Human-targeted zinc finger protein to treat angelman syndrome

Abstract

In Angelman syndrome (AS), loss of UBE3A protein in the brain is typically due to mutation or deletion of the maternal UBE3A allele. Due to genetic imprinting, the paternal UBE3A allele is present but epigenetically silenced by an antisense transcript. As disclosed herein, activation of this paternal allele of UBE3A has great promise to treat the underlying causes of this disorder and bring meaningful improvements to children. Disclosed herein is a programable Artificial Transcription Activator (ATF) that can silence expression of the UBE3A transcript. Also disclosed herein is a method of treating Angelman Syndrome in a subject, the method involving administering to the subject an effective amount of a pharmaceutical composition comprising one or more AAV vectors containing a polynucleotide encoding the disclosed ATF.

Description

HUMAN-TARGETED ZINC FINGER PROTEIN TO TREAT ANGELMAN SYNDROMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 542,152, filed October 3, 2023, which is hereby incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] This application contains a sequence listing filed in ST.26 format entitled “320317-2100” created on April 20, 2024, and having 36,864 bytes. The content of the sequence listing is incorporated herein in its entirety.BACKGROUND OF THE INVENTION

[0003] Angelman syndrome (AS) is a neurodevelopmental disorder that is associated with severe cognitive and motor deficits, epilepsy, sleep-disorder, and an atypical ‘happy’ disposition. Individuals with AS are often diagnosed at 2-3 years of age and have a normal life-span. They require assisted living and medical care throughout their lives. There are currently few treatment options for individuals with AS, most of which involve antiepileptic medications to treat seizures.

[0004] Angelman syndrome is caused by mutations that affect the expression or function of the maternally inherited ubiquitin-protein ligase E3A (UBE3A) gene. Unlike most genes, UBE3A is subject to genomic imprinting, which is a rare, naturally occurring phenomenon that turns-off one allele of a gene while leaving the other allele on. In neurons of the central nervous system (CNS), the paternal UBE3A allele is off, whereas in all other cell types of the body, both alleles of UBE3A are on. Because of this, AS is always caused by mutations that affect the maternally inherited UBE3A allele.

[0005] The paternal UBE3A allele is turned-off by the UBE3A antisense transcript (UBE3A-AS), which is a component of a long RNA transcript that expresses several protein coding and noncoding transcripts. UBE3A-AS is expressed from the paternal allele and only in neurons of the CNS and is both sufficient and necessary to turn-off expression of the paternal UBE3A allele. It’s unclear why UBE3A is imprinted in neurons, but it creates a unique opportunity to treat individuals with AS, because there is a functional, albeit inactive, copy of UBE3A on the paternal chromosome. Studies to date indicate that turning on the paternal UBE3A allele is a viable therapy to treat AS.SUMMARY OF THE INVENTION

[0006] In Angelman syndrome (AS), loss of UBE3A protein in the brain is typically due to mutation or deletion of the maternal UBE3A allele (FIG. 1). Due to genetic imprinting, the paternal UBE3A allele is present but epigenetically silenced by an antisense transcript.As disclosed herein, activation of this paternal allele of UBE3A has great promise to treat the underlying causes of this disorder and bring meaningful improvements to children.

[0007] Disclosed herein is an artificial transcription factor (ATF) involving a transcriptional repressor domain fused to a zinc-finger DNA binding domain, wherein the zinc-finger DNA binding domain has recognition helix regions that bind the target sequences:(1) F3: GCG, F4: CAG, F5: GCG, and F6: GCC;(2) F2: GCG, F3: CAG, F4: GCG, F5: GCC, and F6: GCG;(3) F1 : CAG, F2: CCG, F3: CGG, F4: GCG, F5: CCT, and F6: ACG;(4) F1 : AGG, F2: CAG, F3: CCG, F4: CGG, F5: GCG, and F6: CCT;(5) F1 : CAG, F2: AGG, F3: CAG, F4: CCG, F5: CGG, and F6: GCG;(6) F1 : GGC, F2: GCA, F3: GAG, F4: GCA, F5: GCC, and F6: GCG;(7) F1 : GCT, F2: CAG, F3: AGG, F4: CAG, F5: CCG, and F6: CGG;(8) F1 : TGG, F2: GGC, F3: GCA, F4: GAG, F5: GCA, and F6: GCC;(9) F1 : GGC, F2: GCT, F3: CAG, F4: AGG, F5: CAG, and F6: CCG;(10) F1 : GCA, F2: GAG, F3: GCA, F4: GGC, F5: TGG, and F6: CGC;(11) F1 : CAG, F2: AGG, F3: CAG, F4: GCT, F5: GGC, and F6: GCG;(12) F1 : GCA, F2: CGC, F3: TGG, F4: GGC, F5: GCA, and F6: GAG;(13) F1 : CAT, F2: GCG, F3: GGC, F4: GCT, F5: CAG, and F6: AGG;(14) F1 : GCT, F2: CAT, F3: GCG, F4: GGC, F5: GCT, and F6: CAG;(15) F1 : CCT, F2: CAG, F3: CGC, F4: GCG, F5: CAT, and F6: GAG;(16) F1 : CAG, F2: GCT, F3: CAT, F4: GCG, F5: GGC, and F6: GCT;(17) F1 : CAG, F2: CGC, F3: GCG, F4: CAT, F5: GAG, and F6: CCT;(18) F1 : GCG, F2: CAG, F3: GCT, F4: CAT, F5: GCG, and F6: GGC; or(19) F1 : GGG, F2: GCG, F3: CAG, F4: GCT, F5: CAT, and F6: GCG.

[0008] Therefore, in some embodiments the zinc-finger DNA binding domain has one of the following set of recognition helix regions:(1) F3: RSDDLVR (SEQ ID NO:20), F4: RADNLTE (SEQ ID NO:21), F5: RSDDLVR (SEQ ID NO:20), and F6: DCRDLAR (SEQ ID NO:22);(2) F2: RSDDLVR (SEQ ID NQ:20), F3: RADNLTE (SEQ ID NO:21), F4: RSDDLVR (SEQ ID NQ:20), F5: DCRDLAR (SEQ ID NO:22), and F6: RSDDLVR (SEQ ID NQ:20);(3) F1 : RADNLTE (SEQ ID NO:21), F2: RNDTLTE (SEQ ID NO:23), F3: RSDKLTE (SEQ ID NO:24), F4: RSDDLVR (SEQ ID NQ:20), F5: TKNSLTE (SEQ ID NO:25), and F6: RTDTLRD (SEQ ID NO:26);(4) F1 : RSDHLTN (SEQ ID NO:27), F2: RADNLTE (SEQ ID NO:21), F3: RNDTLTE (SEQ ID NO:23), F4: RSDKLTE (SEQ ID NO:24), F5: RSDDLVR (SEQ ID NQ:20), and F6: TKNSLTE (SEQ ID NO:25);(5) F1 : RADNLTE (SEQ ID N0:21), F2: RSDHLTN (SEQ ID NO:27), F3: RADNLTE (SEQ ID N0:21), F4: RNDTLTE (SEQ ID NO:23), F5: RSDKLTE (SEQ ID NO:24), and F6: RSDDLVR (SEQ ID NQ:20);(6) F1 : DPGHLVR (SEQ ID NO:28), F2: QSGDLRR (SEQ ID NO:29), F3: RSDNLVR (SEQ ID NQ:30), F4: QSGDLRR (SEQ ID NO:29), F5: DCRDLAR (SEQ ID NO:22), and F6: RSDDLVR (SEQ ID NQ:20);(7) F1 : TSGELVR (SEQ ID N0:31), F2: RADNLTE (SEQ ID N0:21), F3: RSDHLTN (SEQ ID NO:27), F4: RADNLTE (SEQ ID N0:21), F5: RNDTLTE (SEQ ID NO:23), and F6: RSDKLTE (SEQ ID NO:24);(8) F1 : RSDHLTT (SEQ ID NO:32), F2: DPGHLVR (SEQ ID NO:28), F3: QSGDLRR (SEQ ID NO:29), F4: RSDNLVR (SEQ ID NQ:30), F5: QSGDLRR (SEQ ID NO:29), and F6: DCRDLAR (SEQ ID NO:22);(9) F1 : DPGHLVR (SEQ ID NO:28), F2: TSGELVR (SEQ ID N0:31), F3: RADNLTE (SEQ ID N0:21), F4: RSDHLTN (SEQ ID NO:27), F5: RADNLTE (SEQ ID N0:21), and F6: RNDTLTE (SEQ ID NO:23);(10) F1 : HTGHLLE (SEQ ID NO:33), F2: RSDHLTT (SEQ ID NO:32), F3: DPGHLVR (SEQ ID NO:28), F4: QSGDLRR (SEQ ID NO:29), F5: RSDNLVR (SEQ ID NQ:30), and F6: QSGDLRR (SEQ ID NO:29);(11) F1 : RSDDLVR (SEQ ID NQ:20), F2: DPGHLVR (SEQ ID NO:28), F3: TSGELVR (SEQ ID NO:31), F4: RADNLTE (SEQ ID NO:21), F5: RSDHLTN (SEQ ID NO:27), and F6: RADNLTE (SEQ ID NO:21);(12) F1 : QSGDLRR (SEQ ID NO:29), F2: HTGHLLE (SEQ ID NO:33), F3: RSDHLTT (SEQ ID NO:32), F4: DPGHLVR (SEQ ID NO:28), F5: QSGDLRR (SEQ ID NO:29), and F6: RSDNLVR (SEQ ID NQ:30);(13) F1 : TSGNLTE (SEQ ID NO:34), F2: RSDDLVR (SEQ ID NQ:20), F3: DPGHLVR (SEQ ID NO:28), F4: TSGELVR (SEQ ID NO:31), F5: RADNLTE (SEQ ID NO:21), and F6: RSDHLTN (SEQ ID NO:27);(14) F1 : TSGELVR (SEQ ID NO:31), F2: TSGNLTE (SEQ ID NO:34), F3: RSDDLVR (SEQ ID NQ:20), F4: DPGHLVR (SEQ ID NO:28), F5: TSGELVR (SEQ ID NO:31), and F6: RADNLTE (SEQ ID NO:21);(15) F1 : TKNSLTE (SEQ ID NO:25), F2: RADNLTE (SEQ ID NO:21), F3: HTGHLLE (SEQ ID NO:33), F4: RSDDLVR (SEQ ID NQ:20), F5: TSGNLTE (SEQ ID NO:34), and F6: RSDNLVR (SEQ ID NQ:30);(16) F1 : RADNLTE (SEQ ID NO:21), F2: TSGELVR (SEQ ID NO:31), F3: TSGNLTE (SEQ ID NO:34), F4: RSDDLVR (SEQ ID NQ:20), F5: DPGHLVR (SEQ ID NO:28), and F6: TSGELVR (SEQ ID NO:31);(17) F1 : RADNLTE (SEQ ID N0:21), F2: HTGHLLE (SEQ ID NO:33), F3: RSDDLVR (SEQ ID NO:20), F4: TSGNLTE (SEQ ID NO:34), F5: RSDNLVR (SEQ ID NQ:30), and F6: TKNSLTE (SEQ ID NO:25);(18) F1 : RSDDLVR (SEQ ID NQ:20), F2: RADNLTE (SEQ ID N0:21), F3: TSGELVR (SEQ ID N0:31), F4: TSGNLTE (SEQ ID NO:34), F5: RSDDLVR (SEQ ID NQ:20), and F6: DPGHLVR (SEQ ID NO:28); or(19) F1 : RSDKLVR (SEQ ID NO:35), F2: RSDDLVR (SEQ ID NQ:20), F3: RADNLTE (SEQ ID NO:21), F4: TSGELVR (SEQ ID NO:31), F5: TSGNLTE (SEQ ID NO:34), and F6: RSDDLVR (SEQ ID NQ:20).

[0009] The ATF can be produced using any number of natural zinc finger proteins as a framework (e.g. Sp1c , Zif268, and F2). In some embodiment, the zinc finger has a Sp1c framework with recognition sites replaced with the recognition models from Table 2 as shown in Table 3. Therefore, in some embodiments, the zinc-finger DNA binding domain is selected from the group consisting of SEQ ID NOs:1 to 19.

[0010] In some embodiments, the effector domain is a repression domain. Therefore, in some embodiments the effector domain is a Kruppel-associated box (KRAB) domain. Other repression domains include Enhancer of zeste homolog 2 (EZH2), I S F%Friend of GATA1 (FOG1), and RING1 and YY1 Binding Protein (RYBP).

[0011] Also disclosed herein is a polynucleotide encoding the disclosed ATF. Also disclosed is an adeno-associated virus (AAV) vector containing a polynucleotide encoding the disclosed ATF. Examples of AAV serotypes include, but are not limited to, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAVrhI O. For example, in some embodiments, the AAV vector is an AAV6 or AAV9 vector. Also disclosed is a pharmaceutical composition comprising one or more AAV vectors containing a polynucleotide encoding the disclosed ATF.

[0012] Also disclosed herein is a method of treating Angelman Syndrome in a subject, the method involving administering to the subject an effective amount of a pharmaceutical composition comprising one or more AAV vectors containing a polynucleotide encoding the disclosed ATF.

[0013] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF FIGURES

[0014] FIG. 1 shows the Angelman locus in mouse. In AS, the maternal allele is deleted or mutated. The paternal allele (shown) has a wild-type Ube3a, but is silenced by theUbe3a-ATS antisense transcript (wiggle line). Two clusters of small nucleolar RNAs (Snords) are shown.

[0015] FIGs. 2A to 2C illustrates Artificial Transcription Factor (ATF) therapy. FIG. 2A shows an engineered zinc-finger-KRAB repressor ATF (“S1K”) silences the entire Ube3a- ATS transcript, allowing physiological Ube3a expression. FIG. 2B shows treatment in AS mice with ATF protein rescues LTP. FIG. 2C shows AAV-ATF rescues motor deficits in open field assay.

[0016] FIGs. 3A and 3B show a single AAV-S1 k administration restored UBE3A in mouse brain.

[0017] FIGs. 4A to 4C show a human targeted zinc-finger protein “555”. FIGs. 4B and 40 show S1 K (FIG. 4B) and 555 (FIG. 4C) have the ability to repress mouse (FIG. 4B) and human (FIG. 40) UBE3A antisense transcript.

[0018] FIGs. 5A and 5B show that 555 demonstrated good on-target binding (FIG. 5A) but also significant off-target binding (FIG. 5B).

[0019] FIG. 6 shows a series of additional human-targeted zinc-finger proteins that target a portion of the SNURF protomer (SEQ ID NO:67).

[0020] FIGs. 7A and 7B show ZFN36 silences UBE3A antisense transcript in LnCap cells.DETAILED DESCRIPTION

[0021] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, 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, since the scope of the present disclosure will be limited only by the appended claims.

[0022] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0024] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0025] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0026] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0027] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0028] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.Definitions

[0029] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0030] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subjectcan be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0031] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0032] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms, which are within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0033] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0034] The term “inhibit” refers to a decrease in an activity, response, condition, disease, or other biological parameter, which those skilled in the art will appreciate may be assessed at a particular point in time, such that in some embodiments, inhibition may be or comprise a delay in onset or reduction in frequency. In some embodiments, inhibition can include, but is not limited to, the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0035] 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 proteinbinding 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 differentprotein or proteins. A binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding and protein-binding activity.

[0036] A “zinc finger DNA binding protein” (or binding domain) is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.Artificial Transcription Activator (ATF)

[0037] Disclosed herein are engineered artificial transcription factors (ATFs) capable of precise regulation of gene expression at a given locus. As used herein, the term “artificial transcription factor” refers to an engineered or “non-naturally occurring” zinc finger protein or fusion protein that binds to an expression control sequence (e.g. promoter) of a gene. Encompassed by the term ATF are engineered zinc fingers comprising at least one zinc finger domain, typically two zinc finger domains, three zinc finger domains, or more. In some embodiments, the zinc finger domain is a Cys2-His2 type, where two, three, or more zinc finger domains are arranged in parallel to constitute a zing finger protein.

[0038] As used herein, the term “non-naturally occurring” means, for example, one or more of the following: (a) a polypeptide comprised of a non-naturally occurring amino acid sequence; (b) a polypeptide having a non-naturally occurring secondary structure not associated with the polypeptide as it occurs in nature; (c) a polypeptide that includes one or more amino acids not normally associated with the species of organism in which that polypeptide occurs in nature; (d) a polypeptide that includes a stereoisomer of one or more of the amino acids comprising the polypeptide, which stereoisomer is not associated with the polypeptide as it occurs in nature; (e) a polypeptide that includes one or more chemical moieties other than one of the natural amino acids; or (f) an isolated portion of a naturally occurring amino acid sequence (e.g., a truncated sequence).

[0039] In general, the disclosed ATFs comprise a zinc finger backbone, an interaction domain (ID) (DNA-binding domain), and an effector domain (transcriptional repressor domain), and can include an optional nuclear localization signal (NLS). Since the ID interacts with target DNA, it can be located at the N-terminus to be functional for dimerization with a second ATF. The other elements may be present in any order.

[0040] A zinc finger domain is a DNA binding motif of DNA binding proteins that are most frequently discovered in eukaryotes, which is discovered in various species from yeast to higher plant life and human beings. The zinc finger domain has been known to function as a transcription repressor that down-regulate the gene expression by itself. Therefore, if fusing the zinc finger domain with the effector domain (up- or down-regulation), the resultedfusing protein, which is a novel transcription factor, may up- or down-regulate the expression of a target gene that is recognized by the zinc finger.

[0041] In the present disclosure, the zinc finger domain may be a Cys2-His2 type, which three or more zinc finger domains are arranged in parallel to constitute a zing finger protein. Because a single zinc finger domain can recognize a target sequence comprising three or four bases, it can prepare a sequence-specific zinc finger that can selectively recognize target sequences of 9-10 bases by properly re-arranging and linking several zinc finger domains each other.

[0042] A zinc finger DNA binding element can be derived or produced from a wild type zinc finger-containing polypeptide by truncation or expansion, or as a variant of a wild type-derived polypeptide by a process of site directed mutagenesis, or by a combination of the procedures. See e.g., U.S. Pat. Nos. 6,242,568; 6,140,466; and 6,140,081. The term “truncated” refers to a zinc finger-nucleotide binding polypeptide that contains less that the full number of zinc finger motifs found in the native zinc finger binding polypeptide or that has been deleted of non-desired sequences. For example, truncation of the zinc finger- nucleotide binding protein TFIIIA, which naturally contains nine zinc finger motifs, might be a polypeptide with only zinc fingers one through three. Expansion refers to a zinc finger polypeptide to which additional zinc finger motifs have been added. For example, TFIIIA may be extended to 12 fingers by adding 3 zinc finger motifs.

[0043] In addition, a zinc finger DNA binding element may include zinc finger motifs from more than one wild type polypeptide, thus resulting in a “hybrid” zinc finger polypeptide. The term “mutagenized” refers to a zinc finger polypeptide that has been obtained by performing any of the known methods for accomplishing random or site-directed mutagenesis of the DNA encoding the protein. Examples of known zinc finger proteins that can be truncated, expanded, and / or mutagenized according to the present invention in order to alter the function of a zinc finger-nucleotide binding motif include TFIIIA and Zif268. Other zinc finger-containing nucleotide binding polypeptides are well known to those of skill in the art.

[0044] A DNA binding element of the present invention typically comprises a plurality of DNA binding domains. Preferably, the DNA binding domains are zinc-finger motifs. Preferably, the DNA binding element contains from 2, 3, 4, 5, 6, 7, 8, 9, or 10 such motifs, more preferably from 2 to 6 such motifs and, most preferably, 3 such motifs. The DNA binding domains are operably linked to each other. In one embodiment, the DNA binding domains are directly linked or bonded together via well known peptide linkages. In another embodiment, the DNA binding domains are operatively linked using a peptide linker containing from 5 to 50 amino acid residues. Preferably, the linker contains from 5 to 40 amino acid residues, more preferably from 5 to 30 amino acid residues and, even morepreferably from 5 to 15 amino acid residues. The linkers are preferably flexible. Exemplary linkers are set forth, for example, in US Patent Application Publication No. 2007 / 0020627.

[0045] Each of the DNA binding zinc finger motifs is preferably designed and made to specifically bind nucleotide target sequences corresponding to the formula 5'-NNN-3', where N is any nucleotide (i.e., A, C, G or T). Such DNA binding domains are well known in the art. See, e.g., U.S. Pat. Nos. 6,242,568, 6,140,466 and 6,140,081 . A known “recognition code” that relates the amino acids of a single zinc finger motif to its associated DNA target can be utilized as a guide for the design of the DNA binding elements. See Table 2 as an example. See also Corbi et al., Biochem Cell Biol. 82:428-36, 2004; Klug, Q. Rev. Biophys. 31 (1): 1 -21 , 2010; Pabo et al., Annu. Rev. Biochem. 70:313-340, 2001 ; Segal et al., Curr. Opin. Biotechnol. 12(6):632-637, 2001 ; Klug, Annu. Rev. Biochem. 79:213-231 , 2010; and Bhakta et al., Methods Mol. Biol. 649:3-30, 2010. This code can be used for modular assembly of DNA binding elements, for example, by combining three separate zinc finger motifs that can each recognize a 3 bp DNA sequence to generate a three zinc-finger DNA binding element that can specifically recognize a 9 bp target site. Alternatively, screening methods or selection strategies can be used to identify zinc finger sequences that specifically bind to a desired DNA sequence. These methods include, for example, phage display, yeast one-hybrid systems, and bacterial one- hybrid and two-hybrid systems and other methods known to one of skill in the art (see, e.g., Maeder et al., Mol. Cell. 31 :294-301 , 2008). Combinations of zinc finger motifs that bind to specific DNA sequences can be obtained from commercial sources or using tools provided by the Zinc Finger Consortium.

[0046] Structural information about known zinc finger motifs can be used to guide the design of DNA binding elements that bind to a desired target sequence. For example, the structure of a three finger polypeptide-DNA complex derived from the mouse immediate early protein Zif268 (also known as Krox-24) has been solved by X-ray crystallography (see, e.g., Pavletich et al., Science, 252:809-817, 1991). Each finger contains an anti-parallel beta-turn, a finger tip region, and a short amphipathic alpha-helix which, in the case of the zinc finger motifs of Zif268, binds in the major groove of DNA. In addition, the conserved hydrophobic amino acids and zinc coordination by the cysteine and histidine residues stabilize the structure of the individual finger domain. The crystal structure of Zif268 indicates that specific histidine (non-zinc coordinating His residues) and arginine residues on the surface of the alpha-helix participate in DNA recognition. Specifically, the charged amino acids immediately preceding the alpha-helix and at helix positions 2, 3, and 6 (immediately preceding the conserved histidine) participate in hydrogen bonding to DNA guanines. In general, modifications in or near the alpha-helix are more likely to affect DNA binding specificity, whereas modifications in framework regions (e.g., beta turns or linker regions)are less likely to affect DNA binding specificity, while they may or may not affect structural integrity (e.g. proper folding) of the protein.

[0047] A zinc finger DNA binding motif typically comprises a unique heptamer (contiguous sequence of 7 amino acid residues) within the alpha-helix of the motif, which largely determines binding specificity to a target nucleotide. The heptameric sequence can be located anywhere within the a-helical domain but it is preferred that the heptamer extend from position -1 to position 6 as the residues are conventionally numbered in the art. In some embodiments, one or more modifications in the key amino acid positions (-1 , +3, and +6) of the zinc finger alpha-helix can enable it to bind the desired DNA target sequence. In some embodiments, one or more modifications in other amino acid positions of the zinc finger alpha-helix can be used to enable it to bind the desired DNA target sequence (e.g., positions +1 , +2, +4, or +5). In other embodiments, changes to residues outside of the heptamer can also be introduced to enable the protein to bind to the desired DNA target sequence. A zinc finger motif can include any p-sheet and framework sequences known in the art to function as part of a zinc finger motif. In some embodiments, the p-sheet and / or framework sequences are not substantially modified during the process of modifying a zinc finger domain to bind to a desired target sequence. Therefore, a zinc finger motif of a DNA binding element of the invention (e.g., in a fusion protein or a modified human transcription factor) may have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a known zinc finger motif, with only a few different amino acid residues, e.g., 1 , 2, 3, 4, 5, 6, 7, or 8 amino acid residues modified from the known zinc finger motif. These modifications are typically located in the alpha-helix or in amino acid residues surrounding the alpha-helix. Residues that are involved structural integrity or proper 3 dimensional protein folding, e.g., in zinc coordination, are disfavored for modification. Methods to determine whether a DNA binding element can bind to a particular DNA sequence are well known to those skilled in the art, and include, e.g., electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation (ChIP), and DNase I protection assays.

[0048] In some embodiments, the effector domain is a repression domain. Therefore, in some embodiments the effector domain is a Kruppel-associated box (KRAB) domain. Other repression domains include Enhancer of zeste homolog 2 (EZH2), I S F% r jyn~gwEsxkjvifxj (e.g. DMNT3A, DMNT3L), tm^sj jfhjy-4xj%j3L34lFH6., Friend Of GATA1 (FOG1), and RING1 and YY1 Binding Protein (RYBP).

[0049] Various linkers may be used to link the DNA binding domains to each other or link the DNA binding domains with the effector domain. In the present disclosure, it may use a linker that links the zinc finger domains in naturally occurring zinc finger proteins.

[0050] Nuclear localization signals (NLS) are amino acid motifs conferring nuclear import through binding to proteins defined by gene ontology G0:0008139, for example clusters of basic amino acids containing a lysine residue (K) followed by a lysine (K) or arginine residue (R), followed by any amino acid (X), followed by a lysine or arginine residue consensus sequence.AAV Vectors and Compositions

[0051] In some embodiments, the disclosed ATFs are delivered to cells in a subject by means of a viral vector, of which many are known and available in the art. The therapeutic vector is desirably non-toxic, minimally immunogenic (i.e., elicits a very mild immune response, if any), easy to produce, and efficient in protecting and delivering DNA into the target cells. In particular embodiments, the viral vector is a recombinant adeno- associated vector (AAV).

[0052] The use of AAVs is a common mode of exogenous delivery of DNA as it is relatively non-toxic, provides efficient gene transfer, and can be easily optimized for specific purposes. More than 30 naturally occurring serotypes of AAV are available. Many natural variants in the AAV capsid exist, allowing identification and use of an AAV with properties specifically suited for muscle cells. AAV viruses may be engineered by conventional molecular biology techniques, making it possible to optimize these particles for cell specific delivery of nucleic acid sequences, for minimizing immunogenicity, for tuning stability and particle lifetime, for efficient degradation, etc. Examples of AAV serotypes include, but are not limited to, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAVrh . See, e.g., International Patent Application WO 2005 / 033321 , for a discussion of various AAV serotypes.

[0053] In several embodiments, expression of artificial transcription factors of the present invention or other genes can be achieved through delivery by recombinantly engineered AAVs or artificial AAVs that comprise nucleic acid sequences encoding the artificial transcription factors or genes that are operably linked to promoters. In certain preferred embodiments, these AAVs are formulated as pharmaceutical compositions for treatment of Angelman Syndrome.

[0054] Desirable AAV fragments for assembly into vectors include the cap proteins, including vp1 , vp2, vp3, and hypervariable regions, the rep proteins, including rep78, rep68, rep52, and rep40, and the sequences encoding these proteins. These fragments may be readily utilized in a variety of vector systems and host cells. Such fragments maybe used, alone, in combination with other AAV serotype sequences or fragments, or in combination with elements from other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, e.g., AAV with a non-naturally occurring capsid protein. Such an artificial capsid may be generated by any suitable technique, using a selected AAV sequence (e.g., afragment of a vp1 capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV serotype, non-contiguous portions of the same AAV serotype, from a non-AAV viral source, or from a non-viral source. An artificial AAV serotype may be, without limitation, a pseudotyped AAV capsid, a chimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAV capsid. Pseudotyped vectors, wherein the capsid of one AAV is replaced with a heterologous capsid protein, are useful in the invention.

[0055] In some embodiments, the vectors contain, at a minimum, sequences encoding a selected AAV serotype capsid, e.g., an AAV8 capsid, or a fragment thereof. In another embodiment, useful vectors contain, at a minimum, sequences encoding a selected AAV serotype rep protein, e.g., AAV8 rep protein, or a fragment thereof. Optionally, such vectors may contain both MV cap and rep proteins. In vectors in which both AAV rep and cap are provided, the AAV rep and AAV cap sequences can both be of one serotype origin, e.g., all AAV8 origin. In other embodiments, the vectors useful in compositions and methods described herein contain, at a minimum, sequences encoding an AAV6 capsid, or a fragment thereof. In some embodiments, useful vectors contain sequences encoding an AAV6 rep protein, or a fragment thereof. In other embodiments, in vectors in which both AAV rep and cap are provided, the AAV rep and AAV cap sequences can be both of AAV6 origin.

[0056] Alternatively, vectors may be used in which the rep sequences are from an AAV serotype which differs from that which is providing the cap sequences. In one embodiment, the rep and cap sequences are expressed from separate sources (e.g., separate vectors, or a host cell and a vector). In another embodiment, these rep sequences are fused in frame to cap sequences of a different AAV serotype to form a chimeric AAV vector, such as AAV2 / 8 described in U.S. Pat. No. 7,282,199, which is incorporated by reference herein.

[0057] A suitable recombinant AAV is generated by culturing a host cell which contains a nucleic acid sequence encoding an AAV serotype capsid protein, or fragment thereof, as defined herein; a functional rep gene; a transgene nucleic acid comprising AAV inverted terminal repeats (ITRs), a muscle-specific promoter sequence, and an artificial transcription factor (e.g., a fusion protein or modified human transcription factor) sequence; and sufficient helper functions to permit packaging of the transgene nucleic acid into the AAV capsid protein. The components required to be cultured in the host cell to package an AAV minigene in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., transgene nucleic acid sequences, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. AAV vectors of the present invention can be produced, for example, by triple transfection of subconfluent HEK293 cells by threeplasmids: AAV cis-plasmid containing the gene of interest, AAV trans-plasmid containing AAV rep and cap genes, and an adenovirus helper plasmid, for example pDF6.

[0058] Such a stable host cell may contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein, in the discussion below of regulatory elements suitable for use with the transgene. In still another alternative, a selected stable host cell may contain selected components under the control of a constitutive promoter and other selected components under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain E1 helper functions under the control of a constitutive promoter), but which contains the rep and / or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.

[0059] The transgene, rep sequences, cap sequences, and helper functions required for producing the recombinant AAV of the invention may be delivered to the packaging host cell in the form of any genetic element which transfers the sequences carried thereon. The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of this invention are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of generating recombinant AAV virions are well known and the selection of a suitable method is not a limitation on the present invention. See, e.g., Fisher et al., J. Virol. 70:520-532, 1992 and U.S. Pat. No. 5,478,745, among others. These publications are incorporated herein by reference.

[0060] Unless otherwise specified, the AAV ITRs, and other selected AAV components described herein, may be readily selected from among any AAV serotype, including, without limitation, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or other known and unknown AAV serotypes. These ITRs or other AAV components may be readily isolated using techniques available to those of skill in the art from an AAV serotype. Such AAV may be isolated or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, Va.). Alternatively, the AAV sequences may be obtained through synthetic or other suitable means by reference to published sequences such as are available in the literature or in databases such as, e.g., GenBank, PubMed, or the like.

[0061] A transgene of the present invention comprises, for example, an artificial transcription factor (e.g., a fusion protein or modified transcription factor) nucleic acidsequence (e.g., Vp16-Jazz, Vp16-Bagly, Vp16-CJ7-UtroUp, JZifl , or JZif2) or other gene desired to be delivered to muscles, as described above, and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). In one desirable embodiment, the ITRs of AAV serotype 8 are used. However, ITRs from other suitable serotypes may be selected. It is this transgene which is packaged into a capsid protein and delivered to a selected host cell.

[0062] The regulatory sequences include conventional control elements which are operably linked to the transgene in a manner which permits its transcription, translation and / or expression in a cell transfected with the vector or infected with the virus produced by the invention. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e. , Kozak consensus sequence); sequences that enhance protein stability; sequences that confer nuclear localization (e.g., nuclear localization sequences) and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences are known in the art and may be utilized. Poly A sequences may be derived from many suitable species, including, without limitation SV-40, humans, and bovines.

[0063] The regulatory sequences useful in the constructs of the present invention may also contain an intron, desirably located between the promoter / enhancer sequence and the gene. One desirable intron sequence is derived from SV-40, and is a 100 bp mini-intron splice donor / splice acceptor referred to as SD-SA. In a preferred embodiment, the intron comprises the first intron of the human alpha-actin gene, including the splice donor site, as well as part of the second intron of the human beta-globin gene and part of the third exon of the human beta-globin gene, including the splice acceptor site.

[0064] In other embodiments, the recombinant AAV vectors may include accessory functional elements including cap signals, sequences coding for epitope tags (e.g., haemmaglutinin (HA) tag, myc tag, maltose-binding protein (MBP) tag, green fluorescent protein (GFP) or any other fluorescent protein, etc.), and / or multicloning sites which contain a plurality of restriction enzyme cutting sites that make it possible to insert into the vector any gene coding for a protein of interest. The amino acid sequence of an exemplary myc tag is shown in SEQ ID NO:75.

[0065] Another regulatory component of the recombinant AAV useful in the invention is an internal ribosome entry site (IRES). An IRES sequence, or other suitable systems, may be used to produce more than one polypeptide from a single gene transcript. An IRES (or other suitable sequence) is used to produce a protein that contains more than one polypeptide chain or to express two different proteins from or within the same cell. An exemplary IRES is the FGF-1 internal ribosome entry sequence, which supports transgeneexpression in skeletal muscle cells. See, e.g., Delluc-Clavieres et al., Gene Ther. 15(15):1090-1098, 2008. Preferably, the IRES is located 3' to the transgene in the recombinant AAV vector.

[0066] The selection of the promoter to be employed in the recombinant AAV may be made from among a wide number of constitutive, inducible, cell-type specific, or tissue-type specific promoters that can express the selected transgene in the desired nerve cell. In a preferred embodiment, the promoter is neuron-specific. An example of a neuron-specific promoter is hSynl .

[0067] The promoter(s) used in the present invention may be derived from any species. In one embodiment, the promoter is of a small size, under 1000 bp, due to the size limitations of the AAV vector. In another embodiment, the promoter is under 400 bp. Examples of constitutive promoters useful in the invention include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the chicken p-actin (CBA) promoter, the phosphoglycerol kinase (PGK) promoter, the EF1 promoter (Invitrogen), and the immediate early CMV enhancer coupled with the CBA promoter.

[0068] Inducible promoters which can be used in the present invention allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied compounds, include, the zinc- inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system; the ecdysone insect promoter, the tetracycline-repressible system, the tetracycline-inducible system, the RU486-inducible system and the rapamycin-inducible system. Other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only. Any type of inducible promoter which is tightly regulated and is specific for the particular target cell type may be used.

[0069] Other regulatory sequences useful in the invention include enhancer sequences. Enhancer sequences useful in the invention include the IRBP enhancer, immediate early cytomegalovirus enhancer, one derived from an immunoglobulin gene orSV40 enhancer, the cis-acting element identified in the mouse proximal promoter, etc. Other enhancer sequences are also well-known in the art.

[0070] Selection of these and other common vector and regulatory elements are conventional and many such sequences are available. See, e.g., Sambrook et al., supra and references cited therein, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989. Of course, not all vectors and expression control sequences will function equally well to express all of the transgenes of this invention. However, one of skill in the art may make a selection among these, and other, expression control sequences without departing from the scope of this invention,Method of Treatment

[0071] In some embodiments, Angelman syndrome (AS) therapy with pharmaceutical compositions described herein is administered to subject(s) suffering from or susceptible to AS. In some embodiments, a subject has been determined to have genetic characteristic associated with a defect in a maternal UBE3A gene. In some embodiments, an AS-associated genetic characteristic is or comprises a maternal deletion. In some embodiments, an AS-associated genetic characteristic is or comprises uniparental disomy. In some embodiments, an AS-associated genetic characteristic is or comprises a L / BE3A mutation. In some embodiments, an AS-associated genetic characteristic is or comprises an imprinting defect.

[0072] In some embodiments, a subject has been determined to have one or more developmental history and / or laboratory finding characteristics that have been associated with AS such as, for example, one or more of: (i) normal prenatal and birth history with normal head circumference and absence of major birth defects; (ii) feeding difficulties as a neonate and / or as an infant; (iii) developmental delay evident by 6-12 months of age, sometimes associated with truncal hypotonus; (iv) unsteady limb movements and / or increased smiling; (v) delayed but forward progression of development (no loss of skills); (vi) normal metabolic, hematologic and chemical laboratory profiles; and / or (vii) structurally normal brain when assessed using MRI or CT (may have mild cortical atrophy or dysmyelination).

[0073] Alternatively or additionally, in some embodiments, a subject has been determined to display one or more clinical features that are consistently associated with AS such as, for example, one or more of: (i) developmental delay, functionally severe; (ii) movement or balance disorder, usually ataxia of gait and / or tremulous movement of limbs. In some embodiments, such movement disorder can be mild. In some embodiments, such movement disorder may not appear as frank ataxia but can be or involve, for example, forward lurching, unsteadiness, clumsiness, or quick, jerky motion; (iii) behavioral uniqueness: any combination of frequent laughter / smiling; apparent happy demeanor; easilyexcitable personality, often with uplifted hand-flapping or waving movements; hypermotoric behavior; and / or (iv) speech impairment, such as for example absent or minimal use of words; alternatively or additionally, receptive and non-verbal communication skills higher than verbal ones.

[0074] Alternatively or additionally, in some embodiments, a subject has been determined to display one or more clinical features that are frequently (e.g., about 80% of the time) associated with AS such as, for example, one or more of: (i) delayed, disproportionate growth in head circumference, usually resulting in microcephaly (<2 S.D. of normal OFC) by age 2 years. In some embodiments, microcephaly is more pronounced in those with 15q11.2-q13 deletions; (ii) seizures, onset usually < 3 yrs. of age ( seizure severity may decrease with age but regardless, in some embodiments, the seizure disorder lasts throughout adulthood); and / or (iii) has abnormal EEG, with a characteristic pattern, as is known in the art. In some embodiments, EEG abnormalities can occur in the first 2 years of life and can precede clinical features, and may not be correlated to clinical seizure events.

[0075] Alternatively or additionally, in some embodiments, a subject has been determined to display one or more clinical features that are sometimes (e.g., about 20-80% of the time) associated with AS such as, for example, one or more of: (i) flat occiput, (ii) occipital groove, (iii) protruding tongue, (iv) tongue thrusting; suck / swallowing disorders, (v) feeding problems and / or truncal hypotonia during infancy, (vi) prognathia, (vii) wide mouth, wide-spaced teeth, (viii) frequent drooling, (ix) excessive chewing / mouthing behaviors, (x) strabismus, (xi) hypopigmented skin, light hair and eye color, in some embodiments determined as compared to family, and typically seen only in deletion cases, (xii) hyperactive lower extremity deep tendon reflexes, (xiii) uplifted, flexed arm position especially during ambulation, (xiv) wide-based gait with pronated or valgus-positioned ankles, (xv) increased sensitivity to heat, (xvi) abnormal sleep wake cycles and diminished need for sleep, (xvii) attraction to / fascination with water; fascination with crinkly items such as certain papers and plastics, (xviii) abnormal food related behaviors, (xix) obesity (in the older child), (xx) scoliosis, and / or (xxi) constipation.

[0076] In some embodiments, a therapeutic regimen for the treatment of AS as described herein involves administration of one or more doses of a pharmaceutical composition that comprises and / or delivers an AAV vector encoding an ATF disclosed herein.

[0077] In some embodiments, a subject to whom a provided therapeutic regimen is administered has suffered or is suffering from one or more seizures and / or is receiving or has received anti-seizure therapy. For example. In some embodiments, a subject may have received or be receiving one or more of valproic acid, clonazepam, phenobarbital, topiramate, carbamazepine, lamotrigine, leveltiracetam, phenytoin, zonisamide,ethosuxaminde, gabapentin, felbatame, oxcarbazepine, tranxene, ACTS, nitrazapam, pregabalin, mysoline, vigabatrin, etc. In some particular embodiments, a subject may have received or be receiving one or more of valproic acid, clonazepam, phenobarbital, topiramate, carbamazepine, lamotrigine, and / or levetiracetam.

[0078] Alternatively or additionally, in some embodiments, a subject may have received or be receiving dietary therapy such as, for example, a ketogenic diet, low glycemic index therapy, etc.

[0079] Still further alternatively or additionally, in some embodiments, a subject may have received or be receiving treatment with a vagal nerve stimulator.

[0080] Also disclosed is the use of an AAV vector encoding an ATF disclosed herein for the manufacture of a medicament. In some embodiments the AAV vector encoding an ATF disclosed herein is for use in a combination treatment with another therapeutic agent. The therapeutic agent can for example be anticonvulsant medication.

[0081] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.EXAMPLESExample 1:

[0082] A programable Artificial Transcription Activator (ATF) was generated (“S1 K) that could silence expression of the Ube3a-ATS transcript in mice (FIG. 2A). This derepression allowed Ube3a to be expressed under its normal physiological promoter, avoiding concerns of overexpression. Treatment with the ATF protein rescued deficits in long-term potentiation in hippocampal slices (FIG. 2B), and treatment with AAV-S1K showed excellent rescue of motor deficits by open field assay total activity (FIG. 2C). In addition, AAV-S1 K administration was shown to restore UBE3A in mouse brain (FIGs. 3A and 3B).

[0083] A human-targeted zinc-finger protein termed “555” was created (FIG. 4A) and shown to repress human UBE3A antisense transcript (FIG. 4C). However, this protein is not suitable for use in humans and has significant off-target binding (FIG. 5B).

[0084] Therefore, the following series of human-targeted zinc-finger proteins were designed that target the SNURF promoter (FIG. 6, Table 1) using the Sp1c framework in which the recognition sites were replaced with the recognition models from Table 2 as shown in Table 3. As disclosed herein, the framework of other natural zinc finger protein could be used. For example, other suitable frameworks include Zif268 and F2. As the sequences of these zinc finger proteins are known in the art, also disclosed are zinc-fingers produced using those frameworks with the disclosed recognition helix regions.

[0085] Zinc-fingers using the Sp1c framework are provided below.

[0086] ZFN1 :EKPYKCPECGKSFSRSDDLVRHQRTHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPY KCPECGKSFSRSDDLVRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTG (SEQ ID NO:1).

[0087] ZFN4:EKPYKCPECGKSFSRSDDLVRHQRTHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPY KCPECGKSFSRSDDLVRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTGEKPYKCP ECGKSFSRSDDLVRHQRTHTG (SEQ ID NO:2)

[0088] ZFN6:EKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSRNDTLTEHQRTHTGEKPY KCPECGKSFSRSDKLTEHQRTHTGEKPYKCPECGKSFSRSDDLVRHQRTHTGEKPYKCPE CGKSFSTKNSLTEHQRTHTGEKPYKCPECGKSFSRTDTLRDHQRTHTG (SEQ ID NO:3).

[0089] ZFN9:EKPYKCPECGKSFSRSDHLTNHQRTHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPY KCPECGKSFSRNDTLTEHQRTHTGEKPYKCPECGKSFSRSDKLTEHQRTHTGEKPYKCPE CGKSFSRSDDLVRHQRTHTGEKPYKCPECGKSFSTKNSLTEHQRTHTG (SEQ ID NO:4).

[0090] ZFN12:EKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSRSDHLTNHQRTHTGEKPY KCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSRNDTLTEHQRTHTGEKPYKCPE CGKSFSRSDKLTEHQRTHTGEKPYKCPECGKSFSRSDDLVRHQRTHTG (SEQ ID NO:5).

[0091] ZFN14:EKPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSQSGDLRRHQRTHTGEKP YKCPECGKSFSRSDNLVRHQRTHTGEKPYKCPECGKSFSQSGDLRRHQRTHTGEKPYKC PECGKSFSDCRDLARHQRTHTGEKPYKCPECGKSFSRSDDLVRHQRTHTG (SEQ ID NO:6).

[0092] ZFN15:EKPYKCPECGKSFSTSGELVRHQRTHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPY KCPECGKSFSRSDHLTNHQRTHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPE CGKSFSRNDTLTEHQRTHTGEKPYKCPECGKSFSRSDKLTEHQRTHTG (SEQ ID NO:7).

[0093] ZFN17:EKPYKCPECGKSFSRSDHLTTHQRTHTGEKPYKCPECGKSFSDPGHLVRHQRTHTGEKPY KCPECGKSFSQSGDLRRHQRTHTGEKPYKCPECGKSFSRSDNLVRHQRTHTGEKPYKCP ECGKSFSQSGDLRRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTG (SEQ ID NO:8).

[0094] ZFN18:EKPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSTSGELVRHQRTHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSRSDHLTNHQRTHTGEKPYKCP ECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSRNDTLTEHQRTHTG (SEQ ID N0:9).

[0095] ZFN20:EKPYKCPECGKSFSHTGHLLEHQRTHTGEKPYKCPECGKSFSRSDHLTTHQRTHTGEKPY KCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSQSGDLRRHQRTHTGEKPYKCP ECGKSFSRSDNLVRHQRTHTGEKPYKCPECGKSFSQSGDLRRHQRTHTG (SEQ ID NO: 10).

[0096] ZFN21 :EKPYKCPECGKSFSRSDDLVRHQRTHTGEKPYKCPECGKSFSDPGHLVRHQRTHTGEKP YKCPECGKSFSTSGELVRHQRTHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCP ECGKSFSRSDHLTNHQRTHTGEKPYKCPECGKSFSRADNLTEHQRTHTG (SEQ ID NO:11).

[0097] ZFN23:EKPYKCPECGKSFSQSGDLRRHQRTHTGEKPYKCPECGKSFSHTGHLLEHQRTHTGEKP YKCPECGKSFSRSDHLTTHQRTHTGEKPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKC PECGKSFSQSGDLRRHQRTHTGEKPYKCPECGKSFSRSDNLVRHQRTHTG (SEQ ID NO:12).

[0098] ZFN24:EKPYKCPECGKSFSTSGNLTEHQRTHTGEKPYKCPECGKSFSRSDDLVRHQRTHTGEKPY KCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSTSGELVRHQRTHTGEKPYKCP ECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSRSDHLTNHQRTHTG (SEQ ID NO: 13).

[0099] ZFN27:EKPYKCPECGKSFSTSGELVRHQRTHTGEKPYKCPECGKSFSTSGNLTEHQRTHTGEKPY KCPECGKSFSRSDDLVRHQRTHTGEKPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKCP ECGKSFSTSGELVRHQRTHTGEKPYKCPECGKSFSRADNLTEHQRTHTG (SEQ ID NO:14).

[0100] ZFN28:EKPYKCPECGKSFSTKNSLTEHQRTHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPY KCPECGKSFSHTGHLLEHQRTHTGEKPYKCPECGKSFSRSDDLVRHQRTHTGEKPYKCPE CGKSFSTSGNLTEHQRTHTGEKPYKCPECGKSFSRSDNLVRHQRTHTG (SEQ ID NO: 15).

[0101] ZFN30:EKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSTSGELVRHQRTHTGEKPY KCPECGKSFSTSGNLTEHQRTHTGEKPYKCPECGKSFSRSDDLVRHQRTHTGEKPYKCPE CGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSTSGELVRHQRTHTG (SEQ ID NO:16).

[0102] ZFN31 :EKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSHTGHLLEHQRTHTGEKPYKCPECGKSFSRSDDLVRHQRTHTGEKPYKCPECGKSFSTSGNLTEHQRTHTGEKPYKCPE CGKSFSRSDNLVRHQRTHTGEKPYKCPECGKSFSTKNSLTEHQRTHTG (SEQ ID NO: 17).

[0103] ZFN33:EKPYKCPECGKSFSRSDDLVRHQRTHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPY KCPECGKSFSTSGELVRHQRTHTGEKPYKCPECGKSFSTSGNLTEHQRTHTGEKPYKCPE CGKSFSRSDDLVRHQRTHTGEKPYKCPECGKSFSDPGHLVRHQRTHTG (SEQ ID NO: 18).

[0104] ZFN36:EKPYKCPECGKSFSRSDKLVRHQRTHTGEKPYKCPECGKSFSRSDDLVRHQRTHTGEKP YKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSTSGELVRHQRTHTGEKPYKCP ECGKSFSTSGNLTEHQRTHTGEKPYKCPECGKSFSRSDDLVRHQRTHTG (SEQ ID NO: 19).

[0105] Of these, ZF36 was shown to silence UBE3A transcript in LnCap cells (FIG. 7B).

[0106] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0107] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.TH Docket No. 320317-2100TH Docket No. 320317-2100TH Docket No. 320317-2100

Claims

CLAIMS1. An artificial transcription factor (ATF) comprising a transcriptional repressor domain fused to a zinc-finger DNA binding domain, wherein the zinc-finger DNA binding domain comprises recognition helix regions that bind the target sequences:(1) F3: GCG, F4: CAG, F5: GCG, and F6: GCC;(2) F2: GCG, F3: CAG, F4: GCG, F5: GCC, and F6: GCG;(3) F1 : CAG, F2: CCG, F3: CGG, F4: GCG, F5: CCT, and F6: ACG;(4) F1 : AGG, F2: CAG, F3: CCG, F4: CGG, F5: GCG, and F6: CCT;(5) F1 : CAG, F2: AGG, F3: CAG, F4: CCG, F5: CGG, and F6: GCG;(6) F1 : GGC, F2: GCA, F3: GAG, F4: GCA, F5: GCC, and F6: GCG;(7) F1 : GCT, F2: CAG, F3: AGG, F4: CAG, F5: CCG, and F6: CGG;(8) F1 : TGG, F2: GGC, F3: GCA, F4: GAG, F5: GCA, and F6: GCC;(9) F1 : GGC, F2: GCT, F3: CAG, F4: AGG, F5: CAG, and F6: CCG;(10) F1 : GCA, F2: GAG, F3: GCA, F4: GGC, F5: TGG, and F6: CGC;(11) F1 : CAG, F2: AGG, F3: CAG, F4: GCT, F5: GGC, and F6: GCG;(12) F1 : GCA, F2: CGC, F3: TGG, F4: GGC, F5: GCA, and F6: GAG;(13) F1 : CAT, F2: GCG, F3: GGC, F4: GCT, F5: CAG, and F6: AGG;(14) F1 : GCT, F2: CAT, F3: GCG, F4: GGC, F5: GCT, and F6: CAG;(15) F1 : CCT, F2: CAG, F3: CGC, F4: GCG, F5: CAT, and F6: GAG;(16) F1 : CAG, F2: GCT, F3: CAT, F4: GCG, F5: GGC, and F6: GCT;(17) F1 : CAG, F2: CGC, F3: GCG, F4: CAT, F5: GAG, and F6: CCT;(18) F1 : GCG, F2: CAG, F3: GCT, F4: CAT, F5: GCG, and F6: GGC; or(19) F1 : GGG, F2: GCG, F3: CAG, F4: GCT, F5: CAT, and F6: GCG.

2. The ATF of claim 1 , wherein the zinc-finger DNA binding domain comprises the recognition helix regions as follows:(1) F3: RSDDLVR (SEQ ID NO:20), F4: RADNLTE (SEQ ID NO:21), F5: RSDDLVR (SEQ ID NO:20), and F6: DCRDLAR (SEQ ID NO:22);(2) F2: RSDDLVR (SEQ ID NQ:20), F3: RADNLTE (SEQ ID NO:21), F4: RSDDLVR (SEQ ID NQ:20), F5: DCRDLAR (SEQ ID NO:22), and F6: RSDDLVR (SEQ ID NQ:20);(3) F1 : RADNLTE (SEQ ID NO:21), F2: RNDTLTE (SEQ ID NO:23), F3: RSDKLTE (SEQ ID NO:24), F4: RSDDLVR (SEQ ID NQ:20), F5: TKNSLTE (SEQ ID NO:25), and F6: RTDTLRD (SEQ ID NO:26);(4) F1 : RSDHLTN (SEQ ID NO:27), F2: RADNLTE (SEQ ID N0:21), F3: RNDTLTE (SEQ ID NO:23), F4: RSDKLTE (SEQ ID NO:24), F5: RSDDLVR (SEQ ID NQ:20), and F6: TKNSLTE (SEQ ID NO:25);(5) F1 : RADNLTE (SEQ ID N0:21), F2: RSDHLTN (SEQ ID NO:27), F3: RADNLTE (SEQ ID N0:21), F4: RNDTLTE (SEQ ID NO:23), F5: RSDKLTE (SEQ ID NO:24), and F6: RSDDLVR (SEQ ID NQ:20);(6) F1 : DPGHLVR (SEQ ID NO:28), F2: QSGDLRR (SEQ ID NO:29), F3: RSDNLVR (SEQ ID NQ:30), F4: QSGDLRR (SEQ ID NO:29), F5: DCRDLAR (SEQ ID NO:22), and F6: RSDDLVR (SEQ ID NQ:20);(7) F1 : TSGELVR (SEQ ID N0:31), F2: RADNLTE (SEQ ID N0:21), F3: RSDHLTN (SEQ ID NO:27), F4: RADNLTE (SEQ ID N0:21), F5: RNDTLTE (SEQ ID NO:23), and F6: RSDKLTE (SEQ ID NO:24);(8) F1 : RSDHLTT (SEQ ID NO:32), F2: DPGHLVR (SEQ ID NO:28), F3: QSGDLRR (SEQ ID NO:29), F4: RSDNLVR (SEQ ID NQ:30), F5: QSGDLRR (SEQ ID NO:29), and F6: DCRDLAR (SEQ ID NO:22);(9) F1 : DPGHLVR (SEQ ID NO:28), F2: TSGELVR (SEQ ID N0:31), F3: RADNLTE (SEQ ID N0:21), F4: RSDHLTN (SEQ ID NO:27), F5: RADNLTE (SEQ ID N0:21), and F6: RNDTLTE (SEQ ID NO:23);(10) F1 : HTGHLLE (SEQ ID NO:33), F2: RSDHLTT (SEQ ID NO:32), F3: DPGHLVR (SEQ ID NO:28), F4: QSGDLRR (SEQ ID NO:29), F5: RSDNLVR (SEQ ID NQ:30), and F6: QSGDLRR (SEQ ID NO:29);(11) F1 : RSDDLVR (SEQ ID NQ:20), F2: DPGHLVR (SEQ ID NO:28), F3: TSGELVR (SEQ ID NO:31), F4: RADNLTE (SEQ ID NO:21), F5: RSDHLTN (SEQ ID NO:27), and F6: RADNLTE (SEQ ID NO:21);(12) F1 : QSGDLRR (SEQ ID NO:29), F2: HTGHLLE (SEQ ID NO:33), F3: RSDHLTT (SEQ ID NO:32), F4: DPGHLVR (SEQ ID NO:28), F5: QSGDLRR (SEQ ID NO:29), and F6: RSDNLVR (SEQ ID NQ:30);(13) F1 : TSGNLTE (SEQ ID NO:34), F2: RSDDLVR (SEQ ID NQ:20), F3: DPGHLVR (SEQ ID NO:28), F4: TSGELVR (SEQ ID NO:31), F5: RADNLTE (SEQ ID NO:21), and F6: RSDHLTN (SEQ ID NO:27);(14) F1 : TSGELVR (SEQ ID NO:31), F2: TSGNLTE (SEQ ID NO:34), F3: RSDDLVR (SEQ ID NQ:20), F4: DPGHLVR (SEQ ID NO:28), F5: TSGELVR (SEQ ID NO:31), and F6: RADNLTE (SEQ ID NO:21);(15) F1 : TKNSLTE (SEQ ID NO:25), F2: RADNLTE (SEQ ID N0:21), F3: HTGHLLE (SEQ ID NO:33), F4: RSDDLVR (SEQ ID NO:20), F5: TSGNLTE (SEQ ID NO:34), and F6: RSDNLVR (SEQ ID NQ:30);(16) F1 : RADNLTE (SEQ ID N0:21), F2: TSGELVR (SEQ ID N0:31), F3: TSGNLTE (SEQ ID NO:34), F4: RSDDLVR (SEQ ID NQ:20), F5: DPGHLVR (SEQ ID NO:28), and F6: TSGELVR (SEQ ID N0:31);(17) F1 : RADNLTE (SEQ ID N0:21), F2: HTGHLLE (SEQ ID NO:33), F3: RSDDLVR (SEQ ID NQ:20), F4: TSGNLTE (SEQ ID NO:34), F5: RSDNLVR (SEQ ID NQ:30), and F6: TKNSLTE (SEQ ID NO:25);(18) F1 : RSDDLVR (SEQ ID NO:20), F2: RADNLTE (SEQ ID N0:21), F3: TSGELVR (SEQ ID N0:31), F4: TSGNLTE (SEQ ID NO:34), F5: RSDDLVR (SEQ ID NQ:20), and F6: DPGHLVR (SEQ ID NO:28); or(19) F1 : RSDKLVR (SEQ ID NO:35), F2: RSDDLVR (SEQ ID NQ:20), F3: RADNLTE (SEQ ID NO:21), F4: TSGELVR (SEQ ID NO:31), F5: TSGNLTE (SEQ ID NO:34), and F6: RSDDLVR (SEQ ID NQ:20).

3. The ATF of claim 2, wherein the zinc-finger DNA binding domain is selected from the group consisting of SEQ ID NOs:1 to 19.

4. The ATF of any one of claims 1 to 3, wherein the transcriptional repressor domain is a Kruppel-associated box (KRAB) domain.

5. A polynucleotide encoding the ATF of any one of claims 1 to 4.

6. An adeno-associated virus (AAV) vector comprising the polynucleotide of claim 5 operably linked to an expression control sequence.

7. The AAV vector of claim 6, wherein the AAV vector is an AAV6 or AAV9 vector.

8. A pharmaceutical composition comprising one or more AAV vectors of claim 6 or 7.

9. A method of treating Angelman Syndrome in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition of claim 8.

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