Gene therapy for Angelman syndrome

Abstract

The present disclosure provides nucleic acids (including AAV expression cassettes), AAV vectors, and compositions for use in methods for treating and / or delaying the onset of diseases associated with mutations in genes such as UBE3A associated with Angelman syndrome. Also provided herein are methods for treating and / or delaying the onset of Angelman syndrome. TIFF2026506583000020.tif55170

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 483,894, filed February 8, 2023, which is incorporated herein by reference in its entirety for all purposes.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (STRD_028_00US_SeqList_ST26.xml; size: 58,915 bytes; created on February 6, 2024) are incorporated herein by reference in their entirety. [Background technology]

[0003] background Angelman syndrome is a genetic disorder characterized by developmental delay, intellectual disability, speech impairment, difficulty walking, frequent smiling or laughing, excitability, and difficulty falling asleep. Other symptoms of the syndrome include seizures, jerky movements, microcephaly, tongue protrusion, hand flapping, and scoliosis. Although developmental delays due to Angelman syndrome may first be noticeable around age 6 months, the clinical features of the syndrome are usually detectable around age 1 year or later.

[0004] Angelman syndrome is usually caused by the loss of function of the maternally inherited ubiquitin protein ligase E3A (UBE3A). The gene encoding UBE3A is located within the region of chromosome 15 known as 15q11-q13. Angelman syndrome has been associated with genetic errors, such as deletions or mutations of one or more nucleic acids in the UBE3A gene or segments of chromosome 15, uniparental disomy, imprinting abnormalities, or translocations, which often result in the maternal copy of the UBE3A gene being absent or not functioning properly.

[0005] Currently, there is no cure for Angelman syndrome. The standard of care for Angelman syndrome focuses on symptom management. Therefore, there is a need for disease-modifying therapeutic compositions and methods for treating Angelman syndrome. Summary of the Invention

[0006] overview The present disclosure provides a nucleic acid molecule comprising an adeno-associated virus (AAV) expression cassette, the AAV expression cassette comprising, from 5' to 3', the following: (i) a 5' AAV inverted terminal repeat (ITR); (ii) a promoter; (iii) a transgene associated with Angelman syndrome; and (iv) a 3' AAV ITR. In some embodiments, the promoter drives expression of a transgene associated with Angelman syndrome. In some embodiments, the promoter is capable of expressing the transgene in neuronal cells. In some embodiments, the promoter comprises a synapsin (SYN) promoter. In some embodiments, the SYN promoter comprises a nucleic acid sequence derived from: (i) a human SYN promoter; (ii) a chicken SYN promoter; (iii) a mouse SYN promoter; or (iv) any combination thereof. In some embodiments, the SYN promoter comprises a human SYN (hSYN) promoter.

[0007] In some embodiments, the hSYN promoter comprises the nucleic acid sequence SEQ ID NO: 3 or a sequence at least 90% identical thereto. In some embodiments, the transgene associated with Angelman syndrome encodes ubiquitin protein ligase E3A (UBE3A). In some embodiments, the transgene associated with Angelman syndrome encodes human UBE3A (hUBE3A). In some embodiments, the transgene associated with Angelman syndrome comprises a mutation capable of removing a predicted cryptic splice site. In some embodiments, the transgene associated with Angelman syndrome comprises the nucleic acid substitution G2556C relative to the nucleic acid sequence of the wild-type human UBE3A gene. In some embodiments, the transgene associated with Angelman syndrome comprises a nucleic acid sequence at least 90% identical to SEQ ID NO: 12, and the nucleic acid substitution G2556C relative to SEQ ID NO: 12. In some embodiments, the transgene associated with Angelman syndrome comprises a nucleic acid sequence at least 90% identical to SEQ ID NO: 5. In some embodiments, the transgene associated with Angelman syndrome comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 5 and the nucleic acid substitution G2556C relative to SEQ ID NO: 12.

[0008] In some embodiments, at least one of the 5'ITR and the 3'ITR is about 110 to about 160 nucleotides in length. In some embodiments, the 5'ITR is the same length as the 3'ITR. In some embodiments, the 5'ITR and the 3'ITR are each about 145 nucleotides in length. In some embodiments, the 5'ITR and the 3'ITR are each about 141 nucleotides in length. In some embodiments, at least one of the 5'ITR and the 3'ITR is isolated from or derived from the genome of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. In some embodiments, the 5'ITR and the 3'ITR are each isolated from or derived from the genome of AAV2. In some embodiments, the 5' ITR comprises the sequence of SEQ ID NO: 2 or SEQ ID NO: 9. In some embodiments, the 3' ITR comprises the sequence of SEQ ID NO: 8 or SEQ ID NO: 10.

[0009] In some embodiments, the AAV expression cassette comprises an intron. In some embodiments, the intron is derived from the human beta globin gene (hBGIN). In some embodiments, the intron comprises one or more of the following mutations relative to SEQ ID NO: 13: (i) a mutation at the 5' end to include an exon 2 splicing donor (AGG); (ii) a mutation at the 3' end to include an exon 3 splicing acceptor (CTC); and (iii) G74T and G205A. In some embodiments, the intron comprises the nucleic acid sequence of SEQ ID NO: 4 or a sequence at least 90% identical thereto.

[0010] In some embodiments, the AAV expression cassette comprises a polyadenylation signal. In some embodiments, the polyadenylation signal is a polyadenylation signal isolated from or derived from one or more of the following genes: simian virus 40 (SV40), rBG, α-globin, β-globin, human collagen, human growth hormone (hGH), polyomavirus, human growth hormone (hGH), or bovine growth hormone (bGH). In some embodiments, the AAV expression cassette comprises a bGH polyadenylation signal. In some embodiments, the bGH polyadenylation signal comprises the nucleic acid sequence of SEQ ID NO: 6 or a sequence at least 90% identical thereto.

[0011] In some embodiments, the AAV expression cassette comprises at least one stuffer sequence. In some embodiments, the at least one stuffer sequence comprises the nucleic acid sequence of SEQ ID NO: 7, or a sequence at least 90% identical thereto. In some embodiments, the AAV expression cassette comprises a Kozak sequence. In some embodiments, the Kozak sequence comprises the nucleic acid sequence of SEQ ID NO: 14, or a sequence at least 90% identical thereto; or the nucleic acid sequence acagccacc, or a sequence at least 90% identical thereto. In some embodiments, the AAV expression cassette comprises an enhancer.

[0012] In some embodiments, the AAV expression cassette comprises the nucleic acid sequence SEQ ID NO: 1, or a sequence at least 90% identical thereto. In some embodiments, the AAV expression cassette comprises the nucleic acid sequence SEQ ID NO: 11, or a sequence at least 90% identical thereto.

[0013] The present disclosure also provides a plasmid comprising any one of the nucleic acid molecules disclosed herein, and a cell comprising any one of the nucleic acid molecules disclosed herein or any one of the plasmids disclosed herein.The present disclosure further provides a method for producing a recombinant AAV vector, comprising contacting an AAV-producing cell with any one of the nucleic acid molecules disclosed herein or any one of the plasmids disclosed herein.The present disclosure also provides a recombinant AAV vector produced by any one of the methods for producing a recombinant AAV vector disclosed herein.

[0014] In some embodiments, the vector is of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. In some embodiments, the recombinant AAV vector is a single-stranded AAV (ssAAV). In some embodiments, the recombinant AAV vector is a self-complementary AAV (scAAV). In some embodiments, the AAV vector comprises capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. In some embodiments, the AAV vector comprises a capsid protein having one or more substitutions or mutations compared to a wild-type AAV capsid protein. In some embodiments, the AAV vector comprises a capsid protein comprising: (i) the amino acid sequence of SEQ ID NO: 15, or a sequence at least 90% identical thereto; or (ii) the amino acid sequence of SEQ ID NO: 16, or a sequence at least 90% identical thereto; or (iii) the amino acid sequence of SEQ ID NO: 17, or a sequence at least 90% identical thereto.

[0015] In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 15, or a sequence at least 90% identical thereto. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 16, or a sequence at least 90% identical thereto. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 17, or a sequence at least 90% identical thereto. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 17.

[0016] The present disclosure provides a composition comprising: (a) any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the cells disclosed herein, or any one of the recombinant AAV vectors disclosed herein; and (b) a pharmaceutically acceptable carrier. The present disclosure provides methods of: A method of expressing a transgene associated with Angelman syndrome in a tissue, comprising the step of contacting the tissue with any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the recombinant AAV vectors disclosed herein, or any one of the compositions disclosed herein, thereby expressing the transgene associated with Angelman syndrome in the tissue.

[0017] In some embodiments, the tissue comprises brain tissue. In some embodiments, the tissue comprises nerve cells. In some embodiments, the contacting step is performed in vitro, ex vivo, or in vivo. In some embodiments, the contacting step is performed in vivo in a subject in need thereof. In some embodiments, the contacting step comprises administering a therapeutically effective amount of a nucleic acid molecule, a plasmid, a recombinant AAV vector, or a composition to the subject. In some embodiments, the subject is suffering from or at risk of developing Angelman syndrome.

[0018] The present disclosure provides the following methods: A method for treating Angelman syndrome in a subject in need thereof, comprising the steps of: administering to the subject a therapeutically effective amount of any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the cells disclosed herein, any one of the recombinant AAV vectors disclosed herein, or any one of the compositions disclosed herein, thereby treating Angelman syndrome in the subject. In some embodiments, the subject has or is at risk of developing Angelman syndrome. In some embodiments, Angelman syndrome is associated with, is driven by, or is caused by a genetic mutation. In some embodiments, the genetic mutation comprises a mutation in the human UBE3A gene. In some embodiments, the genetic mutation comprises a mutation in chromosomal region 15q11-q13.

[0019] In some embodiments, the method includes reducing the severity of Angelman syndrome, delaying the onset or progression of Angelman syndrome, and / or eliminating the symptoms of Angelman syndrome. In some embodiments, the symptoms of Angelman syndrome include: (a) developmental delay, (b) intellectual disability, (c) speech impairment, (d) gait ataxia, (e) tremors in the hands and feet, (f) frequent laughter or smiling, (g) excitability, (h) microcephaly, (i) epileptic seizures, (j) sleep disorders, (k) tongue protrusion, (l) hand flapping, (m) scoliosis, or (n) any combination thereof. In some embodiments, the method includes prolonging the survival of a subject with Angelman syndrome compared to a control subject who has not been administered a therapeutically effective dose, or compared to the expected survival of the subject before administration of a therapeutically effective dose. In some embodiments, the subject is a human subject.

[0020] These and other aspects are described in more detail in the detailed description below. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 shows a schematic diagram of the AAV expression cassette engineered for expression of the human ubiquitin protein ligase E3A (hUBE3A) gene. [Figure 2] 1 is a graph showing hUBE3A mRNA expression levels in induced pluripotent stem cells (iPSCs) when either wild-type (WT) isogenic healthy iPSCs or mutant (MU) UBE3A− / + iPSCs were transduced with the cassettes indicated on the X-axis. [Figure 3] 1 is a graph showing the cell body cluster area of ​​WT and mutant (MU) UBE3A- / + iPSCs when transduced with each cassette or buffer as indicated in the figure legend. [Figure 4] 1 is a graph showing the cell body cluster area of ​​MU UBE3A− / + iPSCs 13 days after transduction with each cassette as indicated in the figure legend. [Figure 5] Graph showing vector copy number (VCN; Y-axis) in the tissues listed on the X-axis (forebrain, hindbrain, and left lateral liver) following administration of AAV particles containing control vehicle or AAV cassettes as indicated in the figure legend and Table A to WT or Ube3a- / + mice. [Figure 6] Graph showing the levels of UBE3A mRNA (Y-axis) in the tissues listed on the X-axis (forebrain, hindbrain, and left lateral liver) resulting from expression of the hUBE3A gene when WT or Ube3a- / + mice were administered AAV particles containing the control vehicle or AAV cassettes indicated in the figure legend and Table A. [Figure 7] Western blot showing UBE3A protein expression (dotted box) in forebrain tissue resulting from expression of the hUBE3A gene when WT or Ube3a- / + mice were administered AAV particles containing a control vehicle or an AAV cassette as indicated in the figure legend and Table A. [Figure 8] Western blot showing UBE3A protein expression (dotted box) in hindbrain tissue resulting from expression of the hUBE3A gene when WT or Ube3a- / + mice were administered AAV particles containing a control vehicle or an AAV cassette as indicated in the figure legend and Table A. [Figure 9] Graph showing quantification of UBE3A protein expression in forebrain, hindbrain, or left lateral liver tissue resulting from hUBE3A gene expression when AAV particles containing a control vehicle or AAV cassette as indicated in the figure legend and Table A were administered to WT or Ube3a- / + mice. [Figure 10-1] Figures 10A-10F are images from immunohistochemical analysis of UBE3A stained with anti-hUBE3A antibody in brain tissue obtained from WT or Ube3a- / + mice administered control vehicle or AAV particles containing the AAV cassettes indicated in the figure legends and Table A. [Figure 10-2] See description of Figure 10-1. [Figure 11]Zoomed-in images from immunohistochemical analysis of UBE3A using staining with anti-hUBE3A antibody are shown for brain tissue obtained from WT or Ube3a − / + mice upon administration of control vehicle or AAV particles containing the AAV cassettes indicated in the figure legend and Table A. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description The present disclosure provides nucleic acids (including AAV expression cassettes), AAV vectors, and compositions for use in methods for treating and / or delaying the onset of diseases associated with mutations in genes such as UBE3A associated with Angelman syndrome. Also provided herein are methods for treating and / or delaying the onset of Angelman syndrome.

[0023] definition The following terms are used in the description and appended claims.

[0024] The singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0025] Additionally, the term "about" as used herein when referring to a measurable value, e.g., a quantity such as the length of a polynucleotide or polypeptide sequence, a dose, a time, a temperature, etc., is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0026] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as no combinations ("or") when interpreted alternatively.

[0027] The term "wild-type" is a term understood by those skilled in the art and refers to the typical form of an organism, strain, gene, protein, or characteristic found in nature, as distinguished from mutant or variant forms. For example, a wild-type protein is the typical form of that protein found in nature.

[0028] The term "mutant protein" is a skilled artisan's term understood by those of skill in the art and refers to a protein that is distinguished from the wild-type form of that protein based on the presence of an amino acid modification, such as, for example, an amino acid substitution, insertion, and / or deletion. The term "mutant gene" is a skilled artisan's term understood by those of skill in the art and refers to a gene that is distinguished from the wild-type form of that gene based on the presence of a nucleic acid modification, such as, for example, a nucleic acid substitution, insertion, and / or deletion. In some embodiments, a mutant gene encodes a mutant protein.

[0029] A "nucleic acid" or "polynucleotide" is a sequence of nucleotide bases, e.g., RNA, DNA, or DNA-RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotides). In some embodiments, nucleic acids of the present disclosure are either single-stranded or double-stranded DNA sequences. Nucleic acids may be 1 to 1,000, 1,000 to 10,000, 10,000 to 100,000, 100,000 to 1 million, or over 1 million nucleotides in length. Nucleic acids generally contain phosphodiester bonds, but also include nucleic acid analogs that may have alternative backbones, including, for example, phosphoramide, phosphorothioate, phosphorodithioate, O-methyl phosphoramidite linkages, and peptide nucleic acid backbones and linkages. Other analogous nucleic acids include positive backbones, non-ionic backbones, and non-ribose backbones. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acid. These ribose-phosphate backbone modifications may facilitate the addition of labels or may increase the stability and half-life of such molecules in physiological environments. The nucleic acids of the present disclosure may be linear or circular (e.g., plasmids).

[0030] As used herein, the term "promoter" refers to one or more nucleic acid control sequences that direct the transcription of an operably linked nucleic acid. A promoter may include a nucleic acid sequence near the transcription start site, such as a TATA element. A promoter may also include a cis-acting polynucleotide sequence that can be bound by a transcription factor.

[0031] A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter or transcription factor binding site arrangement) and a second nucleic acid sequence, where the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0032] An "AAV expression cassette" is a nucleic acid that is packaged into a recombinant AAV vector and contains sequences encoding one or more transgenes. When the AAV vector is contacted with a target cell, the transgenes are expressed by the target cell.

[0033] As used herein, the terms "viral vector," "viral vector," or "gene delivery vector" refer to a viral particle that functions as a nucleic acid delivery vehicle, which contains a nucleic acid (e.g., an AAV expression cassette) packaged within the virion. Exemplary viral vectors of the present disclosure include adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, and retroviral vectors.

[0034] As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAV type rh32.33, AAV type rh8, AAV type rh10, AAV type rh74, AAV type hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAV now known or later discovered. See, e.g., Table 1.

[0035] Table 1. Adeno-associated virus serotypes TIFF2026506583000002.tif164160TIFF2026506583000003.tif245160TIFF2026506583000004.tif101160

[0036] The terms "viral producer cells," "viral producer cell lines," or "viral producer cell lines" refer to cells used to produce viral vectors. HEK293 cells and 293T cells are common viral producer cell lines. Table 2 below lists exemplary viral producer cell lines for various viral vectors.

[0037] Table 2: Exemplary virus-producing cell lines TIFF2026506583000005.tif48128

[0038] "HEK293" refers to a cell line originally derived from human embryonic kidney cells grown in tissue culture. The HEK293 cell line grows easily in culture and is commonly used for virus production. As used herein, "HEK293" can also refer to one or more variant HEK293 cell lines, i.e., cell lines derived from the original HEK293 cell line that additionally contain one or more genetic modifications. Many variant HEK293 lines have been developed and optimized for one or more specific applications. For example, the 293T cell line contains the SV40 large T antigen, which allows episomal replication of transfected plasmids containing the SV40 origin of replication, increasing expression of the desired gene product.

[0039] "Sf9" refers to an insect cell line that is a clonal isolate derived from the parent Spodoptera frugiperda cell line IPLB-Sf-21-AE. Sf9 cells can be grown serum-free and can be cultured either adherently or in suspension.

[0040] "Transfection reagent" refers to a composition that facilitates the transfer of nucleic acids into cells. Some transfection reagents commonly used in the art include nucleic acids and one or more lipids that bind to the cell surface (e.g., Lipofectamine™).

[0041] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are consistent throughout the window of alignment of components, e.g., nucleotides or amino acids. The "percent identity" of an aligned segment of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller, defined portion of the reference sequence. The "percent identity" is the percent identity multiplied by 100. The degree of identity (homology) between two sequences can be determined using computer programs and mathematical algorithms. The percentage identity can be calculated using the alignment program Clustal Omega, available at www.ebi.ac.uk / Tools / msa / clustalo, using default parameters. See Sievers et al., "Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega." (2011 October 11) Molecular systems biology 7:539.

[0042] As used herein, "treatment" or "treating," or "alleviating" or "ameliorating," are used interchangeably. These terms refer to an approach to obtaining a beneficial or desired result, including, but not limited to, a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit refers to any therapeutically relevant improvement or effect on one or more diseases, conditions, or symptoms under treatment. For a prophylactic benefit, the composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet manifested.

[0043] As used herein, the terms "subject," "individual," and "patient" are used interchangeably to refer to vertebrates, such as mammals. A mammal may be, for example, a mouse, rat, rabbit, cat, dog, pig, sheep, horse, non-human primate (e.g., cynomolgus monkey, chimpanzee), or human. Also encompassed are subject tissues, cells, or derivatives thereof, obtained in vivo or cultured in vitro. A human subject may be an adult, teenager, child (2-14 years), infant (1-24 months), or newborn (up to 1 month). In some embodiments, an adult is about 65 years of age or older, or an elderly person about 60 years of age or older. In some embodiments, a subject is a pregnant woman or a woman who wishes to become pregnant.

[0044] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to achieve a certain result, e.g., an amount of an agent sufficient to bring about a beneficial or desired outcome. A therapeutically effective amount may vary depending on one or more of the subject and disease state being treated, the subject's weight and age, the severity of the disease state, the method of administration, etc., and can be readily determined by one of ordinary skill in the art. The specific dose may vary depending on one or more of the particular agent selected, the administration regimen to be followed, whether or not it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system by which the agent is delivered.

[0045] As used herein, the term "gene therapy" refers to the process of introducing genetic material into cells to compensate for an abnormal gene or to make a therapeutic protein.

[0046] AAV expression cassette The present disclosure provides nucleic acid sequences comprising one or more adeno-associated virus (AAV) expression cassettes. In some embodiments, the AAV expression cassette comprises a 5' inverted repeat (ITR), a promoter, a transgene, and a 3' ITR. In some embodiments, the transgene is a gene associated with Angelman syndrome. In some embodiments, the AAV expression cassette comprises a Kozak sequence, a polyadenylation sequence, and / or a stuffer sequence.

[0047] In some embodiments, the AAV expression cassette comprises the nucleic acid sequence of SEQ ID NO: 1 or a sequence at least 70% identical thereto (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%, at least 99%, at least 99.5%, or 100% identical thereto, including all values ​​and subranges therebetween). In some embodiments, the AAV expression cassette comprises the nucleic acid sequence of SEQ ID NO: 1.

[0048] In some embodiments, the AAV expression cassette comprises the nucleic acid sequence of SEQ ID NO: 11 or a sequence at least 70% identical thereto (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%, at least 99%, at least 99.5%, or 100% identical thereto, including all values ​​and subranges therebetween). In some embodiments, the AAV expression cassette comprises the nucleic acid sequence of SEQ ID NO: 11.

[0049] (i) Terminal inverted repeats Inverted terminal repeats (ITRs) mediate AAV proviral integration and AAV DNA packaging into virions. ITRs are involved in various activities in the AAV life cycle. For example, ITRs, which can form hairpin structures, play a role in excision from the plasmid after transfection, vector genome replication, integration, and rescue from the host cell genome.

[0050] The AAV expression cassette of the present disclosure can include a 5' ITR and a 3' ITR. The ITR sequences can be about 110 to about 160 nucleotides in length, e.g., 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 160 nucleotides in length. In some embodiments, the ITR sequences can be about 141 nucleotides in length. In some embodiments, the 5' ITR is the same length as the 3' ITR. In some embodiments, the 5' ITR and the 3' ITR are different lengths. In some embodiments, the 5' ITR is longer than the 3' ITR, and in other embodiments, the 3' ITR is longer than the 5' ITR.

[0051] The ITRs can be isolated or derived from the genome of any AAV, for example, an AAV listed in Table 1. In some embodiments, at least one of the 5'ITR and 3'ITR is isolated or derived from the genome of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. In some embodiments, at least one of the 5'ITR and 3'ITR can be a wild-type or mutated ITR isolated or derived from a member of a parvovirus species other than AAV. For example, in some embodiments, the ITRs can be a wild-type or mutant ITR isolated or derived from a bocavirus or parvovirus B19.

[0052] In some embodiments, the ITRs comprise a modification to facilitate scAAV production. In some embodiments, the modification to facilitate scAAV production is the deletion of a terminal resolution sequence (TRS) from the ITR. In some embodiments, the 5' ITR is a wild-type ITR and the 3' ITR is a mutated ITR lacking a terminal resolution sequence. In some embodiments, the 3' ITR is a wild-type ITR and the 5' ITR is a mutated ITR lacking a terminal resolution sequence. In some embodiments, the terminal resolution sequence is absent from both the 5' ITR and the 3' ITR. In other embodiments, the modification to facilitate scAAV production is the replacement of the ITR with a different hairpin-forming sequence, e.g., a short hairpin (sh) RNA-forming sequence.

[0053] In some embodiments, the 5' ITR may comprise a sequence of SEQ ID NO: 2 or a sequence at least 70% identical thereto (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%, at least 99%, at least 99.5%, or 100% identical thereto, including all values ​​and subranges therebetween). In some embodiments, the 5' ITR may comprise a sequence of SEQ ID NO: 9 or a sequence at least 70% identical thereto (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%, at least 99%, at least 99.5%, or 100% identical thereto, including all values ​​and subranges therebetween).

[0054] In some embodiments, the 3' ITR may comprise the sequence of SEQ ID NO: 8 or a sequence at least 70% identical thereto (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%, at least 99%, at least 99.5%, or 100% identical thereto, including all values ​​and subranges therebetween). In some embodiments, the 3' ITR may comprise the sequence of SEQ ID NO: 10 or a sequence at least 70% identical thereto (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%, at least 99%, at least 99.5%, or 100% identical thereto, including all values ​​and subranges therebetween).

[0055] In some embodiments, the 5' ITR comprises the sequence of SEQ ID NO: 2 and the 3' ITR comprises the sequence of SEQ ID NO: 8. In some embodiments, the 5' ITR comprises the sequence of SEQ ID NO: 9 and the 3' ITR comprises the sequence of SEQ ID NO: 10.

[0056] In some embodiments, the AAV expression cassette contains one or more "surrogate" ITRs, i.e., non-ITR sequences that perform the same function as the ITRs. See, for example, Xie, J. et al., Mol. Ther., 25(6):1363-1374 (2017). In some embodiments, the ITRs in the AAV expression cassette are replaced with surrogate ITRs. In some embodiments, the surrogate ITRs include hairpin-forming sequences. In some embodiments, the surrogate ITRs are shRNA-forming sequences.

[0057] (ii) Promoter In some embodiments, the AAV expression cassettes described herein include a promoter. In some embodiments, the promoter is a synthetic promoter. In some embodiments, the promoter may include a nucleic acid sequence derived from an endogenous promoter and / or an endogenous enhancer.

[0058] In some embodiments, the promoter comprises a nucleic acid sequence derived from one or more promoters commonly used in the art for gene expression. For example, in some embodiments, the promoter further comprises a nucleic acid sequence derived from a CMV promoter, an SV40 early promoter, an SV40 late promoter, a metallothionein promoter, a mouse mammary tumor virus (MMTV) promoter, a Ras sarcoma virus (RSV) promoter, a polyhedrin promoter, a chicken beta-actin (CBA) promoter, a dihydrofolate reductase (DHFR) promoter, and a phosphoglycerol kinase (PGK) promoter. In some embodiments, the promoter comprises a nucleic acid sequence derived from a chicken beta-actin (CBA) promoter, an EF-1α promoter, or an EF-1α short promoter.

[0059] In some embodiments, the promoter can express a transgene in a neuronal cell. In some embodiments, the promoter is a cell-specific promoter, for example, a neuronal cell-specific promoter. As used herein, "cell-specific promoter" refers to a promoter that can express a transgene at a higher level in a specific cell (e.g., a neuronal cell) compared to a control cell (e.g., a non-neuronal cell). Thus, in some embodiments, the AAV expression cassette disclosed herein comprises a promoter that expresses a transgene in a neuronal cell at a higher level than the expression level of the transgene by the promoter in a non-neuronal cell. In some embodiments, a promoter expresses a transgene in a neuronal cell at a level that is at least about 1.2-fold (e.g., about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, or about 100-fold, including all values ​​and subranges therebetween) higher than the level of transgene expression from that promoter in a non-neuronal cell.

[0060] In some embodiments, the promoter may comprise a nucleic acid sequence derived from an endogenous promoter and / or enhancer, e.g., an endogenous promoter and / or enhancer of a gene that is expressed at a higher level in neuronal cells compared to non-neuronal cells.

[0061] In some embodiments, the promoter comprises a synapsin (SYN) promoter. In some embodiments, the SYN promoter comprises a nucleic acid sequence derived from: (i) a human SYN promoter; (ii) a chicken SYN promoter; (iii) a mouse SYN promoter; or (iv) any combination thereof. In some embodiments, the SYN promoter comprises a human SYN (hSYN) promoter.

[0062] In some embodiments, the promoter comprises the sequence of SEQ ID NO: 3 or a sequence at least 70% identical thereto (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%, at least 99%, at least 99.5%, or 100% identical thereto, including all values ​​and subranges therebetween).

[0063] In some embodiments, the AAV expression cassettes described herein further comprise an enhancer. The enhancer can be, for example, a CMV enhancer. In some embodiments, the enhancer comprises the sequence of SEQ ID NO: 18 or a sequence at least 70% identical thereto (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%, at least 99%, at least 99.5%, or 100% identical thereto, including all values ​​and subranges therebetween).

[0064] In some embodiments, the promoter is one of the following promoters: HMG-COA reductase promoter; sterol regulatory element 1 (SRE-1); phosphoenolpyruvate carboxykinase (PEPCK) promoter; human C-reactive protein (CRP) promoter; human glucokinase promoter; cholesterol 7-alpha hydroylase (CYP-7) promoter; β-galactosidase alpha-2,6 sialyltransferase promoter; insulin-like growth factor binding protein (IGFBP-1) promoter; aldolase B promoter; human transferrin promoter; collagen type I promoter; prostatic acid phosphatase (PAP) promoter; prostatic secretory protein 94 (PSP94) promoter; prostate-specific antigen complex promoter; human glandular kallikrein gene promoter (hgt-1); cardiomyocyte-specific enhancer-binding factor MEF-2; muscle creatine kinase promoter; pancreatitis-associated protein promoter (PA P); elastase 1 transcriptional enhancer; pancreas-specific amylase and elastase enhancer promoter; pancreatic cholesterol esterase gene promoter; uteroglobin promoter; cholesterol side-chain cleavage (SCC) promoter; gamma-gamma enolase (neuron-specific enolase, NSE) promoter; neurofilament heavy chain (NF-H) promoter; human CGL-1 / granzyme B promoter; terminal deoxytransferase (TdT), lambda 5, VpreB, and lck (lymphocyte-specific tyrosine protein kinase p561ck) promoters; human CD2 promoter and its 3' transcriptional enhancer; human NK and T cell-specific activation (NKG5) promoter; pp60c-src tyrosine kinase promoter; organ-specific neoantigen (OSN), molecular weight 40 kDa (p40) promoter; colon-specific antigen-P promoter; human α-lactalbumin promoter; phosphoenolpyruvate carboxykinase (PEPCK) promoter;The AAV expression cassette further comprises a nucleic acid sequence derived from any one or more of the following: a HER2 / neu promoter, a casein promoter, an IgG promoter, a choriofetal antigen promoter, an elastase promoter, a porphobilinogen deaminase promoter, an insulin promoter, a growth hormone factor promoter, a tyrosine hydroxylase promoter, an albumin promoter, an alpha-fetoprotein promoter, an acetylcholine receptor promoter, an alcohol dehydrogenase promoter, an alpha- or beta-globin promoter, a T-cell receptor promoter, an osteocalcin promoter, an IL-2 promoter, an IL-2 receptor promoter, a whey (WAP) promoter, and an MHC class II promoter. In some embodiments, the AAV expression cassette disclosed herein further comprises a nucleic acid sequence derived from any one or more of the promoters, enhancers, and / or other sequences described in U.S. Patent No. 8,708,948 B2, U.S. Patent No. 9,1385,96 B2, U.S. Patent No. 10,286,085 B2, and U.S. Patent No. US8,538,520 B2, the contents of each of which are incorporated herein by reference in their entirety. ;

[0065] (iii) Genes associated with Angelman syndrome As used herein, "Angelman syndrome-associated gene" refers to any gene in a subject with Angelman syndrome that can be targeted by gene therapy to alleviate at least one symptom of Angelman syndrome. In some embodiments, subjects with Angelman syndrome have reduced or undetectable levels of a protein encoded by a gene associated with Angelman syndrome. In some embodiments, the gene associated with Angelman syndrome encodes a protein that contributes to normal neuronal function.

[0066] In some embodiments, in a subject with Angelman syndrome, there are one or more mutations in a gene associated with Angelman syndrome (e.g., the UBE3A gene). In some embodiments, in a subject with Angelman syndrome, there is a loss of function of a gene associated with Angelman syndrome (e.g., the UBE3A gene). In some embodiments, one or more mutations in a gene associated with Angelman syndrome; or a reduction or loss of expression or function of a gene associated with Angelman syndrome, are associated with Angelman syndrome, promote Angelman syndrome, or cause Angelman syndrome. In some embodiments, a mutation in a gene associated with Angelman syndrome causes the maternal copy of the UBE3A gene to be absent or to function improperly.

[0067] The type of mutation in a gene (e.g., the UBE3A gene) associated with Angelman syndrome is not limited and can be an insertion, deletion, duplication, and / or substitution. In some embodiments, the mutation in the UBE3A gene is associated with, driven by, or caused by uniparental disomy. In some embodiments, the mutation in the UBE3A gene is associated with, driven by, or caused by an imprinting disorder. In some embodiments, the mutation in the UBE3A gene is associated with, driven by, or caused by one or more translocations. In some embodiments, the mutation in the UBE3A gene is any UBE3A mutation that has been identified in patients with Angelman syndrome. For example, the mutation in the UBE3A gene is selected from one or more UBE3A gene mutations described in Dagli AI, et al. Angelman Syndrome. 1998 Sep 15 GeneReviews, which is incorporated herein by reference for all purposes.

[0068] The present disclosure provides an AAV expression cassette comprising a gene associated with Angelman syndrome. In some embodiments, the AAV expression cassette comprises a gene associated with Angelman syndrome encoding a protein comprising a therapeutic (e.g., for medical or veterinary use) or immunogenic (e.g., for a vaccine) polypeptide. In some embodiments, the AAV expression cassette comprises a gene associated with Angelman syndrome in a mammal. In some embodiments, the AAV expression cassette comprises a gene associated with Angelman syndrome in a human. In some embodiments, the AAV expression cassette comprises a gene associated with Angelman syndrome that encodes ubiquitin protein ligase E3A (UBE3A).

[0069] In some embodiments, the transgene encodes human UBE3A. In some embodiments, the human UBE3A comprises an amino acid sequence at least 70% identical to SEQ ID NO: 19 (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%, at least 99%, at least 99.5%, or 100% identical, including all values ​​and subranges therebetween).

[0070] In some embodiments, the transgene associated with Angelman syndrome comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 12 (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%, at least 99%, at least 99.5%, or 100% identical, including all values ​​and subranges therebetween). In some embodiments, the transgene associated with Angelman syndrome comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 12.

[0071] In some embodiments, the transgene associated with Angelman syndrome comprises a mutation that can remove a predicted cryptic splice site. In some embodiments, the transgene associated with Angelman syndrome comprises the nucleic acid substitution G2556C relative to the nucleic acid sequence of wild-type human UBE3A. In some embodiments, the transgene associated with Angelman syndrome comprises the nucleic acid substitution G2556C relative to the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the transgene associated with Angelman syndrome comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 12 and the nucleic acid substitution G2556C relative to SEQ ID NO: 12.

[0072] In some embodiments, the human UBE3A comprises a nucleic acid sequence at least 70% identical to SEQ ID NO: 5 (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%, at least 99%, at least 99.5%, or 100% identical, including all values ​​and subranges therebetween). In some embodiments, the transgene associated with Angelman syndrome comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 5.

[0073] In some embodiments, the AAV expression cassette comprises a Kozak sequence. The Kozak sequence is a nucleic acid sequence that functions as a protein translation initiation site in many eukaryotic mRNA transcripts. In some embodiments, the Kozak sequence overlaps the start codon. In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 14 or the nucleic acid sequence acagccacc (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%, at least 99%, at least 99.5%, or 100% identity, including all values ​​and subranges therebetween). In some embodiments, the Kozak sequence comprises the nucleic acid sequence of SEQ ID NO: 14 or a sequence at least 90% identical thereto; or the nucleic acid sequence acagccacc or a sequence at least 90% identical thereto.

[0074] (iv) Polyadenylation (PolyA) signal A polyadenylation signal is a nucleotide sequence found in almost all mammalian genes that controls the addition of a stretch of approximately 200 adenosine residues (a poly(A) tail) to the 3' end of a gene transcript. The poly(A) tail contributes to mRNA stability; mRNA lacking a poly(A) tail is rapidly degraded. There is also evidence that the presence of a poly(A) tail positively affects the translatability of mRNA by influencing translation initiation.

[0075] In some embodiments, the AAV expression cassette of the present disclosure comprises a polyadenylation signal, which may be selected from the polyadenylation signals of simian virus 40 (SV40), rabbit beta globin (rBG), alpha-globin, beta-globin, human collagen, human growth hormone (hGH), polyoma virus, human growth hormone (hGH), and bovine growth hormone (bGH).

[0076] In some embodiments, the AAV expression cassette comprises a bGH polyadenylation signal. In some embodiments, the bGH polyadenylation signal comprises a nucleic acid sequence having at least 70% identity to the nucleic acid sequence of SEQ ID NO: 6 (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%, at least 99%, at least 99.5%, or 100% identical, including all values ​​and subranges therebetween). In some embodiments, the bGH polyadenylation signal comprises the nucleic acid sequence of SEQ ID NO: 6 or a sequence at least 90% identical thereto.

[0077] In some embodiments, the polyadenylation signal is an SV40 polyadenylation signal. In some embodiments, the polyadenylation signal is an rBG polyadenylation signal. In some embodiments, the polyadenylation signal comprises the sequence of SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the polyadenylation signal comprises a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 20 or SEQ ID NO: 21.

[0078] (v) Stuffer array AAV vectors typically accept DNA inserts with a defined size range, generally about 4 kb to about 5.2 kb, or slightly larger. Therefore, for shorter sequences, it may be necessary to include additional nucleic acids in the insert fragment to achieve the required length acceptable to the AAV vector. Thus, in some embodiments, the AAV expression cassettes of the present disclosure may include a stuffer sequence. The stuffer sequence can be, for example, a sequence of 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 75, 75 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 400, 400 to 500, 500 to 750, 750 to 1,000, 1,000 to 1,500, 1,500 to 2,000, 2,000 to 2,500, 2,500 to 3,000, 3,000 to 3,500, 3,500 to 4,000, 4,000 to 4,500, or 4,500 to 5,000 or more nucleotides in length. The stuffer sequence can be placed at any desired location within the cassette so as not to interfere with the function or activity of the vector.

[0079] In some embodiments, the AAV cassette comprises at least one stuffer sequence. In some embodiments, the stuffer sequence comprises a nucleic acid sequence having at least 70% identity to the nucleic acid sequence of SEQ ID NO: 7 (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%, at least 99%, at least 99.5%, or 100% identical, including all values ​​and subranges therebetween). In some embodiments, the stuffer sequence comprises the nucleic acid sequence of SEQ ID NO: 7 or a sequence at least 90% identical thereto. In some embodiments, the stuffer sequence comprises the nucleic acid sequence of SEQ ID NO: 7 or a portion thereof. In some embodiments, the stuffer sequence comprises a portion (e.g., a 500 nucleotide long portion) of the nucleic acid sequence of SEQ ID NO: 7 or a sequence at least 90% identical thereto.

[0080] (vi) intron sequences In some embodiments, the AAV expression cassettes of the present disclosure may include intron sequences, hi some embodiments, the inclusion of the intron sequences enhances expression compared to expression in the absence of the intron sequences.

[0081] In some embodiments, the intron sequence is a hybrid or chimeric sequence. In some embodiments, the intron sequence is isolated or derived from one or more intron sequences of SV40 (SV40IN), β-globin, chicken β-actin, minute virus of mice (MVM), factor IX, and / or human IgG (heavy chain or light chain). In some embodiments, the intron sequence is chimeric.

[0082] In some embodiments, the intron is derived from the human beta globin gene (hBGIN). In some embodiments, the intron comprises one or more of the following mutations relative to SEQ ID NO: 13: (i) a mutation at the 5' end to include an exon 2 splicing donor (AGG); (ii) a mutation at the 3' end to include an exon 3 splicing acceptor (CTC); and (iii) G74T and G205A. In some embodiments, the intron comprises a mutation at the 5' end to include an exon 2 splicing donor (AGG). In some embodiments, the intron comprises a mutation at the 3' end to include an exon 3 splicing acceptor (CTC). In some embodiments, the intron comprises the mutations G74T and / or G205A relative to SEQ ID NO: 13. In some embodiments, the intron contains the following mutations relative to SEQ ID NO: 13: (i) a mutation at the 5' end to include an exon 2 splicing donor (AGG); (ii) a mutation at the 3' end to include an exon 3 splicing acceptor (CTC); and (iii) G74T and G205A.

[0083] In some embodiments, the intron sequence comprises a nucleic acid sequence having at least 70% identity to the nucleic acid sequence of SEQ ID NO: 4 (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%, at least 99%, at least 99.5%, or 100% identical, including all values ​​and subranges therebetween). In some embodiments, the intron sequence comprises the sequence of SEQ ID NO: 4 or a sequence at least 90% identical thereto. In some embodiments, the intron sequence comprises the sequence of SEQ ID NO: 4.

[0084] Methods for producing AAV The AAV expression cassettes described herein can be incorporated into vectors (e.g., plasmids or bacmids) using standard molecular biology techniques. The present disclosure provides vectors comprising any one of the AAV expression cassettes described herein. The vectors (e.g., plasmids or bacmids) can further comprise one or more genetic elements used during AAV production, including, for example, the AAV rep and cap genes, and helper virus protein sequences.

[0085] AAV expression cassettes, and vectors (e.g., plasmids) containing the AAV expression cassettes described herein, can be used to produce recombinant AAV vectors.

[0086] The present disclosure provides a method for producing a recombinant AAV vector, comprising contacting an AAV producer cell (e.g., a HEK293 cell) with an AAV expression cassette or vector (e.g., a plasmid) of the present disclosure. The present disclosure further provides a cell comprising any one of the AAV expression cassettes or vectors disclosed herein. In some embodiments, the method further comprises contacting the AAV producer cell with, for example, one or more additional plasmids encoding the AAV rep and cap genes and helper virus protein sequences. In some embodiments, a method for producing a recombinant AAV vector is provided, comprising contacting an AAV producer cell (e.g., an insect cell such as an Sf9 cell) with at least one insect cell-compatible vector comprising an AAV expression cassette of the present disclosure. An "insect cell-compatible vector" is any compound or formulation (biological or chemical) that promotes transformation or transfection of insect cells with nucleic acids. In some embodiments, the insect cell-compatible vector is a baculovirus vector. In some embodiments, the method further comprises maintaining the insect cell under conditions such that AAV is produced.

[0087] The present disclosure provides a recombinant AAV vector produced using any one of the methods disclosed herein. The produced recombinant AAV vector can be of any serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. In some embodiments, the produced recombinant AAV vector can include one or more AAV capsid proteins with one or more amino acid modifications (e.g., substitutions and / or deletions) compared to native AAV capsids. For example, recombinant AAV vector can be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV and bovine AAV derived modified AAV vector.In some embodiments, recombinant AAV vector is single-stranded AAV (ssAAV).In some embodiments, recombinant AAV vector is self-complementary AAV (scAAV).

[0088] In some embodiments, the AAV vector comprises the capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV or bovine AAV. In some embodiments, the AAV vector comprises a capsid protein with one or more substitutions or mutations compared to wild-type AAV capsid protein. The recombinant AAV vector disclosed herein can be used to transduce a transgene sequence into a target cell, for example, by contacting the recombinant AAV vector with the target cell.

[0089] In some embodiments, the AAV vector comprises a capsid protein comprising an amino acid sequence at least 70% identical to SEQ ID NO: 15 (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%, at least 99%, at least 99.5%, or 100% identical, including all values ​​and subranges therebetween). In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 15 or a sequence at least 90% identical thereto. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 15.

[0090] In some embodiments, the AAV vector comprises a capsid protein comprising an amino acid sequence at least 70% identical to SEQ ID NO: 16 (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%, at least 99%, at least 99.5%, or 100% identical, including all values ​​and subranges therebetween). In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 16 or a sequence at least 90% identical thereto. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 16.

[0091] In some embodiments, the AAV vector comprises a capsid protein comprising an amino acid sequence at least 70% identical to SEQ ID NO: 17 (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%, at least 99%, at least 99.5%, or 100% identical, including all values ​​and subranges therebetween). In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 17 or a sequence at least 90% identical thereto. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 17.

[0092] In some embodiments, the AAV vector comprises a capsid protein comprising: (i) the amino acid sequence of SEQ ID NO: 15 or a sequence at least 90% identical thereto; or (ii) the amino acid sequence of SEQ ID NO: 16 or a sequence at least 90% identical thereto; or (iii) the amino acid sequence of SEQ ID NO: 17 or a sequence at least 90% identical thereto.

[0093] Methods of Expression and Treatment The present disclosure provides a composition comprising any one of the nucleic acids, AAV expression cassettes, plasmids, cells, or recombinant AAV vectors disclosed herein.In some embodiments, the compositions disclosed herein comprise at least one pharmaceutically acceptable carrier, excipient, and / or vehicle, such as a solvent, buffer solution, solution, dispersion medium, coating agent, antibacterial and antifungal agent, isotonicity agent, absorption delaying agent.In some embodiments, the pharmaceutically acceptable carrier, excipient, and / or vehicle may comprise saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. In some embodiments, pharmaceutically acceptable carriers, excipients, and / or vehicles include phosphate buffered saline, sterile saline, lactose, sucrose, calcium phosphate, dextran, agar, pectin, peanut oil, sesame oil, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), or suitable mixtures thereof. In some embodiments, the compositions disclosed herein further contain minor amounts of emulsifying or wetting agents, or pH buffering agents.

[0094] In some embodiments, the compositions disclosed herein further comprise other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, for example, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, or albumin. In some embodiments, the compositions disclosed herein may further comprise antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, or thimerosal; isotonic agents, for example, sugars or sodium chloride; and / or absorption delaying agents, for example, aluminum monostearate and gelatin.

[0095] The present disclosure provides the following methods: A method for expressing a transgene associated with Angelman syndrome in a cell, comprising the steps of: contacting the cell with any one of the nucleic acid molecules, plasmids, cells, recombinant AAV vectors, or compositions disclosed herein, thereby expressing a transgene associated with Angelman syndrome in the cell.

[0096] The present disclosure provides the following methods: A method for expressing a transgene associated with Angelman syndrome in a tissue, comprising: contacting the tissue with any one of the nucleic acid molecules, plasmids, cells, recombinant AAV vectors, or compositions disclosed herein, thereby expressing the transgene associated with Angelman syndrome in the tissue. In some embodiments, the tissue comprises at least one cell.

[0097] In some embodiments, the cell is a neuronal cell. In some embodiments, the cell is a dividing cell, such as a cultured cell in cell culture. In some embodiments, the cell is a non-dividing cell. In some embodiments, the Angelman syndrome-associated gene is delivered to the cell in vitro, e.g., to produce an Angelman syndrome-associated polypeptide in vitro or for ex vivo gene therapy.

[0098] In some embodiments, the contacting step is performed in vitro, ex vivo, or in vivo. In some embodiments, the contacting step is performed in vivo in a subject in need thereof. In some embodiments, the contacting step comprises administering a therapeutically effective amount of a nucleic acid molecule, a plasmid, a recombinant AAV vector, or a composition to the subject. In some embodiments, the subject is suffering from or at risk of developing Angelman syndrome.

[0099] The present disclosure provides the following methods: A method for treating Angelman syndrome in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of any one of the nucleic acid molecules, plasmids, cells, recombinant AAV vectors, or compositions disclosed herein, thereby treating Angelman syndrome in the subject. In some embodiments, the subject is suffering from or at risk of developing Angelman syndrome. In some embodiments, Angelman syndrome is associated with, promoted by, or caused by a genetic alteration. In some embodiments, the genetic alteration comprises one or more genetic alterations (e.g., one or more deletions, insertions, duplications, and / or substitutions) to the UBE3A gene compared to the wild-type UBE3A gene, and / or altered expression and / or activity of the UBE3A protein compared to the wild-type UBE3A protein. In some embodiments, the subject at risk of developing Angelman syndrome is a newborn identified as carrying a mutation in the UBE3A gene. In some embodiments, a gene associated with Angelman syndrome (eg, UBE3A) is targeted by gene therapy to increase its expression and / or function.

[0100] In some embodiments, the method comprises reducing the severity of Angelman syndrome, delaying the onset or progression of Angelman syndrome, and / or eliminating the symptoms of Angelman syndrome. In some embodiments, the symptoms of Angelman syndrome comprise: (a) developmental delay, (b) intellectual disability, (c) speech impairment, (d) gait ataxia, (e) tremors in the hands and feet, (f) frequent laughter or smiling, (g) excitability, (h) microcephaly, (i) epileptic seizures, (j) sleep disorders, (k) tongue protrusion, (l) hand flapping, (m) curvature of the spine, or (n) any combination thereof.

[0101] In some embodiments, the method comprises increasing the survival time of the subject relative to a control subject with Angelman syndrome who has not been administered a therapeutically effective dose. In some embodiments, the method comprises increasing the survival time of the subject relative to the expected survival time of the subject prior to administration of the therapeutically effective dose. In some embodiments, the method comprises increasing the survival time of the subject by a value ranging from about 3 months to about 50 years (e.g., about 6 months, about 1 year, about 5 years, about 10 years, about 15 years, about 20 years, about 25 years, about 30 years, about 35 years, about 40 years, about 45 years, about 50 years, including all values ​​and subranges therebetween) relative to (i) a control subject with Angelman syndrome who has not been administered a therapeutically effective dose, or (ii) the expected survival time of the subject prior to administration of the therapeutically effective dose.

[0102] The dosage of the recombinant AAV vector to be administered to a subject depends on the mode of administration, the disease or condition to be treated and / or prevented, the condition of the individual subject, the particular viral vector or capsid, the nucleic acid to be delivered, etc., and can be determined by routine methods. An exemplary dose to achieve a therapeutic effect is at least about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 , about 10 12 , about 10 13 , about 10 14 , about 10 15 transducing units, optionally about 10 8 ~about 10 13 where is the transducing unit, is the titer, .

[0103] In certain embodiments, more than one administration (e.g., two, three, four or more administrations) may be employed to achieve desired gene expression levels over various intervals, e.g., daily, weekly, monthly, yearly, etc.

[0104] In some embodiments, the subject is a human subject. Exemplary administration modes include oral, transmucosal, intrathecal, transdermal, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular (including administration to skeletal muscle, diaphragm, and / or cardiac muscle), intradermal, intrapleural, intracerebral, and intraarticular), intracerebroventricular (ICV) injection (e.g., bilateral ICV injection), intralymphatic, etc., and direct injection into tissue or organ (e.g., into the liver, skeletal muscle, cardiac muscle, diaphragm, or brain). Delivery to target tissue can also be achieved by delivering a depot containing a viral vector and / or capsid.

[0105] In some embodiments, the methods disclosed herein may include administering to a subject a therapeutically effective amount of any one of the nucleic acids, AAV expression cassettes, plasmids, cells, recombinant AAV vectors, or compositions disclosed herein in combination with one or more second therapies targeting Angelman syndrome. In some embodiments, the methods disclosed herein for treating at least one symptom of Angelman syndrome and / or delaying its onset in a subject may further include administering one or more second therapies targeting Angelman syndrome. As used herein, the term "administered in combination" is understood to mean that two (or more) different treatments are delivered to a subject such that the effects of the treatments on the patient overlap at some point during the subject's life with a disorder (e.g., Angelman syndrome). In certain embodiments, one treatment is still being delivered when the second treatment is initiated, thereby resulting in an overlap in administration. This may be referred to herein as "simultaneous" or "concurrent" delivery. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins, which may be referred to as "sequential" delivery.

[0106] In some embodiments, treatment is more effective because it is combined administration.For example, the second treatment is more effective, or the second treatment is less effective but has the same effect, or the second treatment is more effective than the first treatment when the second treatment is administered without the first treatment, or the same situation as the first treatment.The effect of two treatments can be partially additive, completely additive, or greater than additive (synergistic).

[0107] All articles, publications, and patents cited herein are incorporated by reference to the same extent as if each individual article, publication, or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. However, mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not an admission or in any way implied that they constitute valid prior art or form part of the general knowledge anywhere in the world, and should not be treated as such.

[0108] Unless the context indicates otherwise, it is specifically contemplated that the various features described herein can be used in any combination.

[0109] Unless otherwise defined, 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.

[0110] It is to be understood that the above description and the examples that follow are intended to illustrate, but not limit, the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. [Example]

[0111] The following examples are included herein for illustrative purposes only and are not intended to be limiting.

[0112] Example 1: Design of an adeno-associated virus (AAV) cassette encoding human UBE3A To test expression of the human ubiquitin protein ligase E3A (hUBE3A) gene and production of functional UBE3A protein, several AAV cassettes containing elements in various orders and combinations were generated (Figure 1). Each cassette shown in Figure 1 contains a hUBE3A gene containing the nucleic acid substitution G2556C. This mutant hUBE3A gene is referred to herein as hUBE3Av2 and comprises the nucleic acid sequence of SEQ ID NO: 5. Without being bound by theory, it is believed that the nucleic acid substitution G2556C acts as a silent mutation that removes a strongly predicted cryptic splice site.

[0113] Each cassette also contains one or more stuffer sequences, such as a human albumin (hAlb) stuffer sequence, and / or an intron (e.g., a human β-globin intron (hBGIN) or an SV40 intron), inserted upstream and / or downstream of the hUBE3Av2 gene, as shown in Figure 1. Without being bound by theory, it is believed that the inclusion of one or more stuffer sequences promotes transgene expression. Each cassette also contains a bovine growth hormone polyA signal (bGHpA; SEQ ID NO: 6), a 5' internal terminal repeat (ITR; SEQ ID NO: 2), and a 3' ITR (SEQ ID NO: 8). Expression of hUBE3Av2 is driven by the human synapsin (hSyn) promoter (SEQ ID NO: 3; used in cassettes P-T223 and P-T224) or the human putative endogenous promoter 1 (hP1 promoter containing the sequence of SEQ ID NO: 24, e.g., in cassettes P-T225 and P-T226). Without being bound by theory, it is believed that the hSyn promoter drives tissue-specific expression of the gene, e.g., in the brain. Each cassette contains approximately 4.7 kilobases (kB) of packaged genome.

[0114] The hBGIN (containing the nucleic acid sequence of SEQ ID NO: 4) used in these cassettes was mutated to include the hBGIN exon 2 splicing donor (AGG) and the hBGIN exon 3 splicing acceptor (CTC) at the 5' and 3' ends, respectively. Without being bound by theory, it is believed that these mutations in hBGIN enable efficient splicing. Additionally, hBGIN was mutated with G74T and G205A to remove a strongly predicted splice acceptor site. Without being bound by theory, it is believed that the G205A mutation in hBGIN prevents premature splicing.

[0115] Example 2: Expression of an AAV cassette encoding human UBE3A in induced pluripotent stem cells (iPSCs) The following AAV cassettes: P-T116, P-T178, P-T223, P-T224, P-T225, and P-T226, were packaged into AAV particles and then used to transduce iPSCs. The transduced iPSCs were transduced, lysed, and analyzed for mRNA expression by RT-qPCR to test for hUBE3Av2 expression. The arrangement of elements in the P-T116 cassette is: pTR141-hP1-SV40IN-hUBE3Av1-SV40pA, and the P-T116 cassette contains the nucleic acid sequence of SEQ ID NO: 22. The arrangement of elements in the P-T178 cassette is: pTR141-hSyn-SV40IN-hUBE3Av1-SV40pA. P-T116 and P-T178 differ only in the promoter, and the P-T178 cassette comprises the nucleic acid sequence of SEQ ID NO:23.

[0116] To measure gene expression, WT iPSCs and mutant (MU) UBE3A - / + We transduced each cassette into both WT and MU iPSCs, and measured hUBE3A mRNA expression by RT-qPCR (Figure 2). Of the six cassettes tested, the highest levels of hUBE3A mRNA were observed to be expressed from cassette P-T224 (containing the nucleic acid sequence of SEQ ID NO: 1) in both WT and MU iPSCs. These results indicate that the elements present in the P-T224 cassette, and their specific order and combination, promote efficient expression of the hUBE3A transgene from P-T224. These results also suggest that, because P-T224 is the only cassette with the mutated hUBE3A sequence disclosed herein, inclusion of this sequence results in higher hUBE3A mRNA expression from the P-T224 cassette.

[0117] To assess whether expression of AAV cassettes encoding hUBE3A could rescue the phenotype of cells with loss of hUBE3A function, we transfected each cassette into MU UBE3A - / + Transduced iPSCs and cell body cluster area (mm 2) was measured (Figures 3 and 4). The cell body cluster area is a measure of the area occupied by cell bodies (cell bodies are distinct from neurites) in a given well. Without being bound by theory, measurement of this marker is believed to be due to the transduced AAV-mediated UBE3A - / + This may represent a rescue of UBE3A function in iPSCs.

[0118] The cell body cluster area was measured using WT or MU UBE3A - / + Figure 4 shows that by day 13, transduction of the P-T223, P-T224, and P-T226 cassettes significantly outperformed transduction of (i) the other three cassettes, (ii) WT iPSCs, or (iii) MU UBE3A. - / + This shows that compared to iPSCs, it provided the lowest cell body cluster area (Figure 4).

[0119] Collectively, the above results demonstrate successful expression of hUBE3A mRNA from all six cassettes, while UBE3Av2 expression was highest from cassette P-T224 compared to the other five cassettes (P-T116, P-T178, P-T223, P-T225, and P-T226). The results also demonstrate that AAV-mediated expression of hUBE3A rescues loss-of-function of UBE3A, as evidenced by a reduction in soma cluster area.

[0120] Overall, the results indicate that the elements present in the P-T224 cassette, and their particular order and combination, promote efficient expression of the hUBE3A transgene from P-T224. Furthermore, these results demonstrate that AAV-mediated expression of the mutated versions of hUBE3A disclosed herein using the expression cassette elements disclosed herein can rescue the phenotype of cells deficient in UBE3A function.

[0121] Example 3: Administration of an AAV vector containing P-T224 inhibits UBE3A - / + Restoring wild-type UBE3A protein levels in mouse brains The P-T223, P-T224, P-T225, and P-T226 cassettes were tested for expression of UBE3A mRNA and protein in mice. Each of the AAV cassettes was packaged into an AAV capsid containing the AAV capsid protein (SEQ ID NO: 16), and the resulting AAV particles were then expressed as UBE3A. - / + P1 neonatal mice bearing the genotype, while a control vehicle was administered to wild-type (WT) or UBE3A - / + Mice with either genotype (heterozygous, HET) were administered 1.6x10 AAV particles via bilateral intracerebroventricular (ICV) injection on postnatal day 1 (PND1). 11 The dose was 2 μL per ventricle (4 μL total, flow rate: 1 μL / min) at a dose of 100 μg / vg. Three weeks after injection, brain (anterior and posterior) and liver tissue samples from the mice were assessed by molecular biological analysis and histology.

[0122] (Table A) TIFF2026506583000006.tif60158

[0123] Ube3a - / + The mouse model is a partial knockout (i.e., the paternal allele is not mutated). Without being bound by theory, it is believed that in neurons, the paternal imprinting and the mutated maternal allele result in a complete UBE3A knockout; however, this does not occur in other tissues (e.g., liver), where UBE3A protein is reduced but still detectable.

[0124] Administration of AAV particles containing each AAV cassette encoding UBE3A resulted in higher vector copy numbers (VCN) across the three tissue samples tested (forebrain, hindbrain, and left lateral liver) compared to administration of the WT and HET vehicle controls (Figure 5). These results demonstrate that all AAV particles tested can successfully transduce the tissues tested.

[0125] To assess UBE3A expression in these tissues, RT-qPCR was performed on tissue samples to measure UBE3A mRNA levels. Surprisingly, although AAV particles containing each tested AAV cassette transduced the tested tissues at similar levels, administration of AAV particles containing cassette P-T224 (containing hSyn and hBGIN; see Figure 5 and Table A) resulted in higher levels of UBE3A mRNA expression in both anterior and posterior brain tissue samples compared with P-T223, P-T225, and P-T226 (Figure 6). Without being bound by theory, we believe that the inclusion of the hBGIN intron contributes to the approximately 1-log higher mRNA levels upon expression from the P-T224 cassette. Expression of all tested AAV cassettes was lower in liver tissue compared to brain tissue, demonstrating tissue-specific expression of the cassettes.

[0126] To assess UBE3A protein expression in these tissues, we measured UBE3A protein levels in anterior brain tissue (Figure 7) and posterior brain tissue (Figure 8) by Western blot analysis. As shown in Figures 7 and 8, the levels of UBE3A protein expressed from AAV particles containing P-T224 in brain tissue were comparable to UBE3A levels in wild-type mice, indicating that the P-T224 cassette can effectively drive gene expression in neurons in brain tissue. Quantification of expressed UBE3A protein levels further confirmed that expression from the cassette P-T224 achieved restoration of UBE3A protein levels close to WT levels throughout both brain tissues (Figure 9). Similar levels of UBE3A were observed to be expressed in the liver of all the various mouse groups listed in Table A.

[0127] To further evaluate the expression and localization of UBE3A protein in brain tissue, we performed the following experiment. Sagittal brain sections were analyzed by immunohistochemistry (IHC) using anti-hUBE3A antibody staining, revealing that AAV-mediated expression of UBE3A from the P-T224 cassette produced detectable UBE3A protein in both the anterior and posterior brain regions (Figures 10A-10F). UBE3A protein concentrations were higher in the anterior brain compared to the posterior brain regions (Figure 11).

[0128] These results indicate that AAV-mediated expression of UBE3A from the P-T224 cassette results in superior UBE3A mRNA expression levels, UBE3A protein expression levels, and precise localization of UBE3A in target brain tissues in mice compared to other AAV cassettes tested. Without being bound by theory, it is believed that the unique combination of elements in the P-T224 cassette, such as the hSyn promoter and the mutated hBGIN intron sequence disclosed herein, combined with the mutated hUBE3Av2 gene disclosed herein, contributes to the effective expression of the target gene from the P-T224 cassette, which can facilitate successful rescue of one or more symptoms characteristic of Angelman syndrome.

[0129] array TIFF2026506583000007.tif237161TIFF2026506583000008.tif240161TIFF2026506583000009.tif225161TIFF202 6506583000010.tif247161TIFF2026506583000011.tif221161TIFF2026506583000012.tif245161TIFF20265065830 00013.tif234161TIFF2026506583000014.tif236161TIFF2026506583000015.tif245161TIFF2026506583000016.t if245161TIFF2026506583000017.tif245161TIFF2026506583000018.tif245161TIFF2026506583000019.tif169161

[0130] Numbered Aspects The following list of aspects is included herein for illustrative purposes only and is not intended to be exhaustive or limiting. Claimed subject matter is expressly not limited to the following aspects. Embodiment 1. A nucleic acid molecule comprising an adeno-associated virus (AAV) expression cassette, the AAV expression cassette comprising, from 5' to 3', the following: (i) 5'AAV inverted terminal repeat (ITR); (ii) promoters; (iii) a transgene associated with Angelman syndrome; and (iv) 3' AAV ITR A nucleic acid molecule comprising: Embodiment 2. The nucleic acid molecule of embodiment 1, wherein the promoter drives expression of a transgene associated with Angelman syndrome. Embodiment 3. The nucleic acid molecule of embodiment 1 or 2, wherein the promoter is capable of expressing the transgene in a neuronal cell. Aspect 4. The nucleic acid molecule according to any one of Aspects 1 to 3, wherein the promoter comprises a synapsin (SYN) promoter. Embodiment 5. The nucleic acid molecule of embodiment 4, wherein the SYN promoter comprises a nucleic acid sequence derived from: (i) a human SYN promoter; (ii) a chicken SYN promoter; (iii) a mouse SYN promoter; or (iv) any combination thereof. Embodiment 6 The nucleic acid molecule of embodiment 5, wherein the SYN promoter comprises a human SYN (hSYN) promoter. Embodiment 7. The nucleic acid molecule of any one of Embodiments 4 to 6, wherein the hSYN promoter comprises the nucleic acid sequence SEQ ID NO: 3, or a sequence at least 90% identical thereto. Aspect 8. The nucleic acid molecule of any one of Aspects 1 to 7, wherein the Angelman syndrome-associated transgene encodes ubiquitin protein ligase E3A (UBE3A). Aspect 9. The nucleic acid molecule of any one of Aspects 1 to 8, wherein the Angelman syndrome-associated transgene encodes human UBE3A (hUBE3A). Embodiment 10. The nucleic acid molecule of embodiment 8 or 9, wherein the Angelman syndrome-associated transgene comprises a mutation capable of removing a predicted cryptic splice site. Embodiment 11. The nucleic acid molecule of embodiment 10, wherein the Angelman syndrome-associated transgene comprises the nucleic acid substitution G2556C relative to the nucleic acid sequence of the wild-type human UBE3A gene. Embodiment 12. The nucleic acid molecule of embodiment 11, wherein the Angelman syndrome-associated transgene comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 12, and a nucleic acid substitution G2556C relative to SEQ ID NO: 12. Embodiment 13. The nucleic acid molecule of any one of Embodiments 1 to 12, wherein the Angelman syndrome-associated transgene comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 5. Embodiment 14. The nucleic acid molecule of any one of embodiments 1 to 13, wherein the Angelman syndrome-associated transgene comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 5 and a nucleic acid substitution G2556C relative to SEQ ID NO: 12. Aspect 15: The nucleic acid molecule according to any one of Aspects 1 to 14, wherein at least one of the 5' ITR and the 3' ITR is about 110 to about 160 nucleotides in length. Aspect 16: The nucleic acid molecule according to any one of Aspects 1 to 15, wherein the 5' ITR is the same length as the 3' ITR. Embodiment 17. The nucleic acid molecule of any one of embodiments 1 to 16, wherein the 5' ITR and the 3' ITR are each about 145 nucleotides in length. Embodiment 18. The nucleic acid molecule of any one of embodiments 1 to 16, wherein the 5' ITR and the 3' ITR are each about 141 nucleotides in length. Embodiment 19. The nucleic acid molecule of any one of embodiments 1 to 18, wherein at least one of the 5' ITR and the 3' ITR is isolated from or derived from the genome of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. Embodiment 20. The nucleic acid molecule of any one of embodiments 1 to 19, wherein the 5' ITR and the 3' ITR are each isolated from or derived from the genome of AAV2. Embodiment 21. The nucleic acid molecule of any one of embodiments 1 to 20, wherein the 5' ITR comprises the sequence of SEQ ID NO: 2 or SEQ ID NO: 9. Embodiment 22. The nucleic acid molecule of any one of embodiments 1 to 21, wherein the 3' ITR comprises the sequence of SEQ ID NO: 8 or SEQ ID NO: 10. Embodiment 23. The nucleic acid molecule of any one of embodiments 1 to 22, wherein the AAV expression cassette comprises an intron. Embodiment 24. The nucleic acid molecule of embodiment 23, wherein the intron is derived from the human β-globin gene (hBGIN). Embodiment 25. The nucleic acid molecule of embodiment 24, wherein the intron comprises one or more of the following mutations relative to SEQ ID NO: 13: (i) a mutation at the 5' end to include an exon 2 splicing donor (AGG); (ii) a mutation at the 3' end to include an exon 3 splicing acceptor (CTC); and (iii) G74T and G205A. Embodiment 26. The nucleic acid molecule of embodiment 24 or embodiment 25, wherein the intron comprises the nucleic acid sequence of SEQ ID NO: 4, or a sequence at least 90% identical thereto. Embodiment 27. The nucleic acid molecule of any one of embodiments 1 to 26, wherein the AAV expression cassette comprises a polyadenylation signal. Embodiment 28. The nucleic acid molecule of embodiment 27, wherein the polyadenylation signal is a polyadenylation signal isolated from or derived from one or more of the following genes: simian virus 40 (SV40), rBG, α-globin, β-globin, human collagen, human growth hormone (hGH), polyoma virus, human growth hormone (hGH), or bovine growth hormone (bGH). Embodiment 29 The nucleic acid molecule of embodiment 27 or embodiment 28, wherein the AAV expression cassette comprises a bGH polyadenylation signal. Embodiment 30. The nucleic acid molecule of embodiment 29, wherein the bGH polyadenylation signal comprises the nucleic acid sequence of SEQ ID NO: 6, or a sequence at least 90% identical thereto. Embodiment 31 The nucleic acid molecule of any one of embodiments 1 to 30, wherein the AAV expression cassette comprises at least one stuffer sequence. Embodiment 32. The nucleic acid molecule of embodiment 31, wherein the at least one stuffer sequence comprises the nucleic acid sequence of SEQ ID NO: 7, or a sequence at least 90% identical thereto. Embodiment 33. The nucleic acid molecule of any one of embodiments 1 to 32, wherein the AAV expression cassette comprises a Kozak sequence. Embodiment 34. The nucleic acid molecule of embodiment 33, wherein the Kozak sequence comprises the nucleic acid sequence of SEQ ID NO: 14, or a sequence at least 90% identical thereto; or the nucleic acid sequence acagccacc, or a sequence at least 90% identical thereto. Embodiment 35. The nucleic acid molecule of any one of embodiments 1 to 34, wherein the AAV expression cassette comprises an enhancer. Embodiment 36 The nucleic acid molecule of any one of embodiments 1 to 35, wherein the AAV expression cassette comprises the nucleic acid sequence SEQ ID NO: 1, or a sequence at least 90% identical thereto. Embodiment 37. The nucleic acid molecule of any one of embodiments 1 to 35, wherein the AAV expression cassette comprises the nucleic acid sequence SEQ ID NO: 11, or a sequence at least 90% identical thereto. Embodiment 38: A plasmid comprising the nucleic acid molecule of any one of embodiments 1 to 37. Aspect 39: The nucleic acid molecule according to any one of Aspects 1 to 37, or the plasmid according to Aspect 38. including, cells. Embodiment 40. A method for producing a recombinant AAV vector, comprising the step of contacting an AAV producer cell with a nucleic acid molecule according to any one of embodiments 1 to 37, or a plasmid according to embodiment 38. Embodiment 41. A recombinant AAV vector produced by the method of embodiment 40. Aspect 42. The recombinant AAV vector of Aspect 41, wherein the vector is of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, and bovine AAV. Embodiment 43. The recombinant AAV vector of embodiment 41 or embodiment 42, which is a single-stranded AAV (ssAAV). Embodiment 44. The recombinant AAV vector of embodiment 41 or embodiment 42, which is a self-complementary AAV (scAAV). Aspect 45. The recombinant AAV vector according to any one of Aspects 41 to 44, wherein the AAV vector comprises capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. Embodiment 46. A recombinant AAV vector according to any one of embodiments 41 to 45, wherein the AAV vector comprises a capsid protein having one or more substitutions or mutations compared to a wild-type AAV capsid protein. 47. The AAV vector comprises: (i)(i) the amino acid sequence of SEQ ID NO: 15 or a sequence at least 90% identical thereto; or (ii)(ii) the amino acid sequence of SEQ ID NO: 16 or a sequence at least 90% identical thereto; or (iii)(iii) the amino acid sequence of SEQ ID NO: 17 or a sequence at least 90% identical thereto 47. The recombinant AAV vector of any one of aspects 41 to 46, comprising a capsid protein comprising: Embodiment 48 The recombinant AAV vector of embodiment 47, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 15, or a sequence at least 90% identical thereto. Aspect 49. The recombinant AAV vector of Aspect 48, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 15. Embodiment 50 The recombinant AAV vector of embodiment 47, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 16, or a sequence at least 90% identical thereto. Embodiment 51. The recombinant AAV vector of embodiment 50, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 16. Embodiment 52 The recombinant AAV vector of embodiment 47, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 17, or a sequence at least 90% identical thereto. Aspect 53. The recombinant AAV vector of Aspect 52, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 17. Embodiment 54. A composition comprising: (a) a nucleic acid molecule according to any one of embodiments 1 to 37, a plasmid according to embodiment 38, a cell according to embodiment 39, or a recombinant AAV vector according to any one of embodiments 41 to 53; and (b) a pharmaceutically acceptable carrier. Embodiment 55. A method for expressing a transgene associated with Angelman syndrome in a tissue, the method comprising the step of contacting the tissue with a nucleic acid molecule according to any one of embodiments 1 to 37, a plasmid according to embodiment 38, a recombinant AAV vector according to any one of embodiments 41 to 53, or a composition according to embodiment 54, thereby expressing the transgene associated with Angelman syndrome in the tissue. Embodiment 56 The method of embodiment 55, wherein the tissue comprises brain tissue. Embodiment 57 The method of embodiment 55 or embodiment 56, wherein the tissue comprises nerve cells. Embodiment 58. The method of any one of embodiments 55 to 57, wherein the contacting step is carried out in vitro, ex vivo, or in vivo. Embodiment 59 The method of embodiment 58, wherein said contacting step is performed in vivo in a subject in need thereof. Embodiment 60 The method of embodiment 59, wherein the contacting step comprises administering to the subject a therapeutically effective amount of a nucleic acid molecule, a plasmid, a recombinant AAV vector, or a composition. Embodiment 61 The method of embodiment 59 or embodiment 60, wherein the subject has or is at risk of developing Angelman syndrome. Embodiment 62. A method for treating Angelman syndrome in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a nucleic acid molecule according to any one of embodiments 1 to 37, a plasmid according to embodiment 38, a cell according to embodiment 39, a recombinant AAV vector according to any one of embodiments 41 to 53, or a composition according to embodiment 54, thereby treating Angelman syndrome in the subject. Embodiment 63 The method of embodiment 62, wherein the subject has or is at risk of developing Angelman syndrome. Embodiment 64 The method of any one of embodiments 61 to 63, wherein the Angelman syndrome is associated with, driven by, or caused by a genetic mutation. 65. The method of claim 64, wherein the genetic mutation comprises a mutation in the human UBE3A gene. Embodiment 66 The method of embodiment 64, wherein the genetic mutation comprises a mutation in chromosomal region 15q11-q13. Embodiment 67. The method of any one of embodiments 61 to 66, comprising reducing the severity of Angelman syndrome, delaying the onset or progression of Angelman syndrome, and / or eliminating the symptoms of Angelman syndrome. Aspect 68. The method of Aspect 67, wherein the symptoms of Angelman syndrome include: (a) developmental delay, (b) intellectual disability, (c) speech impairment, (d) gait ataxia, (e) tremors in the limbs, (f) frequent laughter or smiling, (g) excitability, (h) microcephaly, (i) epileptic seizures, (j) sleep disorders, (k) tongue protrusion, (l) hand flapping, (m) curvature of the spine, or (n) a combination thereof. Embodiment 69. The method of any one of embodiments 61 to 68, comprising prolonging survival of the subject having Angelman syndrome compared to a control subject who has not been administered the therapeutically effective dose, or compared to expected survival of the subject prior to administration of the therapeutically effective dose. Embodiment 70. The method of any one of embodiments 60 to 69, wherein the subject is a human subject.

Claims

1. 1. A nucleic acid molecule comprising an adeno-associated virus (AAV) expression cassette, the AAV expression cassette comprising, from 5′ to 3′: (i) 5'AAV inverted terminal repeat (ITR); (ii) a promoter; (iii) a transgene associated with Angelman syndrome; and (iv) 3' AAV ITR The nucleic acid molecule comprising:

2. 2. The nucleic acid molecule of claim 1, wherein the promoter drives expression of the Angelman syndrome-associated transgene.

3. 3. The nucleic acid molecule of claim 1, wherein the promoter drives expression of the transgene in a neuronal cell.

4. The nucleic acid molecule of any one of claims 1 to 3, wherein the promoter comprises a synapsin (SYN) promoter.

5. 5. The nucleic acid molecule of claim 4, wherein the SYN promoter comprises a nucleic acid sequence derived from: (i) a human SYN promoter; (ii) a chicken SYN promoter; (iii) a mouse SYN promoter; or (iv) any combination thereof.

6. The nucleic acid molecule of claim 5, wherein the SYN promoter comprises a human SYN (hSYN) promoter.

7. 7. The nucleic acid molecule of any one of claims 4 to 6, wherein the hSYN promoter comprises the nucleic acid sequence SEQ ID NO: 3 or a sequence at least 90% identical thereto.

8. 8. The nucleic acid molecule of any one of claims 1 to 7, wherein the Angelman syndrome-associated transgene encodes ubiquitin protein ligase E3A (UBE3A).

9. 9. The nucleic acid molecule of any one of claims 1 to 8, wherein the Angelman syndrome-associated transgene encodes human UBE3A (hUBE3A).

10. 10. The nucleic acid molecule of claim 8 or 9, wherein the Angelman syndrome-associated transgene comprises a mutation capable of removing a predicted cryptic splice site.

11. 11. The nucleic acid molecule of claim 10, wherein the Angelman syndrome-associated transgene comprises a nucleic acid substitution G2556C relative to the nucleic acid sequence of the wild-type human UBE3A gene.

12. 12. The nucleic acid molecule of claim 11, wherein the Angelman syndrome-associated transgene comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 12 and a nucleic acid substitution G2556C relative to SEQ ID NO:

12.

13. 13. The nucleic acid molecule of any one of claims 1 to 12, wherein the Angelman syndrome-associated transgene comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:

5.

14. 14. The nucleic acid molecule of any one of claims 1 to 13, wherein the Angelman syndrome-associated transgene comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 5 and a nucleic acid substitution G2556C relative to SEQ ID NO:

12.

15. 15. The nucleic acid molecule of any one of claims 1 to 14, wherein at least one of the 5' ITR and the 3' ITR is about 110 to about 160 nucleotides in length.

16. 16. The nucleic acid molecule of any one of claims 1 to 15, wherein the 5' ITR is the same length as the 3' ITR.

17. 17. The nucleic acid molecule of any one of claims 1 to 16, wherein the 5' ITR and the 3' ITR are each about 145 nucleotides in length.

18. 17. The nucleic acid molecule of any one of claims 1 to 16, wherein the 5' ITR and the 3' ITR are each about 141 nucleotides in length.

19. 19. The nucleic acid molecule of any one of claims 1 to 18, wherein at least one of the 5' ITR and the 3' ITR is isolated from or derived from the genome of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV.

20. 20. The nucleic acid molecule of any one of claims 1 to 19, wherein the 5'ITR and the 3'ITR are each isolated or derived from the genome of AAV2.

21. 21. The nucleic acid molecule of any one of claims 1 to 20, wherein the 5'ITR comprises the sequence of SEQ ID NO: 2 or SEQ ID NO:

9.

22. 22. The nucleic acid molecule of any one of claims 1 to 21, wherein the 3' ITR comprises the sequence of SEQ ID NO: 8 or SEQ ID NO:

10.

23. 23. The nucleic acid molecule of any one of claims 1 to 22, wherein the AAV expression cassette comprises an intron.

24. 24. The nucleic acid molecule of claim 23, wherein the intron is derived from the human beta globin gene (hBGIN).

25. 25. The nucleic acid molecule of claim 24, wherein the intron comprises one or more of the following mutations relative to SEQ ID NO: 13: (i) a mutation at the 5' end to include an exon 2 splicing donor (AGG); (ii) a mutation at the 3' end to include an exon 3 splicing acceptor (CTC); and (iii) G74T and G205A.

26. 26. The nucleic acid molecule of claim 24 or claim 25, wherein the intron comprises the nucleic acid sequence of SEQ ID NO: 4 or a sequence at least 90% identical thereto.

27. 27. The nucleic acid molecule of any one of claims 1 to 26, wherein the AAV expression cassette comprises a polyadenylation signal.

28. 28. The nucleic acid molecule of Claim 27, wherein the polyadenylation signal is a polyadenylation signal isolated from or derived from one or more of the following genes: simian virus 40 (SV40), rBG, alpha-globin, beta-globin, human collagen, human growth hormone (hGH), polyoma virus, human growth hormone (hGH), or bovine growth hormone (bGH).

29. 29. The nucleic acid molecule of claim 27 or claim 28, wherein the AAV expression cassette comprises a bGH polyadenylation signal.

30. 30. The nucleic acid molecule of claim 29, wherein the bGH polyadenylation signal comprises the nucleic acid sequence of SEQ ID NO: 6 or a sequence at least 90% identical thereto.

31. 31. The nucleic acid molecule of any one of claims 1 to 30, wherein the AAV expression cassette comprises at least one stuffer sequence.

32. 32. The nucleic acid molecule of claim 31, wherein the at least one stuffer sequence comprises the nucleic acid sequence of SEQ ID NO: 7 or a sequence at least 90% identical thereto.

33. 33. The nucleic acid molecule of any one of claims 1 to 32, wherein the AAV expression cassette comprises a Kozak sequence.

34. 34. The nucleic acid molecule of claim 33, wherein the Kozak sequence comprises the nucleic acid sequence of SEQ ID NO: 14 or a sequence at least 90% identical thereto; or the nucleic acid sequence acagccacc or a sequence at least 90% identical thereto.

35. 35. The nucleic acid molecule of any one of claims 1 to 34, wherein the AAV expression cassette comprises an enhancer.

36. 36. The nucleic acid molecule of any one of claims 1 to 35, wherein the AAV expression cassette comprises the nucleic acid sequence SEQ ID NO: 1 or a sequence at least 90% identical thereto.

37. 36. The nucleic acid molecule of any one of claims 1 to 35, wherein the AAV expression cassette comprises the nucleic acid sequence SEQ ID NO: 11 or a sequence at least 90% identical thereto.

38. A plasmid comprising the nucleic acid molecule of any one of claims 1 to 37.

39. A nucleic acid molecule according to any one of claims 1 to 37 or a plasmid according to claim 38. including, cells.

40. 39. A method for producing a recombinant AAV vector, comprising contacting an AAV-producing cell with a nucleic acid molecule according to any one of claims 1 to 37, or a plasmid according to claim 38.

41. 41. A recombinant AAV vector produced by the method of claim 40.

42. 42. The recombinant AAV vector of claim 41, wherein the vector is of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, and bovine AAV.

43. 43. The recombinant AAV vector of claim 41 or claim 42, which is a single-stranded AAV (ssAAV).

44. 43. The recombinant AAV vector of claim 41 or claim 42, which is a self-complementary AAV (scAAV).

45. 42. The recombinant AAV vector of claim 41, wherein the AAV vector comprises capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV.

46. The recombinant AAV vector of claim 41, wherein the AAV vector comprises a capsid protein having one or more substitutions or mutations compared to a wild-type AAV capsid protein.

47. The AAV vector comprises: a. (i) the amino acid sequence of SEQ ID NO: 15 or a sequence at least 90% identical thereto; or b.(ii) the amino acid sequence of SEQ ID NO: 16 or a sequence at least 90% identical thereto; or c. (iii) the amino acid sequence of SEQ ID NO: 17 or a sequence at least 90% identical thereto 42. The recombinant AAV vector of claim 41, comprising a capsid protein comprising:

48. The recombinant AAV vector of claim 47, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 15 or a sequence at least 90% identical thereto.

49. The recombinant AAV vector of claim 48, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO:

15.

50. The recombinant AAV vector of claim 47, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 16 or a sequence at least 90% identical thereto.

51. The recombinant AAV vector of claim 50, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO:

16.

52. The recombinant AAV vector of claim 47, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 17 or a sequence at least 90% identical thereto.

53. The recombinant AAV vector of claim 52, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO:

17.

54. 52. A composition comprising: (a) a nucleic acid molecule according to any one of claims 1 to 37, a plasmid according to claim 38, a cell according to claim 39, or a recombinant AAV vector according to any one of claims 41 to 53; and (b) a pharmaceutically acceptable carrier.

55. 1. A method for expressing a transgene associated with Angelman syndrome in a tissue, the method comprising contacting the tissue with a nucleic acid molecule of any one of claims 1 to 37, a plasmid of claim 38, a recombinant AAV vector of any one of claims 41 to 53, or a composition of claim 54, thereby expressing the transgene associated with Angelman syndrome in the tissue.

56. 56. The method of claim 55, wherein the tissue comprises brain tissue.

57. 57. The method of claim 55 or claim 56, wherein the tissue comprises nerve cells.

58. 58. The method of any one of claims 55 to 57, wherein the contacting step is carried out in vitro, ex vivo, or in vivo.

59. 59. The method of claim 58, wherein said contacting step is performed in vivo in a subject in need thereof.

60. 60. The method of Claim 59, wherein the contacting step comprises administering to the subject a therapeutically effective amount of the nucleic acid molecule, the plasmid, the recombinant AAV vector, or the composition.

61. 61. The method of claim 59 or claim 60, wherein the subject has or is at risk of developing Angelman syndrome.

62. 1. A method for treating Angelman syndrome in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the nucleic acid molecule of any one of claims 1 to 37, the plasmid of claim 38, the cell of claim 39, the recombinant AAV vector of any one of claims 41 to 53, or the composition of claim 54, thereby treating Angelman syndrome in the subject.

63. 63. The method of claim 62, wherein the subject has or is at risk of developing Angelman syndrome.

64. 64. The method of any one of claims 61 to 63, wherein the Angelman syndrome is associated with, driven by, or caused by a genetic mutation.

65. 65. The method of claim 64, wherein the genetic mutation comprises a mutation in the human UBE3A gene.

66. 65. The method of claim 64, wherein the genetic mutation comprises a mutation in chromosomal region 15q11-q13.

67. 67. The method of any one of claims 61 to 66, comprising reducing the severity of Angelman syndrome, delaying the onset or progression of Angelman syndrome, and / or eliminating the symptoms of Angelman syndrome.

68. 68. The method of claim 67, wherein the symptoms of Angelman syndrome include: (a) developmental delay, (b) intellectual disability, (c) speech impairment, (d) gait ataxia, (e) tremors in the hands and feet, (f) frequent laughing or smiling, (g) excitability, (h) microcephaly, (i) seizures, (j) sleep disorders, (k) tongue protrusion, (l) hand flapping, (m) curvature of the spine, or (n) a combination thereof.

69. 69. The method of any one of Claims 61-68, comprising prolonging survival of the subject with Angelman syndrome compared to a control subject who has not been administered the therapeutically effective dose, or compared to expected survival of the subject prior to administration of the therapeutically effective dose.

70. 70. The method of any one of claims 60 to 69, wherein the subject is a human subject.

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