Synthetic transcription factors and methods of making and using thereof

Abstract

Constructs comprising transactivation domains (TADs) for activating gene expression are disclosed herein, along with methods of using the same. Nucleotides encoding the same and vectors for the delivery of the nucleotides are also disclosed. In particular, synthetic transcription factors comprising a TAD fused to a heterologous DNA binding domain, and methods of using thereof are provided.

Description

Atty. Docket No.5724-108WO1 / / P.0279.WO1 SYNTHETIC TRANSCRIPTION FACTORS AND METHODS OF MAKING AND USING THEREOF FIELD

[0001] The present disclosure relates to transcriptional activation domain (TAD) compositions and their use for treating disease. CROSS REFERENCE TO RELATED APPLICATION

[0002] The present application claims priority from U.S. Provisional Application 63 / 642,565, filed on May 3, 2024, the content of which is incorporated herein by reference in its entirety. SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 2, 2025, is named P0279 WO1 Sequence Listing.xml and is 45,056 bytes in size. BACKGROUND

[0004] Gene therapy offers new promise for the treatment of previously intractable conditions. Previous efforts to engineer synthetic transcription factors utilized afinite set of activation and repressor domains. Alerasool, N. et al. (Molecular Cell 82, 677–695, February 3, 2022) and Tycko, J. et al. (Cell 183, 2020–2035, December 23, 2020) report several such activation and repressor domains. There remains, however, a need for additional transcription regulatory domains such as activation and repressor domains to expand this toolbox. SUMMARY OF THE INVENTION

[0005] Provided herein are synthetic transcription factors comprising an amino acid sequence of a FOXO3 PFAMflanked S626D (e.g., the amino acid sequence of SEQ ID NO: 1), wherein the synthetic transcription factor does not comprise the amino acid sequence of SEQ ID NO:2. In some aspects, the synthetic transcription factor is linked to a heterologous DNA binding domain (e.g., a zincfinger protein, TALE protein, dCas9, or dCas12a).

[0006] In some aspects the heterologous DNA binding domain is an inducible DNA binding domain. When the heterologous DNA binding domain is a zincfinger protein, the zincfinger protein may be selected from SEQ ID NOs: 6, 15, 24, & 33.Atty. Docket No.5724-108WO1 / / P.0279.WO1

[0007] Also provided herein are nucleic acids encoding the synthetic transcription factors provided herein. In some aspects, the nucleic acids may be provided in the context of an expression cassette or a vector, such as a viral vector.

[0008] Also provided herein are cells comprising the synthetic transcription factors and / or the nucleic acids (e.g., the vectors) described herein. In some aspects, the cell comprises at least two synthetic transcription factors (e.g., two synthetic transcription factors described herein, or a mix of a synthetic transcription factor described herein and a synthetic transcription factor described elsewhere).

[0009] Also provided herein are compositions comprising a synthetic transcription factor, a nucleic acid (e.g., a vector), and / or a cell as described herein. Such compositions are therapeutically useful for treating diseases and disorders, such as disease and disorders of the nervous system. In some aspects, the composition comprises two or more synthetic transcription factors, nucleic acids (e.g., vectors), and / or cells (e.g., two synthetic transcription factors described herein, or a mix of a synthetic transcription factor described herein and a synthetic transcription factor described elsewhere).

[0010] Also provided herein are kits comprising at least one synthetic transcription factor, nucleic acid (e.g., vector), cell, and / or composition described herein.

[0011] Methods of modulating the expression of at least one target gene in a host cell are also provided herein. These methods may also be characterized as uses of a synthetic transcription factor, nucleic acid (e.g., vector), cell, or composition to modulate the expression of at least one target gene in a host cell. These methods comprise introducing into the host cell at least one synthetic transcription factor, nucleic acid (e.g., vector), cell, or composition as described herein. In some aspects, the at least one target gene whose expression is to be modulated is an endogenous gene, an exogenous gene, or a combination thereof. In some aspects, the host cell is in a subject. In some aspects, the method comprises administering the at least one synthetic transcription factor, nucleic acid (e.g., vector), or composition to the subject.

[0012] Methods of treating a symptom of a disease are also provided herein. These methods may also be characterized as uses of a synthetic transcription factor, nucleic acid (e.g., vector), cell, or composition to treat a symptom of a disease. These methods comprise modulating the expression of at least one target gene in a cell of a subject having the disease, by introducing into the cell at least one synthetic transcription factor, nucleic acid (e.g., vector), cell, or composition as described herein. In some aspects, the disease is Dravet Syndrome and the gene is SCN1A. In some aspects, the synthetic transcription factor is linked to a zincfinger proteinAtty. Docket No.5724-108WO1 / / P.0279.WO1 selected from SEQ ID NOs: 6, 15, 24, & 33. In some aspects, the synthetic transcription factor is administered with a viral vector. In some aspects, the disease is an autism spectrum disorder. In some aspects the disease is a neurological disorder such as an autism spectrum disorder and the gene is SCN2a, RAI1 or SHANK3. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 depicts four FOXO3 transcriptional activation domain constructs. Panel A displays the full-length FOXO3a protein (aa 1–673). Panel B displays the PFAM fragment “FOXO3 PFAM 16676” (aa 604–644) (SEQ ID NO: 43). Panel C displays a segment of the FOXO3 protein that includes the PFAM fragment with an additional 19aa and 20aaflanked on each end referred to as “FOXO3 PFAMflanked” (aa 585–664). Panel D displays the FOXO3 PFAM flanked segment with the S626D phosphomimetic mutation. The location of conserved region CR3 (SEQ ID NO: 42) is provided to show relation of these TADs to FOXO3a.

[0014] FIG.2A shows SCN1A gene expression following transduction of induced pluripotent stem cell-derived (iPSC) human neurons by adeno-associate virus (AAV) vectors carrying a p65-based synthetic transcription factor. FIG. 2B shows SCN1A gene expression following transduction of a FOXO3 S626D-based synthetic transcription factor using AAV6. FIG. 2C shows SCN1A gene expression following transduction of a FOXO3 S626D-based synthetic transcription factor targeted with ZFP3. FIG. 2D shows SCN1A gene expression following transduction of a FOXO3 S626D-based synthetic transcription factor targeted with ZFP4. FIG. 2E shows SCN1A gene expression following transduction of a FOXO3 S626D-based synthetic transcription factor targeted with ZFP5. FIG. 2F shows SCN1A gene expression following transduction of a FOXO3 S626D-based synthetic transcription factor targeted with ZFP6. The broken line crossing the y-axis at 1 graph indicates no change in SCN1A expression in comparison to neurons treated with a negative control AAV. The y-axis in each panel is human SCN1A mRNA expression normalized to the mean of two housekeeping genes (ATP5b and EIF4a2). The x-axis in each panel is the multiplicity of infection (MOI).

[0015] FIG. 3A shows a volcano plot of genes whose expression is suppressed (leftward plume) or enhanced (rightward plume) by expression of a p65-based synthetic transcription factor in iPSC human neurons in comparison to expression of a non-targeting ZFP fused to a KRAB-based synthetic transcription factor in iPSC human neurons. There are 934 genes suppressed and 799 genes enhanced in this panel. FIG.3B shows a volcano plot of genes whose expression is suppressed (leftward plume) or enhanced (rightward plume) by expression of a FOXO3 S626D-based synthetic transcription factor in comparison to expression of a non-Atty. Docket No.5724-108WO1 / / P.0279.WO1 targeting ZFP fused to a KRAB-based synthetic transcription factor in iPSC human neurons. There are 2 genes suppressed, including the KRAB control gene expressed by the negative control AAV, and 2 genes enhanced in this panel. FIG.3C shows a volcano plot of genes whose expression is suppressed (leftward plume) or enhanced (rightward plume) by expression of a FOXO3 S626D-based synthetic transcription factor targeted with ZFP3 in iPSC human neurons. There are 3 genes suppressed, including the KRAB gene expressed by the negative control AAV, SPP1, and OR2M3, and 4 genes enhanced in this panel, including NEUROD6, FRMPD3-AS1, BTN3A2, ARSK, plus the ZFA-targeted gene, SCN1A. FIG.3D shows a volcano plot of genes whose expression is suppressed (leftward plume) or enhanced (rightward plume) by expression of a FOXO3 S626D-based synthetic transcription factor targeted with ZFP4 in iPSC human neurons. There is 1 gene suppressed, the KRAB gene expressed by the negative control AAV, and no genes enhanced in this panel, except for the ZFA-targeted gene, SCN1A. FIG. 3E shows a volcano plot of genes whose expression is suppressed (leftward plume) or enhanced (rightward plume) by expression of a FOXO3 S626D-based synthetic transcription factor targeted with ZFP5 in iPSC human neurons. There is 1 gene suppressed, the KRAB gene expressed by the negative control AAV, and 1 gene enhanced, CXCL14, in addition to plus the ZFA-targeted gene, SCN1A, in this panel. FIG. 3F shows a volcano plot of genes whose expression is suppressed (leftward plume) or enhanced (rightward plume) by expression of a FOXO3 S626D-based synthetic transcription factor targeted with ZFP6 in iPSC human neurons. There is one gene suppressed, the KRAB gene expressed by the negative control AAV, and no genes enhanced in this panel, except for the ZFA-targeted gene, SCN1A. The dot for SCN1A is indicated by an arrow in each panel, and the broken line in each panel indicates the i the cutoff for statistically significant changes in gene expression with an FDR p-value of <0.05.

[0016] FIG. 4A shows Scn1a gene expression following transduction of primary mouse (Scn1a+ / –) cortical neurons by AAV vectors carrying a FOXO3 S626D-based synthetic transcription factor targeted with ZFP3. FIG. 4B shows Scn1a gene expression following transduction of a FOXO3 S626D-based synthetic transcription factor targeted with ZFP4. FIG. 4C shows Scn1a gene expression following transduction of a FOXO3 S626D-based synthetic transcription factor targeted with ZFP5. FIG. 4D shows Scn1a gene expression following transduction of a FOXO3 S626D-based synthetic transcription factor targeted with ZFP6. The y-axis in each panel is mouse Scn1a mRNA expression normalized to the mean of two housekeeping genes (Atp5b and Eif4a2). The broken line in the middle of each panel indicatesAtty. Docket No.5724-108WO1 / / P.0279.WO1 the normalized “100%” from Scn1a+ / + neurons of wild-type mice that were transduced with a negative control AAV (grey bar). The x-axis in each panel is the MOI.

[0017] FIG. 5A shows Scn1a gene expression following transduction of primary mouse (Scn1a+ / -) hippocampal neurons by AAV vectors carrying a FOXO3 S626D-based synthetic transcription factor targeted with ZFP3. FIG. 5B shows Scn1a gene expression following transduction of a FOXO3 S626D-based synthetic transcription factor targeted with ZFP4. FIG. 5C shows Scn1a gene expression following transduction of a FOXO3 S626D-based synthetic transcription factor targeted with ZFP5. FIG. 5D shows Scn1a gene expression following transduction of a FOXO3 S626D-based synthetic transcription factor targeted with ZFP6. The y-axis in each panel is human Scn1a mRNA expression normalized to the mean of two housekeeping genes (Atp5b and Eif42a). The broken line in the middle of each panel indicates the normalized “100%” from Scn1a+ / + neurons of wild-type mice that were transduced with a negative control AAV (grey bar). The x-axis in each panel is the MOI.

[0018] FIG.6 reports intensity of Scn1a gene expression relative to negative control (animals treated with non-targeting ZFP fused to FOXO3 S626D-based synthetic transcription factor) determined by a mean Spot total intensity parameter, which quantifies the RNAscope signal detected for Scn1a transcripts. The data are presented for NeuN+neurons in the cortex, thalamus, midbrain, hippocampus, pons, and medulla that were categorized as having ZFP- TAD expression based on the RNAscope signal detected using a ZFP-TAD specific probe. ZFP-TADs are scaled to the negative control group. The horizontal line represents the normalized Scn1a level in negative control animals. N=4. For each brain region, a 1-way ANOVA with Dunnett’s multiple comparisons test, p-values represent a comparison to negative control; *p<0.05, **P<0.005, ***p<0.001.

[0019] FIG. 7A shows Scn2a gene expression following transfection of Neuro2a cells by mRNA encoding a FOXO3 S626D-based synthetic transcription factor targeted with ZFP7. FIG. 7B shows Shank3 gene expression following transfection of Neuro2a cells by mRNA encoding a FOXO3 S626D-based synthetic transcription factor targeted with ZFP8. FIG.7C shows RAI1 gene expression following transfection of SKNMC cells by mRNA encoding a FOXO3 S626D- based synthetic transcription factor targeted with ZFP10. The broken line crossing the axis at 1 on each graph indicates no change in target gene expression in comparison to cells treated with a negative control ZFP. The y-axis in each panel is mRNA expression of the target gene normalized to the mean of two housekeeping genes (Atp5b and Eif42a). The x-axis in each panelAtty. Docket No.5724-108WO1 / / P.0279.WO1 is the multiplicity of infection (MOI). DETAILED DESCRIPTION

[0020] The present disclosure provides synthetic transcription factors that are linked to a heterologous DNA binding domain and uses for modulating expression of at least one target gene in a cell.

[0021] Definitions. In the present disclosure, the following terms have the following meanings:

[0022] “About” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0023] A “transcription effector domain” refers to a protein or polypeptide that interacts— directly or indirectly—with specific DNA sequences to block or recruit RNA polymerase activity to the promoter site for a gene or set of genes. A transcriptional activator domains (TAD) is a transcription effector domain that activates polymerization, while transcriptional repressor domains (TRD) is a transcription effector domain that blocks polymerization.

[0024] An “expression cassette” as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to a nucleotide sequence of interest, which is—optionally—operably linked to termination signals and / or other regulatory elements. An expression cassette may also comprise sequences required for proper translation of the nucleotide sequence.

[0025] A “vector” refers to an expression cassette operatively linked sufficient cis-acting elements for expression and propagation. Vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0026] “Identity” or “identical,” when used herein to describe the relationship between two or more amino acid sequences, or between two or more nucleic acid sequences, refers to the degree of sequence relatedness between the compared sequences. “Identity” measures the percentage of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e.,Atty. Docket No.5724-108WO1 / / P.0279.WO1 “algorithms”). “Percent sequence identity” refers to the percentage of nucleotides or nucleotide analogs in a nucleic acid sequence, or amino acids in an amino acid sequence, that is identical with the corresponding nucleotides or amino acids in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. In case a polypeptide or polynucleotide is longer than a reference sequence, additional amino acids or nucleotides in the sequence, that do not align with the reference sequence, are not taken into account for determining sequence identity. Identity of related amino acid sequences or nucleic acid sequences are calculated by BLAST methods described in Altschul et al. (1990) J Mol Biol. 215(3):403-10. The BLAST program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md.20894).

[0027] “Subject” includes living organisms in which an immune response can be elicited (e.g., mammals, human). In one embodiment, a subject may be a “patient,” i.e., a warm- blooded animal (e.g., human), who / which is awaiting the receipt of, or is receiving medical care or was / is / will be the object of a medical procedure, or is monitored for the development of the targeted disease or condition, such as, for example, a neurological disorder or seizure disorder or an autism spectrum disorder. In one embodiment, the subject is an adult (for example a subject above the age of 18). In another embodiment, the subject is a child (for example a subject below the age of 18). In one embodiment, the subject is a male. In another embodiment, the subject is a female. In one embodiment, the subject is affected (e.g., diagnosed) with a neurological disorder or seizure disorder or an autism spectrum disorder. In one embodiment, the subject is at risk of developing a neurological or seizure disorder. Examples of risks factor include, but are not limited to, genetic predisposition, or familial history of a neurological disorder or seizure disorder or an autism spectrum disorder.

[0028] As used herein, the terms “providing,” “administering,” and “introducing,” are used interchangeably to convey the placement of the transcription factors and nucleic acids of the present disclosure into a subject by a method or route which results in at least partial localization to a desired site. The transcription factors and nucleic acids can be administered by any appropriate route which results in delivery to a desired location in the subject.

[0029] “Therapeutically effective amount” refers to the level or amount of an TAD as described herein that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of a disease, disorder, or condition; (2) slowingAtty. Docket No.5724-108WO1 / / P.0279.WO1 down or stopping the progression, aggravation, or deterioration of one or more symptoms of the disease, disorder, or condition; (3) bringing about ameliorations of the symptoms of the disease, disorder, or condition; (4) reducing the severity or incidence of the disease, disorder, or condition; or (5) curing the disease, disorder, or condition. A therapeutically effective amount may be administered prior to the onset of the disease, disorder, or condition, for a prophylactic or preventive action. Alternatively or additionally, the therapeutically effective amount may be administered after initiation of the disease, disorder, or condition, for a therapeutic action.

[0030] “Treating” or “treatment” or “alleviation” refers to therapeutic treatment, wherein the objective is to reverse or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. In one embodiment, a subject is successfully “treated” for a disease or disorder if, after receiving a therapeutic amount of TAD according to the present disclosure, the subject shows at least one of the following: increase in expression of a relevant protein; relief to some extent of one or more of the symptoms associated with the disease or disorder to be treated; reduced morbidity and mortality; and improvement in quality-of-life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

[0031] “Specifically binds” refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific. Such interactions are generally characterized by a dissociation constant (Kd) of 10-6M-1or lower. “Non-specific binding” refers to, non-covalent interactions that occur between any molecule of interest (e.g. an engineered nuclease) and a macromolecule (e.g. DNA) that are not dependent on sequence- specific binding interactions.

[0032] A “binding protein” is a protein that is able to bind non-covalently to another molecule. A binding protein can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein) and / or a protein molecule (a protein- binding protein). In the case of a protein-binding protein, it can bind to itself (to form homodimers, homotrimers, etc.) and / or it can bind to one or more molecules of a differentAtty. Docket No.5724-108WO1 / / P.0279.WO1 protein or proteins. A binding protein can have more than one type of binding activity. For example, zincfinger proteins have DNA-binding, RNA-binding and protein-binding activity.

[0033] A “DNA binding molecule” is a molecule that can bind to DNA. Such DNA binding molecule can be a polypeptide, a domain of a protein, a domain within a larger protein, or a polynucleotide. In some aspects, the polynucleotide is DNA, while in other aspects, the polynucleotide is RNA. In some aspects, the DNA binding molecule is a protein domain of a nuclease.

[0034] A “DNA binding protein” (or binding domain) is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner, for example through one or more zinc fingers or through interaction with one or more RVDs in a zincfinger protein, respectively. A DNA binding domain is often abbreviated “DBD.”

[0035] A “zincfinger protein” or “ZFP” refers to a protein having a DNA-binding domain that is stabilized by zinc. ZFPs bind to DNA in a sequence-specific manner. The individual DNA-binding unit of a ZFP is referred to as a “zincfinger.” Eachfinger contains a DNA- binding “recognition helix” that is typically comprised of seven amino acid residues and determines DNA binding specificity. A ZFP domain has at least onefinger and eachfinger binds from two to four base pairs of nucleotides, typically three or four base pairs of DNA (contiguous or noncontiguous). Each zincfinger typically comprises approximately 28–30 amino acids and chelates zinc.

[0036] “Wild-type” refers to a gene or a gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene. In contrast, “modified,” “mutant,” or “polymorphic” refer to a gene or gene product that displays modifications in sequence and or functional properties (e.g., altered characteristics) when compared to the wild-type gene or gene product. Naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.

[0037] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice orAtty. Docket No.5724-108WO1 / / P.0279.WO1 testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of, cardiology, medicine, medicinal & pharmaceutical chemistry, and cell biology described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art, or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0038] Transcription Activation Domains. The present disclosure provides synthetic transcription factors comprising one or more transcriptional effector domains linked to a heterologous DNA binding domain.

[0039] In some aspects, the synthetic transcription factor comprises a TAD with an amino acid sequence derived from the Pfam PF16676 domain of the human FOXO3 protein. In some aspects, the TAD comprises a phosphomimetic mutation at FOXO3 position S626 of UniProt O43524, for example, an S626D phosphomimetic mutation. In some aspects, the TAD has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO:1. FIG.1 exemplifies a series of TADs that can be used in some aspects of the present disclosure.

[0040] In some aspects, the TAD is fused to a heterologous DNA binding domain. In some aspects the synthetic transcription factor comprises—in addition to the TAD of SEQ ID NO:1—one or more additional TADs, or a combination thereof. In some aspects, the one or more additional TADs are selected from among those found described in WO 23 / 23553, the entire contents of which are herein incorporated by reference.

[0041] In some aspects, two or more transcription effector domains can be fused to the DNA binding domain in any orientation, and may be separated from each other with an amino acid linker. In select aspects, the synthetic transcription factor comprises two or more transcription effector domains (e.g., TADs, , or a combination thereof) fused to a heterologous DBD. In some aspects, protein or amino acid binding may be via protein-protein affinity interactions, hydrophobic interactions, disulfide linkage(s), cross-link(s), covalent bond(s), chemical linkage(s), isopeptide bonds, or any other type of binding between two amino acids or two polypeptides.

[0042] In some aspects, the DBD can be any polypeptide which is capable of binding double- or single-stranded DNA, generally or with sequence specificity. By way of non-limiting examples, suitable DBDs include those polypeptides having helix-turn-helix motifs, zincAtty. Docket No.5724-108WO1 / / P.0279.WO1 fingers, leucine zippers, HMG-box (high mobility group box) domains, winged helix region, winged helix-tum-helix region, helix-loop-helix region, immunoglobulin fold, B3 domain, Wor3 domain, TAL effector (TALE) DNA-binding domain, and the like. In some aspects, the heterologous DBD comprises a programmable DNA binding domain, e.g., a DNA binding domain engineered, for example by altering one or more amino acid of a natural DNA binding domain to bind to a predetermined nucleotide sequence. In some aspects, the DBD is capable of binding directly to the target DNA sequences.

[0043] In some aspects, the DBD may be a zincfinger protein (ZFP). For example a ZFP for use in the synthetic transcription factors described herein may have: at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO:6; at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO:15; at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO:24; or at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO:33.

[0044] The DBD may be derived from domains found in naturally occurring Transcription activator-like effectors (TALEs), such as AvrBs3, Hax2, Hax3 or Hax4. TALEs have a modular DNA-binding domain consisting of repetitive sequences of residues. Each repeat region consists of 34 amino acids. A pair of residues at positions 12 & 13 of each repeat region determines the nucleotide specificity. Combining the regions allows synthesis of sequence- specific TALE DBDs

[0045] The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene editing system has been described extensively elsewhere (see, e.g., WO 16 / 36754). In some aspects, the DBD derives from CRISPR associated (Cas) protein (e.g., catalytically dead Cas9 or Cas12a, also known as dCas9 or dCas12a) and associates with the target DNA through a guide RNA (gRNA). The gRNA itself comprises a sequence complementary to one strand of the DNA target sequence and a scaffold sequence which binds and recruits dCas9 or dCas12a to the target DNA sequence.

[0046] The present disclosure also provides synthetic transcription factors comprising one or more transcriptional effector domains fused to an exogenous factor which associates with a second exogenous factor comprising a DBD such that the DBD functions as an inducible DBD.Atty. Docket No.5724-108WO1 / / P.0279.WO1 Such inducible systems include, but not limited to, tetracycline Tet, / DOX inducible systems, light inducible systems, Abscisic acid (ABA) inducible systems, cumate systems, 40HT / estrogen inducible systems, ecdysone-based inducible systems, and FKBP12 / FRAP (FKBP12-rapamycin complex) inducible systems.

[0047] Nucleic acids. The present disclosure also provides nucleic acids encoding a synthetic transcription factor (e.g., TAD) as disclosed herein. In some aspects, the nucleic acid may encode a TAD having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO:1. In some aspects, the nucleic acid encoding the TAD may be joined in frame to a nucleic acid encoding a heterologous DBD. In some aspects, the nucleic acid may comprise the sequence of SEQ ID NO:5. In some aspects, the nucleic acid may encode a ZFP fused to the TAD. For example, the nucleic acid may encode a ZFP having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO:6; at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO:15; at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO:24; or at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO:33, and fused to a TAD. In some aspects, the nucleic acid encoding the ZFP may comprise the sequence of any one of SEQ ID Nos: 13, 22, 31, or 40.

[0048] The nucleic acids disclosed herein may exist in the context of expression cassettes comprising coding sequences encoding the TADs of the present disclosure operably linked to regulatory sequences (e.g., promoters and / or terminators) that can drive the expression of the coding sequence. In certain aspects, the promoters are constitutive promoters (e.g., the cytomegalovirus or “CMV” immediate early promoter). In certain aspects, the promoters are inducible (e.g., the yeast GAL10 promoter). Expression cassettes of the present disclosure can comprise any of a number of promoters known to the art, wherein the promoter is constitutive, regulatable, or inducible, cell type specific, tissue-specific, or species specific. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences, introns). Many promoter / regulatory sequences useful forAtty. Docket No.5724-108WO1 / / P.0279.WO1 driving constitutive expression of a gene are available in the art and include, but are not limited to, for example, CMV (cytomegalovirus promoter), EF1A (human elongation factor 1α promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human β-actin promoter, rodent β-actin promoter, CBh (chicken β-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken β actin promoter, and rabbit β-globin splice acceptor), TRE (Tetracycline response element promoter), U6 (human U6 small nuclear promoter), and the like. Additional promoters that can be used for expression of the components of the present composition, include, without limitation, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming vims (SFFV) LTR, the simian vims 40 (SV40) early promoter, herpes simplex tk virus promoter. Additional promoters include any constitutively active promoter. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within a cell.

[0049] Moreover, inducible expression can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible promoter / regulatory sequence. Promoters that are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, may also be used in the expression cassettes disclosed herein.

[0050] The present disclosure also provides for vectors (e.g., expression vectors) containing the nucleic acids and cells containing the nucleic acids or expression cassettes disclosed herein. The vectors may be used to propagate the nucleic acid in an appropriate cell and / or to allow expression from the nucleic acid. The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.

[0051] To construct cells that express the present transcription factors, expression vectors for stable or transient expression of the molecules of the present disclosure may be constructed via conventional methods and introduced into cells. For example, nucleic acids encoding the transcription factors of the present disclosure, may be cloned into a suitable expression vector, such as a plasmid or a viral vector in operable linkage to a suitable promoter. The selection of expression vectors / plasmids / viral vectors should be suitable for integration and replication in eukaryotic cells. In certain aspects, the vector is a baculovirus, a herpesvirus, an adenovirus, or an adeno-associated virus (e.g., AAV.PHP.B).Atty. Docket No.5724-108WO1 / / P.0279.WO1

[0052] Cells useful for expressing the transcription factors of the present disclosure include, by way of non-limiting example, yeast cell (e.g., S. pombe), insect cells (e.g., Sf9 cells), and mammalian cells (e.g., CHO cells, HeLa cells, Vero cells, etc.). Many suitable cell lines are available from the American Type Culture Collection.

[0053] In certain aspects, vectors of the present disclosure can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman & al. (1987) EMBO J. 6:187). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g.. Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0054] The vectors of the present disclosure may direct the expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Such regulatory elements include promoters that may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., neuronal tissue) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue. The term “cell type specific” as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell (e.g., neurons) in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue.

[0055] Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene for selection of stable or transient transfectants in host cells; transcription termination and RNA processing signals; 5'- and 3'-untranslated regions; internal ribosome binding sites (IRESes); multiple cloning sites with a wide selection of restriction endonuclease recognition sequences; and a reporter gene for assessing expression of the chimeric receptor. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Selectable markers include chloramphenicol resistance,Atty. Docket No.5724-108WO1 / / P.0279.WO1 tetracycline resistance, spectinomycin resistance, neomycin resistance, streptomycin resistance, erythromycin resistance, rifampicin resistance, bleomycin resistance, thermally adapted kanamycin resistance, gentamycin resistance, hygromycin resistance, trimethoprim resistance, dihydrofolate reductase (DHFR), GPT; and the URA3, HIS4, LEU2, & TRP1 genes of S. cerevisiae.

[0056] In some aspects, the vectors may be maintained in a cell as an autonomously replicating sequence or extrachromosomal element. Additionally or alternatively, the vectors may be integrated into host DNA.

[0057] The disclosure further provides for cells comprising a synthetic transcription factor, a nucleic acid, an expression cassette or a vector, as disclosed herein.

[0058] Conventional viral and non-viral based gene transfer methods can be used to introduce the nucleic acids into cells, tissues, or a subject. Such methods can be used to administer the nucleic acids (including the expression cassettes and vectors) to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., encapsulated in a liposome), and a nucleic acid complexed with a delivery vehicle.

[0059] Viral vector delivery systems include DNA and RNA viruses. In some aspects, the viral vector delivers episomal nucleic acids to the cell. In some aspects, the viral vector delivers nucleic acids that integrate into the host genome. A variety of viral constructs may be used to deliver the present nucleic acids to the cells, tissues, and / or subjects. Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex viral vectors. Nonlimiting examples of such recombinant viruses include recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, recombinant herpes simplex viruses, recombinant poxviruses, phages, etc. The present disclosure provides vectors capable of integration in the host genome, such as retrovirus or lentivirus. See, e.g., Ausubel & al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989.

[0060] In certain embodiments, the viral vector is capable of crossing the blood brain barrier for delivery of the present nucleic acids to cells in the central nervous system, and more specifically the brain. Suitable vectors for use in achieving delivery through the blood brain barrier (BBB) include but are not limited to: those described in PCT / US2024 / 029507 (filed May 15, 2024 and published as WO 2024 / 238684), U.S. Publication No. 2021 / 0380969,Atty. Docket No.5724-108WO1 / / P.0279.WO1 Nonenmacher et al. (2020) Mol Ther Methods Clin Dev 20:366–78, and U.S. Publication No. 2022 / 0089650, the entire contents of each of which is incorporated herein by reference for all purposes. Accordingly, in some aspects, the vector is a blood-brain barrier-penetrant vector that is capable of crossing the BBB.

[0061] The nucleic acids or transcription factors may be delivered by any suitable means. In certain aspects, the nucleic acids or proteins thereof are delivered in vivo. In other aspects, the nucleic acids or proteins thereof are delivered to isolated / cultured cells in vitro or ex vivo to provide modified cells useful for in vivo delivery to patients suffering a disease or condition.

[0062] Vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of host cells. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate co- precipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, etc. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and / or translation) of sequences delivered by the viral vector genome. In the case of a viral vector, “transduction” conveys nucleic acid delivery from the vector into the cell and expression of the nucleic acid.

[0063] Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment). Similarly, the construct containing the one or more transgenes can be delivered by any method appropriate for introducing nucleic acids into a cell. In some aspects, the construct or the nucleic acid encoding the components of the present disclosure is a DNA molecule. In some aspects, the nucleic acid encoding the components of the present disclosure is a DNA vector and may be electroporated to cells. In some aspects, the nucleic acid encoding the components of the present disclosure is an RNA molecule, which may be electroporated to cells.

[0064] Additionally, delivery vehicles such as nanoparticle- and lipid-based delivery systems can be used. Additional, non-limiting examples of delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics.

[0065] In some aspects, the composition further comprises a gRNA. The gRNA may be encoded on the same nucleic acid as a synthetic transcription factor or a different nucleic acid.Atty. Docket No.5724-108WO1 / / P.0279.WO1 In some aspects, the vector encoding a synthetic transcription factor may further encode a gRNA, under the same or different promoter. In some aspects, the gRNA is encoded on its own vector, separated from that of the transcription factor.

[0066] Methods of Modulating Gene Expression. The present disclosure also provides methods of modulating the expression of at least one target gene in a cell, the method comprising introducing into the cell at least one synthetic transcription factor, nucleic acid, vector, or composition as described herein. In some aspects, the gene expression of at least two genes is modulated.

[0067] In some aspects, the method comprises introducing at least one synthetic transcription factor as described herein into a cell to be treated. In some aspects, the method comprises introducing a nucleic acid as described herein (e.g., a vector) into a cell to be treated, so that the cell might produce the synthetic transcription factor. In some aspects, the cell will be modified to express more than one synthetic transcription factor as described herein, e.g., two, three, four, or more such synthetic transcription factors.

[0068] In some aspects, the gene whose expression is to be modulated is an endogenous gene. In some aspects, the gene whose expression is to be modulated is an exogenous gene. In some aspects, the gene is on an exogenous vector. In some aspects, the exogenous gene was introduced into the cell as part of a gene therapy regime. For example, in some aspects the synthetic transcription factor activates or augments SCN1A expression to remedy or alleviate Dravet syndrome. Therefore, the synthetic transcription factors and nucleic acids disclosed herein are also disclosed for use in a method of alleviating a symptom of a disease or disorder, such as Dravet syndrome. In some aspects the synthetic transcription factor activates or augments gene expression to remedy or alleviate an autism spectrum disorder. In some aspects the disease is a neurological disorder such as an autism spectrum disorder and the gene is SCN2a, RAI1 or SHANK3.

[0069] Modulation of expression comprises increasing or decreasing gene expression compared to normal gene expression for the target gene. When the expressions of at least two genes are modulated, both genes may have increased gene expression, both gene may have decreased gene expression, or one gene may have increased gene expression and the other may have decreased gene expression.

[0070] The cell in which expression is modulated may be prokaryotic or eukaryotic. In some aspects, the cell is eukaryotic. In some aspects, the modulation occurs in vitro. In some aspects,Atty. Docket No.5724-108WO1 / / P.0279.WO1 the modulation occurs ex vivo. In some embodiment, the synthetic transcription factor or the nucleic acid is introduced into the cell in an ex vivo context, and the modified cell is then introduced into the organism to be treated (e.g., a human subject).

[0071] In some aspects, the cell in which expression is modulated is in an organism or host, such that introducing the disclosed compositions, vectors, and / or nucleic acids into the cell comprises administration to a subject. The method may comprise providing or administering to the subject, in vivo, or by transplantation of ex vivo treated cells, at least one synthetic transcription factor, nucleic acid, and / or vector as described herein.

[0072] A subject may be human or non-human and may include, for example, animal strains or species used as model systems for research purposes, such a mouse model as described herein. Likewise, the subject may be either adult or juvenile (e.g., a child). Moreover, the subject may be any living organism, for example a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non- human primates such as chimpanzees, and other apes and monkey species; bears; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs; and the like. Examples of non-mammals include, but are not limited to, birds,fish, and the like. In one embodiment of the methods and compositions provided herein, the mammal is a human.

[0073] Where the subject is a human, the dose of viral vector to be administered can be any suitable dose in a quantity sufficient to modulate gene expression. For example, the dosages administered may be sufficient to increase gene expression by >100%, for example >110%, >120%, >130%, >140%, >150%, >160%, >175%, >200%, >225%, >250%, etc. of normal expression levels without administration of viral vector. By way of non-limiting example, suitable dosages may involve viral vectors at anywhere from 1.0×1012viral genomes per kilogram of body mass (VG / kg) to 1.0×1015VG / kg—e.g., 5.0×1012VG / kg, 1.0×1013VG / kg, 1.5×1013VG / kg, 2.0×1013VG / kg, 2.5×1013VG / kg, 3.0×1013VG / kg, 3.5×1013VG / kg, 4.0×1013VG / kg, 4.5×1013VG / kg, 5.0×1013VG / kg, 1.0×1014VG / kg, 5.0×1014VG / kg, or more. Additionally or alternatively, dosage can be defined in terms of viral genomes per gram of brain mass (VG / gbrain). By way of non-limiting example, suitable dosages may involve viral vectors at anywhere from 7.5109VG / gbrainto 2.1×1012VG / gbrain, for example 3.0×1010VG / gbrainto 3.0×1011or 5.0×1010VG / gbrainto 1.85×1011VG / gbrain, for example 7.5×1010VG / gbrain, 8.0×1010Atty. Docket No.5724-108WO1 / / P.0279.WO1

[0074] Kits. The present disclosure also provides kits including a nucleic acid encoding a TAD of the present disclosure, or a DNA binding domain, or a combination thereof. In some aspects, the kit includes a vector as described herein. In some aspects, the kit includes a cell comprising a nucleic acid as described herein.

[0075] The kits can also comprise instructions for using the components of the kit. The instructions are relevant materials or methodologies pertaining to the kit. The materials may include any combination of the following: background information, list of components, brief or detailed protocols for using the compositions, trouble-shooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate component. In some aspects, the instructions are supplied as a paper. In some aspects, the instructions are supplied as an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. In some aspects, the instructions can comprise a description of use of the components for the methods of identifying repressor domains or methods of modulating gene expression.

[0076] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars,flexible packaging, and the like.

[0077] Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above.

[0078] The kit may further comprise a device for holding or administering the present composition. The device may include an infusion device, an intravenous solution bag, a hypodermic needle, a vial, and / or a syringe.

[0079] The TAD of the present disclosure has several advantages for applications that rely on synthetic transcription factors including that it is functional when packaged and delivered in viral vectors and is constitutively active as a transcription activator as further exemplified below.Atty. Docket No.5724-108WO1 / / P.0279.WO1 NUMBERED ITEMS

[0080] The present disclosure includes any one or combination of any of the following numbered items. 1. A synthetic transcription factor, wherein the synthetic transcription factor comprises an amino acid sequence of (FOXO3 PFAMflanked S626D) SEQ ID NO: 1, and wherein the synthetic transcription factor does not comprise SEQ ID NO:2. 2. The synthetic transcription factor of item 1, wherein the synthetic transcription factor is linked to a heterologous DNA binding domain. 3. The synthetic transcription factor of item 2, wherein the heterologous DNA binding domain is a zincfinger protein, TALE protein, dCas9, or dCas12a. 4. The synthetic transcription factor of item 3, wherein the heterologous DNA binding domain is a zincfinger protein. 5. The synthetic transcription factor of item 4, wherein the zincfinger protein is selected from SEQ ID NOs: 6, 15, 24, and 33. 6. The synthetic transcription factor of any one of items 1–5, wherein the heterologous DNA binding domain is an inducible DNA binding domain. 7. A nucleic acid encoding the synthetic transcription factor of any one of items 1–5. 8. The nucleic acid of item 7, comprising a nucleotide sequence according to SEQ ID NO:5. 9. The nucleic acid of item 7 or 8, comprising a nucleotide sequence selected from SEQ ID NOs: 13, 22, 31, and 40. 10. A vector comprising a nucleic acid of any one of items 7 to 9. 11. The vector of item 10, wherein the vector is a viral vector. 12. The vector of item 11, wherein the vector is capable of delivering the nucleic acid across a blood-brain barrier. 13. A cell comprising the synthetic transcription factor of any one of items 1–6, the nucleic acid of any one of items 7–9, or the vector of any one of items 10–12.Atty. Docket No.5724-108WO1 / / P.0279.WO1 14. The cell of item 13, wherein the cell comprises at least two synthetic transcription factors where at least one of the synthetic transcription factors is the synthetic transcription factor of any one of items 1–6. 15. A composition comprising a synthetic transcription factor of any one of items 1–6, the nucleic acid of any one of items 7–9, the vector of any one of items 10–12, or the cell of item 13 or item 14. 16. The composition of item 15, wherein the composition comprises two or more synthetic transcription factors, nucleic acids, vectors, or cells, wherein at least one of the synthetic transcription factors is the synthetic transcription factors of any one of items 1–6. 17. A kit comprising at least one synthetic transcription factor of any one of items 1–6, the nucleic acid of any one of items 7–9, or the vector of any one of items 10–12, the cell of item 13 or item 14, or the composition of item 15 or item 16. 18. A method of modulating the expression of at least one target gene in a host cell, the method comprising introducing into the host cell at least one synthetic transcription factor of any one of items 1–6, the nucleic acid of any one of items 7–9, the vector of any one of items 10–12, the cell of item 13 or item 14, or the composition of item 15 or item 16. 19. The method of item 18, wherein the at least one target gene is an endogenous gene, an exogenous gene, or a combination thereof. 20. The method of item 18 or 19, wherein the host cell is in a subject. 21. The method of any one of items 18 to 20, wherein the method comprises administering the at least one synthetic transcription factor, the nucleic acid, the vector, or the composition to the subject. 22. Use of a synthetic transcription factor of any one of items 1–6, the nucleic acid of any one of items 7–9, the vector of any one of items 10–12, the cell of item 13 or item 14, or the composition of item 15 or item 16 for modulating the expression of at least one target gene in a cell. 23. The use of item 22, wherein the at least one target gene is an endogenous gene, an exogenous gene, or a combination thereof.Atty. Docket No.5724-108WO1 / / P.0279.WO1 24. The use of item 22 or 23, wherein modulating the expression treats a symptom of a disease in a patient. 25. The use of item 24, wherein the disease is Dravet Syndrome and the at least one target gene is SCN1A. 26. The use of any one of items 22 to 25, wherein the synthetic transcription factor is administered with a viral vector at a dose of 5×1012viral genomes per kilogram body mass (VG / kg) to 5×1013VG / kg, e.g.1×1013VG / kg. 27. The use of item 26, wherein the viral vector is a recombinant adeno-associated virus (rAAV) vector. 28. A method of treating a symptom of a disease comprising modulating the expression of at least one target gene in a cell of a subject having the disease, by introducing into the cell at least one synthetic transcription factor of any one of items 1–6, the nucleic acid of any one of items 7–9, the vector of any one of items 10–12, the cell of item 13 or item 14, or the composition of item 15 or item 16. 29. The method of item 28, wherein the disease is Dravet Syndrome and the at least one target gene is SCN1A. 30. The method of item 28 or 29, wherein the synthetic transcription factor is linked to a zinc finger protein selected from SEQ ID NOs: 6, 15, 24, and 33. 31. The method of any one of items 28 to 30, wherein the synthetic transcription factor is administered with a viral vector. 32. The method of item 31, wherein the viral vector is an rAAV vector. 33. The method of item 31 or 32, wherein the viral vector comprises a nucleotide sequence selected from SEQ ID NOs: 13, 22, 31, and 40. 34. The method of any one of items 31 to 33 wherein the viral vector comprises a nucleotide sequence according to SEQ ID NO:5. 35. The method of any one of items 31 to 33, wherein the viral vector is administered at a dose of 5×1012VG / kg to 5×1013VG / kg. 36. The method of item 35, wherein the viral vector is administered at a dose of 1×1013VG / kg.Atty. Docket No.5724-108WO1 / / P.0279.WO1 37. The method of any one of items 31 to 36, wherein the viral vector is administered intravenously. 38. The method of any one of items 31 to 36, wherein the viral vector is administered intracranially. 39. The method of any one of items 31 to 38, wherein the viral vector is administered at a dose sufficient to increase SCN1A expression more than 100% in the thalamus, cortex, and hippocampus of the subject compared to SCN1A expression before transduction with the viral vector. For example, the expression is >100%, >110%, >120%, >130%, >140%, >150%, >160%, >170%, >180%, >190%, >200%, >210%, or more. For example, the expression is 110%-600%, 110%-400%, 140%-350%, 170%-300%, or any range encompassed by the present disclosures. 40. The method of item 39, wherein the viral vector is administered at a dose sufficient to increase SCN1A expression more than 150% in the thalamus, cortex, and hippocampus of the subject compared to SCN1A expression before transduction with the viral vector. 41. The use or method of any preceding item, wherein the vector is a blood-brain barrier- penetrant vector. EXAMPLES

[0081] The invention is further described in the following examples, which do not limit the scope of the claims.

[0082] Example 1. Production of Adeno-Associated Virus (AAV) Vectors. Recombinant adeno-associated virus (rAAV) vectors were generated by the triple transfection method. Briefly, HEK293 cells were plated in ten-layer CellSTACK®chambers (Corning, Acton, Mass.) and grown for three days to a density of 80%. Three plasmids—(i) an AAV Helper plasmid containing the Rep and Cap genes, (ii) an Adenovirus Helper plasmid containing the adenovirus helper genes, and (iii) a transgene plasmid containing the ZFP-TAD sequence to be packagedflanked by AAV2 inverted terminal repeats—were transfected into the cells using calcium phosphate. The ZFP coding sequences were SEQ ID Nos: 13, 22, 31, & 40 and the FOXO3 PFAMflanked S626D TAD coding sequence was SEQ ID NO:5. After three days, the cells were harvested.

[0083] The cells were lysed by three rounds of freeze / thaw and the cell debris was removed by centrifugation. The rAAV was precipitated using polyethylene glycol.Atty. Docket No.5724-108WO1 / / P.0279.WO1

[0084] After resuspension, the virus was purified by ultracentrifugation overnight on a cesium chloride gradient. The virus was formulated by dialysis and thenfilter-sterilized. After adjusting the titer (viral genomes / mL) of all AAV batches by dilution with PBS + 0.001% Pluronic F-68, the AAVs were aliquoted to single use doses and stored at -80°C until use. After thawing, no aliquot was refrozen.

[0085] Example 2. Improved Off-Target Profiles in Human Cells. To assess the specificity of the TAD of SEQ ID NO:1, induced pluripotent cell-derived (iPSC) neurons were transduced in vitro with AAV6 vectors from Example 1 above.

[0086] Human iPSC-derived GABAergic neurons were purchased from Cellular Dynamics International and plated onto poly-L-ornithine- and laminin-coated 96-well plates at a density of 4×104cells per well and maintained according to the manufacturer’s instructions. The cells were infected with AAV expressing the desired ZFP-TAD (bearing either the TAD of p65 or FOXO3 PFAMflanked S626D) at the MOIs indicated in FIG.248 hours after plating. Infected cultures were maintained for up to 21 days (50–75% media changes performed every 3–5 days).

[0087] The cells were harvested at the end of the experimental period. RNA was isolated and reverse transcribed. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed for gene expression analysis. FIG.2A shows normalized SCN1A expression levels in iPSC-derived neuronal culture following AAV delivery of the p65 ZFA-TAD, while FIG. 2B shows the normalized SCN1A expression levels following AAV delivery of the FOXO3 PFAM flanked S626D ZFA-TAD.

[0088] For microarray analysis, the cells were transduced with 1×105VGs / cell 48 hours after plating and harvested 19 days after viral transduction. Microarray analysis (Clariom S Array) was performed on total RNA isolated from these cultures. Microarray analyses were performed following the manufacturer’s protocol (Thermo Fisher Scientific), and the assay results were analyzed using TAC4 software. The results of this analysis are shown in FIG.3A (p65 ZFA- TAD) and FIG.3B (FOXO3 PFAMflanked S626D ZFA-TAD). Colored dots represent genes whose expression was statistically different as compared to the control (i.e., neurons transduced with a KRAB-based synthetic transcription factor fused to a non-targeting ZFP). The dot representing the targeted gene (SCN1A) is indicated with a label and arrow. Dots below the dashed line showed no statistically significant change in gene expression as compared to the control.

[0089] To test the specificity of the choice of ZFP on the TAD of SEQ ID NO:1, the same experiment was repeated with four different ZFP-TAD constructs (ZFA3, ZFA4, ZFA5, &Atty. Docket No.5724-108WO1 / / P.0279.WO1 ZFA6, see Table 1). FIGs.2C–2F show normalized SCN1A expression in human iPSC-derived neurons from these four ZFP-TAD constructs, while FIGs. 3C–3F show volcano plots illustrating the transcriptomes of neurons expressing the ZFP-TAD in comparison to neurons expressing a non-targeting ZFP with a KRAB-based synthetic transcription factor.

[0090] Example 3. Scn1a Upregulation in Mouse Neuronal Cells. Primary mouse cortical and hippocampal brain tissues were obtained from embryonic day 18 Scn1a+ / -and isogenic Scn1a+ / +F1 hybrid mice that were generated at Jackson Laboratory by crossing heterozygous 129SScn1atm1Kea / Mmjax (SCN1A KO, Stock #024761) male mice with C57BL / 6J (Stock#000664) female mice. The brain tissues were dissociated into individual cells using a papain dissociation kit according to the manufacturer’s instructions. Cells were plated onto poly-D-lysine (PDL)-coated 96-well plates at 55,000 cells / well and maintained according to the manufacturer’s specifications using Gibco Neurobasal Medium containing GlutaMAX™ I supplement, B27 supplement, and penicillin / streptomycin. 48 hours after plating, 5.5×104cells / well in 96-well plates were infected with AAV-ZFP-TAD at the MOIs indicated in FIGs. 4 & 5 and harvested 7 days later. 50% media exchanges were performed every 3–4 days. Harvested RNA was analyzed by RT-qPCR for Scn1a gene expression.

[0091] The results of these analyses are shown in FIGs. 4 & 5. FIGs. 4A–4D show normalized Scn1a expression from these four ZFP-TAD constructs in mouse cortical Scn1a+ / -neurons, while FIGs. 5A–5D show normalized Scn1a expression from these four ZFP-TAD constructs in mouse hippocampal Scn1a+ / -neurons. The data are scaled relative to Scn1a+ / +neurons from wild-type mice that were transduced with a negative control.

[0092] Example 4: In Vivo Tolerability and Functionality Study in Wildtype Mice. The tolerability and functionality of introducing AAV.PHP.B vectors encoding ZFP-TADs as described herein to target the mouse Scn1a gene in wildtype C57BL / 6 mice was evaluated in the brain via RT-qPCR analysis on neuroinflammatory (Gfap, Iba1) and neuronal (NeuN, Tubb3) markers. Cohorts of 4 mice each were intravenously administered vehicle (formulation buffer) or test articles (AAV.PHP.B encoding ZFP-TADs). An AAV.PHP.B encoding a ZFP- TAD with a non-targeting control ZFP was administered as a negative control. The mice were euthanized 4 weeks after dosing.

[0093] To harvest tissue for subsequent analysis, euthanized mice were perfused with PBS. Micro-dissected brain tissue was treated with RNALater. After 24 hours, the RNALater was removed, and tissues wereflash-frozen in liquid nitrogen. Frozen tissues were maintained at - 80°C until analysis.Atty. Docket No.5724-108WO1 / / P.0279.WO1

[0094] Reverse transcription was performed using the High-Capacity RT Kit (Thermo Fisher Scientific) kit following the manufacturer’s instructions. TaqMan qPCR was used to measure the expression levels of the ZFP-TAD, Gfap, Aif11, Rbfox3, and Tubb3 RNAs extracted from the tissues. Gene expression levels were normalized to the mean of the expression levels of the housekeeping genes Atp5b and Eif4a2, then scaled to the average value from the Vehicle- treated animals. No significant differences were observed for neuroinflammatory or cellular toxicity markers compared to vehicle treated animals (data not shown).

[0095] Brain hemispheres were removed (including the olfactory bulb and cerebellum) from all euthanized mice and placed in 10% neutral buffered formalin (NBF) at room temperature for 22–24 hrs. Afterfixation, brain hemispheres were transferred to 0.01M PBS + 0.02% Sodium Azide, pH 7.4 ± 0.2 at 2–8ºC. Brain hemispheres were embedded into paraffin blocks and sectioned for multiplexed RNAscope-immunohistochemistry single cell analysis. A multiplexed approach combining in situ hybridization (ISH) to detect mRNA transcripts (i.e. ZFP-TAD and mouse Scn1a) and immunohistochemistry (IHC) to label putative neurons (NeuN) was used. AAV-PHP.B-hSYN1-ZFP-TADs achieved >150% Scn1a upregulation relative to the negative control group in neurons of thalamus, cortex, and hippocampus at 1×1013VG / kg doses (FIG.6).

[0096] Example 5: Upregulation of various genes in neuroblastoma cells with ZFP-TAD constructs containing a ZFP designed to target the specified gene. Neuro2a or SKNMC cells were nucleofected with mRNA encoding the ZFP-TAD at the doses indicated in the figures. Cells were nucleofected in Amaxa SF solution on the Lonza Nucleofector II device. Following nucleofection, cells were seeded into a 96-well plate containing complete medium and harvested 10 hours later. Harvested RNA was analyzed by RT-qPCR for target gene expression indicated in the figures. The results of these analyses are shown in FIGs.7A-7C.

[0097] All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0098] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.Atty. Docket No.5724-108WO1 / / P.0279.WO1 Table 1. Sequences of the disclosure Q G T D G A Q M L R I L L SAtty. Docket No.5724-108WO1 / / P.0279.WO1 a g g a g T Q H H G S c a a g a g g g c g t c t c c g a c g a T Q H H Y DAtty. Docket No.5724-108WO1 / / P.0279.WO1 c a a g a g g g c c t c a c c t g c c g T C H C T D c a a g a c t t c g g t a cAtty. Docket No.5724-108WO1 / / P.0279.WO1 c g g c a c T C H K S L c a a g a c t t c c c g c a c a a g t a S

Claims

Atty. Docket No.5724-108WO1 / / P.0279.WO1 CLAIMS 1. A synthetic transcription factor, wherein the synthetic transcription factor comprises an amino acid sequence of (FOXO3 PFAMflanked S626D) SEQ ID NO: 1, and wherein the synthetic transcription factor does not comprise SEQ ID NO:

2.

2. The synthetic transcription factor of claim 1, wherein the synthetic transcription factor is linked to a heterologous DNA binding domain.

3. The synthetic transcription factor of claim 2, wherein the heterologous DNA binding domain is a zincfinger protein, TALE protein, dCas9, or dCas12a.

4. The synthetic transcription factor of claim 3, wherein the heterologous DNA binding domain is a zincfinger protein.

5. The synthetic transcription factor of claim 4, wherein the zincfinger protein is selected from SEQ ID NOs: 6, 15, 24, and 33.

6. The synthetic transcription factor of any one of claims 1–5, wherein the heterologous DNA binding domain is an inducible DNA binding domain.

7. A nucleic acid encoding the synthetic transcription factor of any one of claims 1–5.

8. The nucleic acid of claim 7, comprising a nucleotide sequence according to SEQ ID NO:

5.

9. The nucleic acid of claim 7 or 8, comprising a nucleotide sequence selected from SEQ ID NOs: 13, 22, 31, and 40.

10. A vector comprising a nucleic acid of any one of claims 7 to 9.

11. The vector of claim 10, wherein the vector is a viral vector.

12. The vector of claim 11, wherein the vector is capable of delivering the nucleic acid across a blood-brain barrier.

13. A cell comprising the synthetic transcription factor of any one of claims 1–6, the nucleic acid of any one of claims 7–9, or the vector of any one of claims 10–12.

14. The cell of claim 13, wherein the cell comprises at least two synthetic transcription factors where at least one of the synthetic transcription factors is the synthetic transcription factor of any one of claims 1–6.Atty. Docket No.5724-108WO1 / / P.0279.WO1 15. A composition comprising a synthetic transcription factor of any one of claims 1–6, the nucleic acid of any one of claims 7–9, the vector of any one of claims 10–12, or the cell of claim 13 or claim 14.

16. The composition of claim 15, wherein the composition comprises two or more synthetic transcription factors, nucleic acids, vectors, or cells, wherein at least one of the synthetic transcription factors is the synthetic transcription factors of any one of claims 1–6.

17. A kit comprising at least one synthetic transcription factor of any one of claims 1–6, the nucleic acid of any one of claims 7–9, or the vector of any one of claims 10–12, the cell of claim 13 or claim 14, or the composition of claim 15 or claim 16.

18. A method of modulating the expression of at least one target gene in a host cell, the method comprising introducing into the host cell at least one synthetic transcription factor of any one of claims 1–6, the nucleic acid of any one of claims 7–9, the vector of any one of claims 10– 12, the cell of claim 13 or claim 14, or the composition of claim 15 or claim 16.

19. The method of claim 18, wherein the at least one target gene is an endogenous gene, an exogenous gene, or a combination thereof.

20. The method of claim 18 or 19, wherein the host cell is in a subject.

21. The method of any one of claims 18 to 20, wherein the method comprises administering the at least one synthetic transcription factor, the nucleic acid, the vector, or the composition to the subject.

22. Use of a synthetic transcription factor of any one of claims 1–6, the nucleic acid of any one of claims 7–9, the vector of any one of claims 10–12, the cell of claim 13 or claim 14, or the composition of claim 15 or claim 16 for modulating the expression of at least one target gene in a cell.

23. The use of claim 22, wherein the at least one target gene is an endogenous gene, an exogenous gene, or a combination thereof.

24. The use of claim 22 or 23, wherein modulating the expression treats a symptom of a disease in a patient.

25. The use of claim 24, wherein the disease is Dravet Syndrome and the at least one target gene is SCN1A.Atty. Docket No.5724-108WO1 / / P.0279.WO1 26. The use of any one of claims 22 to 25, wherein the synthetic transcription factor is administered with a viral vector at a dose of 5×1012viral genomes per kilogram body mass (VG / kg) to 5×1013VG / kg, e.g.1×1013VG / kg.

27. The use of claim 26, wherein the viral vector is a recombinant adeno-associated virus (rAAV) vector.

28. A method of treating a symptom of a disease comprising modulating the expression of at least one target gene in a cell of a subject having the disease, by introducing into the cell at least one synthetic transcription factor of any one of claims 1–6, the nucleic acid of any one of claims 7–9, the vector of any one of claims 10–12, the cell of claim 13 or claim 14, or the composition of claim 15 or claim 16.

29. The method of claim 28, wherein the disease is Dravet Syndrome and the at least one target gene is SCN1A.

30. The method of claim 28 or 29, wherein the synthetic transcription factor is linked to a zinc finger protein selected from SEQ ID NOs: 6, 15, 24, and 33.

31. The method of any one of claims 28 to 30, wherein the synthetic transcription factor is administered with a viral vector.

32. The method of claim 31, wherein the viral vector is an rAAV vector.

33. The method of claim 31 or 32, wherein the viral vector comprises a nucleotide sequence selected from SEQ ID NOs: 13, 22, 31, and 40.

34. The method of any one of claims 31 to 33 wherein the viral vector comprises a nucleotide sequence according to SEQ ID NO:

5.

35. The method of any one of claims 31 to 33, wherein the viral vector is administered at a dose of 5×1012VG / kg to 5×1013VG / kg.

36. The method of claim 35, wherein the viral vector is administered at a dose of 1×1013VG / kg.

37. The method of any one of claims 31 to 36, wherein the viral vector is administered intravenously.Atty. Docket No.5724-108WO1 / / P.0279.WO1 38. The method of any one of claims 31 to 36, wherein the viral vector is administered intracranially.

39. The method of any one of claims 31 to 38, wherein the viral vector is administered at a dose sufficient to increase SCN1A expression more than 100% in the thalamus, cortex, and hippocampus of the subject compared to SCN1A expression before transduction with the viral vector.

40. The method of claim 39, wherein the viral vector is administered at a dose sufficient to increase SCN1A expression more than 150% in the thalamus, cortex, and hippocampus of the subject compared to SCN1A expression before transduction with the viral vector.

41. The use or method of any preceding claim, wherein the vector is a blood-brain barrier- penetrant vector.

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