprises one or more engineered proteins that regulate gene expression, including both activation and suppression of gene expression, and engineered DNA sequences that are regulated by these engineered proteins. Both the proteins and the cognate DNA sequences are modular in design, enabling tuning of the quantitative performance of the system and / or to multiplex these elements to build sophisticated, customized, cellular functions.

[0153] Using, among other things, a minimal zinc-finger protein architecture, the present disclosure provides genetically compact regulators of transgene expression for use in AAV vectors, including delivery to neurons. This disclosure is the first to show novel and non-obvious improvements to minimal zinc finger transcription factors based on the COMET system that can be used to make novel miniature synthetic transcription factors (mini-synTFs) that are controllable by small molecules, e.g., FDA-approved drugs. In particular, the present disclosure shows the generation and characterization of a library of drug-regulated mini-synTFs. As described in the Examples herein, mini-synTF systems were built and validated in AAV transgene expression systems. The present disclosure also shows evaluation of mini-synTF control in neuronal cells, e.g., with a profile of mini-synTF control of transgene expression in neuronal cell lines.

[0154] Among other things, the present disclosure demonstrates the feasibility of multiple modalities of minimal, drug-controllable regulation of transgene expression using mini-synTFs. The presently disclosed mini-synTF controllers are more compact than any existing system (<1.8 kb), leaving substantial capacity for one or more payload transgenes. Further, the disclosed mini-synTF controllers exhibit desirable performance characteristics when deployed in AAV vectors (e.g., minimal background expression, induced expression comparable to constitutive promoters). In some embodiments, the disclosed mini-synTF controllers can be optimized, based on design choice, for any drug-controllable system.T. Definitions

[0155] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0156] Technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise defined. Unless otherwise specified, materials and / or methodologies known to those of ordinary skill in the art can be utilized in carrying out the methods described herein, based on the guidance provided herein.

[0157] A: As used herein, the singular terms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Reference to an object in the singular is not intended to mean “one and only one” unless explicitly so stated, but rather “one or more.”

[0158] About: As used herein, “about” when used with a numerical value means the numerical value stated as well as plus or minus 10% of the numerical value. For example, “about 10” should be understood as both “10” and “9-11.”

[0159] A / B: As used herein, a phrase in the form “A / B” or in the form “A and / or B” means (A), (B), or (A and B); a phrase in the form “at least one of A, B, and C” means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C).

[0160] Agent: As used herein, the term “agent”, may refer to a physical entity or phenomenon. In some embodiments, an agent may be characterized by a particular feature and / or effect. In some embodiments, as will be clear from context, the term agent may be used to refer to an entity (e.g., for example, a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc., or complex, combination, mixture or system [e.g., cell, tissue, organism] thereof) and / or a phenomenon (e.g., heat, electric current or field, magnetic force or field, etc., or combination thereof). In some embodiments, the term “agent” may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymeric moiety. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantially free of any polymer or polymeric moiety. In some embodiments, an agent may be or comprise a system or device. In someembodiments, an agent may be or comprise information, e.g., a piece or collection of input or output data. In some embodiments, an agent may be or comprise a force such as an electric force, a gravitational force, a magnetic force, etc. In many embodiments of the present disclosure, an agent is an entity (e.g., a small molecule) whose presence correlates with activity of an engineered transcriptional modulator as described herein.

[0161] Associated: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and / or form of one is correlated with that of the other. For example, in some embodiments, of the present disclosure, activity of an engineered transcriptional modulator is associated with presence of a particular small molecule agent. In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0162] Compact: As used herein, the terms “compact” and “spaced” architecture refer to the spacing between two or more mini-synTF binding sites. A “compact” architecture has less than about 10 bp between mini-synTF binding sites, and a “spaced” architecture has greater than about 20 bp between mini-synTF binding sites. For example, pHIE467 has 6 “compact” mini-synTF binding sites, spaced approximately 7 bp apart, whereas pAC317 has 6 “spaced” mini-synTF binding sites, spaced approximately 23 bp apart. See Table 4 and Table 5 for more information on which constructs have “compact” or “spaced” mini-synTF binding sites.

[0163] Comparable: As used herein, the term “comparable” refers to two or more agents (e.g., entities or set(s) of conditions), situations, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, whatdegree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0164] Divergent: As used herein, the term “divergent” refers to configuration of a constitutively expressed mini-synTF construct and an inducible reporter construct within an AAV vector, as shown, for example, in FIG. 3B. Divergent configurations have constructs inserted into a vector in opposite 5’-3 ’ directions. “Upstream” configurations (i.e., “upstream tandem” (UT) and “upstream divergent” (UD) have a mini-synTF construct inserted into a vector before a reporter construct (in the 5’-3’ direction), and “downstream” configurations (i.e., “downstream tandem” (DT) and “downstream divergent” (DD) have a reporter construct inserted into a vector before a mini-synTF construct (in the 5’-3’ direction).

[0165] Engineered: In general, the term “engineered” refers to the aspect of having been designed and / or manipulated by the hand of man. For example, a polynucleotide or polypeptide is considered to be “engineered” when two or more nucleic acid or amino acid sequences that are not linked together in that order in nature are designed and / or manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide or polypeptide and / or when a particular residue in a polynucleotide or polypeptide is non-naturally occurring and / or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature. For example, in some embodiments described and / or utilized herein, an engineered polynucleotide comprises a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence. In some embodiments, a polypeptide may be considered to be “engineered” if encoded by or expressed from an engineered polynucleotide, and / or if produced other than by natural expression (e.g., of an endogenous gene) in a cell. Analogously, a cell or organism is considered to be “engineered” if it has been subjected to a manipulation, so that its genetic, epigenetic, and / or phenotypic identity is altered relative to an appropriate reference cell such asotherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation, so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). In some embodiments, an engineered cell is one that has been manipulated so that it contains and / or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and / or a particular form thereof) in an altered amount (e.g., at all) and / or according to altered timing relative to such an appropriate reference cell. As is common practice and is understood by those in the art, progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a parent entity.

[0166] Mini-synTF: As used herein, the term “mini-synTF” refers to minimal inducible synthetic transcription factors of the present disclosure comprising an activation domain and a DNA binding element. In some embodiments, a mini-synTF further comprises a regulatory element. A DNA binding element (e.g., a zinc finger element) in a mini-synTF can bind to a transcription factor binding site, e.g., a particular DNA sequence on a nucleic acid sequence encoding a payload. Mini-synTF’s ability to modulate, e.g., initiate, transcription is regulated by responsiveness of a regulatory element in a mini-synTF to small molecules, e.g., drugs, optionally FDA-approved drugs, which can result in increased or decreased: (i) degradation, (ii) nuclear export, (iii) nuclear translocation, and / or (iv) activity, of the mini-synTF. A mini-synTF is also referred to herein as an engineered transcriptional modulator.

[0167] Polypeptide: As used herein, the term “polypeptide,” which is interchangeably used herein with the term “protein,” refers to a polymer of at least three amino acid residues. In some embodiments, a polypeptide comprises one or more, or all, natural amino acids. In some embodiments, a polypeptide comprises one or more, or all non-natural amino acids. In some embodiments, a polypeptide comprises one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, a polypeptide comprises one or more modifications such as acetylation, amidation, aminoethylation, biotinylation, carb amyl ati on, carbonylation, citrullination, deamidation, deimination,eliminylation, glycosylation, lipidation, methylation, pegylation, phosphorylation, sumoylation, or combinations thereof. In some embodiments, a polypeptide may participate in one or more intra- or inter-molecular disulfide bonds. In some embodiments, a polypeptide participates in non-covalent complex formation by non-covalent or covalent association with one or more other polypeptides (e.g., as in an antibody). In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a member of a polypeptide class or family shows sequence homology or significant sequence identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class. For example, in some embodiments, a member polypeptide shows an overall degree of sequence identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may in some embodiments comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region has a function, e.g., an evolutionarily conserved function. In some embodiments, a useful polypeptide may comprise a fragment of or a variant of a parent polypeptide. In some embodiments, a useful polypeptide may comprise a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and / or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.

[0168] Recipient Cell: The term “recipient cell” is used herein to refer to a cell that has received a heterologous nucleic acid or polypeptide. In some embodiments, a recipient cell is a cell into which a nucleic acid construct encoding an engineered transcriptional modulator and / or one or more payloads as described herein has been introduced, for example by gene therapy.

[0169] Reference: As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will recognize when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0170] Small molecule: As used herein, the term “small molecule” means a low molecular weight organic and / or inorganic compound. In general, a “small molecule” is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, a small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not and / or does not comprise a protein or polypeptide (e g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not and / or does not comprise a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not and / or does not comprise a polysaccharide; for example, in some embodiments, a small molecule is not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, a small molecule is not a lipid. Those of ordinary skill in the art, reading the present disclosure, will appreciate that certain small molecule compounds described herein may be provided and / or utilized in any of a variety of forms such as, for example, crystal forms, salt forms, protected forms, pro-drug forms, ester forms, isomeric forms (e g., optical and / or structural isomers), isotopic forms, etc. Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more stereoisomeric forms. In some embodiments, such a small molecule may be utilizedin accordance with the present disclosure in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers; in some embodiments, such a small molecule may be utilized in accordance with the present disclosure in a racemic mixture form. Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more tautomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual tautomer, or in a form that interconverts between tautomeric forms. Those of skill in the art will appreciate that certain small molecule compounds have structures that permit isotopic substitution (e.g.,2H or3H for H;11C,13C or14C for 12C;13N or15N for 14N;17O or18O for 16O;36Cl for35Cl;18F for19F;131I for127I; etc.). In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in one or more isotopically modified forms, or mixtures thereof. In some embodiments, reference to a particular small molecule compound may relate to a specific form of that compound. In some embodiments, a particular small molecule compound may be provided and / or utilized in a salt form (e.g., in an acid-addition or base-addition salt form, depending on the compound); in some such embodiments, the salt form may be a pharmaceutically acceptable salt form. In some embodiments, where a small molecule compound is one that exists or is found in nature, that compound may be provided and / or utilized in accordance in the present disclosure in a form different from that in which it exists or is found in nature. In some embodiments of the present disclosure, a small molecule compound has been evaluated and / or approved for use in humans and / or domestic animals, for example, by an appropriate regulatory agency such as the United States Food and Drug Administration. In some embodiments of the present disclosure, an agent that regulates an engineered transcriptional modulator as described herein is a small molecule; in some such embodiments, such small molecule has been evaluated and / or approved for use in humans and / or domestic animals, for example, by an appropriate regulatory agency such as the United States Food and Drug Administration. In some embodiments, such a small molecule has been approved for administration to humans, for example by the United States Food and Drug Administration.

[0171] Spaced: As used herein, the terms “compact” and “spaced” architecture refers to the spacing between two or more mini-synTF binding sites. A “compact” architecture has less than about 10 bp between mini-synTF binding sites, and a “spaced” architecture has greater thanabout 20 bp between mini-synTF binding sites. For example, pHIE467 has 6 “compact” mini-synTF binding sites, spaced approximately 7 bp apart, whereas pAC317 has 6 “spaced” mini-synTF binding sites, spaced approximately 23 bp apart. See Table 4 and Table 5 for more information on which constructs have “compact” or “spaced” mini-synTF binding sites.

[0172] Tandem: As used herein, the term “tandem” refers to configuration of a constitutively expressed mini-synTF construct and an inducible reporter construct within an AAV vector, as shown, for example, in FIG. 3B. Tandem configurations have constructs inserted into a vector in the same 5 ’-3’ direction.II. Engineered Transcriptional Modulators

[0173] The present disclosure provides engineered transcriptional modulators (e.g., synthetic transcription factors), as described herein.

[0174] Among other things, the present disclosure provides engineered transcriptional modulators characterized by one or more of: (a) compact size; (b) responsiveness to exogenous control, e.g., presence, level, form, etc. of a particular agent such as a small molecule compound which, in certain embodiments, may be approved for administration to humans and / or domestic animals or which can be used in experimental animal models; (c) human or humanized elements (some or all), and (d) potential for multiplexing using distinct transcriptional modulators targeting distinct regulatory sequences.

[0175] Typically, an engineered transcriptional modulator provided by and / or utilized in accordance with the present disclosure includes each of a DNA binding element, a transcription modulation element, a regulatory element, and optionally one or more linkers.

[0176] In some embodiments, one or more elements included in an engineered transcriptional modulator as described herein is a human element, or a humanized element, e.g., so that predicted immunogenicity may be reduced if such engineered transcriptional modulator is utilized in a human subject. In some embodiments, all elements included in an engineered transcriptional modulator are human or humanized. Alternatively, if a particular engineered transcriptional modulator is for use in a non-human animal, one or more, or in some embodiments, all elements of an engineered transcriptional modulator are from such non-human animal, or are modified to reduce immunogenicity in such non-human animal (e.g., are canine orcaninized for use in dogs, feline or felinized for use in cats, equine or equinized for use in horses, etc).

[0177] Provided engineered transcriptional modulators are characterized by compact size -typically below 70 kDa. In some embodiments, engineered transcriptional modulators are approximately 45 kDa in size. The present disclosure surprisingly documents the feasibility of achieving effective, exogenously-controlled transcriptional modulation with such a small engineered transcriptional modulator, and provides various representative examples of effective systems.

[0178] In some embodiments, length of a nucleic acid sequence encoding an engineered transcriptional modulator includes length of a nucleic acid sequence encoding an engineered transcriptional modulator and, optionally, one or more additional elements. In some embodiments, a nucleic acid encodes an engineered transcriptional modulator comprising (a) a DNA binding element and (b) a transcription modulation element, and optionally (c) one or more regulatory elements. In some embodiments, a nucleic acid encodes (i) an engineered transcriptional modulator comprising (a) a DNA binding element and (b) a transcription modulation element, and optionally (c) one or more regulatory elements, and further encodes (iii) a promoter and / or one or more regulatory elements for (a)-(c) and / or one or more payloads.

[0179] In some embodiments, a length of a nucleic acid sequence encoding an engineered transcriptional modulator and optionally one or more additional elements is less than about 1.8kb. In some embodiments, a length of a nucleic acid sequence encoding an engineered transcriptional modulator and optionally one or more additional elements is less than about 1.8kb, less than about 1.7kb, less than about 1.6kb, less than about 1.5kb, less than about 1.4kb, less than about 1.3kb, less than about 1.2kb, less than about l.lkb, less than about Ikb, less than about 0.9kb, less than about 0.8kb, less than about 0.7kb, less than about 0.6kb, or less than about 0.5kb.

[0180] In some embodiments, a length of a nucleic acid sequence encoding an engineered transcriptional modulator and optionally one or more additional elements is less than 1.8kb. In some embodiments,, a length of a nucleic acid sequence encoding an engineered transcriptional modulator and optionally one or more additional elements is less than 1.8kb, less than 1.7kb, lessthan 1,6kb, less than 1,5kb, less than 1 4kb, less than 1,3kb, less than 1,2kb, less than 1.1 kb, less than Ikb, less than 0.9kb, less than 0.8kb, less than 0.7kb, less than 0.6kb, or less than O.5kb.

[0181] In some embodiments, a length of a nucleic acid sequence encoding an engineered transcriptional modulator and optionally one or more additional elements is between about 0.5kb to about 3.5kb, including the endpoints of the range. In some embodiments, a length of a nucleic acid sequence encoding an engineered transcriptional modulator and optionally one or more additional elements is about 0.5kb, about 0.6kb, about 0.7kb, about 0.8kb, about 0.9kb, about l. Okb, about l.lkb, about 1.2kb, about 1.3kb, about 1.4kb, about 1.5kb, about 1.6kb, about 1.7kb, about 1.8kb, about 1.9kb, about2.0kb, about 2. Ikb, about 2.2kb, about 2.3kb, about 2.4kb, about 2.5kb, about 2.6kb, about 2.7kb, about 2.8kb, about 2.9kb, about 3.0kb, about 3. Ikb, about 3.2kb, about 3.3 kb, about 3.4kb or about 3.5kb.

[0182] In some embodiments, a length of a nucleic acid sequence encoding an engineered transcriptional modulator and optionally one or more additional elements is between 0.5kb and 3.5kb, including the endpoints of the range. In some embodiments, a length of a nucleic acid sequence encoding an engineered transcriptional modulator and optionally one or more additional elements is 0.5kb, 0.6kb, 0.7kb, 0.8kb, 0.9kb, l. Okb, l.lkb, 1.2kb, 1.3kb, 1.4kb, 1.5kb, 1.6kb, 1.7kb, 1.8kb, 1.9kb, 2.0kb, 2.1kb, 2.2kb, 2.3kb, 2.4kb, 2.5kb, 2.6kb, 2.7kb, 2.8kb, 2.9kb, 3.0kb, 3. Ikb, 3.2kb, 3.3 kb, 3.4kb or3.5kb.

[0183] The present disclosure documents that, in certain embodiments, provided compact engineered transcriptional modulators can be developed that achieve transgene expression levels comparable to that achieved with an appropriate constitutive control system. Alternatively or additionally, in some embodiments, provided compact engineered transcriptional modulators can achieve dramatic activation, in some embodiments 200-fold or more.

[0184] In some embodiments, provided compact engineered transcriptional modulators are characterized by significant differences in expression and / or activity of a regulated gene of interest (a regulated transgene) as described herein between an “off-” state as compared with an “on-” state. For example, in some embodiments, detected expression and / or activity is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, atleast 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 90-fold, at least 95-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold or more that detected in the “off’ state. In some embodiments, detected expression and / or activity is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold or more that detected in the “off’ state.

[0185] Among other things, the present disclosure provides insights relating to design of particularly effective and / or useful compact engineered transcriptional modulators, including documenting that certain design feature choices can confer surprising combinatorial effects not obvious or reasonably predictable based upon contributions of individual choices.

[0186] Without wishing to be bound by any particular theory, the present disclosure proposes that certain features of provided compact engineered transcriptional modulators, including in some instances, combinatorial impacts of design features, may be driven by geometric constraints for co-factor and / or DNA binding, which may, in some embodiments, vary with activation domain choice. For example, as described herein, in some instances, presence and / or one or more features (e.g., length, flexibility vs rigidity, orientation, etc.) of a linker between relevant elements (e.g., between a DNA binding element and a transcription modulation element and / or between a DNA binding element or a transcription modulation element and a regulatory element) may impact ability of an engineered transcriptional modulator as described herein to accomplish DNA binding, transcriptional modulation (e.g., recruitment of and / or other interaction with one or more component of the transcriptional machinery and its co-factors), and / or responsiveness to regulation, e.g., by a small molecule agent as described herein.DNA Binding Elements

[0187] Those skilled in the art are aware of a variety of polypeptide structures that impart DNA-binding capability, including many with well-characterized binding sites. Exemplary DNA-binding structures include, for example, helix-turn-helix, homeodomain, basic region leucine zipper, nuclear hormone receptor, and zinc finger structures. See, for example, Lambert et al.,Cell 172:650, 2018). Further, those with ordinary skill in the art will appreciate that polypeptide structures with particular DNA binding capabilities can bind to particular sequences in DNA, e.g., a motif or a consensus.

[0188] While teachings of the present disclosure need not be limited to engineered transcriptional modulators with a zinc finger DNA binding element, the present disclosure documents particular effectiveness of such zinc finger binding elements, and their surprising effectiveness even when sufficiently small to be included in a compact engineered transcriptional modulator as described herein, and particularly in a compact engineered transcriptional modulator that includes a regulatory element (e.g., a small-molecule responsive regulatory element) as described herein.

[0189] The present disclosure demonstrates the surprising results that a compact engineered transcriptional modulator comprising ZF6 outperformed a compact engineered transcriptional modulator comprising ZF1 across modalities. For example, as shown in Example 3 in the “Sensitivity Analysis of Mini-synTF Design Choices.”

[0190] In some embodiments, a DNA binding element utilized in accordance with the present disclosure is a zinc-finger element. In some embodiments, a utilized zinc-finger element is or comprises one or more ofZFl, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, and / or ZF19. In some embodiments, a zing-finger element is or comprises a combination of two or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, and / or ZF19.

[0191] In some embodiments, a zinc finger DNA binding element comprises one or more ZF 1. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF1, e.g., one or more ofZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19. In some embodiments, ZF1 comprises the amino acid sequence of SEQ ID NO: 97 or an amino acid sequence having at least 85% identity thereto.

[0192] In some embodiments, a zinc finger DNA binding element comprises one or more ZF2. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF2, e.g., one or more ofZFl, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0193] In some embodiments, a zinc finger DNA binding element comprises one or more ZF3. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF3, e.g., one or more ofZFl, ZF2, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0194] In some embodiments, a zinc finger DNA binding element comprises one or more ZF4. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF4, e.g., one or more ofZFl, ZF2, ZF3, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0195] In some embodiments, a zinc finger DNA binding element comprises one or more ZF5. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF5, e.g., one or more ofZFl, ZF2, ZF3, ZF4, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0196] In some embodiments, a zinc finger DNA binding element comprises one or more ZF6. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF6, e.g., one or more ofZFl, ZF2, ZF3, ZF4, ZF5, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19. In some embodiments, ZF6 comprises the amino acid sequence of SEQ ID NO: 84 or an amino acid sequence having at least 85% identity thereto.

[0197] In some embodiments, a zinc finger DNA binding element comprises one or more ZF7. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF7, e.g., one or more ofZFl, ZF2, ZF3, ZF4, ZF5, ZF6, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0198] In some embodiments, a zinc finger DNA binding element comprises one or more ZF8. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF8, e.g., one or more ofZFl, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0199] In some embodiments, a zinc finger DNA binding element comprises one or more ZF9. In some embodiments, a zinc finger DNA binding element further comprises one or more otherZFs other than ZF9, e.g., one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0200] In some embodiments, a zinc finger DNA binding element comprises one or more ZF10. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF10, e.g., one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0201] In some embodiments, a zinc finger DNA binding element comprises one or more ZF11. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF 11, e.g., one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0202] In some embodiments, a zinc finger DNA binding element comprises one or more ZF12. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF12, e.g., one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0203] In some embodiments, a zinc finger DNA binding element comprises one or more ZF13. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF 13, e.g., one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0204] In some embodiments, a zinc finger DNA binding element comprises one or more ZF14. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF 14, e.g., one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0205] In some embodiments, a zinc finger DNA binding element comprises one or more ZF15. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF15, e.g., one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF16, ZF17, ZF18, or ZF19.

[0206] In some embodiments, a zinc finger DNA binding element comprises one or more ZF16. In some embodiments, a zinc finger DNA binding element further comprises one or more otherZFs other than ZF16, e.g., one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF17, ZF18, or ZF19.

[0207] In some embodiments, a zinc finger DNA binding element comprises one or more ZF17. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF17, e.g., one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF18, or ZF19.

[0208] In some embodiments, a zinc finger DNA binding element comprises one or more ZF18. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF 18, e.g., one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, or ZF19.

[0209] In some embodiments, a zinc finger DNA binding element comprises one or more ZF19. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF19, e.g., one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, or ZF18.

[0210] In some embodiments, a zinc finger DNA binding element binds to a 9-mer motif in a nucleic acid sequence. In some embodiments, a zinc finger DNA binding element (e.g., ZF1) binds to a GAGTGAGGA (SEQ ID NO: 101) sequence. In some embodiments, a zinc finger DNA binding element (e.g., ZF6) binds to a GTGTAGGGG (SEQ ID NO: 102) sequence.Transcription Modulation Element

[0211] An engineered transcriptional modulator for use in accordance with the present disclosure includes a transcription modulation element - i.e., an element that, when recruited to a relevant DNA binding site, e.g., by virtue of association with a DNA binding element as described herein, modulates (e.g., activates or represses) transcription of a transcribable sequence (i.e., a gene sequence) operatively associated with a DNA sequence to which the DNA binding element binds. In some embodiments, a transcription modulation element is a transcription activation element. In some embodiments, a transcription modulation element is a transcription repression element. Typically, when a transcription modulation element is a transcription activation element, binding of an engineered transcriptional modulator to its cognate DNA site correlates with increased expression of the operatively associated gene; when a transcription modulationelement is a transcription repression element, binding of an engineered transcriptional modulator including such transcription modulation element correlates with decreased expression.

[0212] Without wishing to be bound by any particular theory, it is proposed that, in many embodiments, a transcription modulation element binds to (e.g., directly) one or more elements of the transcription machinery.

[0213] Those skilled in the art are aware of a variety of polypeptide elements, including human and / or humanized polypeptide elements, that are described as having transcription modulation activity. For example, described transcription activation domains include those found in: VP64, p65.1, p65.2, RTA.2, VP16, VPR, p300, CBP, Hsfl, Swi / Snf, MED15, E1A, Gal4-AD, B42, and p65; described transcription repression domains include KRAB Domain (Kriippel-associated box), SID Domain (Sin3 Interaction Domain), POZ / BTB Domain, LSD1-binding Domain, Groucho / TLE Interaction Domain, embryonic ectoderm development domain (EED domain), DNA methyltransferase 3B domain (DNMT3B domain), histone deacetylase 4 domain (HDAC4 domain). In some embodiments, an engineered transcriptional modulator (e.g., a compact engineered transcriptional modulator) includes one or more of these transcription activation domains.

[0214] In some embodiments, a transcription activation domain is a VP64 domain, a p65.1 domain, a p65.2 domain, a RTA.2 domain, a VP16 domain, a VPR domain, a p300 domain, a CBP domain, a Hsfl domain, a Swi / Snf domain, a MED15 domain, a E1A domain, a Gal4-AD domain, a B42 domain, a p65 domain, or any biological equivalent thereof, or any variant thereof or any fragment thereof (e.g., functional fragment comprising transcriptional activation activity).

[0215] Certain mechanosensitive transcription factors (MTFs) are capable of modulating transcription in response to a mechanical cue or in response to an external ligand, wherein, as a result of stimulation, an MTF is transported (e.g., shuttled) to the nucleus, where it modulates target gene expression (e.g., by changing RNA polymerase II or histone modifiers). See, e.g., Mahata et al. (2023) Nature Methods, 20: 1716-1728. In some embodiments, a transcription activation domain is a mechanosensitive transcription element. In some embodiments, a mechanosensitive transcription element is YAP, TAZ, SRF, MRTF-A and -B, or MYOCD.Example 5 of the present disclosure shows activity, and effective small molecule control thereof,of compact engineered transcriptional modulators comprising mechanosensitive modulation elements.

[0216] In some embodiments, a transcription repressor domain is a KRAB Domain (Kriippel-associated box), SID Domain (Sin3 Interaction Domain), POZ / BTB Domain, LSD1-binding Domain, Groucho / TLE Interaction Domain, embryonic ectoderm development domain (EED domain), DNA methyltransferase 3B domain (DNMT3B domain), histone deacetylase 4 domain (HDAC4 domain), or any biological equivalent thereof, or any variant thereof or any fragment thereof (e.g., functional fragment comprising transcriptional activation activity).

[0217] In some embodiments, a transcriptional modulation element is a human element, or a humanized element, or a fragment of either of the foregoing.

[0218] The present disclosure demonstrates the surprising results that a compact engineered transcriptional modulator comprising a p65 transcriptional activation domain (e.g., human p65) outperformed compact engineered transcriptional modulators comprising other transcription activations domains, e.g., VP64 or RTA.2, e.g., as shown in FIG. 2D-2E.

[0219] This disclosure further demonstrates that the presence and / or size of a linker can modulate the activity of a compact engineered transcriptional modulator. The presence and / or size of a linker between a regulatory element and a DNA binding element or a linker between a regulatory element a transcription modulation element can modulate the activity of a compact engineered transcriptional modulator. For example, as shown in FIG. 2F, the presence and / or size of a linker between a regulatory element and a DNA binding element or between a regulatory element a transcription modulation element surprisingly altered the activity of the compact engineered transcriptional modulator even when the compact engineered transcriptional modulator had the same transcription modulation element and the only different is the presence and / or size of a linker.

[0220] In some embodiments, a transcription activation domain is or comprises a p65.1 domain or a fragment or variant thereof. In some embodiments, a transcription activation domain comprises the sequence of SEQ ID NO: 98, or a sequence having at least 85% identity thereto.

[0221] In some embodiments, a transcription activation domain is or comprises a VP64 domain or a fragment or variant thereof. In some embodiments, a transcription activation domain comprises the sequence of SEQ ID NO: 90, or a sequence having at least 85% identity thereto.

[0222] In some embodiments, a transcription activation domain is or comprises a p65.2 domain or a fragment or variant thereof. In some embodiments, a transcription activation domain comprises the sequence of SEQ ID NO: 91, or a sequence having at least 85% identity thereto.

[0223] In some embodiments, a transcription activation domain is or comprises a RTA.2 domain or a fragment or variant thereof. In some embodiments, a transcription activation domain comprises the sequence of SEQ ID NO: 99, or a sequence having at least 85% identity thereto. Regulatory Element

[0224] One feature of the present disclosure is that, in some embodiments, an engineered transcriptional modulator (e.g., a compact engineered transcriptional modulator) provided by and / or used in accordance with the present disclosure includes a regulatory element that confers upon the engineered transcriptional modulator responsiveness to exogenous control (e.g., to a small molecule agent, and in particular to a drug approved by one or more appropriate regulatory agencies for use in humans).

[0225] Those skilled in the art will be aware of various contexts in which exogenous control has been engineered into an aspect of gene expression. For example, drug-induced control of mRNA splicing has been described, including in the context of AAV-delivered gene therapy. See, for example, Monteys, et al. Nature 596:291, 2021; Chen, et al. bioRxiv, 2024.2007.2001.601517, 2024 (doi.org / 10.1101 / 2024.07.01.601517). Small-molecule-regulatable control of transcription factors has also been achieved, for example, through conformational change (e.g., the classic bacterial TetR-based systems (see, for example, Breger et al., in Gene Therapy for Neurological Disorders: Methods and Protocols (ed Fredric P. Manfredsson), 57-66 (Springer New York, 2016), dimerization (e.g., rapamycin control; see, for example, Donahue et al., Nature Comm. 11:779, 2020), drug-regulated control of integrated viral proteases (see, for example, Tague et al., Nature Met. 15:519, 2018; Li etal., Science 378:1227, 2022), and hormone regulated nuclear localization (e.g., via estrogen inhibitor Afimoxifene; see, for example, Li et al., Science 378:1227, 2022).

[0226] Moreover, specifically in the context of transcriptional modulators, certain polypeptide regulatory elements have been described that can impart exogenous regulation. That is, Khalil et al. (US Patent Number 11,530,246 to Boston University, issued Dec. 20, 2022 with a prior publication on Dec. 3, 2020 as US2020 / 0377564) have described “synthetic transcription factors” (’’synTFs”) that include (a) a DNA binding domain, (b) and effector domain; and (c) what is referred to as a “regulator protein” that is said to “contro[l] 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 or repress translation (sic, transcription) machinery to the promoter to regulate gene expression of a gene of interest”. See column 2, lines 8-15 of US Patent Number 11,530,246. Khalil etal., specifically describe as regulator proteins: (a) proteases (e.g., self-cleaving proteases whose self-cleavage is inhibited by a small molecule); (b) a pair of proximity domains (e g., that may become associated upon interaction with a small molecule; or (c) a translocation domain (e.g., a cytosolic sequestering protein whose activity is modulated by a small molecule). Khalil etal. specifically mention SMASh domains as induced degradation domains, e.g., comprising a self-cleaving protease domain and a degron domain, (e.g., a SMASh domain).

[0227] The present disclosure identifies the source of a problem with such small-molecule-responsive systems in that they are not well-suited for use in AAV-mediated gene therapies. For example, TetR systems often show high levels of off-state transgene expression, or “leakiness”, which is undesirable for most clinical applications. Moreover, packaging requirements limit the length of DNA that can be delivered (up to 4.7 kb); and the large size of many inducible transcription factor-based systems makes them unsuitable for use in AAV as it would unduly restrict transgene size.

[0228] The present disclosure also appreciates the validation of certain small-molecule responsive systems, specifically including the regulator protein strategies employed by Khalil et al in the context of full-length transcriptional modulators. See US Patent Number 11,530,246 to Boston University, issued Dec. 20, 2022 with a prior publication on Dec. 3, 2020 as US2020 / 0377564. The present disclosure surprisingly demonstrates that such strategies can be adapted for incorporation into compact engineered transcriptional modulators as described herein.

[0229] In some particular embodiments, the present disclosure combines validated strategies imparting exogenous responsiveness to small molecule agents, including to certain small molecule agents that are FDA approved drugs (e.g., grazoprevir (GZV) and 4-hydroxytamoxifen / tamoxifen (4OHT).

[0230] The present disclosure exemplifies three different drug-controllable modalities, and documents their successful use to control on- and off-switching of AAV-delivered transgene expression.

[0231] For example, the present disclosure demonstrates the surprising results that a compact engineered transcriptional modulator comprising a regulatory element such as a protease element or a nuclear localization signal is responsive to a small molecule regulator resulting in altered, e g., increased or decreased payload expression.

[0232] For example, a compact engineered transcriptional modulator comprising a regulatory domain comprising a NS3p protease element when used in the presence of the small molecule GZV resulted in over 200-fold increase in payload expression, see e.g., FIG. 3H. Such a system is also referred to herein as a GZV-on system.

[0233] As another example, a compact engineered transcriptional modulator comprising a regulatory domain comprising a protease element (e.g., aNS3p protease element) and one or more destabilization elements, e g., one or more degradation domains (e.g., degradation tag (degron)) or a nuclear export signal, can be used to turn off payload expression whereby in the presence of the small molecule GZV, payload expression is reduced with such a compact engineered transcriptional modulator, see e.g., FIG. 31. Such a system is also referred to herein as a GZV-off system.

[0234] As yet another example, use of a regulatory element comprising a nuclear localization signal, e.g., ERT2, in a compact engineered transcriptional modulator resulted in about 200-fold increase in payload expression in the presence of the small molecule 4-OHT (as shown in FIG.3G). Such a system is also referred to herein as a 4OHT-on system.

[0235] In some embodiments, an engineered transcriptional modulator disclosed herein comprises a regulatory element, e.g., as described herein. In some embodiments, regulatoryelement in an engineered transcriptional modulator polypeptide is situated between a DNA binding element and a transcription modulation element.

[0236] In some embodiments, a regulatory element confers upon an engineered transcriptional modulator responsiveness to exogenous control (e.g., to a small molecule agent, and in particular to a drug approved by one or more appropriate regulatory agencies for use in humans). In some embodiments, a regulatory element is responsive to a small molecule. For example, responsiveness of a regulatory element to a small molecule can include: stabilization of a regulatory element, change in (e.g., inhibition of) activity of a regulatory element, change in conformation (e.g., dimerization) of a regulatory element, change in localization of a regulatory element, or any combination thereof.

[0237] In some embodiments, a regulatory element can stabilize an engineered transcriptional modulator polypeptide in the presence of a small molecule by inhibiting degradation of an engineered transcriptional modulator polypeptide. In some such embodiments, a regulatory element is or comprises a protease (e.g., NS3p) that results in constitutive degradation of an engineered transcriptional modulator polypeptide. In the presence of a small molecule that blocks the activity of the protease, degradation of an engineered transcriptional modulator polypeptide is prevented resulting in stabilization of an engineered transcriptional modulator polypeptide. In some embodiments, a regulatory element comprises a NS3p protease or a fragment or variant thereof. In some embodiments, a small molecule that can be useful in stabilizing an engineered transcriptional modulator polypeptide comprising a NS3p protease as a regulatory element is one that can inhibit the protease activity of NS3p, e.g., grazoprevir.

[0238] In some embodiments, a regulatory element can destabilize an engineered transcriptional modulator polypeptide in the presence of a small molecule by promoting degradation of an engineered transcriptional modulator polypeptide. In some such embodiments, a regulatory element is or comprises a protease element (e.g., NS3p) and one or more destabilization elements (e.g., a degradation domain (e.g., degradation tag (degron)) or a nuclear export signal) wherein the one or more destabilization elements are cleaved by the protease resulting in constitutive expression of an engineered transcriptional modulator polypeptide. In the presence of a small molecule that blocks the activity of the protease (e.g., prevents degradation of one or more destabilization domains by the protease), destabilization of an engineered transcriptionalmodulator polypeptide by one or more destabilization elements is induced resulting in degradation of an engineered transcriptional modulator polypeptide and reduced expression of a payload. In some embodiments, a regulatory element comprises a NS3p protease or a fragment or variant thereof and one or more destabilization elements. In some embodiments, a destabilization element comprises a degradation domain, e.g., a ODC-1 degron. In some embodiments, a destabilization element comprises a nuclear export signal. In some embodiments, a small molecule that can be useful in destabilizing an engineered transcriptional modulator polypeptide comprising a NS3p protease and one or more destabilization elements as a regulatory element is one that can induce the protease activity of NS3p, e.g., grazoprevir.

[0239] In some embodiments, a NS3p protease comprises an amino acid sequence of SEQ ID NO: 85, or a sequence with at least 85% identity thereto.

[0240] In some embodiments, a regulatory element can induce dimerization of an engineered transcriptional modulator polypeptide, e.g., by promoting binding of one or more engineered transcriptional modulator polypeptides to each other in the presence of a small molecule.

[0241] In some embodiments, a regulatory element can alter the localization of an engineered transcriptional modulator polypeptide, e.g., by translocating an engineered transcriptional modulator polypeptide from a cytoplasm to a nucleus. In some such embodiments, a regulatory element is or comprises a peptide in the cytoplasm that upon binding to a ligand can translocate into the nucleus of a cell. In some embodiments, a regulatory element comprises a human estrogen receptor (ERT2) or a fragment or variant thereof. In some embodiments, a small molecule that can be useful in inducing nuclear localization of an engineered transcriptional modulator polypeptide comprising a regulatory element comprising ERT2 is small molecule that can bind to ERT2 and translocate it to the nucleus, e.g., 4-hydroxytamoxifen (4OHT).

[0242] In some embodiments, an ERT2 polypeptide comprises an amino acid sequence of SEQ ID NO: 86, or a sequence with at least 85% identity thereto.Linkers

[0243] Among insights provided by the present disclosure is that the use of one or more linkers or varying lengths can impact the activity and / or effectiveness of a compact engineered transcriptional modulator disclosed herein. This insight is particularly non-obvious based uponthe contributions of individual choice elements, highlighting the importance of performing sensitivity analyses, e.g., as described herein. This is especially relevant for features modulating mini-synTF geometry, which can impact binding to DNA and recruitment of transcriptional machinery and co-factors. For example, the GZV-on modality was more impacted by linker additions than the GZV-off modality, potentially indicating that addition of linkers between the NS3p and synTF parts can provide needed flexibility to enable coordination of transcriptional activation.

[0244] This disclosure further demonstrates that the presence and / or size of a linker can modulate the activity of a compact engineered transcriptional modulator. The presence and / or size of a linker between a regulatory element and a DNA binding element or a linker between a regulatory element a transcription modulation element can modulate the activity of a compact engineered transcriptional modulator. For example, as shown in FIG. 2F, the presence and / or size of a linker between a regulatory element and a DNA binding element or between a regulatory element a transcription modulation element surprisingly altered the activity of the compact engineered transcriptional modulator even when the compact engineered transcriptional modulator had the same transcription modulation element and the only different is the presence and / or size of a linker.

[0245] In some embodiments, a linker, e.g., depending on a size of a linker, can modulate the flexibility of a compact engineered transcriptional modulator. In some embodiments, a linker, e.g., depending on a size of a linker, modulate spatial configuration of compact engineered transcriptional modulator. In some embodiments, flexibility and / or spatial configuration of a compact engineered transcriptional modulator can be important for (i) providing access to a DNA binding site for binding by a DNA binding element, (ii) binding of a compact engineered transcriptional modulator to one or more co-factors and / or accessory proteins, (iii) recruitment of one or more factors for initiating transcription, or any combination thereof.

[0246] In some embodiments, an engineered transcriptional modulator comprising: (i) a DNA binding element (e.g., as described herein) and (ii) a transcriptional modulator element, comprises one or more linkers between (i) and (ii). In some embodiments, an engineered transcriptional modulator comprising: (i) a DNA binding element (e.g., as described herein), (ii) a transcriptional modulator element (e.g., as described herein), and (iii) a regulatory element(e g., as described herein), comprises one or more linkers disposed between (i) and (ii), (i) and (iii) and / or (ii) and (iii).

[0247] In some embodiments, a linker comprises about 2 to about 50, about 2 to about 45, about 2 to about 40, about 2 to about 35, about 2 to about 30, about 2 to about 25, about 2 to about 20, about 2 to about 15, about 2 to about 10, about 2 to about 5, about 3 to about 50, about 5 to about 50, about 10 to about 50, about 15 to about 50, about 20 to about 50, about 25 to about 50, about 30 to about 50, about 35 to about 50, about 40 to about 50, about 45 to about 50 amino acids.

[0248] In some embodiments, a linker is an unstructured linker.

[0249] Any suitable linker can be used in an engineered transcriptional modulator disclosed herein. Exemplary linkers known in the art that can be useful in an engineered transcriptional modulator include Gly-Ser linkers, and permutations thereof.

[0250] In some embodiments, a linker has an amino acid sequence according to any one of SEQ ID NO: 92, SEQ ID NO: 93, or SEQ ID NO: 95.

[0251] As used herein, the terms “solid,” “rigid,” and “flexible” refer to properties of linkers connecting various domains of a mini-synTF. It is well known in the art that the rigidity or flexibility of a linker is affected by factors such as length and / or amino acid composition of the linker. For example, linkers with higher serine content are predicted to be more rigid than linkers with less serine content.Other Elements

[0252] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, an engineered transcriptional modulator as provided by and / or utilized in accordance with the present disclosure may include one or more additional elements such as, for example, a localization signal, a pro element, a tag (e.g., a detectable tag, a purification tag, or another typically short oligopeptide, often a ligand for a binding agent such as an antibody).

[0253] In some embodiments, however, no other elements beyond a DNA binding element, a transcription modulation element, a regulatory element, and one or more linkers, is included in an engineered transcriptional modulator provided by and / or utilized in accordance with the present disclosure, given the priority of compact size as described herein.Exemplary Embodiments

[0254] In some embodiments, an engineered transcriptional modulator (e.g., a compact engineered transcriptional modulator) as provided by and / or utilized in accordance with the present disclosure includes a degradation domain (e.g., degradation tag (degron)) such as, for example, an ODC-1 sequence incorporated as 1, 2, 3, or 4 tandem repeats. In some embodiments, a degradation domain or an NES is cleaved off by a protease, thereby stabilizing the mini-synTF or retaining the mini-synTF in the nucleus. In some embodiments, such a protease is inhibited by an FDA-approved drug. In some embodiments, such a protease is an NS3 protease. In some embodiments, a protease is inhibited by GZV, and the mini-synTF is destabilized or exported from the nucleus. In some embodiments, a protease is located proximal to the AD. In other embodiments, the protease is located proximal to the DBD. In some embodiments, a mini-synTF does not comprise a degradation domain or NES, but includes a protease that cleaves off the AD or DBD, thereby destabilizing the mini-synTF. In some embodiments, such a protease is inhibited, thereby stabilizing the mini-synTF. In some embodiments, such a protease is inhibited by GVZ.

[0255] In other embodiments, a mini-synTF comprises a nuclear translocation domain proximal to a transcription modulation element (e.g., a transcription activation domain) or one or more DNA binding elements. In some embodiments, a nuclear translocation domain is activated by an FDA-approved drug, and a mini-synTF is translocated to the nucleus. In some embodiments, a nuclear translocation domain comprises an ERT2 sequence, and a corresponding FDA approved drug is 4OHT.

[0256] In some embodiments, a mini-synTF comprises a linker between a transcription modulation element (e.g., a transcription activation domain), one or more DNA binding elements, and one or more regulatory elements (e g., a degradation domain or NES, protease, and / or nuclear translocation domain). In some embodiments, a linker comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In some embodiments, an amino acid sequence of a mini-synTF comprises 1, 2, 3, 4, or 5 tandem repeats of amino acids GGS or GGGGS. In some embodiments, a linker is solid or rigid. In some embodiments, a linker is flexible.

[0257] The present disclosure provides synthetic promoters capable of binding mini-synTFs disclosed herein. In some embodiments, a synthetic promoter comprises 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or 12 mini-synTF binding sites before a minimal promoter. In some embodiments, mini-synTF binding sites are arranged in a compact architecture. In some embodiments, mini-synTF binding sites are arranged in a spaced architecture.

[0258] In some embodiments, the synTF has a protease configured to cleave the activation domain from the zinc finger domain, and the protease is inhibited by the drug to induce gene expression. In some embodiments, use of the drug is discontinued to suppress gene expression. In other embodiments, the protease is constitutively inactive, and the drug activates the protease to suppress gene expression. In some embodiments, use of the drug is discontinued to induce gene expression.

[0259] In some embodiments, an engineered transcriptional modulator polypeptide comprises an amino acid sequence provided in Table 1, or a sequence with at least 85% identity thereto.

[0260] In some embodiments, an engineered transcriptional modulator polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 1 - 37, or a sequence with at least 85% identity thereto.

[0261] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 1, or a sequence with at least 85% identity thereto.

[0262] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 2, or a sequence with at least 85% identity thereto.

[0263] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 3, or a sequence with at least 85% identity thereto.

[0264] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 4, or a sequence with at least 85% identity thereto.

[0265] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 5, or a sequence with at least 85% identity thereto.

[0266] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 6, or a sequence with at least 85% identity thereto.

[0267] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 7, or a sequence with at least 85% identity thereto.

[0268] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 8, or a sequence with at least 85% identity thereto.

[0269] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 9, or a sequence with at least 85% identity thereto.

[0270] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 10, or a sequence with at least 85% identity thereto.

[0271] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 11, or a sequence with at least 85% identity thereto.

[0272] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 12, or a sequence with at least 85% identity thereto.

[0273] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 13, or a sequence with at least 85% identity thereto.

[0274] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 14, or a sequence with at least 85% identity thereto.

[0275] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 15, or a sequence with at least 85% identity thereto.

[0276] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 16, or a sequence with at least 85% identity thereto.

[0277] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 17, or a sequence with at least 85% identity thereto.

[0278] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 18, or a sequence with at least 85% identity thereto.

[0279] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 19, or a sequence with at least 85% identity thereto.

[0280] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 20, or a sequence with at least 85% identity thereto.

[0281] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 21, or a sequence with at least 85% identity thereto.

[0282] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 22, or a sequence with at least 85% identity thereto.

[0283] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 23, or a sequence with at least 85% identity thereto.

[0284] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 24, or a sequence with at least 85% identity thereto.

[0285] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 25, or a sequence with at least 85% identity thereto.

[0286] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 26, or a sequence with at least 85% identity thereto.

[0287] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 27, or a sequence with at least 85% identity thereto.

[0288] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 28, or a sequence with at least 85% identity thereto.

[0289] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 29, or a sequence with at least 85% identity thereto.

[0290] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 30, or a sequence with at least 85% identity thereto.

[0291] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 31, or a sequence with at least 85% identity thereto.

[0292] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 32, or a sequence with at least 85% identity thereto.

[0293] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 33, or a sequence with at least 85% identity thereto.

[0294] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 34, or a sequence with at least 85% identity thereto.

[0295] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 35, or a sequence with at least 85% identity thereto.

[0296] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 36, or a sequence with at least 85% identity thereto.

[0297] In some embodiments, an engineered transcriptional modulator polypeptide comprises the amino acid sequence of SEQ ID NO: 37, or a sequence with at least 85% identity thereto.III. Expression ConstructsNucleic acid construct encoding compact engineered transcriptional modulators

[0298] The present disclosure demonstrates the surprising feasibility of achieving effective transcriptional modulation with delivery and / or expression systems comprising an unusually compact sized engineered transcriptional modulators and a payload of interest. In some embodiments, an engineered transcriptional modulator disclosed herein is sufficiently compact that it can be encoded by a nucleic acid construct that also includes one or more payloads, e.g., gene of interest, and regulatory sequences sufficient to render such payloads responsive to such encoded engineered transcriptional modulator while remaining small enough to be delivered by AAV-based technologies. In particular, this compact size of delivery and / or expression systems comprising a nucleic acid construct encoding an engineered transcriptional modulator (e.g., encoded by a nucleic acid sequence of less than about 1.8kb in length) and one or more payloads is in contrast with existing systems having tunable transcription factors and payloads that are much larger in size.

[0299] In some embodiments, a nucleic acid construct comprising a nucleic acid sequence encoding an engineered transcriptional modulator described herein is less than 1.8kb in length. In some embodiments, a nucleic acid construct comprising a nucleic acid sequence encoding an engineered transcriptional modulator described herein is about 0.1kb, about 0.2 kb, about 0.3 kb, about 0.4 kb, about 0.5 kb, about 0.6 kb, about 0.7 kb, about 0.8 kb, about 0.9 kb, about 1 kb,about 1.1 kb, about 1.2 kb, about 1.3 kb, about 1.4 kb, about 1.5 kb, about 1.6 kb, about 1.7 kb, about 1.8 kb in length.

[0300] In some embodiments, provided herein are nucleic acid constructs comprising a sequence encoding engineered transcriptional modulators described herein (e.g., compact engineered transcriptional modulators). In some embodiments, nucleic acid constructs provided herein comprise a sequence encoding an engineered transcriptional modulator comprising: (i) a DNA binding element (e.g., as described herein) and (ii) a transcriptional modulator element (e.g., as described herein). In some embodiments, nucleic acid constructs provided herein comprise a sequence encoding an engineered transcriptional modulator that further comprises one or more additional elements, e.g., as described herein, e.g., a regulatory element. In some embodiments, nucleic acid constructs provided herein comprise a sequence encoding an engineered transcriptional modulator comprising: (i) a DNA binding element (e.g., as described herein), (ii) a transcriptional modulator element (e.g., as described herein), and (iii) a regulatory element (e.g., as described herein).

[0301] In some embodiments, nucleic acid constructs provided herein comprise a sequence encoding an engineered transcriptional modulator comprising a DNA binding element. In some embodiments, a DNA binding element comprises one or more domains (e.g., peptide structures) that impart DNA binding capability to a polypeptide.

[0302] In some embodiments, a DNA binding element comprises a plurality of domains (e.g., peptide structures) that impart DNA binding capability to a polypeptide. In some embodiments, a DNA binding element comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 domains (e.g., peptide structures) that impart DNA binding capability to a polypeptide. In some embodiments, a DNA binding element comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 domains (e.g., peptide structures) that impart DNA binding capability to a polypeptide.

[0303] In some embodiments, a DNA binding element comprises one or more of: a helix-turn-helix (HLH) domain, a homeodomain, a basic region leucine zipper, a nuclear hormone receptor, a zinc finger domain, or any combination thereof.

[0304] In some embodiments, a DNA binding element is or comprises one or more zinc finger domains, e.g., zinc finger DNA binding elements. In some embodiments, a zinc finger DNAbinding element comprises ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, and / or ZF19. In some embodiments, a zinc finger DNA binding element comprises a combination of two or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, and / or ZF19.

[0305] In some embodiments, a zinc finger DNA binding element comprises one or more ZF 1. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF1, e.g., one or more ofZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19. In some embodiments, ZF1 comprises the amino acid sequence of SEQ ID NO: 97 or an amino acid sequence having at least 85% identity thereto.

[0306] In some embodiments, a zinc finger DNA binding element comprises one or more ZF2. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF2, e.g., one or more ofZFl, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0307] In some embodiments, a zinc finger DNA binding element comprises one or more ZF3. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF3, e.g., one or more ofZFl, ZF2, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0308] In some embodiments, a zinc finger DNA binding element comprises one or more ZF4. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF4, e.g., one or more ofZFl, ZF2, ZF3, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0309] In some embodiments, a zinc finger DNA binding element comprises one or more ZF5. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF5, e.g., one or more ofZFl, ZF2, ZF3, ZF4, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0310] In some embodiments, a zinc finger DNA binding element comprises one or more ZF6. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF6, e.g., one or more ofZFl, ZF2, ZF3, ZF4, ZF5, ZF7, ZF8, ZF9, ZF10, ZF11,ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19. In some embodiments, ZF6 comprises the amino acid sequence of SEQ ID NO: 84 or an amino acid sequence having at least 85% identity thereto.

[0311] In some embodiments, a zinc finger DNA binding element comprises one or more ZF7. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF7, e.g., one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0312] In some embodiments, a zinc finger DNA binding element comprises one or more ZF8. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF8, e.g., one or more ofZFl, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0313] In some embodiments, a zinc finger DNA binding element comprises one or more ZF9. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF9, e.g., one or more ofZFl, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0314] In some embodiments, a zinc finger DNA binding element comprises one or more ZF10. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF10, e g., one or more ofZFl, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0315] In some embodiments, a zinc finger DNA binding element comprises one or more ZF11. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF 11, e.g., one or more ofZFl, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0316] In some embodiments, a zinc finger DNA binding element comprises one or more ZF12. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF 12, e.g., one or more ofZFl, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0317] In some embodiments, a zinc finger DNA binding element comprises one or more ZF13. In some embodiments, a zinc finger DNA binding element further comprises one or more otherZFs other than ZF13, e.g., one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF14, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0318] In some embodiments, a zinc finger DNA binding element comprises one or more ZF14. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other thanZF14, e.g., one or more ofZFl, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF15, ZF16, ZF17, ZF18, or ZF19.

[0319] In some embodiments, a zinc finger DNA binding element comprises one or more ZF15. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF 15, e.g., one or more ofZFl, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF16, ZF17, ZF18, or ZF19.

[0320] In some embodiments, a zinc finger DNA binding element comprises one or more ZF16. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF16, e.g., one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF17, ZF18, or ZF19.

[0321] In some embodiments, a zinc finger DNA binding element comprises one or more ZF17. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF 17, e.g., one or more ofZFl, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF18, or ZF19.

[0322] In some embodiments, a zinc finger DNA binding element comprises one or more ZF18. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other than ZF 18, e.g., one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, or ZF19.

[0323] In some embodiments, a zinc finger DNA binding element comprises one or more ZF19. In some embodiments, a zinc finger DNA binding element further comprises one or more other ZFs other thanZF19, e.g., one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, or ZF18.

[0324] In some embodiments, a DNA binding element is or comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 zinc finger domains, e.g., zinc finger DNA binding elements. In some embodiments, a DNA binding elementis or comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 zinc finger domains, e.g., zinc finger DNA binding elements.

[0325] In some embodiments, nucleic acid constructs provided herein comprise a sequence encoding an engineered transcriptional modulator comprising a transcriptional modulator element, e.g., as described herein.

[0326] In some embodiments, a transcriptional modulator element is or comprises a transcription activation domain. In some embodiments, a transcription activation domain is or comprises a VP64 domain, a p65.1 domain, a p65.2 domain, a RTA.2 domain, a VP16 domain, a VPR domain, a p300 domain, a CBP domain, a Hsfl domain, a Swi / Snf domain, a MED 15 domain, a El A domain, a Gal4-AD domain, a B42 domain, a p65 domain, or any biological equivalent thereof, or a variant thereof, or a fragment thereof.

[0327] In some embodiments, a transcription activation domain is or comprises a p65.1 domain or a fragment or variant thereof. In some embodiments, a transcription activation domain comprises the sequence of SEQ ID NO: 98, or a sequence having at least 85% identity thereto.

[0328] In some embodiments, a transcription activation domain is or comprises a VP64 domain or a fragment or variant thereof. In some embodiments, a transcription activation domain comprises the sequence of SEQ ID NO: 90, or a sequence having at least 85% identity thereto.

[0329] In some embodiments, a transcription activation domain is or comprises a p65.2 domain or a fragment or variant thereof. In some embodiments, a transcription activation domain comprises the sequence of SEQ ID NO: 91, or a sequence having at least 85% identity thereto.

[0330] In some embodiments, a transcription activation domain is or comprises a RTA.2 domain or a fragment or variant thereof. In some embodiments, a transcription activation domain comprises the sequence of SEQ ID NO: 99, or a sequence having at least 85% identity thereto.

[0331] In some embodiments, a transcription activation domain is a mechanosensitive transcription element. In some embodiments, a mechanosensitive transcription element is YAP, TAZ, SRF, MRTF-A or -B, or MYOCD or any biological equivalent thereof, or a variant thereof, or a fragment thereof.

[0332] In some embodiments, a transcriptional modulator element is or comprises a transcription repressor domain. In some embodiments, a transcription repressor domain comprises a KRABDomain (Kriippel-associated box), SID Domain (Sin3 Interaction Domain), POZ / BTB Domain, LSDl-binding Domain, Groucho / TLE Interaction Domain, embryonic ectoderm development domain (EED domain), DNA methyltransferase 3B domain (DNMT3B domain), histone deacetylase 4 domain (HDAC4 domain), or any biological equivalent thereof, or a variant thereof, or a fragment thereof.

[0333] In some embodiments, nucleic acid constructs provided herein comprise a sequence encoding an engineered transcriptional modulator comprising a regulatory element, e.g., as described herein. In some embodiments, a regulatory element confers upon an engineered transcriptional modulator responsiveness to exogenous control (e.g., to a small molecule agent, and in particular to a drug approved by one or more appropriate regulatory agencies for use in humans). In some embodiments, a regulatory element is responsive to a small molecule. For example, responsiveness of a regulatory element to a small molecule can include: stabilization of a regulatory element, change in (e.g., inhibition of) activity of a regulatory element, change in conformation (e.g., dimerization) of a regulatory element, change in localization of a regulatory element, or any combination thereof.

[0334] In some embodiments, a regulatory element can stabilize an engineered transcriptional modulator polypeptide in the presence of a small molecule by inhibiting degradation of an engineered transcriptional modulator polypeptide. In some such embodiments, a regulatory element is or comprises a protease domain (e g., a self-cleaving protease domain (e.g., NS3p)) that results in constitutive degradation of an engineered transcriptional modulator polypeptide. In the presence of a small molecule that blocks the activity of the protease, degradation of an engineered transcriptional modulator polypeptide is prevented resulting in stabilization of an engineered transcriptional modulator polypeptide. In some embodiments, a regulatory element comprises a NS3p protease or a fragment or variant thereof. In some embodiments, a small molecule that can be useful in stabilizing an engineered transcriptional modulator polypeptide comprising aNS3p protease as a regulatory element is one that can inhibit the protease activity of NS3p, e.g., grazoprevir.

[0335] In some embodiments, a regulatory element can destabilize an engineered transcriptional modulator polypeptide in the presence of a small molecule by promoting degradation of an engineered transcriptional modulator polypeptide. In some such embodiments, a regulatoryelement is or comprises a protease element (e.g., NS3p) and one or more destabilization elements (e.g., a degradation domain (e.g., degradation tag (degron)) or a nuclear export signal) wherein the one or more destabilization elements are cleaved by the protease resulting in constitutive expression of an engineered transcriptional modulator polypeptide. In the presence of a small molecule that blocks the activity of the protease (e.g., prevents degradation of one or more destabilization domains by the protease), destabilization of an engineered transcriptional modulator polypeptide by one or more destabilization elements is induced resulting in degradation of an engineered transcriptional modulator polypeptide and reduced expression of a payload. In some embodiments, a regulatory element comprises a NS3p protease or a fragment or variant thereof and one or more destabilization elements. In some embodiments, a destabilization element comprises a degradation domain, e.g., a ODC-1 degron. In some embodiments, a destabilization element comprises a nuclear export signal. In some embodiments, a small molecule that can be useful in destabilizing an engineered transcriptional modulator polypeptide comprising a NS3p protease and one or more destabilization elements as a regulatory element is one that can induce the protease activity of NS3p, e.g., grazoprevir.

[0336] In some embodiments, a NS3p protease comprises an amino acid sequence of SEQ ID NO: 85, or a sequence with at least 85% identity thereto.

[0337] In some embodiments, a regulatory element can induce dimerization of an engineered transcriptional modulator polypeptide, e.g., by promoting binding of one or more engineered transcriptional modulator polypeptides to each other in the presence of a small molecule.

[0338] In some embodiments, a regulatory element can alter the localization of an engineered transcriptional modulator polypeptide, e.g., by translocating an engineered transcriptional modulator polypeptide from a cytoplasm to a nucleus. In some such embodiments, a regulatory element is or comprises a peptide in the cytoplasm that upon binding to a ligand can translocate into the nucleus of a cell. In some embodiments, a regulatory element comprises a human estrogen receptor (ERT2) or a fragment or variant thereof. In some embodiments, a small molecule that can be useful in inducing nuclear localization of an engineered transcriptional modulator polypeptide comprising a regulatory element comprising ERT2 is small molecule that can bind to ERT2 and translocate it to the nucleus, e.g., 4-hydroxytam oxifen (4OHT).

[0339] In some embodiments, an ERT2 polypeptide comprises an amino acid sequence of SEQ ID NO: 86, or a sequence with at least 85% identity thereto.

[0340] In some embodiments, nucleic acid constructs provided herein comprise a sequence encoding an engineered transcriptional modulator comprising: (i) a DNA binding element (e.g., as described herein) and (ii) a transcriptional modulator element, and a sequence encoding one or more linkers between (i) and (ii). In some embodiments, nucleic acid constructs provided herein comprise a sequence encoding an engineered transcriptional modulator comprising: (i) a DNA binding element (e.g., as described herein), (ii) a transcriptional modulator element (e.g., as described herein), and (iii) a regulatory element (e.g., as described herein), and a sequence encoding one or more linkers disposed between (i) and (ii), (i) and (iii) and / or (ii) and (iii).

[0341] In some embodiments, a linker comprises about 2 to about 50, about 2 to about 45, about 2 to about 40, about 2 to about 35, about 2 to about 30, about 2 to about 25, about 2 to about 20, about 2 to about 15, about 2 to about 10, about 2 to about 5, about 3 to about 50, about 5 to about 50, about 10 to about 50, about 15 to about 50, about 20 to about 50, about 25 to about 50, about 30 to about 50, about 35 to about 50, about 40 to about 50, about 45 to about 50 amino acids.

[0342] In some embodiments, a linker is an unstructured linker.

[0343] In some embodiments, a nucleic acid construct comprising a sequence encoding an engineered transcriptional modulator comprises one or more transcriptional regulatory elements. In some embodiments, one or more transcriptional regulatory elements comprise a promoter, an enhancer, a chimeric intron, a poly A tail, or any combination thereof.

[0344] In some embodiments, a promoter is or comprises a constitutive promoter, or a tissue specific promoter. Exemplary constitutive promoters are known in the field and include, but are not limited to, a CMV promoter, a CBA promoter, a CAG promoter, a SFFV promoter, a EFla promoter, a PGK promoter or functional fragments of any of the foregoing. Exemplary tissue specific promoters can be readily obtained by those with ordinary skill in the pertinent art based on the tissue that is intended to be targeted by delivery of an engineered transcriptional modulator described herein.Nucleic acid constructs comprising engineered transcriptional modulator and payloads (e.g., gene of interest)

[0345] Also provided herein are nucleic acid constructs comprising a first sequence encoding an engineered transcriptional modulator (e.g., as described herein) and a second sequence encoding one or more payloads (e.g., genes of interest under the control of one or more transcriptional regulatory elements). In some embodiments, such nucleic acid constructs comprising a sequence encoding an engineered transcriptional modulator (e.g., as described herein) and a second sequence encoding one or more payloads (e.g., genes of interest under the control of one or more transcriptional regulatory elements) is also referred to as a nucleic acid construct system.

[0346] In some embodiments, one or more payloads are associated with, e.g., under the control of, one or more transcriptional regulatory elements situated in a nucleic acid sequence (e.g., a second sequence). In some embodiments, one or more transcriptional regulatory elements comprise a promoter, an enhancer, a chimeric intron, a poly A tail, one or more binding sites recognized by a DNA binding element, or any combination thereof. In some embodiments, a promoter and / or enhancer comprises one or more binding sites recognized by a DNA binding element (e.g., as described herein).

[0347] In some embodiments, a promoter is or comprises a constitutive promoter, or a tissue specific promoter. Exemplary constitutive promoters are known in the field and include, but are not limited to, a CMV promoter, a CBA promoter, a CAG promoter, a SFFV promoter, a EFla promoter, a PGK promoter or functional fragments of any of the foregoing. Exemplary tissue specific promoters can be readily obtained by those with ordinary skill in the pertinent art based on the tissue that is intended to be targeted by delivery of an engineered transcriptional modulator described herein.

[0348] In some embodiments, one or more transcriptional regulatory elements comprise one or more binding sites recognized by a DNA binding element. In some embodiments, one or more binding sites recognized by a DNA binding element is a nucleic acid sequence that is recognized by a DNA binding element, e.g., in an engineered transcriptional modulator described herein. Exemplary binding sites recognized by DNA binding elements include nucleic acid sequences that can be recognized by DNA binding elements such as a helix-turn-helix (HLH) domain, ahomeodomain, a basic region leucine zipper, a nuclear hormone receptor, a zinc (Zn) finger domain.

[0349] In some embodiments, a second sequence (e.g., encoding one or more payloads) comprises one or more binding sites recognized by a zinc finger DNA binding element. In some embodiments, a zinc finger DNA binding element binds to a 9-mer motif in a nucleic acid sequence. In some embodiments, a zinc finger DNA binding element (e.g., ZF1) binds to a GAGTGAGGA (SEQ ID NO: 101) sequence. In some embodiments, a zinc finger DNA binding element (e.g., ZF6) binds to a GTGTAGGGG (SEQ ID NO: 102) sequence. In some embodiments, a second sequence comprises one or more binding sites comprising the sequence of SEQ ID NO: 101 or SEQ ID NO: 102.

[0350] In some embodiments, a second sequence (e.g., encoding one or more payloads) comprises one or more binding sites recognized by a DNA binding element. In some embodiments, a second sequence (e.g., encoding one or more payloads) comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 binding sites recognized by a DNA binding element. In some embodiments, a second sequence (e.g., encoding one or more payloads) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 binding sites recognized by a DNA binding element. In some embodiments, one or more binding sites recognized by a DNA binding element are contiguous. In some embodiments, one or more binding sites recognized by a DNA binding element are separated by at least one nucleotide.

[0351] In some embodiments, a first sequence (e.g., encoding engineered transcriptional modulator described herein) and a second sequence (e.g., encoding one or more payloads and / or one or more transcriptional regulatory elements) are situated on the same nucleic acid construct.

[0352] In some embodiments, a first sequence (e.g., encoding engineered transcriptional modulator described herein) and a second sequence (e.g., encoding one or more payloads and / or one or more transcriptional regulatory elements) are situated between one or more viral elements, e.g., inverted terminal repeats (ITRs) or long terminal repeats (LTRs) in a nucleic acid construct.

[0353] In some embodiments, a first sequence (e.g., encoding engineered transcriptional modulator described herein) and a second sequence (e.g., encoding one or more payloads and / or one or more transcriptional regulatory elements) are situated between two inverted terminal repeats (ITRs) in a nucleic acid construct. In some embodiments, two ITRs in a nucleic acidconstruct are or are derived from a known AAV serotype, e.g., AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 (rh10), AAV11, AAV12, AAV13, AAV-DJ, or AAV-PHP.eB.

[0354] In some embodiments, a nucleic acid construct comprising a first sequence encoding an engineered transcriptional modulator (e.g., as described herein) and a second sequence encoding one or more payloads (e.g., under the control of one or more transcriptional regulatory elements) further comprises one or more additional elements. In some embodiments, one or more additional elements are situated between a first sequence and a second sequence. In some embodiments, one or more additional elements comprises a spacer, or an insulator element. In some embodiments, a spacer comprises one or more nucleotides. In some embodiments, an insulator element is or comprises a cHS4 insulator. In some embodiments, sequence for a cHS4 insulator is provided in SEQ ID NO: 100. Additional exemplary insulator elements that can be useful in nucleic acid constructs disclosed herein include those known in the field and can be readily obtained by one with ordinary skill.

[0355] In some embodiments, a nucleic acid construct comprising a first sequence (e.g., encoding engineered transcriptional modulator described herein) and a second sequence (e.g., encoding one or more payloads and / or one or more regulatory elements) is about Ikb to about 5kb in length.Nucleic acid construct encapsidated in AAV particle

[0356] In some embodiments, a nucleic acid construct comprising a first sequence encoding an engineered transcriptional modulator (e.g., as described herein) and / or a second sequence encoding one or more payloads (e.g., under the control of one or more transcriptional regulatory elements) can be packaged in an AAV particle.

[0357] In some embodiments, a nucleic acid construct comprising a first sequence encoding an engineered transcriptional modulator (e.g., as described herein) and a second sequence encoding one or more payloads (e.g., under the control of one or more transcriptional regulatory elements) comprises one or more sequence of an AAV genome.

[0358] In some embodiments, an AAV particle can be produced by introducing: (i) a nucleic acid construct comprising an AAV genome (as described herein), and (ii) one or more additionalnucleic constructs encoding components necessary and / or sufficient for viral replication, integration and / or packaging. In some embodiments, one or more additional nucleic acid constructs encode a Rep polypeptide, a Cap polypeptide and one or more helper proteins.

[0359] In some embodiments, a nucleic acid construct, e.g., that is an AAV genome, comprises two inverted terminal repeats (ITRs).

[0360] In some embodiments, a first sequence encoding an engineered transcriptional modulator (e g., as described herein) and a second sequence encoding one or more payloads (e.g., under the control of one or more transcriptional regulatory elements) is flanked by a pair of ITRs.

[0361] In some embodiments, a pair of ITRs is symmetrical.

[0362] In some embodiments, a pair of ITRs is asymmetrical.

[0363] In some embodiments, a pair of ITRs comprises two ITRs that are of the same length.

[0364] In some embodiments, a pair of ITRs comprises two ITRs that are not of the same length.

[0365] In some embodiments, a pair of ITRs comprises two ITRs from the same AAV serotype.

[0366] In some embodiments, a pair of ITRs comprises two ITRs from different AAV serotypes.

[0367] In some embodiments, a pair of ITRs in a nucleic acid construct are or are derived from a known AAV serotype, e.g., AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10 (rhlO), AAV11, AAV 12, AAV13, AAV-DJ, or AAV-PHP.eB.

[0368] In some embodiments, a nucleic acid construct comprising an AAV genome is not more than 4.8kb in length.

[0369] In some embodiments a nucleic acid construct comprising an AAV genome is less than 4.8kb in length.

[0370] In some embodiments, a nucleic acid construct comprising an AAV genome which comprises a first sequence encoding an engineered transcriptional modulator (e.g., as described herein) and a second sequence encoding one or more payloads (e.g., under the control of one or more transcriptional regulatory elements) can be packaged into an AAV particle.

[0371] In some embodiments, an AAV particle comprises: (i) an AAV capsid; and (ii) an AAV genome (e.g., comprising a first sequence encoding an engineered transcriptional modulator(e g., as described herein) and a second sequence encoding one or more payloads (e.g., under the control of one or more transcriptional regulatory elements).

[0372] In some embodiments, an AAV capsid is or comprises a capsid of a naturally occurring AAV serotype.

[0373] In some embodiments, an AAV capsid is or comprises a capsid of an engineered AAV particle.

[0374] In some embodiments, an AAV capsid is or comprises a capsid of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 (rh10), AAV11, AAV12, AAV13, AAV-DJ, or AAV-PHP.eB, or a variant of any of the foregoing.Nucleic acid construct configurations

[0375] Nucleic acid constructs provided herein that encode an engineered transcriptional modulator and one or more payloads (e.g., under the control of one or more transcriptional regulatory elements) can be in any configuration that would render useful such nucleic acid construct to achieve effective transcriptional modulation (e.g., when delivered with a delivery and / or expression system disclosed herein). Such nucleic acid constructs can further include one or more additional elements, e.g., one or more spacers and / or linkers.

[0376] In some embodiments, a nucleic acid construct disclosed herein comprises a sequence encoding an engineered transcriptional modulator. In some embodiments, a nucleic acid construct disclosed herein comprises one or more copies of such sequences encoding an engineered transcriptional modulator. In some embodiments, said one or more copies of sequences are in tandem. In some embodiments, said one or more copies of sequences are separated by a linker or spacer. In some embodiments, said one or more copies of sequences are not separated by a linker or spacer.

[0377] In some embodiments, in a nucleic acid construct comprising a sequence encoding an engineered transcriptional modulator, sequences encoding the following elements are situated in any order: (i) a DNA binding element (e.g., as described herein), (ii) a transcriptional modulator element (e.g., as described herein), and (iii) a regulatory element (e.g., as described herein). In some embodiments, in a nucleic acid construct comprising a sequence encoding an engineered transcriptional modulator a sequence encoding (iii) a regulatory element (e.g., as describedherein) is situated between a sequence encoding (i) a DNA binding element and a sequence encoding (iii) a regulatory element. In some embodiments, a linker or spacer is situated between any one or all of (i), (ii) and (iii).

[0378] In some embodiments, in a nucleic acid construct comprising a sequence encoding an engineered transcriptional modulator, sequences encoding the following elements are situated in a 5’ to 3’ orientation: (i) a DNA binding element (e.g., as described herein), (ii) a transcriptional modulator element (e.g., as described herein), and (iii) a regulatory element (e.g., as described herein). In some embodiments, a linker or spacer is situated between any one or all of (i), (ii) and (iii).

[0379] In some embodiments, in a nucleic acid construct comprising a sequence encoding an engineered transcriptional modulator, sequences encoding the following elements are situated in a 5’ to 3’ orientation: (i) a DNA binding element (e.g., as described herein), (iii) a regulatory element (e.g., as described herein) and (ii) a transcriptional modulator element (e.g., as described herein). In some embodiments, a nucleic acid construct further comprises a sequence encoding (iv) one or more additional elements, e.g., situated between any of (i), (ii) and / or (iii). In some embodiments, one or more additional elements comprise a degradation domain, a nuclear export signal, a nuclear localization signal or any combination thereof. In some embodiments, a linker or spacer is situated between any one or all of (i), (ii) (iii), and (iv).

[0380] In some embodiments, in a nucleic acid construct comprising a sequence encoding an engineered transcriptional modulator, sequences encoding the following elements are situated in a 5’ to 3’ orientation: (ii) a transcriptional modulator element (e.g., as described herein), (iii) a regulatory element (e.g., as described herein) and (i) a DNA binding element (e.g., as described herein). In some embodiments, a nucleic acid construct further comprises a sequence encoding (iv) one or more additional elements, e.g., situated between any of (i), (ii) and / or (iii). In some embodiments, one or more additional elements comprise a degradation domain, a nuclear export signal, a nuclear localization signal or any combination thereof. In some embodiments, a linker or spacer is situated between any one or all of (i), (ii) (iii), and (iv).

[0381] In some embodiments, a nucleic acid construct comprising a sequence encoding an engineered transcriptional modulator comprises sequences encoding: (i) a DNA binding element (e.g., as described herein), (ii) a transcriptional modulator element (e.g., as described herein),(iii) a regulatory element (e.g., as described herein), and (iv) one or more additional elements. In some embodiments, one or more additional elements comprise a degradation domain, a nuclear export signal, a nuclear localization signal or any combination thereof. In some embodiments, a linker or spacer is situated between any one or all of (i), (ii) (iii), and (iv).

[0382] In some embodiments, in a nucleic acid construct comprising a sequence encoding an engineered transcriptional modulator, sequences encoding the following elements are situated in any order: (i) a DNA binding element (e.g., as described herein), (ii) a transcriptional modulator element (e.g., as described herein), (iii) a regulatory element (e.g., as described herein), and (iv) one or more additional elements. In some embodiments, a linker or spacer is situated between any one or all of (i), (ii) (iii), and (iv).

[0383] In some embodiments, a nucleic acid construct disclosed herein comprises a sequence encoding one or more payloads (e.g., under the control of one or more transcriptional regulatory elements). In some embodiments, a nucleic acid construct disclosed herein comprises one or more copies of such sequences encoding one or more payloads (e.g., under the control of one or more transcriptional regulatory elements). In some embodiments, said one or more copies of sequences are in tandem. In some embodiments, said one or more copies of sequences are separated by a linker or spacer. In some embodiments, said one or more copies of sequences are not separated by a linker or spacer.

[0384] In some embodiments, a nucleic acid construct disclosed herein comprises a first sequence (e.g., encoding an engineered transcriptional modulator described herein) and a second sequence (e.g., encoding one or more payloads and / or one or more transcriptional regulatory elements). In some embodiments, a first sequence (e.g., encoding an engineered transcriptional modulator described herein) and a second sequence (e.g., encoding one or more payloads and / or one or more transcriptional regulatory elements) are situated on the same nucleic acid construct.

[0385] In some embodiments, a nucleic acid construct disclosed herein comprises one or more copies of a first sequence and one or more copies of a second sequence. In some embodiments, a nucleic acid construct disclosed herein comprises one copy of a first sequence and one or more copies of a second sequence. In some embodiments, a nucleic acid construct disclosed herein comprises one or more copies of a first sequence and one copy of a second sequence. In someembodiments, one or more copies of a first sequence and one or more copies of a second sequence can each be separated by a linker or spacer.

[0386] In some embodiments, a nucleic acid construct disclosed herein comprises a first sequence encoding an engineered transcriptional modulator (e.g., as described herein) and a second sequence encoding one or more payloads (e.g., genes of interest under the control of one or more transcriptional regulatory elements). In some embodiments, such a nucleic acid construct can further comprise one or more additional elements, e.g., spacers, linkers, insulators, etc.

[0387] In some embodiments, a first sequence is situated 5’ of a second sequence.

[0388] In some embodiments, a first sequence is situated 3’ of a second sequence.

[0389] In some embodiments, a nucleic acid construct comprises one or more copies of a first sequence and / or one or more copies of a second sequence.

[0390] In some embodiments, in a nucleic acid construct comprising a first sequence encoding an engineered transcriptional modulator (e.g., as described herein) and a second sequence encoding one or more payloads (e.g., genes of interest under the control of one or more transcriptional regulatory elements), a first sequence and a second sequence are positioned in tandem. In some embodiments, a first sequence is upstream of a second sequence. In some embodiments, a first sequence is downstream of a second sequence. In some embodiments, a sequence encoding an additional element, e.g., an insulator element, is positioned between a first sequence and a second sequence. An exemplary schematic of a tandem configuration is provided in the middle and lower panels of FIG. 3B.

[0391] In some embodiments, in a nucleic acid construct comprising a first sequence encoding an engineered transcriptional modulator (e.g., as described herein) and a second sequence encoding one or more payloads (e.g., genes of interest under the control of one or more transcriptional regulatory elements), a first sequence and a second sequence are positioned in a divergent configuration. In some embodiments, in a divergent configuration a promoter controlling transcription of a first sequence and a promoter controlling transcription of a second sequence are placed in opposite orientations. In some embodiments, a sequence encoding an additional element, e.g., an insulator element, is positioned between a first sequence and a second sequence.

[0392] Exemplary schematics of nucleic acid constructs having divergent configurations is provided in the middle and lower panels of FIG. 3B. In some embodiments, a nucleic acid construct comprising a first sequence encoding an engineered transcriptional modulator (e.g., as described herein) and a second sequence encoding one or more payloads (e.g., genes of interest under the control of one or more transcriptional regulatory elements), is in an upstream divergent configuration, e.g., as shown in the middle panel of FIG. 3B. In some embodiments, a nucleic acid construct comprising a first sequence encoding an engineered transcriptional modulator (e.g., as described herein) and a second sequence encoding one or more payloads (e.g., genes of interest under the control of one or more transcriptional regulatory elements), is in a downstream divergent configuration, e.g., as shown in the lower panel of FIG.3B.

[0393] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence provided in Table 2 or a sequence having at least 85% identity to a nucleic acid sequence in Table 2.

[0394] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 39 or a sequence having at least 85% identity thereto.

[0395] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 40 or a sequence having at least 85% identity thereto.

[0396] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 41 or a sequence having at least 85% identity thereto.

[0397] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 42 or a sequence having at least 85% identity thereto.

[0398] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 43 or a sequence having at least 85% identity thereto.

[0399] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 44 or a sequence having at least 85% identity thereto.

[0400] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 45 or a sequence having at least 85% identity thereto.

[0401] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 46 or a sequence having at least 85% identity thereto.

[0402] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 47 or a sequence having at least 85% identity thereto.

[0403] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 48 or a sequence having at least 85% identity thereto.

[0404] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 49 or a sequence having at least 85% identity thereto.

[0405] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 50 or a sequence having at least 85% identity thereto.

[0406] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 51 or a sequence having at least 85% identity thereto.

[0407] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 52 or a sequence having at least 85% identity thereto.

[0408] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 53 or a sequence having at least 85% identity thereto.

[0409] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 54 or a sequence having at least 85% identity thereto.

[0410] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 55 or a sequence having at least 85% identity thereto.

[0411] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 56 or a sequence having at least 85% identity thereto.

[0412] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 57 or a sequence having at least 85% identity thereto.

[0413] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 58 or a sequence having at least 85% identity thereto.

[0414] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 59 or a sequence having at least 85% identity thereto.

[0415] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 60 or a sequence having at least 85% identity thereto.

[0416] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 61 or a sequence having at least 85% identity thereto.

[0417] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 62 or a sequence having at least 85% identity thereto.

[0418] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 63 or a sequence having at least 85% identity thereto.

[0419] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 64 or a sequence having at least 85% identity thereto.

[0420] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 65 or a sequence having at least 85% identity thereto.

[0421] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 66 or a sequence having at least 85% identity thereto.

[0422] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 67 or a sequence having at least 85% identity thereto.

[0423] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 68 or a sequence having at least 85% identity thereto.

[0424] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 69 or a sequence having at least 85% identity thereto.

[0425] In some embodiments, a nucleic acid construct disclosed herein comprises a nucleic acid sequence of SEQ ID NO: 70 or a sequence having at least 85% identity thereto.IV. Genes or payloads of interest

[0426] An expression system comprising an engineered transcriptional modulator described herein can include a nucleic acid construct comprising a sequence encoding an engineeredtranscriptional modulator and a sequence encoding one or more payloads (e.g., genes of interest or transgenes).

[0427] In some embodiments, one or more payloads is an RNA payload. In some embodiments, an RNA payload is or comprises a messenger RNA, a non-coding RNA, a circular RNA, a IncRNA, a gRNA, an antisense RNA, an snRNA or an aptamer.

[0428] In some embodiments, one or more payloads is a polypeptide payload. In some embodiments, a polypeptide payload is an enzyme. In some embodiments, a polypeptide payload is an antibody. In some embodiments, a polypeptide payload is a secreted protein.

[0429] In some embodiments, a polypeptide payload is or comprises a protein that can be useful in protein replacement therapy. Protein replacement therapy may be beneficial in the treatment of diseases or disorders characterized by deficient or dysregulated protein expression. Non-limiting examples of such diseases or disorders include rare diseases, genetic disorders, metabolic disorders, cancers, clotting disorders, and infectious diseases.

[0430] In some embodiments, a protein replacement therapy comprises factor VIII for hemophilia A or factor IX, e.g., for treating hemophilia B.

[0431] In some embodiments, a protein replacement therapy comprises human al -antitrypsin (Al AT), e.g., for treating emphysema.

[0432] In some embodiments, a polypeptide payload is or comprises a protein that can be useful in enzyme replacement therapy. Enzyme replacement therapy may be beneficial in the treatment of diseases or disorders characterized by deficient or dysregulated enzyme expression. Nonlimiting examples of such diseases or disorders include genetic disorders, metabolic disorders, cancers, clotting disorders, and infectious diseases.

[0433] In some embodiments, enzyme therapy comprises adenosine deaminase (ADA).

[0434] In certain embodiments, a polypeptide payload useful in an engineered transcriptional modulator disclosed herein comprises one or more of the following: phenylalanine hydroxylase, a-L-iduronidase, iduronate-2-sulphatase, N-acetylgalactosamine-6-sulphatase, N-acetylgalactosamine-4-sulphatase, or adenosine deaminase.

[0435] In some embodiments, a polypeptide payload useful in an engineered transcriptional modulator disclosed herein comprises a CRISPR-Cas protein, a Zinc finger protein, a TAL, a base editor, a prime editor, a meganuclease, or any combination thereof.

[0436] In some embodiments, one or more payloads is a therapeutic payload (e.g., an RNA payload or a polypeptide payload).

[0437] In some embodiments, one or more payloads is a diagnostic payload (e.g., an RNA payload or a polypeptide payload).

[0438] In some embodiments, one or more payloads is a reporter molecule (e.g., an RNA payload or a polypeptide payload).V. Gene Therapy Technologies

[0439] Among other things, provided herein are gene therapy technologies that can be useful in delivering a nucleic acid construct disclosed herein to cells.

[0440] In some embodiments, a gene therapy is useful for delivering a nucleic acid construct disclosed herein to cells in vitro. In some embodiments, a gene therapy is useful for delivering a nucleic acid construct disclosed herein to cells in vivo.

[0441] In some embodiments, a gene therapy of the present disclosure is useful for delivering a nucleic acid construct disclosed herein to cells ex vivo, such that cells are engineered ex vivo for delivery (e.g., via injection) to a subject. In some embodiments, a gene therapy of the present disclosure is used for a cell therapy (e.g., for a CAR T cell therapy).Delivery Systems

[0442] Those skilled in the art will be aware of a variety of technologies utilized to introduce nucleic acids of interest into cells, including for example, viral-, lipid- and / or polymer-based delivery systems. The present disclosure appreciates that such systems are often constrained in the size of nucleic acid that can be delivered. For example, most gene therapy applications utilize adeno-associated viral (AAV) systems. See e.g., Dunbar, C. E. et al. Gene therapy comes of age. Science 359, eaan4672 (2018); Wang, D., Tai, P. W. L. & Gao, G. Adeno-associated virus vector as a platform for gene therapy delivery. Nature Reviews Drug Discovery 18, 358-378(2019); Cring, M. R. & Sheffield, V C. Gene therapy and gene correction: targets, progress, and challenges for treating human diseases. Gene therapy 29, 3-12 (2022); and Mendell, J. R. et al. Current clinical applications of in vivo gene therapy with AAVs. Molecular Therapy 29, 464-488 (2021), the entire contents of each of which are hereby incorporated by reference. AAV systems cannot encapsidate a genome greater than about 4.7 or 4.8 kb in size.

[0443] In some embodiments, a delivery system comprises a nucleic acid construct disclosed herein. In some embodiments, a delivery system can be used to deliver a nucleic acid construct to a cell, tissue or subject.

[0444] In some embodiments, a delivery system comprises an engineered transcriptional modulator polypeptide. In some embodiments, a delivery system can be used to deliver an engineered transcriptional modulator polypeptide to a cell, tissue or subject.

[0445] In some embodiments, a delivery system comprises a vector. In some embodiments, a delivery system comprises a transposon. In some embodiments, a delivery system comprises a DNA template (e.g., for recombinase or homology-directed repair (HDR) mediated genomic insertion). In some embodiments, a delivery system comprises a Prime editor and its genetic payload (template).

[0446] In some embodiments, a delivery system is or comprises a viral vector. In some embodiments, a viral vector is an AAV vector, a lenti viral vector and / or a retroviral vector. In some embodiments, a viral vector is a lentiviral vector. In some embodiments, a viral vector is a all-DNA viral vector (e.g., a herpes virus vector). In some embodiments, a viral vector is an AAV vector.

[0447] In some embodiments, an AAV vector refers to a nucleic acid construct comprising one or more elements of an AAV genome that can be encapsidated into an AAV particle. In some embodiments, an AAV particle comprises a capsid and a nucleic acid construct (e.g., as disclosed herein or comprising one or more elements of an AAV genome).

[0448] In some embodiments, a delivery system is a lipid based delivery system. In some embodiments, a lipid based delivery system comprises a liposome or a lipid nanoparticle.Exemplary lipid-based delivery systems are known in the field, e.g., as described in Shrestha H, et al., Lipid-Based Drug Delivery Systems. J P harm (Cairo), 2014; PMID: 26556202.

[0449] In some embodiments, a delivery system is a polymer based delivery system.Expression Systems

[0450] Those skilled in the art will appreciate that many gene therapy systems employ constitutive or tissue-specific promoters to achieve high-level, sustained expression of therapeutic biomolecules in the body. These often include ubiquitous strong promoters of viral or eukaryotic origin, such as CMV, CBA and CAG, or human promoters known to be highly active in target tissues. The present disclosure appreciates, however, that in many clinical applications, the ability to fine-tune transgene expression in response to patient-specific needs can be beneficial, or even necessary, for optimizing therapeutic efficacy. The present disclosure appreciates, for example, that dynamic control over gene expression may allow for dose adjustment and / or spatial and / or temporal regulation of therapeutic activity, which in some cases may reduce risk of under- or over-treatment, and / or of one or more undesired side effects.

[0451] The present disclosure appreciates that in certain circumstances, constitutive expression of a transgene (e.g., a therapeutic transgene) can pose safety challenges, especially in nondividing cells. For example, in regions such as the central nervous system (CNS) tissue, where transduced cells may be non-proliferative and long-lived, indefinite expression of a transgene (e.g., of the cargo it encodes and / or delivers) might have adverse effects on cell health.

[0452] The present disclosure therefore identifies the source of certain problems associated with may current gene expression systems, particularly including current gene therapy technologies, for example which utilize constitutive, tissue specific, and / or otherwise insufficiently tunable expression elements. Furthermore, the present disclosure provides certain technologies that achieve exogenous control of genes (e.g., transgenes such as may have been delivered by gene therapy technologies) in target (e.g., recipient cells). In some embodiments, provided technologies achieve small molecule control of regulated expression systems such as transgene systems.Transgenes

[0453] Those skilled in the art, reading the present disclosure, will appreciate its applicability to a range of transgenes. For example, a transgene that can be useful in a gene therapy systemdisclosed herein encodes a polypeptide. As another example, a transgene that can be useful in a gene therapy system disclosed herein is a polynucleotide.

[0454] In some embodiments, one or more transgenes is DNA.

[0455] In some embodiments, one or more transgenes is RNA. In some embodiments, an RNA transgene is or comprises a messenger RNA, a non-coding RNA, a circular RNA, a IncRNA, a gRNA, an antisense RNA, an snRNA or an aptamer.

[0456] In some embodiments, one or more transgenes encodes a polypeptide payload. In some embodiments, a polypeptide is an enzyme. In some embodiments, a polypeptide is an antibody. In some embodiments, a polypeptide is a secreted protein.

[0457] In some embodiments, a polypeptide is or comprises a protein that can be useful in protein replacement therapy. Protein replacement therapy may be beneficial in the treatment of diseases or disorders characterized by deficient or dysregulated protein expression. Non-limiting examples of such diseases or disorders include rare diseases, genetic disorders, metabolic disorders, cancers, clotting disorders, and infectious diseases. In some embodiments, a protein replacement therapy comprises factor Vlll for hemophilia A or factor IX, e.g., for treating hemophilia B. In some embodiments, a protein replacement therapy comprises human al-antitrypsin (A1AT), e.g., for treating emphysema.

[0458] In some embodiments, a polypeptide is or comprises a protein that can be useful in enzyme replacement therapy. Enzyme replacement therapy may be beneficial in the treatment of diseases or disorders characterized by deficient or dysregulated enzyme expression. Non-limiting examples of such diseases or disorders include genetic disorders, metabolic disorders, cancers, clotting disorders, and infectious diseases. In some embodiments, enzyme therapy comprises adenosine deaminase (ADA).

[0459] In certain embodiments, a polypeptide encoded by a transgene comprises one or more of the following: phenylalanine hydroxylase, a-L-iduronidase, iduronate-2-sulphatase, N-acetylgalactosamine-6-sulphatase, N-acetylgalactosamine-4-sulphatase, or adenosine deaminase.

[0460] In some embodiments, a polypeptide encoded by a transgene comprises a CRISPR-Cas protein, a Zinc finger protein, a TAL, a base editor, a prime editor, a meganuclease, or any combination thereof.

[0461] In some embodiments, a polypeptide encoded by a transgene is a therapeutic polypeptide.

[0462] In some embodiments, a polypeptide encoded by a transgene is a diagnostic polypeptide.

[0463] In some embodiments, a transgene is or encodes a reporter molecule.VI. Uses

[0464] Provided herein are methods of treating a disease, disorder or condition responsive to a payload polypeptide. In some embodiments, a method of treating a disease, disorder, or condition responsive to a payload polypeptide comprises administering to a subject suffering from or susceptible to such disease disorder or condition a pharmaceutical composition of the present disclosure that comprises or delivers the payload polypeptide.

[0465] The present disclosure provides methods of treating a disease, disorder or condition responsive to expression of a gene under control of at least one transcriptional regulatory sequence that is recognized by a DNA binding element. In some embodiments, a method of treating a disease, disorder or condition responsive to expression of a gene under control of at least one transcriptional regulatory sequence that is recognized by a DNA binding element comprising administering to a subject suffering from or susceptible to such disease, disorder or condition, a pharmaceutical composition of the present disclosure that comprises or delivers an engineered transcriptional modulator.

[0466] In some embodiments, an engineered transcriptional modulator used in a method of the present disclosure is characterized in that exposure to a small molecule regulator modulates the stabilization, localization and / or conformation of the engineered transcriptional modulator, as compared to stabilization, localization and / or conformation of an engineered transcriptional modulator in the absence of, or before exposure to the small molecule regulator.

[0467] In some embodiments, exposure to a small molecule regulator increases expression and / or activity of a payload, as compared to expression and / or activity of a payload in the absence of, or before exposure to the small molecule regulator.VII. Cells

[0468] Provided herein, among other things, are cells engineered to express an engineered transcriptional modulator polypeptide and / or a payload. In some embodiments, cells described herein can include mammalian cells, e.g., cells in or from a mammal, that are contacted with a nucleic acid construct disclosed herein, or an expression system or a delivery system comprising the same. As another example, cells described herein are contacted with an engineered transcriptional modulator polypeptide or an expression system or a delivery system comprising the same.

[0469] In some embodiments, provided herein are cells comprising a nucleic acid construct that comprises a sequence encoding a payload operatively linked with a promoter and at least one transcriptional regulatory sequence. In some embodiments, a nucleic acid construct comprises a binding site for a DNA binding element. In some embodiments, a nucleic acid construct further comprises a transcriptional modulator polypeptide. In some embodiments, a cell is further engineered to express a transcriptional modulator polypeptide.

[0470] In some embodiments, a cell disclosed herein comprises an engineered transcriptional modulator, e.g., as disclosed herein. In some embodiments, an engineered transcriptional modulator comprises: (a) a DNA binding element comprising one or more zinc finger DNA binding elements; (b) a transcription modulation element comprising a transcription activation element or a transcription repression element; (c) a regulatory element that confers responsiveness to a small molecule regulator. In some embodiments, a cell further comprises at least one payload sequence operatively linked with a promoter and at least one transcriptional regulatory sequence that includes a binding site for the DNA binding element.

[0471] In some embodiments, at least one payload is regulated by presence of a small molecule regulator.

[0472] In some embodiments, a cell is contacted with a small molecule regulator. In some embodiments, contacting a cell with a small molecule regulator results in binding of a small molecule regulator to a regulatory element (e.g., comprising a protease domain or a nuclear localization domain and / or one or more destabilization domains (e.g., degron or NES)). In some embodiments, where a regulatory element comprises a protease domain, binding of a smallmolecule that inhibits the activity of the protease results in increased payload expression. In some embodiments, where a regulatory element comprises a protease domain and one or more destabilization domains (e.g., degron or NES)), binding of a small molecule that inhibits the activity of the protease results in decreased payload expression. In some embodiments, where a regulatory element comprises a nuclear localization element, binding of a small molecule that induces nuclear localization, results in increased payload expression.

[0473] In some embodiments, binding of a small molecule regulator to a regulatory element results in a change in stabilization, a change in localization, a change in conformation and / or a change in activity of an engineered transcriptional modulator polypeptide.

[0474] In some embodiments, expression of a payload is modulated responsive to a small molecule regulator.

[0475] In some embodiments, expression of a payload is modulated responsive to a small molecule regulator.

[0476] In some embodiments, a cell is in vitro, ex vivo or in vivo.

[0477] In some embodiments, a cell is a mammalian cell, optionally wherein the cell is a human cell.

[0478] In some embodiments, a cell is a cell from a central nervous system, optionally wherein the central nervous system cell is or comprises a CNS epithelial cell, a nerve cell, a CNS connective tissue cell, a stem cell, a progenitor cell, a CNS immune cell, a spinal cord cell, a cell that lines one or more brain ventricles, a nerve support cell, a glial cell, a fat cell, a meninges cell, or a combination thereof.

[0479] In some embodiments, a cell is in a subject.

[0480] In some embodiments, cells disclosed herein can comprise a population of cells.

[0481] In some embodiments, cells disclosed herein can be contacted with an AAV particle comprising: (i) a nucleic acid construct comprising a first sequence encoding an engineered transcriptional modulator and a second sequence encoding one or more payloads (e.g., under the control of one or more transcriptional regulatory elements), and (ii) an AAV capsid. In some embodiments, a nucleic acid construct encapsidated in an AAV particle is less than 4.8 kb. In some embodiments, a cell contacted with such an AAV particle, expresses an engineeredtranscriptional modulator and one or more payloads. In some embodiments, exposure of such a cell to a small molecule regulator can modulate the expression of one or more payloads expressed by the cell.VIII. Experimental Animal Models Engineered with Engineered Transcriptional Modulators

[0482] Provided herein, among other things, are genetically engineered animal models in which the expression of one or more genes of interest is placed under the control of a synthetic transcription factor (synTF) system disclosed herein (also referred to as an engineered transcriptional modulator system). In some embodiments, use of a synTF system in engineered animal models enables spatial and temporal regulation of gene expression of one or more genes of interest, facilitating the study of one or more genes of interest in specific tissues, developmental stages, or physiological states. As described herein, a synTF system comprises an engineered transcriptional modulator system that is responsive to a small molecule regulator thereby allowing modulation of expression of one or more target genes in vivo.

[0483] In some embodiments, genetically engineered animal models can be generated by introducing an engineered transcriptional modulator (as described herein) into the germline of said animal. Techniques for generating engineered animal models are known in the field and any relevant technique can be used to generate a genetically engineered animal model having an engineered transcriptional modulator as described herein. Further as would be appreciated by one with ordinary skill in the field, a variety of experimental animal models commonly used in biomedical research can be used. These include, but are not limited to, mice, zebrafish, rats, marmosets, and other vertebrate or invertebrate species suitable for genetic manipulation. In some embodiments, such genetically engineered animal models and can be useful for a variety of applications, e.g., for comparative studies, translational research, and the development of disease-relevant in vivo systems.

[0484] In some embodiments, a genetically engineered animal model disclosed herein is engineered to express an engineered transcriptional modulator and / or one or more genes of interest that are under the control of an engineered transcriptional modulator. In someembodiments, a gene of interest is under the control of a tissue-specific promoter, allowing for localized gene regulation.

[0485] In some embodiments, a gene of interest is expressed in a state-specific manner, using promoters responsive to cellular or environmental cues. These may include promoters activated by developmental signals, metabolic states, cytokine exposure, hypoxia, or other physiological stimuli. Such configurations allow for dynamic control of gene expression in response to internal or external changes, enabling the modeling of disease progression, stress responses, or regenerative processes.

[0486] In some embodiments, one or more genes of interest regulated by a synTF system (an engineered transcriptional modulator system), e.g., as disclosed herein in an engineered animal model can be a gene that is associated with a particular disease or disorder; or a gene that is useful in understanding a particular disease state, disease etiology, developmental stage, cellular stage, etc; or a gene that is useful in studying a particular biologically relevant question. In some embodiments the regulated gene of interest is a native gene. In some embodiments the regulated gene of interest is a mutated, xenotypic, or synthetic gene. In some embodiments, one or more genes of interest can encode a polypeptide, e.g., as disclosed herein. In some embodiments, one or more genes of interest can encode a polynucleotide, e.g., an RNA, e.g., a coding RNA, a noncoding RNA, a IncRNA, a miRNA, etc.EXEMPLARY SEQUENCESTable 1. Exemplary amino acid sequences.Table 2. Exemplary nucleic acid sequences.EXAMPLESExample 1: Materials and Methods

[0488] The present Example describes materials and methods used in subsequent examples. General DNA assembly

[0489] Plasmid cloning was performed primarily using standard PCR and restriction enzyme cloning with Phusion DNA Polymerase (NEB M0530L), Q5 High-Fidelity DNA Polymerase (NEB M0491 S), restriction enzymes (NEB), T4 DNA Ligase (NEB M0202L), Antarctic Phosphatase (NEB M0289L) and T4 Polynucleotide Kinase (NEB M0201L). Golden Gate assembly was also used for some constructs. Plasmids with pcDNA-based backbones were transformed into chemically competent TOP 10 Escherichia coli (Thermo Fisher C404010), and cells were grown at 37°C. Plasmids with AAV integration vector backbones were transformed into chemically competent NEB Stable Escherichia coli (NEB C3040H), and cells were grown at 30°C. A complete list of plasmids used in this study can be found in Table 3 - Table 7 and plasmid sequences can be found herein.Table 3: mini-synTF transfection plasmids.Table 4: mini-synTF AAV vectors.Table 5: Reporter plasmids.Table 6: AAV packaging plasmids.Table 7: Transfection controls.Source vectors for DNA assembly

[0490] DsRed-Express2 was obtained by site directed mutagenesis of pDsRed2-Nl, which was a gift from David Schaffer (University of California, Berkeley). pAAV2 backbones were derived from pOTTC1032 - pAAV EFla Nuc-flox(mCherry)-EGFP, which was a gift from Brandon Harvey (Addgene plasmid #112677). pAAV2 / 5 was a gift from Melina Fan (Addgene plasmid #104964). The cHS4 insulator was sourced from PhiC31-Neo-ins-5xTetO-pEF-H2B-Citrin-ins, which was a gift from Michael Elowitz (Addgene plasmid #78099)28. The hybrid between chicken P-actin (CBA) and minute virus of mice (MVM) intron was sourced from pU6-(BbsI)_CBh-Cas9-T2A-BFP-P2A-Ad4E4orf6, which was a gift from Ralf Kuehn (Addgene plasmid # 64220). VP64, p65, and RTA were sourced from SP-dCas9-VPR, which was a gift from George Church (Addgene plasmid #63798). The coding sequence for mTagBFP2 was codon optimized and custom synthesized.Cloning mini-synTFs

[0491] Mini-synTFs were cloned into a pcDNA backbone to confer high expression in HEK293FT cells (Addgene #138749)13. PCR was used to amplify the coding regions of each part of the mini-synTF (activation domain, DNA-binding domain, NS3p, ERT2, etc.), appending a nuclear localization sequence (NLS) if necessary and restriction enzyme-based cloning was used to insert the products into pcDNA to form the different topologies and designs. Linker and degradation domain inserts were synthesized as 15-90 bp oligonucleotides by Integrated DNA Technologies (some inserts required multiple oligos). The coding and reverse oligonucleotideswere synthesized separately and designed to anneal, resulting in dsDNA with a 4 nt sticky end overhang on each side. The coding and reverse oligonucleotides were mixed (6 pL H2O, 1 pL T4 Ligase Buffer, 1 pL T4 PNK (10 U / pL; NEB), 1 pL of each 100 pM oligonucleotide) and phosphorylated at 37°C for 1 h. They were then denatured at 95°C for 5 min and cooled slowly to room temperature (approximately 22°C) to allow for annealing. The mix was then diluted 100-fold and used as an insert in ligation reactions. In general, restriction sites were chosen to facilitate modular swapping of parts via restriction enzyme cloning.Cloning reporters

[0492] Golden Gate assembly was used to construct all synthetic TF-responsive reporters in a TUPV backbone (pGGB022) that includes a pair of B sal restriction sites upstream of aYB TATA minimal promoter and a DsRedExpress-2 reporter gene29. Promoter inserts containing TF binding sites were synthesized as 15-90 bp oligonucleotides (some promoters were long enough to require multiple inserts) by Integrated DNA Technologies. The coding and reverse strands were synthesized separately and designed to anneal, resulting in dsDNA with a 4 nt sticky end overhang on each side. The coding and reverse oligonucleotides were mixed (6 pL H2O, 1 pL T4 Ligase Buffer, 1 pL T4 PNK (10 U / pL; NEB), 1 pL of each 100 pM oligonucleotide) and phosphorylated at 37°C for 1 h. They were then denatured at 95°C for 5 min and cooled slowly to room temperature (approximately 22°C) to allow for annealing. The mix was then diluted 500-fold to make a 20 nM stock and included in the Golden Gate reaction. Golden Gate reaction mixtures comprise 1 pL T4 ligase buffer, 1 pL lOx BSA (1 mg / mL), 0.5 pL Bsal-HF (20 U / pL; NEB), 0.5 pL T4 Ligase (400 U / pL; NEB), 10 fmol of vector, 1 pL of each insert (diluted to 20 nM), and water to a total volume of 10 pL. The reaction was incubated at 37°C for 1 h, 55°C for 15 min, and 80°C for 20 min, and then cooled to room temperature. Then, 3 pL of the reaction was immediately transformed into 50 pL of chemically competent TOP 10 E. coli.Cloning AA V vectors

[0493] The different transcriptional units (mini-synTF unit and reporter unit) were cloned separately into modified versions of the pcDNA backbone, adding restriction enzyme sites to enable assembly of full AAV vectors via restriction enzyme-based cloning. The mini-synTF transcriptional unit was cloned in several steps, first using PCR to amplify the different elementsof the unit: mini-synTF and polyA-tail, and restriction enzymes to insert into the pcDNA backbone (while removing the backbone’s CMV promoter and polyA-tail). Then, the CBA promoter was inserted upstream of the mini-synTF. The CBA promoter was synthesized as 15-90 bp oligonucleotides by Integrated DNA Technologies (required multiple oligos). The coding and reverse oligonucleotides were synthesized separately and designed to anneal, resulting in dsDNA with a 4 nt sticky end overhang on each side. The coding and reverse oligonucleotides were mixed (6 pL H2O, 1 pL T4 Ligase Buffer, 1 pL T4 PNK (10 U / pL; NEB), 1 pL of each 100 pM oligonucleotide) and phosphorylated at 37°C for 1 h. They were then denatured at 95°C for 5 min and cooled slowly to room temperature (here, approximately 22°C) to allow for annealing. The mix was then diluted 100-fold and used as an insert in ligation reactions. The reporter transcriptional unit was cloned using PCR to amplify the different elements of the unit: synthetic promoter, hybrid intron, dsRedExpress2, polyA-tail. Restriction enzyme cloning was used to assemble all inserts into a pcDNA backbone (while removing the backbone’s CMV promoter and polyA-tail).

[0494] Once the transcriptional units were cloned, they were inserted via restriction enzyme cloning into a modified AAV2 backbone derived from pOTTC1032 (Addgene #112677), designed to contain restriction enzyme sites matching those flanking the transcriptional units. Three pAAV2 “placement vectors” were cloned, each with the restriction enzyme sites arranged in a different order, to allow for cloning of the different vector architectures (upstream tandem, upstream divergent, downstream divergent). The placement vectors were assembled by PCR of the cHS4 insulator, adding the corresponding combination of restriction enzyme sites at the ends. Restriction enzyme cloning was used to insert the PCR products into the modified pAAV2 backbone. Subsequent versions of AAV vectors with alternative design choices were cloned via modular swapping of parts using restriction enzyme-based cloning.Plasmid preparation

[0495] TOP 10 or NEB Stable E. coli were grown overnight, shaking at 37°C or 30°C, respectively, in 50-100 mb of LB media with the appropriate selective antibiotic. The next day, DNA was prepped using a ZymoPURE II Plasmid Midiprep Kit (Zymo D4201) by following the manufacturer’s instructions.Cell culture

[0496] The HEK293FT cell line was purchased from Thermo Fisher / Life Technologies (RRID: CVCL_6911). The AAV293 cell line was a gift from David Schaffer (University of California, Berkeley). HEK293FT and AAV293 cells were cultured in DMEM (Gibco 31600-091), supplemented with 10% FBS (Gibco 16140-071), 6 mM L-glutamine (2 mM from Gibco 31600-091 and 4 mM from additional Gibco 25030-081), penicillin (100 U / pL), and streptomycin (100 pg / mL) (Gibco 15140122), in a 37°C incubator with 5% CO2. HEK293FT and AAV293 cells were subcultured at a 1:5 to 1:20 ratio every 2-4 d using Trypsin-EDTA (Gibco 25300-054). AAV293 cells were kept under 50% confluency during subculture.

[0497] The SH-SY5Y cell line was purchased from the American Type Culture Collection (ATCC, Cat: CRL-2266). SH-SY5Y cells were cultured in a medium base of 50% EMEM (ATCC 30-2003) and 50% Ham’s F-12 Nutrient Mix (Gibco 11765-054), supplemented with 10% FBS (Gibco 16140-071), penicillin (lOOU / pL), and streptomycin (100 pg / mL) (Gibco 15140122), in a 37°C incubator with 5% CO2. SH-SY5Y cells were subcultured at a 1:2 to 1:5 ratio every 3-4 d using Trypsin-EDTA (Gibco 25300-054).

[0498] The HuH-7 cell line was purchased from Applied Biological Materials (ABM, Cat:T8973). HuH-7 cells were cultured in RPMI 1640 Medium (Gibco 31800-105), supplemented with 10% FBS (Gibco 16140-071), 6 mM L-glutamine (2 mM from Gibco 1600-091 and 4 mM from additional Gibco 25030-081), penicillin (lOOU / pL), and streptomycin (100 pg / mL) (Gibco 15140122), in a 37°C incubator with 5% CO2. HuH-7 cells were subcultured at a 1:5 to 1:10 ratio every 3-4 d using Trypsin-EDTA (Gibco 25300-054).Transient transfection of HEK293FTs

[0499] Transient transfection of HEK293FT cells was conducted using the calcium phosphate method. Cells were plated at a minimum density of 1. Ox 105cells per well in a 24-well plate in 0.5 mb DMEM, supplemented as described above. For conditions with ligand treatment, the corresponding small molecule (Grazoprevir, GZV - MedChemExpress HY- 15298; or 4-hydroxytamoxifen / tamoxifen, 4OHT - Sigma Aldrich H7904) was added at the concentrations indicated in each panel. For conditions with vehicle control treatment, the corresponding solvent(DMSO or 200-proof EtOH, respectively) was added at equivalent volume as supplied for ligand conditions.

[0500] After about 24 h, by which time the cells had adhered to the plate and grown to about 50-70% confluency in the well, the cells were transfected. Plasmids (500-600 ng DNAfor 24-well plates) were mixed in H2O, and 2 M CaCh was added to a final concentration of 0.3 M CaCh, for a total of 50 pL per 24-well. This mixture was added dropwise to an equal-volume solution of 2x HEPES-buffered saline (280 mM NaCl, 0.05 M HEPES, 1.5 mM Na2HPO4). After 4 min, the solution was mixed vigorously by pipetting ten times. Next, 100 pl of this mixture was added dropwise to the plated cells in 24-well plates, and the plates were gently swirled.

[0501] For co-transfection assays with separate mini-synTF and reporter plasmids, the mini-synTF plasmid masses were calculated by normalizing to a copy number of 1.66* 1010, approximately 100 ng per 24-well. The reporter plasmid masses were calculated by normalizing to a copy number of 3.63 xlO10, approximately 200 ng per 24-well. These doses of plasmid were determined empirically in prior experiments involving COMET synTFs13. For AAV transfer vector transfection assays, the transfer vector plasmid masses were calculated by normalizing to a copy number of 1.34x 1010, approximately 100 ng per 24-well. All transfection mixes also contained 200 ng of a constitutive mTagBFP2 transfection proxy plasmid. The total mass of all samples in an experiment was held constant by supplementing with empty vector filler DNA (L3928).

[0502] The next morning, the medium was aspirated and replaced with fresh medium, with small molecule or vehicle added where appropriate as detailed above. At 36-48 h post-transfection, cells were harvested for flow cytometry by washing with PBS pH 7.4 and using 0.05% Trypsin-EDTA (Thermo Fisher Scientific 25300120) for 5 min followed by quenching with cold phenol red-free DMEM (Sigma D2902), supplemented with 10% FBS (Gibco 16140-071), 6 mM L-glutamine (2 mM from Gibco 31600-091 and 4 mM from additional Gibco 25030-081), penicillin (lOOU / pL), and streptomycin (100 pg / mL) (Gibco 15140122). Cell suspensions were pipetted and added to 1 mL of FACS buffer (PBS pH 7.4, 2-5 mM EDTA, 0.1% BSA) in 5 mL polystyrene tubes (Fisher 14-961-10A). Cells were spun at 150*g for 5 min, supernatant was decanted, and one drop of fresh FACS buffer was added.A A V production and purification

[0503] AAV293 cells were seeded in 10 cm dishes at a density of 5* 106cells per plate. After 24 h, by which time the cells had adhered to the plate and grown to 70-90% confluency, the cells were transfected. Equimolar amounts (20 pg total) of AAV transfer vector plasmid, rep / cap plasmid (pAAV2 / 5, Addgene #104964; or pXX2, a gift from David Schaffer (University of California, Berkeley)), and pHelper plasmid (a gift from David Schaffer (University of California, Berkeley)) were mixed in H2O, and 2 M CaCb was added to a final concentration of 0.3 M CaCh, for a total of 1 mL per plate. This mixture was added dropwise to an equal-volume solution of 2x HEPES -buffered saline (280 mM NaCl, 0.05 M HEPES, 1.5 mM Na2HPO4). After 4 min, the solution was mixed vigorously by pipetting ten times. Next, 2 mL of this mixture was added dropwise to the plated cells in 10 cm dishes, the plates were gently swirled and placed back in the incubator. The next morning, the medium was aspirated and replaced with fresh medium. After 72-96 h post-transfection, the cells were scraped from the bottom of the plate and pipetted up and down a few times to remove all cells from the bottom. The cell suspension was placed in 15 mL conical tubes and spun at 1500 rpm and 4°C for 15 min. The supernatant was aspirated, and the cell pellets were placed on ice for immediate use or stored at -20°C for later use.

[0504] If frozen, cell pellets were thawed prior to use. Cells were lysed by resuspending in 1 mL lysis buffer30(20 mM Tris, 150 mM NaCl, 10 mM MgCh, pH 7.5) and freeze-thawing 3 times.250 U of Benzonase Nuclease (Millipore 70746-3) and Triton X-100 (BioRad 161-0407) to a final concentration of 0.1% were added to the lysate, and lysate was incubated at 37°C, shaking for 90 min. Lysate was then vortexed and spun at 3500 rpm for 15 min to pellet precipitate. Supernatant was processed through a 0.8 pm filter (in some cases), followed by a 0.45 pm filter. Clarified material was stored at 4°C in microcentrifuge tubes until ready for tittering via qPCR or further processing.

[0505] Dynabeads CaptureSelect AAVX Magnetic Beads (Thermo Scientific 2853522005) were used for purification of AAV2 and AAV5 samples. 40-60 pL of bead slurry was used for each sample, following manufacturer instructions. Beads were washed with 500 pL PBS twice prior to loading, using a magnetic stand to remove discard; then 500-800 pL of sample containing virus was added, incubating for 30 min at room temperature and rocking. A magnetic stand was usedto remove crude lysate “flow through” (which can be kept for further analysis if needed). Loaded beads were washed with 500 pL of PBS twice, and virus was eluted using 50 pL elution buffer (50 mM citric acid, pH 2.5-3), incubating at room temperature for 10 min and pipetting a few times during incubation. After eluate was removed from the beads using a magnetic stand, neutralization buffer (1 M Tris-HCl, pH 8.7) was added immediately at 10% of total eluate volume. Purified material was stored at 4°C in microcentrifuge tubes until ready for use or tittering via qPCR.AAV genomic titer estimation via qPCR

[0506] First, 5 pL of the purified AAV samples or clarified crude lysates were treated with 1 U of DNAse (ThermoFisher EN0521) in lOx DNAse buffer (ThermoFisher B43) to eliminate plasmid DNA, incubating at 37°C for 1 hour followed by a 15 min inactivation step at 75°C. Samples were placed on ice. Then, viral genomic DNA (vgDNA) was extracted using the High Pure Viral Nucleic Acid Kit (Roche 11858874001), following manufacturer instructions. qPCRs were performed by amplifying a sequence within the dsRedExpress2 reporter using primers GACTAC A AGA AGCTGTCC TT CC (forward ), an d CTTC ACGT GGT AG AT GAAGG T G (reverse), which were designed using IDT’s PrimerQuest Tool A plasmid containing the dsRedExpress2 protein (L3509) was used as a standard, serial dilutions were prepared using EASY dilution (TaKara 9160), plating each dilution in duplicate. The vgDNA samples were diluted in NF-HzO, plating 4 serial dilutions per sample (in duplicate). For each qPCR reaction (each in a 96-well), 5 pL of vgDNA sample were mixed with 10 pL Universal S YBR Master Mix 2x (ThermoFisher K0221 ), 0 1 pL forward primer, 0.15 pL reverse primer, and 4.7 pL of NF -H2O. qPCR plates were sealed and spun at 500xg for 2 min before placing in Bio-Rad Cl 000 Thermal Cycler with CFX96 Real-Time System. Samples were heated to 98°C for 3 min, followed by 40 cycles of: 98°C for.15 s, 62°C for 30 s. Then, a final step of 0.5°C increments every 5 s from 65°C to 95°C.

[0507] The number of molecules in each sample was calculated using the Bio-Rad CFX Manager 3.1 software, based on the reported molecules in the standards. For each vgDNA sample dilution, the duplicates were averaged to calculate the number of molecules, which was then multiplied by the corresponding dilution factor, and the four dilutions for each sample wereaveraged and multiplied by 2 (to account for ssDNA) to calculate the genomic titer (number of viral genomes per mb of virus sample).AAV transduction ofHEK293FT, SH-SY5Y, and HuH-7 cells

[0508] Cells were seeded at the densities indicated in figure captions, in either 24-well or 48-well plates. AAV samples were mixed with media and normalized to the concentrations indicated for each panel. 50 pL of media with virus was added to each well. Cells were incubated for 3-7 d at 37°C and 5% CCh. In cases where cells received ligand or vehicle treatment, indicated amounts of small molecule or its corresponding solvent were added at time of plating and 2-3 d post-transduction. Cells were harvested for flow cytometry by washing with PBS pH 7.4 and using 0.05% Trypsin-EDTA (Thermo Fisher Scientific 25300120) for 5 min followed by quenching with cold phenol red-free DMEM (Sigma D2902), supplemented with 10% FBS (Gibco 16140-071), 6 mM L-glutamine (2 mM from Gibco 31600-091 and 4 mM from additional Gibco 25030-081), penicillin (100 U / pL), and streptomycin (100 pg / mL) (Gibco 15140122). Cell suspensions were pipetted and added to 1 mb of FACS buffer (PBS pH 7.4, 2-5 mM EDTA, 0.1% BSA) with 3 pM DAP1 (Thermo Scientific 62247) in 5 mL polystyrene tubes (Fisher 14-961-10A). Cells were spun at 150*g for 5 min, supernatant was decanted, and one drop of fresh FACS buffer with DAPI was added.Analytical flow cytometry

[0509] Flow cytometry was run on a BD LSR Fortessa Special Order Research Product (Robert H. Lurie Cancer Center Flow Cytometry Core). Approximately 3,000-10,000 single, transfected cells were analyzed per sample in transfection experiments. Transfected cells were identified using a separate, single transfection control fluorescent protein (mTagBFP2).

[0510] Samples were analyzed using FlowJo vlO software (FlowJo, LLC). Fluorescence data were compensated for spectral bleed-through. For transfection experiments, as shown in FIG. 6A - FIG. 6F, the HEK293FT cell population was identified by SSC-A versus FSC-A gating, and singlets were identified by FSC-A versus FSC-H gating. To distinguish transfected from nontransfected cells, a control sample of cells was generated by transfecting cells with a mass of pcDNA (empty vector) equivalent to the mass of DNAused in other samples in the experiment. For the single-cell subpopulation of the pcDNA-only sample, a gate was made to identify cellsthat were positive for the constitutive fluorescent protein used as a transfection control in other samples, such that the gate included no more than 1% of the non-fluorescent cells.

[0511] For transduction experiments, as shown in FIG. 6G - 6K, the HEK293FT, SH-SY5Y or HuH-7 cell population was identified by SSC-A versus FSC-A gating, and singlets were identified by FSC-A versus FSC-H gating. Live cells were identified by inclusion of a DAPI viability stain such that DAPI+ (dead) cells were excluded from analysis. Finally, the active reporter gate was set such that the highest 0.1% of non-transduced cells were included.Quantification of reporter output

[0512] AAV transgene expression was quantified by measuring the expression of a fluorescent reporter protein, DsRed-Express2, regulated by the mini-synTF-inducible promoter. To calculate reporter expression, MFI in the PE-Texas Red channel was averaged across three biological replicates, following the gating strategies described above. The MFI was then converted to Molecules of Equivalent PE-Texas Red (MEPTRs). As shown in FIG. 6L- FIG. 6M, to determine conversion factors for MFI to MEPTRs, UltraRainbow Calibration Particles (Spherotech URCP-100-2H) were run with each flow cytometry experiment. These reagents contain nine subpopulations of beads, each with a known number of various fluorophores. The total bead population was identified by FSC-A vs. SSC-A gating, and bead subpopulations were identified through two fluorescent channels. MEPTR values corresponding to each subpopulation were supplied by the manufacturer. A calibration curve was generated for the experimentally determined MFI vs. the manufacturer supplied MEPTRs, and a linear regression was performed with the constraint that 0 MFI equals 0 MEPTRs. The slope from the regression was used as the conversion factor, and error was propagated. Fold differences were calculated by dividing reporter expression with ligand treatment by the reporter expression without ligand treatment. Standard error was propagated through all calculations.Statistical analyses

[0513] All raw data processing and statistical analysis was done using GraphPad Prism version 10.4.1. Comparisons between ligand-treated and vehicle-treated conditions were made using an unpaired Welch’s / -test, which is a version of Student’s / -test in which the variance between samples is treated as not equal. To control for the family -wise error rate when performingmultiple comparisons, the Holm-Sidak method was applied to each set of tests per figure panel (a = 0.05); in all tests, the null hypothesis was rejected for adjusted / ^-values < 0.05. To evaluate the effect of experiment variables, single- or mutli-variable ANOVAs was performed, using Welch’s method along with Dunnett’s T3 multiple comparisons test. In all tests, the null hypothesis was rejected for adjusted / ^-values < 0.05.Mini-synTF Sequences

[0514] Certain exemplary mini-synTF amino acid sequences are shown with annotation of various elements in FIG. 8A - FIG. 8C. Additional exemplary amino acid sequences and nucleic acid sequences are found in Table 1 and Table 2, respectively.Example 2: Design, construction, and validation of mini-synTF Systems

[0515] The present disclosure provides methods for construction of gene and cell therapies in which transgene expression can be controlled by administration of FDA-approved drugs. These methods are particularly distinguished by genetic compactness, rendering them compatible with numerous vectors, including but not limited to size-limited vectors such as those based upon AAV (adeno-associated virus). Conditional transgene expression is a desirable characteristic for AAV-based gene therapies and confers clinical advantages as compared to constitutive transgene expression. Existing and validated strategies for inducible transgene control require transduction of a large DNA cargo, which often exceeds the packaging limitations of AAV vectors. Therefore, a compact, potent, and inducible transgene expression system is needed for gene therapy applications. Towards this end, the present disclosure provides a small drug-inducible minimal synthetic transcription factor (mini-synTF) system for use in AAV (and other applications where genetic compactness is at a premium). The systems and methods presented herein have applications in gene therapy using a DNA based vector (AAV, herpes, plasmid, mini-circle, etc.), biomanufacturing (e.g., stable cell lines to produce biologies under inducible control), cell therapy (e.g., drug-regulable control of transgene expression from therapeutic cell engineered in situ or ex vivo), and regulated control of gene expression from genetic vectors for research applications. Some systems (e.g., those regulated by 4OHT) have particular utility in the CNS because this drug crosses the blood-brain barrier.

[0516] The technology disclosed herein combines compact COMET synthetic transcription factors (synTFs) with validated strategies for controlling larger synTFs using FDA-approved drugs grazoprevir (GZV) and 4-hydroxytamoxifen / tamoxifen (4OHT). The systems are designed to be more potent and compact than any existing transcription factor system (<1.5 kb), while conferring the ability to control activity with FDA-approved drugs. Three modalities for small molecule-regulated control of gene expression were tested (FIG. 1A- FIG. 1C): (FIG. 1A) GZV-induced stabilization (GZV-on); (FIG. IB) GZV-induced destabilization (GZV-off); and (FIG. 1C) 4OHT-induced nuclear localization (4OHT-on). For the GZV-on modality, the NS3 protease (NS3p) was inserted between the DNA binding and activation domains of the synTF, such that the mini-synTF is only active when GZV (an NS3p inhibitor) is present. When GZV is not present, the synTF gets cleaved in half and gene expression is not induced; when GZV is added, the protease activity is inhibited, and transgene expression is induced. GZV-induced destabilization works in a similar mechanism, but in this case the NS3 protease is located on one end of the synTF, and GZV controls the cleavage of a destabilization domain (either a nuclear export sequence or a degradation tag / degron). The GZV-off modality was constructed by inserting the NS3p between the synTF and either a degradation tag (degron) or a nuclear export sequence (NES); thus, this mini-synTF is only active when GZV is absent. Finally, the 4OHT-on modality was achieved by fusion of ERT2 (mutant human estrogen receptor with enhanced ligand sensitivity) to the synTF such that 4OHT induces nuclear localization, inducing transgene expression.

[0517] For each small molecule control modality, multiple mini-synTF topologies for constructing fusion proteins were explored (FIG. 1A- FIG. 1C), testing the hypothesis that geometric constraints might cause different orientations to vary in their ability to bind DNA and recruit transcriptional machinery. The function of these designs was validated via transient cotransfection of mini-synTF and reporter plasmids in HEK293FT cells. In all modalities, designs exhibiting desired performance characteristics were identified, including high levels of drug-controllable reporter expression and low levels of off-state reporter expression, or “leakiness” (FIG. ID - FIG. IF). Multiple synthetic promoter topologies were also explored (FIG. 1G), comparing compact and spaced architectures. Without wishing to be bound to any particular theory, it was hypothesized that since the mini-synTFs include larger subdomains (e.g., NS3p and ERT2) than did previously characterized synTFs, the compact promoter architecture couldresult in steric hindrance when all of the binding sites are occupied by TFs. However, the compact promoter architecture resulted in higher reporter expression and fold inductions compared to a more spaced out promoter across all small molecule control modalities (FIG. 1H - FIG. IK), concordant with previous characterizations of the constitutively-active zinc finger TFs. These results suggest that if any steric hindrance occurs with the new mini-synTFs, those costs are outweighed by the benefits of the compact architecture. Furthermore, on-state reporter expression was directly compared for the different drug-controllable modalities to a constitutively active COMET synTF containing the same activation domain (AD) and zinc-finger DNA-binding domain (DBD) (FIG. IL). All modalities exhibited comparable magnitudes of on-state reporter expression, and in some cases, this was higher than that conferred by their equivalent constitutively active COMET synTF. This suggests that drug-regulated control parts (NS3p and ERT2) can be introduced without compromising the potency of the synTF / promoter system.Example 3: Exemplary Small-Molecule-Controllable Transcription Regulators Amenable to AAV Delivery

[0518] The present Example describes development of a set of compact, potent, small-molecule-controllable transcriptional regulators amenable for delivery via an AAV vector. Among other things, the present disclosure appreciates that an effective platform of composable mammalian elements of transcription (COMET) has been described (see, for example, WO2018 / 175865; see also, Donahue et al., Nature Comm. 11:779, 2020), and that certain effective embodiments of such COMET transcriptional regulators utilize zinc-finger binding domains linked to transcription activation domains to achieve regulated expression of transcribable sequences associated with an appropriate zinc-finger binding site.

[0519] The present Example specifically describes improvements that include, among other things, development of engineered transcriptional modulators whose activity can be exogenously controlled, for example via a small molecule agent; particular utilized small molecule agents are FDA-approved drugs. Described improvements further include development of engineered transcriptional modulators sufficiently small in size that a gene encoding such engineeredtranscriptional modulator and a regulator-responsive gene (e.g., including an appropriate DNA binding site(s) specifically recognized by such engineered transcriptional modulator) operatively associated with a transcribable sequence (e.g., a transgene of interest) can fit in a single AAV genome.

[0520] Thus, the present disclosure surprisingly demonstrates successful development of engineered transcriptional modulators sufficiently compact that a gene encoding them can be included in a single construct, together with a gene of interest (e.g., a transgene) and appropriate regulatory sequences so that transcription of the gene of interest is modulated by the engineered transcriptional modulator (i.e., by binding of the engineered transcriptional modulator to its binding site in the regulatory sequences), and that entire construct remains sufficiently small to be amenable to AAV-type delivery.

[0521] It is particularly surprising that sufficiently compact transcriptional modulators were successfully developed when a regulatory element, conferring responsiveness to an exogenous agent (e.g., a small molecule agent), was included in the engineered transcriptional modulator.

[0522] The present Example specifically describes development of certain compact engineered transcriptional modulators that are zinc-finger synthetic transcription factors (synTFs), and in particular compact synTFs (mini-synTFs) that are responsive to FDA-approved drugs such as grazoprevir (GZV) and 4-hydroxytamoxifen / tamoxifen (4OHT).

[0523] The present Example specifically documents three different drug-controllable modalities for controlling mini-synTF activity, demonstrating impact on expression of a gene of interest (e.g., a transgene). Systems documented in the present Example are more potent and compact than other existing transcription factor systems, and furthermore confer an ability to be controlled with an FDA-approved drug.

[0524] Of note, certain provided engineered transcriptional modulators, including certain drug-controlled engineered transcriptional modulators are demonstrated to be effective in neuronal cells and / or upon AAV delivery.

[0525] In certain embodiments, systems disclosed herein (i.e., in the present Example and elsewhere) are designed to be more potent and compact than any existing transcription factor system, while conferring the ability to control activity with FDA-approved drugs.

[0526] Materials and methods used in the present Example are provided in Example 1.Mini-synTF AA V Vector Design and Validation

[0527] To adapt the mini-synTF designs for deployment in an AAV architecture, key vector design considerations were first evaluated. The AAV vectors utilized in the present disclosure that were regulated by mini-synTFs contained two transcriptional units: one unit encoding the mini-synTF and driven by a constitutive promoter, and a second inducible unit encoding the transgene with expression driven by the mini-synTF via its cognate engineered (also referred to in this example as “synthetic”) promoter (FIG. 3A).

[0528] One consideration relevant to performance of mini-synTF designs was the relative orientation of the transcriptional units when including a constitutive and inducible unit in the same vector. To address this question, three different AAV vector configurations were tested, termed upstream tandem (UT), upstream divergent (UD), and downstream divergent (DD) (FIG.3B). To mitigate bleed-through from the constitutive promoter into the inducible promoter and thus reduce leakiness of the circuit, the effects of including insulator sequence(s) (in this particular case, a pair of cHS4 insulators) between the two transcriptional units was also compared. Some design choices were fixed (e.g., choice of constitutive promoter, poly-A tails, and transgene introns) for convenience.

[0529] Accordingly, in some embodiments, an AAV vector provided by and / or utilized in accordance with the present disclosure comprises a constitutive mini-synTF unit and an inducible transgene with a synthetic promoter. In some embodiments, the constitutive mini-synTF unit and the inducible transgene with a synthetic promoter are separated by an insulator sequence. In some embodiments, the insulator sequence is a cHS4 insulator sequence. In some embodiments, the constitutive mini-synTF unit and the inducible transgene with a synthetic promoter are configured in an upstream tandem (UT), upstream divergent (UD), or downstream divergent (DD) configuration. In some embodiments, the constitutive mini-synTF unit has a pCBA promoter and bGH polyAtail. In some embodiments, the inducible transgene with a synthetic promoter has a hybrid intron and a SV40 polyA tail.

[0530] Vector configurations assessed in the present Example were validated by transient transfection of the AAV transfer vector (ITR-containing) plasmids into HEK293FT cells. Alldrug-regulated modalities qualitatively exhibited the desired expression behaviors, and the overall magnitude of transgene expression was comparable to that conferred by transfection of a similar CMV promoter-driven AAV transfer vector (FIG. 3G - FIG. 3 J). For the drug-on modalities, the upstream divergent architecture conferred higher levels of transgene expression than did other architectures. However, the downstream divergent architecture performed best for the GZV-off modality.

[0531] As expected, removing the cHS4 insulator increased leakiness in most cases, except for upstream tandem configurations; notably, the promoter sequences are further apart in the latter case. For some configurations, removing the insulator resulted in only slight increases of on-state reporter expression.

[0532] Interestingly, some of the modalities conferred higher on-state reporter expression than did a CMV-driven control transfer vector. Since all vector architectures performed as desired in transient transfection, they were carried forward to testing in AAV transduction before choosing an optimal implementation, since it was unclear whether performance trends observed in transfection analysis would hold when control systems were delivered by AAV transduction.

[0533] Mini-synTF driven inducible transgene expression in AAV transduction of HEK293FT, the cell type previously used for transfection-based analyses, was then evaluated. The mini-synTF transfer vectors were packaged into AAV2 capsids and viral dosing was normalized based on viral genomic DNA counts, quantified by qPCR. All three vector configurations were tested in transduction of HEK293FT cells, including constructs with and without the cHS4 insulator sequence, as well as a CMV-driven reporter control vector. For both drug-on modalities (4OHT-on and GZV-on) a significant difference in reporter expression levels was observed between drug-treated and vehicle (solvent only)-treated cells at the whole population level, i.e., including both transduced and non-transduced cells (FIG.3C and FIG. 3E). The 4OHT-on modality showed the best performance overall, with the highest reporter expression levels and transduction levels (up to 70%) in drug-treated cells. In all vector designs, the background (vehicle-treatment) reporter expression was minimal, resulting in high fold inductions across all different configurations. The GZV-on modality showed slightly lower reporter expression levels than did the 4OHT-on modality, although differences between drug-treated and vehicle-treated cells were still significant. Consistently for both modalities, removing the cHS4 insulator coresometimes resulted in increased leakiness (for the divergent configurations only), without providing substantial improvements in on-state reporter expression. Overall, the upstream divergent modality showed high performance (vis-a-vis on-state reporter expression and fold induction) across modalities. When comparing the AAV transfer vector transfection (FIG. 3G -FIG. 3J) with the AAV transduction results, trends were conserved for the GZV-on modality but not for the 4OHT-on modality. Without wishing to be bound to any particular theory, it was hypothesized that since the 4OHT-on modality relies on endogenous cell mechanisms of nuclear import / export (driven by ERT2), it might be more sensitive to changes in copy number regimes-from high-copy levels in transfection to a low-copy regime in AAV transduction. The GZV-on modality, on the other hand, is more independent of endogenous cellular processes since it only relies on NS3p activation, which could make it less sensitive to this copy number effect.Interestingly, none of the GZV-off vectors exhibited a significant difference in reporter expression between drug-treated and vehicle-treated cells (FIG.3K and FIG. 3L), even though signal magnitude was on-par with other modalities.

[0534] These findings also provided insight into how functional AAV titer may vary across constructs. It was noted that a CMV-driven reporter vector control conferred both higher levels of reporter expression (up to an order of magnitude greater than mini-synTF constructs) and high transduction efficiency (almost 100%) (FIG. 3M). This significant difference was surprising given that the mini-synTF modalities had consistently performed on par with, and sometimes out-performed, the CMV control in all transfection-based experiments. Without wishing to be bound by any particular theory, it is proposed that one possible explanation may be that a difference in potency between the mini-synTF and CMV control AAV vectors might derive from differences in genome size (rather than differences in performance of the drug-control modalities versus the constitutive promoter, given the transfection-based analyses). The CMV control genome is about 2 kb smaller than the mini-synTF genomes, which could result in more efficient packaging and transduction. Since the size of the CMV control vector would allow for two genomes to be packaged into the same viral capsid, there is also a possibility that double genomes or even self-complementary genomes are being packaged, which are widely-known for improving transduction efficiency and transgene expression levels, effectively shifting the relationship between physical and functional titer for such constructs27.

[0535] Additionally, effects of different mini-synTF design choices in the context of AAV transduction of HEK293FT cells were explored. Design parameters predicted to have the most impact on reporter expression, based on the prior transfection-based characterization, were prioritized. First, the activation domain choice in both on-switch modalities was examined, comparing the four ADs previously characterized: VP64, p65.1, p65.2, and RTA.2 (FIG. 4A and FIG. 4B). Both VP64 and p65.2 showed significant inducible reporter expression levels, performing similarly across both on-switch modalities. Interestingly, the magnitudes of reporter expression were comparable between the two ADs, which was not the case in transfection experiments, where it was observed that p65.2 outperformed VP64 (especially in the GZV-on modality).

[0536] Next, effects of the number of synTF binding sites in the inducible synthetic promoter driving the expression of the transgene (fluorescent reporter in this case) was examined (FIG.4C and FIG. 4D) All promoter variants conferred similar levels of on-state reporter expression, with differing fold inductions driven by variations in off-state reporter expression. Promoters with 6 synTF binding sites showed the best inducibility, with larger arrays increasing control system size without marginal improvements in performance. For the GZV-off modality, different destabilization domains were examined in an analysis focused on reducing off-state reporter expression (FIG. 4E). Only the human ODC-1 sequence incorporated as 3-tandem repeats reduced leaky expression enough to produce a statistically significant difference in reporter signal between on- and off-states. Overall, the findings indicate that the engineered on-switch mini-synTF modalities successfully provide drug-regulatable control of AAV transgene expression in HEK293FT cells, with inductions up to 200-fold.Sensitivity Analysis of Mini-synTF Design Choices

[0537] Following the initial mini-synTF evaluation, a local sensitivity analysis was performed with respect to certain design choices. To this end, variants of all three mini-synTF modalities were engineered to evaluate different DNA binding elements (also referred to in the Examples as DNA binding domains or DBDs), transcription modulation elements (e.g., transcriptional activation domains (ADs)), regulatory elements (e.g., nuclear export sequences (NES) or degradation domains (e.g., for GZV-off modality) or nuclear translocation domains), and linkers.To enable a higher-throughput scan across these design choices, performance was evaluated via transient co-transfection of mini-synTF and reporter plasmids into HEK293FT cells.

[0538] Two previously characterized zinc-finger DBDs, ZF1 and ZF6, were tested. Designs employing ZF6 yielded higher reporter expression in all modalities (FIG. 2A- FIG. 2C), which concords with previously observed trends but was not obvious a priori given the novel regulatory elements included in presently disclosed mini-synTFs. In addition, for both drug-on modalities, a panel of compact activation domains previously employed in AAV-mediated delivery of CRISPR-Cas9 activators were tested and compared to the VP64 activation domain used in the prototyping experiments. Such activation domains were of interest due to their size and human origin (e g., p65), as compared to VP64’s viral origin. Without wishing to be bound by any particular theory, it is proposed that use of a human activation domain may reduce immunogenicity. For both modalities, a large increase in both on-state reporter expression and fold induction for constructs including the p65.2 AD were observed relative to those employing other activation domains (FIG.2E and FIG. 2F). It was surprising that p65.2 significantly outperformed the other ADs, especially in the GZV-on modality, given that these activation domains all showed similar performance in a CRISPR-Cas9 study.

[0539] Even though the GZV-on modality on-state greatly increased with the p65.2 AD, the reporter expression levels were still lower than the 4OHT-on and GZV-off modalities, which suggested that the GZV-on modality could be further optimized to increase on-state reporter expression. Without wishing to be bound to any particular theory, it was hypothesized that inserting the NS3p between the synTF DNA-binding domain and activation domain could be causing a geometric constraint for binding to DNA and / or transcriptional co-factors, thus reducing the transcriptional potency of the construct. To investigate this, a panel of GZV-on constructs was engineered with linkers of varying length and flexibility, inserted either between the DBD and NS3p, between the NS3p and AD, or both. It was found that linker location can have a significant impact on mini-synTF performance, in a manner that was dependent on AD choice, while linker identity had a smaller effect on performance (FIG. 2F and FIG.2G). For constructs with a VP64 AD, adding a linker between the NS3p and the AD greatly increased reporter expression, while the addition of a linker between the NS3p and the DBD had little effect on reporter expression. The addition of both linkers resulted in a greater increase in reporter expression than the inclusion of either linker individually. For constructs with a p65.2AD, all linker additions explored resulted in a reduced reporter expression compared to the base case. Across these explorations, linker location was more impactful than linker length and flexibility, with a slight preference towards longer and unstructured linkers. It was also found that the benefits of linker additions were dependent on AD choice. This supports the hypothesis that the geometry of the mini-synTF parts is important, especially for recruiting transcriptional co-factors, which varies across different ADs.

[0540] For the GZV-off system, several linkers with varying length and flexibility were also evaluated. Without wishing to be bound to any particular theory, it was hypothesized that including more space and / or flexibility between the NS3p cleavage site and the destabilization domain could improve mini-synTF performance, by allowing better function of the destabilization domain and thus reduction of the off-state leakiness. In general, linker choice minimally impacted GZV-off mini-synTF performance, with some potential advantages conferred by use of longer, more unstructured linkers (FIG. 2J) as seen in the GZV-on panel. Next, adding additional repeats of the degron sequence to enhance the degradation activity and reduce off-state leakiness was explored. For the original design, a degradation sequence derived from the widely used murine ODC-1 protein was used. Two segments of the mODC-1 protein that were identified as degradation tags were compared, with one of them having been validated by others as an efficient degradation tag, and the one showing better performance was selected (FIG. 2K). Constructs containing 2 and 3 repeats of the mODC-1 degron were engineered, as well as constructs with the homologous sequence of the human ODC-1 protein (also with 2 or 3 repeats), which has not been previously explored for this purpose, to humanize the constructs. The human ODC-1 degron performed similarly to the murine ODC-1 degron (FIG. 2H), validating a new humanized sequence for degradation applications. Moreover, including degron multimers increased the degradation activity for both types of tags, increasing mini-synTF fold induction without significant reductions in on-state reporter expression.

[0541] Finally, how synthetic promoter design choices affected mini-synTF performance was evaluated. Specifically, the effect of the number of synTF binding sites in the synthetic promoter on mini-synTF reporter expression and fold induction was evaluated. Based on previous work, reporter expression was expected to increase along with increasing number of synTF binding sites, and so elucidating the tradeoffs between promoter size and reporter expression was of interest. Up to this point, promoters with 6 binding sites were tested, so a new panel of promotersranging from 2-12 binding sites was engineered. For the 4OHT-on modality, reporter expression increased with number of binding sites and plateaued (and even slightly decreased) when the number of binding sites exceeded 8 (FIG. 21). This characterization enables one to evaluate the tradeoff between output magnitude and genetic control system “footprint” (i.e., within an AAV vector). Notably, mini-synTF -mediated activation of promoters with varying binding site counts followed trends observed with constitutive COMET synTFs (FIG. 2L), with 4OHT-inducible mini-synTFs conferring even higher expression levels for the same DBD and AD. Overall, the optimization results showed that mini-synTF systems are tunable, similar with some trends observed with COMET synTFs, providing a varied range of output magnitudes and fold inductions.Example 4: Evaluation of Mini-synTF Control of Transgene Expression in Neuronal Cells

[0542] The present Example documents activity, and effective small molecule control thereof, of three-different modalities of compact engineered transcriptional modulators in neuronal cells.

[0543] Materials and methods used in the present Example are described in Example 1.

[0544] Whether mini-synTF engineered transcriptional modulators provided herein (e.g., as exemplified in Example 1) could confer drug-controllable transgene expression in a target cell line of interest (beyond the HEK293FT testbed) was examined. The human neuroblastoma cell line SH-SY5Y was selected, which is representative of neuronal cells. Since the 4OHT-on modality showed the best performance in HEK293FTs, and since 4OHT can cross the bloodbrain barrier, different 4OHT-on AAV vector configurations were tested in transduction of SH-SY5Y cells (FIG. 5A- FIG. 5C). Even though transduction efficiencies were lower in this first experiment (compared to transductions of HEK293FTs), a substantial difference between drug-treated and vehicle-treated cells at the whole population level, i.e., including both transduced and non-transduced cells, was still observed. When analyzing only cells that were transduced (i.e., to control for low transduction efficiency), reporter on-state expression levels comparable to those conferred by a CMV-driven vector was observed. While this analysis may over-estimate the level of vehicle-induced transgene expression (background) given the gating strategy, even with this potential artifact the difference between on and off states was substantial. It is expected thatimproved transduction efficiencies in SH-SY5Y cells will result in larger differences between ligand and vehicle-treated cells at the whole-population level.

[0545] Applicant has therefore developed multiple modalities of drug-controllable regulation of transgene expression using ultra-compact mini-synTFs and validated their use in AAV vectors. Using FDA-approved small-molecule drugs, over 200-fold activation of an AAV-delivered transgene was achieved, with minimal off-state transgene expression, an important feature for clinical applications. In some cases, mini-synTF vectors conferred transgene levels comparable to a constitutive control vector, highlighting the potency of mini-synTFs described herein. Furthermore, the presently disclosed mini-synTF controllers are more compact than any existing system, leaving substantial capacity for a payload transgene.

[0546] A sensitivity analysis of mini-synTF design choices was performed to identify promising compositions for each drug-controllable system. Features such as activation domain, zinc-finger domain, and linker location had a significant effect on system performance. In certain respects, the present findings from use of mini-synTFs were similar to those from use of constitutively-active COMET synTFs. For example, ZF6 outperformed ZF1 across modalities, and compact promoter binding sites consistently resulted in higher reporter expression than did spaced promoter binding sites. On the other hand, certain mini-synTF feature choices conferred combinatorial effects that would not have been predictable based upon the contributions of individual choices, highlighting the importance of performing such sensitivity analyses. For example, engineering of features modulating mini-synTF geometry can impact binding to DNA and recruitment of transcriptional machinery and co-factors, as observed with the GZV-on modality, which was more impacted by linker additions than the GZV-off modality. Without wishing to be bound by any particular theory, this finding may indicate that addition of linkers between the NS3p and synTF parts can provide needed flexibility to enable coordination of transcriptional activation. This effect varied based on activation domain choice, supporting a hypothesis that performance differences are driven by geometric constraints for co-factor and DNA binding, which would vary with activation domain choice.

[0547] In addition to generating novel drug-regulated synthetic transcription factor modalities, their utility in driving expression of an AAV-delivered transgene was validated. Designing AAV vectors containing all system parts was complex and required assessment of various vectordesign choices. In particular, mini-synTF vectors integrated two transcriptional units: one encoding for the mini-synTF and driven by a constitutive promoter, and a second unit encoding for the transgene (a fluorescent reporter protein in this study) and driven by a mini-synTF regulated synthetic promoter. Different configurations for arranging these transcriptional units was explored, and it was found that even though all exhibited some desirable performance, the upstream divergent configuration consistently outperformed the rest. In general, addition of a cHS4 insulator between the transcriptional units aided in reducing system leakiness, without affecting on-state transgene expression levels. Interestingly, transfection-based analysis of regulatory systems within AAV transfer vectors predicted the rank-order performance of different vector designs when evaluated by vector transduction, but this was only true for GZV-regulated modalities. Without wishing to be bound to any particular theory, it was hypothesized that since 4OHT-based modalities require interaction with endogenous nuclear import mechanisms, they are more sensitive to changes in copy number and thus performance at high copies of mini-synTFs (transfection) differs from performance under lower copies of mini-synTFs (transduction). Overall, several mini-synTF AAV vector designs exhibited desirable performance characteristics for each of the drug-regulated modalities investigated.

[0548] Options for future development of mini-synTF systems for AAV transgene expression are also identified herein. For GZV modalities, some applications could raise immunogenicity concerns8due to the use of the viral (or more importantly, non-human) NS3 protease.Humanizing the sequence could help to mitigate such risks. 4OHT modalities could offer some advantage in this respect, since their regulation is mediated by ERT2, which is of human origin. The best performing activation domain in all modalities was identified as a truncation of p65, also of human origin. Such considerations could also benefit from the novel human ODC-1 degradation tags characterized in this study. Additionally, in some AAV implementations, mini-synTF systems showed reduced potency (i.e., infectious units per physical viral genome) compared to a CMV control vector. This effect was observed when transducing two different cell lines, and without wishing to be bound to any particular theory, it was hypothesized that the difference may be driven by differences in genome size which can impact viral packaging efficiency and infectivity, rather than any direct limitations of mini-synTF control modalities.Example 5: Exemplary Small-Molecule-Controllable Transcription Regulators Comprising Mechanosensitive Transcription Modulation Elements

[0549] The present Example documents activity, and effective small molecule control thereof, of two different modalities of compact engineered transcriptional modulators comprising mechanosensitive transcription modulation elements.

[0550] Materials and methods used in the present Example are provided in Example 1.

[0551] Use of different transcription activation domains was evaluated for ability to modulate performance of GZV-on transcription factors. Five transcription activation domains previously used with mechanosensitive transcription factors (such as, e.g., MRTF-A) were used in place of activation domains evaluated in the preceding Examples. See, e.g., Mahata et al. (2023) Nature Methods, 20: 1716-1728. Transcription factors employing MRTF-A outperformed all alternatives in this set, including transcription factors employing p65.2 (FIG. 7A). Performance of MRTF-A synthetic transcription factors (including both GZV-on and 4OHT-on modalities) was also evaluated when delivered by AAV transduction of HEK293FT cells (FIG. 7B and FIG. 7C). MRTF-A conferred better performance with the GZV-on modality than did p65.2 (FIG. 7B), consistent with the prior transfection-based evaluation (see, e.g. Example 3), but for 4OHT-on transcription factors, p65.2 conferred better performance than did MRTF-A (FIG. 7C). Without wishing to be bound by any particular theory, it is proposed that this finding supports the idea that interaction between activation domain choice and control modality impacts performance.EQUIVALENTS

[0552] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual embodiments of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0553] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.REFERENCES1. Dunbar, C. E. et al. Gene therapy comes of age. Science 359, eaan4672 (2018).2. Wang, D., Tai, P. W. L. & Gao, G. Adeno-associated virus vector as a platform for gene therapy delivery. Nature Reviews Drug Discovery 18, 358-378 (2019). doi.org / 10.1038 / s41573-019-0012-93. Cring, M. R. & Sheffield, V. C. Gene therapy and gene correction: targets, progress, and challenges for treating human diseases. Gene therapy 29, 3-12 (2022).4. Mendell, J. R. et al. Current clinical applications of in vivo gene therapy with AAVs. Molecular Therapy 29, 464-488 (2021).5. Burdett, T. & Nuseibeh, S. Changing trends in the development of AAV-based gene therapies: a meta-analysis of past and present therapies. 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Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors. Human gene therapy 22, 1143-1153 (2011).32. Donahue, P. S. et al. The COMET toolkit for composing customizable genetic programs in mammalian cells. Nature communications 11, 779 (2020).33. Van Rosmalen, M., Krom, M. & Merkx, M. Tuning the flexibility of glycine-serine linkers to allow rational design of multidomain proteins. Biochemistry 56, 6565-6574 (2017). 34. O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic acids research 45, 9901-9916 (2017).35. Maeder, M. L. et al. CRISPR RNA-guided activation of endogenous human genes.Nature methods 10, 977-979 (2013).36. Rechsteiner, M. & Rogers, S. W. PEST sequences and regulation by proteolysis. Trends in Biochemical Sciences 21, 267-271 (1996).37. 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Claims

WHAT IS CLAIMED IS:

1. An engineered transcriptional modulator polypeptide, comprising:(a) one or more DNA binding elements comprising one or more zinc finger DNA binding elements;(b) one or more transcription modulation elements comprising one or more transcription activation elements or one or more transcription repression elements;(c) one or more regulatory elements that confers responsiveness to a small molecule regulator; and(d) optionally one or more linker elements,the engineered transcriptional modulator polypeptide being characterized in that it has a sufficiently compact length such that it can be encoded by a nucleic acid construct having a total length of not more than 4.8 kb, wherein the nucleic acid construct comprises:(i) a nucleic acid sequence that encodes the engineered transcriptional modulator polypeptide, operatively linked with a first promoter and, optionally, a first set of transcription regulatory sequences; and(ii) a nucleic acid sequence encoding one or more genes of interest operatively linked with a second promoter and transcription regulatory sequences including at least one binding site recognized by the DNA binding element.

2. The engineered transcriptional modulator polypeptide of claim 1, wherein the one or more regulatory elements comprise a protease element, a localization element, a pair of proximity elements, or fragments, or variants or any combination thereof.

3. The engineered transcriptional modulator polypeptide of claim 1 or 2, wherein the one or more transcription modulation elements is a transcription activation element.

4. The engineered transcriptional modulator polypeptide of claim 1 or 2, wherein the one or more transcription modulation elements is a transcription repression element.

5. The engineered transcriptional modulator polypeptide of claim 1 or 2, wherein the one or more transcription modulation elements comprise a VP64 domain, a p65.1 domain, a p65.2 domain, a RTA.2 domain, a VP16 domain, a VPR domain, a p300 domain, a CBP domain, a Hsfl domain, a Swi / Snf domain, a MED15 domain, a E1A domain, a Gal4-AD domain, a B42 domain, a p65 domain, or any biological equivalent thereof, or any variant thereof or any fragment thereof.

6. The engineered transcriptional modulator polypeptide of any one of the preceding claims, wherein the one or more zinc finger DNA binding elements comprise one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, and / or ZF19.

7. The engineered transcriptional modulator polypeptide of any one of the preceding claims, wherein the one or more zinc finger DNA binding elements comprises one or more ZF6.

8. The engineered transcriptional modulator polypeptide of any one of claims 1-7, characterized in that, when present in a cell that also includes a nucleic acid construct in which one or more genes of interest is operatively linked with at least one binding site recognized by the one or more DNA binding elements, expression of the one or more genes of interest is modulated responsive to the small molecule regulator.

9. The engineered transcriptional modulator polypeptide of claim 8, wherein the nucleic acid construct comprises: (i) a nucleic acid sequence encoding the engineered transcriptional modulator polypeptide operatively linked with an engineered promoter, and optionally with one or more regulatory sequences, and (ii) the one or more genes of interest operatively linked with the at least one binding site recognized by the DNA binding element.

10. The engineered transcriptional modulator polypeptide of any one of claims 1-9, wherein the one or more transcriptional modulation elements is a human element, or a humanized element, or a fragment of either of the foregoing.

11. The engineered transcriptional modulator polypeptide of any one of claims 1-10, wherein the one or more regulatory elements is or comprises a protease element.

12. The engineered transcriptional modulator polypeptide of claim 11, wherein the protease element is or comprises an NS3p protease, optionally wherein the NS3p has an amino acid sequence of SEQ ID NO: 85 or a sequence having at least 85% identity thereto.

13. The engineered transcriptional modulator polypeptide of claim 12, wherein when the protease element is or comprises NS3p, the small molecule regulator comprises grazoprevir.

14. The engineered transcriptional modulator polypeptide of any one of claims 1-10, wherein the one or more regulatory elements is or comprises a localization element.

15. The engineered transcriptional modulator polypeptide of claim 14, wherein the localization element comprises a nuclear localization element.

16. The engineered transcriptional modulator polypeptide of claim 14 or 15, wherein the localization element is or comprises ERT2, optionally wherein the ERT2 has an amino acid sequence of SEQ ID NO: 86 or a sequence having at least 85% identity thereto.

17. The engineered transcriptional modulator polypeptide of claim 16, wherein when the localization element is or comprises ERT2, the small molecule regulator comprises 4-hydroxytamoxifen.

18. The engineered transcriptional modulator polypeptide of any one of claims 1-17, wherein the one or more regulatory elements further comprises one or more destabilization elements, e.g., one or more degradation domains (e.g., degradation tag (degron)) or a nuclear export signal.

19. The engineered transcriptional modulator polypeptide of any one of claims 1-18, wherein (c) is situated between (a) and (b).

20. The engineered transcriptional modulator polypeptide of any one of claims 1-18, wherein a linker element is present between (a), (b), and / or (c).

21. The engineered transcriptional modulator polypeptide of any one of claims 1-20, wherein (a) is situated at the N terminus of (b), or (a) is situated at the C terminus of (b).

22. The engineered transcriptional modulator polypeptide of any one of claims 1-21, wherein the engineered transcriptional modulator polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 1 - 37, or a sequence with at least 85% identity thereto.

23. An expression system comprising an engineered transcriptional modulator polypeptide of any one of claims 1-22 or a plurality of polypeptides of any one of claims 1-22.

24. A delivery system comprising an engineered transcriptional modulator polypeptide of any one of claims 1-22 or a plurality of polypeptides of any one of claims 1-22.

25. A nucleic acid construct encoding an engineered transcriptional modulator polypeptide of any one of claims 1-22, or a plurality of polypeptides of any one of claims 1-22.

26. A nucleic acid construct comprising:(a) a first sequence encoding an engineered transcriptional modulator polypeptide, operatively linked with a promoter and, optionally with one or more transcriptional regulatory sequences; and(b) a second sequence encoding one or more payloads operatively linked with a promoter and at least one transcriptional regulatory sequence that includes a binding site for a DNA binding element in the engineered transcriptional modulator.

27. The nucleic acid construct of claim 26, wherein the nucleic acid construct comprises one or more additional elements.

28. The nucleic acid construct of claim 27, wherein the one or more additional elements comprises an insulator element.

29. The nucleic acid construct of claim 28, wherein the insulator element is situated between the first sequence and the second sequence.

30. The nucleic acid construct of claim 28 or 29, wherein the insulator element comprises a sequence of SEQ ID NO: 100 or a fragment or a variant thereof.

31. The nucleic acid construct of any one of claims 26-30, wherein the engineered transcriptional modulator polypeptide comprises:(a) one or more DNA binding elements comprising one or more zinc finger DNA binding elements;(b) one or more transcription modulation elements comprising one or more transcription activation elements or one or more transcription repression elements;(c) one or more regulatory elements that confer responsiveness to a small molecule regulator; and(d) optionally one or more linker elements.

32. The nucleic acid construct of claim 31, wherein the one or more regulatory elements comprise a protease element, a localization element, a pair of proximity elements, or fragments, or variants or any combination thereof.

33. The nucleic acid construct of claim 31 or 32, wherein the one or more transcription modulation elements is a transcription activation element.

34. The nucleic acid construct of claim 31 or 32, wherein the one or more transcription modulation elements is a transcription repression element.

35. The nucleic acid construct of any one of claims 31-34, wherein the one or more transcription modulation elements comprise a VP64 domain, a p65.1 domain, a p65.2 domain, a RTA.2 domain, a VP16 domain, a VPR domain, a p300 domain, a CBP domain, a Hsfl domain,a Swi / Snf domain, a MED15 domain, a E1A domain, a Gal4-AD domain, a B42 domain, a p65 domain, or any biological equivalent thereof, or any variant thereof or any fragment thereof.

36. The nucleic acid construct of any one of claims 31-35, wherein the one or more zinc finger DNA binding elements comprise one or more of ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, ZF11, ZF12, ZF13, ZF14, ZF15, ZF16, ZF17, ZF18, and / or ZF19.

37. The nucleic acid construct of any one of claims 31-36, wherein the one or more zinc finger DNA binding elements comprise one or more ZF6.

38. The nucleic acid construct of any one of claims 31-37, wherein the one or more transcriptional modulation elements is a human element, or a humanized element, or a fragment of either of the foregoing.

39. The nucleic acid construct of any one of claims 26-38, wherein the one or more payloads is a polypeptide payload.

40. The nucleic acid construct of any one of claims 26-38, wherein the one or more payloads is a nucleic acid payload (e.g., an RNA payload).

41. The nucleic acid construct of any one of claims 26-40, wherein the first sequence is positioned 5’ of the second sequence in the nucleic acid construct.

42. The nucleic acid construct of any one of claims 26-40, wherein the first sequence is positioned 3’ of the second sequence in the nucleic acid construct.

43. The nucleic acid construct of any one of claims 26-42, wherein the first sequence and the second sequence are positioned in opposite directions to each other in the nucleic acid construct.

44. The nucleic acid construct of any one of claims 26-40, wherein the first sequence and the second sequence are in a divergent configuration in the nucleic acid construct.

45. The nucleic acid construct of claim 44, wherein the first sequence is 5’ to the second sequence.

46. The nucleic acid construct of claim 44, wherein the first sequence is 3’ to the second sequence.

47. The nucleic acid construct of any one of claims 26-46, wherein the first sequence and the second sequence are separate transcription units.

48. The nucleic acid construct of any one of claims 26-47, wherein the nucleic acid construct is no more than 4.8 kb in length.

49. The nucleic acid construct of any one of claims 26-48, wherein the nucleic acid construct has a sequence of any one of SEQ ID NOs: 39 to 70, or a sequence with at least 85% identity thereto.

50. An expression system comprising the nucleic acid construct of any one of claims 25-49, or a plurality of nucleic acid constructs of any one of claims 25-49.

51. A delivery system comprising the nucleic acid construct of any one of claims 25-49, or a plurality of nucleic acid constructs of any one of claims 25-49.

52. The delivery system of claim 51, comprising a vector (e.g., a viral vector), a lipid-based system and / or a polymer-based system.

53. A recombinant adeno-associated virus (rAAV) particle comprising:(i) an AAV capsid protein; and(ii) a nucleic acid construct of any one of claims 25-49.

54. The rAAV particle of claim 53, wherein the nucleic acid construct comprises one or more ITR sequences.

55. The rAAV particle of claim 54, wherein the nucleic acid construct comprises a pair of AAV ITRs flanking:(i) the nucleic acid sequence encoding the engineered transcriptional modulator polypeptide, and(ii) the nucleic acid sequence encoding one or more genes of interest.

56. The rAAV particle of any one of claims 53-55, wherein the AAV capsid protein comprises a capsid protein from: AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 (rh10), AAV11, AAV12, AAV13, AAV-DJ, or AAV-PHP.eB, optionally wherein the AAV capsid protein comprises an AAV2 capsid protein.

57. A cell comprising one or more nucleic acid constructs of any one of claims 25-49.

58. A cell engineered to express one or more engineered transcriptional modulator polypeptides of any one of claims 1-22.

59. A cell engineered to express a payload from a nucleic acid construct that comprises a sequence encoding the payload operatively linked with a promoter and at least one transcriptional regulatory sequence.

60. The cell of claim 59, wherein the nucleic acid construct comprises a binding site for a DNA binding element.

61. The cell of claim 59 or 60, wherein the nucleic acid construct further comprises a transcriptional modulator polypeptide.

62. The cell of any one of claims 59-61, wherein the cell is engineered to express a transcriptional modulator polypeptide of any one of claims 1-22, or a plurality of transcriptional modulator polypeptides of any one of claims 1-22.

63. A cell comprising an engineered transcriptional modulator, wherein the engineered transcriptional modulator comprises: (a) a DNA binding element comprising one or more zinc finger DNA binding elements; (b) a transcription modulation element comprising a transcription activation element or a transcription repression element; (c) a regulatory element that confers responsiveness to a small molecule regulator.

64. The cell of any one of claims 57-63, wherein the cell further comprises at least one payload sequence operatively linked with a promoter and at least one transcriptional regulatory sequence that includes a binding site for the DNA binding element.

65. The cell of claim 64, wherein expression of the at least one payload is regulated by presence of the small molecule regulator.

66. The cell of claim 65, wherein the cell is contacted with the small molecule regulator.

67. The cell of claim 66, wherein contacting the cell with the small molecule regulator results in binding of the small molecule regulator to a regulatory element.

68. The cell of claim 67, wherein binding of the small molecule regulator to a regulatory element results in stabilization, a change in localization, a change in conformation and / or a change in activity of an engineered transcriptional modulator polypeptide.

69. The cell of any one of claims 64-68, wherein expression of the payload is modulated responsive to the small molecule regulator.

70. The cell of any one of claims 57-69, wherein the cell is in vitro, ex vivo or in vivo.

71. The cell of any one of claims 57-70, wherein the cell is a mammalian cell, optionally wherein the cell is a human cell.

72. The cell of claim 71, wherein the cell is a cell from a central nervous system, optionally wherein the central nervous system cell is or comprises a CNS epithelial cell, a nerve cell, a CNS connective tissue cell, a stem cell, a progenitor cell, a CNS immune cell, a spinal cord cell, a cell that lines one or more brain ventricles, a nerve support cell, a glial cell, a fat cell, a meninges cell, or a combination thereof.

73. The cell of claim 71 or 72, wherein the cell is in a subject.

74. A composition comprising one or more engineered transcriptional modulator polypeptides of any one of claims 1-22.

75. A composition comprising one or more nucleic acid constructs of any one of claims 25-49.

76. The composition of claim 74 or 75, further comprising an expression system.

77. The composition of claim 74 or 75, further comprising a delivery system.

78. A pharmaceutical composition comprising the composition of any one of claims 74-77 and one or more pharmaceutically acceptable excipients.

79. A method comprising, delivering one or more engineered transcriptional modulator polypeptides of any one of claims 1-22, to a cell tissue or subject.

80. A method, comprising delivering one or more nucleic acid constructs of any one of claims 25-49, to a cell tissue or subject.

81. The method of claim 79, comprising introducing into a cell, tissue or subject a nucleic acid construct encoding an engineered transcriptional modulator polypeptide.

82. The method of any one of claims 79-81, wherein delivering comprises administering.

83. The method of any one of claims 79-82, wherein the method is a treatment method.

84. The method of any one of claims 79-82, wherein the method is a prevention method.

85. The method of any one of claims 79-84, wherein the method delivers a payload to a cell, tissue or subject.

86. The method of claim 85, wherein the payload is a polypeptide payload.

87. The method of claim 85, wherein the payload is a polynucleotide payload (e.g., an RNA payload).

88. The method of any one of claims 85-87, wherein the payload is a therapeutic payload or a diagnostic payload.

89. The method of any one of claims 79-88, further comprising delivering (e.g., administering) a small molecule regulator to the cell, tissue or subject.

90. The method of claim 89, wherein the method delivers a payload and expression of the payload is modulated responsive to the small molecule regulator.

91. A method of expressing a payload in a cell, comprising introducing into a cell, a nucleic acid construct comprising a sequence encoding a payload operatively linked with a promoter and at least one transcriptional regulatory sequence.

92. The method of claim 91, wherein the nucleic acid construct comprises a binding site for a DNA binding element.

93. The method of claim 91 or 92, wherein an engineered transcriptional modulator polypeptide comprising a DNA binding element that recognizes the binding site is present in the cell, tissue or subject.

94. The method of any one of claims 91-93, wherein payload expression level is controlled by modulating exposure to the small molecule regulator.

95. The method of claim 94, wherein contacting the cell, tissue or the subject with the small molecule regulator induces expression of the payload.

96. A method of treating a disease, disorder or condition responsive to a payload polypeptide, comprising administering to a subject suffering from or susceptible to such disease disorder or condition a pharmaceutical composition of claim 78 that comprises or delivers the payload polypeptide.

97. A method of treating a disease, disorder or condition responsive to expression of a gene under control of at least one transcriptional regulatory sequence that is recognized by a DNA binding element, comprising administering to a subject suffering from or susceptible to such disease, disorder or condition, a pharmaceutical composition of claim 78 that comprises or delivers an engineered transcriptional modulator.

98. The method of any one of claims 79-97, wherein the engineered transcriptional modulator is characterized in that exposure to a small molecule regulator modulates the stabilization, localization and / or conformation of the engineered transcriptional modulator, as compared to stabilization, localization and / or conformation of an engineered transcriptional modulator in the absence of, or before exposure to the small molecule regulator.

99. The method of any one of claims 79-98, wherein exposure to a small molecule regulator increases expression and / or activity of a payload, as compared to expression and / or activity of a payload in the absence of, or before exposure to the small molecule regulator.

100. A kit comprising an expression system of claim 23 or 50.

101. A kit comprising a delivery system of any one of claims 24 and 51-52.

102. An organism engineered to express a cell of any one of claims 57- 59, one or more nucleic acid constructs of any one of claims 25-49, or one or more engineered transcriptional modulator polypeptides of any one of claims 1-22.

103. The organism of claim 102, wherein the organism is a non-human animal.

104. The organism of claim 103, wherein the non-human animal is a rodent (e.g., a rat or a mouse).

105. The delivery system of any one of claims 24 and 51-52, wherein the delivery system comprises an AAV, a lentiviral vector, an all-DNA viral vector, a transposon, or a DNA template (e g., for recombinase or HDR mediated genomic insertion).

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