MODIFIED UBE3A GENE FOR A GENE THERAPY APPROACH FOR ANGELMAN SYNDROME

Abstract

A novel vector, composition and method of treating a neurological disorder characterized by deficient UBE3A is presented. The UBE3A gene, which encodes for E6-AP, a ubiquitin ligase, was found to be responsible for Angelman syndrome (AS). A unique feature of this gene is that it undergoes maternal imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal gene. A UBE3A protein construct was generated with additional sequences that allow the secretion from cells and uptake by neighboring neuronal cells. This UBE3A vector may be used in gene therapy to confer a functional E6-AP protein into the neurons and rescue disease pathology.

Claims

1. A UBE3A vector, comprising: a transcription initiation sequence; a UBE3A sequence disposed downstream of the transcription initiation sequence, or a homologous sequence; a secretion sequence disposed downstream of the transcription initiation sequence, or a homologous sequence; and a cell uptake sequence disposed downstream of the transcription initiation sequence, wherein the cell uptake sequence is penetrin, R6W3, pVEC, or a homologous sequence.

2. The vector of claim 1, wherein the transcription initiation sequence is a cytomegalovirus chicken-beta actin hybrid promoter or human ubiquitin c promoter.

3. The vector of claim 2, further comprising a cytomegalovirus immediate-early enhancer sequence disposed upstream of the transcription initiation sequence.

4. The vector of claim 1, further comprising a woodchuck hepatitis post-transcriptional regulatory element.

5. The vector of claim 1, further comprising a plasmid, wherein the plasmid is a recombinant adeno-associated virus serotype 2-based plasmid, and wherein the recombinant adeno-associated virus serotype 2-based plasmid lacks DNA integration elements.

6. The vector of claim 1, wherein the secretion sequence is disposed upstream of the UBE3A sequence.

7. The vector of claim 1, wherein the cell uptake sequence is disposed upstream of the UBE3A sequence and downstream of the secretion sequence.

8. The vector of claim 1, wherein the secretion sequence is insulin, GDNF, or IgK.

9. The vector of claim 1, wherein the UBE3A sequence is SEQ ID No:9, SEQ ID No:14, SEQ ID No: 15, SEQ ID No:17, a cDNA of SEQ ID No: 10, a cDNA of SEQ ID No: 16, or a homologous sequence.

10. A method of treating a neurodegenerative disorder, comprising the steps: administering the UBE3A vector of claim 1 to a patient suffering from a neurodegenerative disorder.

11. The method of claim 10, wherein the UBE3A vector is administered to the patient via injection in a brain of the patient.

12. A composition for use in treating a neurodegenerative disorder characterized by deficient UBE3A comprising: the UBE3A vector of claim 1; and a pharmaceutically acceptable carrier.

13. The composition of claim 12, wherein the neurodegenerative disorder is Angelman syndrome.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] For a fuller understanding of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which:

[0022] FIG. 1 is a dot blot of anti-GFP on media from HEK293 cells transfected with GFP clones containing signal peptides as indicated.

[0023] FIG. 2 is a map of the mouse UBE3A vector construct used in the present invention. Major genes are noted.

[0024] FIG. 3 is a Western blot showing secretion of E6-AP protein from plasmid transfected HEK293 cells. Culture media taken from control cells transfected cell culture media (cnt txn), media from Ube3a transfected cells (Ube3a txn); and media from untransfected cells (cnt untxn) were run on an acrylamide gel and anti-E6-AP antibody.

[0025] FIG. 4 is a graph of percentage area staining for E6-AP protein. Nontransgenic (Ntg) control mice shows the level of Ube3a expression in a normal mouse brain. Angelman syndrome mice (AS) show staining level in those mice (aka background staining). Injection of AAV4-STUb into the lateral ventricles of an AS mouse shows the level of E6-AP protein staining is increased as compared to an AS mouse. n=2.

[0026] FIG. 5 is a microscopic image of anti-E6-AP staining in a nontransgenic mouse. GFP (green fluorescent protein) is a cytosolic protein which is not secreted. This suggests that the Ube3a is being released from the ependymal cells and taken up in the parenchyma.

[0027] FIG. 6 is a microscopic image of anti-E6-AP staining in a nontransgenic mouse showing higher magnification images of the ventricular system (Lateral ventricle (LV), 3.sup.rd ventricle). GFP (green fluorescent protein) is a cytosolic protein which is not secreted. This suggests that the Ube3a is being released from the ependymal cells and taken up in the parenchyma.

[0028] FIG. 7 is a microscopic image of anti-E6-AP staining in an uninjected AS mouse.

[0029] FIG. 8 is a microscopic image of anti-E6-AP staining in an uninjected AS mouse. showing higher magnification images of the ventricular system (Lateral ventricle (LV), 3.sup.rd ventricle).

[0030] FIG. 9 is a microscopic image of anti-E6-AP staining in an AS mouse injected into the lateral ventricle with AAV4-STUb. Expression can be seen in the ependymal cells but staining is also observed in the parenchyma immediately adjacent to the ventricles (indicated with arrows). GFP (green fluorescent protein) is a cytosolic protein which is not secreted. This suggests that the Ube3a is being released from the ependymal cells and taken up in the parenchyma.

[0031] FIG. 10 is a microscopic image of anti-E6-AP staining in an AS mouse injected into the lateral ventricle with AAV4-STUb showing higher magnification images of the ventricular system (Lateral ventricle (LV), 3.sup.rd ventricle). Expression can be seen in the ependymal cells but staining is also observed in the parenchyma immediately adjacent to the ventricles (indicated with arrows). GFP (green fluorescent protein) is a cytosolic protein which is not secreted. This suggests that the Ube3a is being released from the ependymal cells and taken up in the parenchyma.

[0032] FIG. 11 is a microscopic image of anti-E6-AP staining in an AS mouse injected into the lateral ventricle with AAV4-STUb. Higher magnification images of the ventricular system (Lateral ventricle (LV)) of Ube3a expression after AAV4-STUb delivery. Expression can be seen in the ependymal cells but staining is also observed in the parenchyma immediately adjacent to the ventricles (indicated with arrows). GFP (green fluorescent protein) is a cytosolic protein which is not secreted. This suggests that the Ube3a is being released from the ependymal cells and taken up in the parenchyma.

[0033] FIG. 12 is a microscopic image of anti-E6-AP staining in an AS mouse injected into the lateral ventricle with AAV4-STUb. Higher magnification images of the ventricular system (3.sup.rd ventricle) of Ube3a expression after AAV4-STUb delivery. Expression can be seen in the ependymal cells but staining is also observed in the parenchyma immediately adjacent to the ventricles (indicated with arrows). GFP (green fluorescent protein) is a cytosolic protein which is not secreted. This suggests that the Ube3a is being released from the ependymal cells and taken up in the parenchyma.

[0034] FIG. 13 is a microscopic image of anti-E6-AP staining in a nontransgenic mouse transfected with GFP. Expression is not observed with the AAV4-GFP injections, which shows only transduction of the ependymal and choroid plexus cells. GFP (green fluorescent protein) is a cytosolic protein which is not secreted. This suggests that the Ube3a is being released from the ependymal cells and taken up in the parenchyma.

[0035] FIG. 14 is a microscopic image of anti-E6-AP staining in an AS mouse injected into the lateral ventricle with AAV4-STUb. Sagittal cross section of the brain of Ube3a expression after AAV4-STUb delivery.

[0036] FIG. 15 is a microscopic image of anti-E6-AP staining in an AS mouse injected into the lateral ventricle with AAV4-STUb. Sagittal cross section of the lateral ventricle (LV) in the brain showing Ube3a expression after AAV4-STUb delivery.

[0037] FIG. 16 is a microscopic image of anti-E6-AP staining in an AS mouse injected into the lateral ventricle with AAV4-STUb. Sagittal cross section of the 3.sup.rd ventricle (3V) in the brain showing Ube3a expression after AAV4-STUb delivery.

[0038] FIG. 17 is a microscopic image of anti-E6-AP staining in an AS mouse injected into the lateral ventricle with AAV4-STUb. Sagittal cross section of the interior horn of the lateral ventricle (LV) in the brain showing Ube3a expression after AAV4-STUb delivery.

[0039] FIG. 18 is a microscopic image of anti-E6-AP staining in an AS mouse injected into the lateral ventricle with AAV4-STUb. Sagittal cross section of the lateral ventricle (4V) in the brain showing Ube3a expression after AAV4-STUb delivery.

[0040] FIG. 19 is a microscopic image of anti-E6-AP staining in an AS mouse injected into the lateral ventricle with AAV4-STUb. Sagittal cross section of the fourth ventricle (LV) in the brain showing Ube3a expression after AAV4-STUb delivery.

[0041] FIG. 20 is a microscopic image of anti-E6-AP staining in an AS mouse injected into the lateral ventricle with AAV4-STUb. Sagittal cross section of the brain with higher magnification images of the ventricular system on the lateral ventricle (LV), and (C) 3.sup.rd ventricle (3V) of Ube3a expression after AAV4-STUb delivery.

[0042] FIG. 21 is a map of the human UBE3A vector construct used in the present invention. Major genes are noted.

[0043] FIG. 22 is a Western blot of HEK293 cell lysate transfected with hSTUb construct. The proteins were stained with anti-E6AP.

[0044] FIG. 23 is a dot blot with Anti-E6AP of HEK293 cells transfected with hSTUb construct with GDNF signal or insulin signal, shows insulin signal works better for expression and secretion.

[0045] FIG. 24 is a dot blot confirming insulin signal secretion using anti-HA tag antibody.

[0046] FIG. 25(A) is an illustration of the plasmid construct f for the GFP protein.

[0047] FIG. 25(B) is an image of gel electrophoresis result for the GFP protein.

[0048] FIG. 25(C) is a dot blot for different secretion signals using the GFP construct. The construct with the secretion signal was transduced into cell cultures and two clones obtained from each. The clones were cultured and media collected.

[0049] FIG. 26(A) is an illustration of the plasmid construct f for the E6-AP protein.

[0050] FIG. 26(B) is an image of gel electrophoresis result for the E6-AP protein.

[0051] FIG. 26(C) is a dot blot for different secretion signals using the E6-AP construct. The construct with the secretion signal was transduced into cell cultures and two clones obtained from each. The clones were cultured and media collected.

[0052] FIG. 27 is a Western blot showing the efficacy of cellular peptide uptake signals in inducing reuptake of the protein by neurons in transfected HEK293 cells. The cell lyses were added to new cell cultures of HEK293 cells and the concentration of E6-AP in these cells after incubation measured via Western blot.

[0053] FIG. 28(A) is a graph showing field excitatory post-synaptic potentials. A construct of Ube3A version 1 (hUbev1), a secretion signal, and the CPP TATk was transduced via an rAAV vector into mouse models of AS. Long-term potentiation of the murine brain was measured via electrophysiology post-mortem and compared to GFP-transfected AS model control mice and wild-type control mice.

[0054] FIG. 28(B) is a graph showing field excitatory post-synaptic potentials. A construct of Ube3A version 1 (hUbev1), a secretion signal, and the CPP TATk was transduced via an rAAV vector into mouse models of AS. Long-term potentiation of the murine brain was measured via electrophysiology post-mortem and compared to GFP-transfected AS model control mice and wild-type control mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0055] As used herein, the singular forms a, an and the include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a polypeptide includes a mixture of two or more polypeptides and the like.

[0056] 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference in their entirety to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0057] All numerical designations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied up or down by increments of 1.0 or 0.1, as appropriate. It is to be understood, even if it is not always explicitly stated that all numerical designations are preceded by the term about. It is also to be understood, even if it is not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art and can be substituted for the reagents explicitly stated herein.

[0058] As used herein, the term comprising is intended to mean that the products, compositions and methods include the referenced components or steps, but not excluding others. Consisting essentially of when used to define products, compositions and methods, shall mean excluding other components or steps of any essential significance. Thus, a composition consisting essentially of the recited components would not exclude trace contaminants and pharmaceutically acceptable carriers. Consisting of shall mean excluding more than trace elements of other components or steps.

[0059] As used in the specification and claims, the singular form a, an and the include plural references unless the context clearly dictates otherwise. For example, the term a vector includes a plurality of vectors.

[0060] As used herein, about means approximately or nearly and in the context of a numerical value or range set forth means 15% of the numerical.

[0061] Adeno-associated virus (AAV) vector as used herein refers to an adeno-associated virus vector that can be engineered for specific functionality in gene therapy. In some instances, the AAV can be a recombinant adeno-associated virus vector, denoted rAAV. While AAV4 is described for use herein, any suitable AAV known in the art can be used, including, but not limited to, AAV9, AAV5, AAV1 and AAV4.

[0062] Administration or administering is used to describe the process in which compounds of the present invention, alone or in combination with other compounds, are delivered to a patient. The composition may be administered in various ways including injection into the central nervous system including the brain, including but not limited to, intrastriatal, intrahippocampal, ventral tegmental area (VTA) injection, intracerebral, intracerebellar, intramedullary, intranigral, intraventricular, intracisternal, intracranial, intraparenchymal including spinal cord and brain stem; oral; parenteral (referring to intravenous and intraarterial and other appropriate parenteral routes); intrathecal; intramuscular; subcutaneous; rectal; and nasal, among others. Each of these conditions may be readily treated using other administration routes of compounds of the present invention to treat a disease or condition.

[0063] Treatment or treating as used herein refers to any of: the alleviation, amelioration, elimination and/or stabilization of a symptom, as well as delay in progression of a symptom of a particular disorder. For example, treatment of a neurodegenerative disease may include any one or more of the following: amelioration and/or elimination of one or more symptoms associated with the neurodegenerative disease, reduction of one or more symptoms of the neurodegenerative disease, stabilization of symptoms of the neurodegenerative disease, and delay in progression of one or more symptoms of the neurodegenerative disease.

[0064] Prevention or preventing as used herein refers to any of: halting the effects of the neurodegenerative disease, reducing the effects of the neurodegenerative disease, reducing the incidence of the neurodegenerative disease, reducing the development of the neurodegenerative disease, delaying the onset of symptoms of the neurodegenerative disease, increasing the time to onset of symptoms of the neurodegenerative disease, and reducing the risk of development of the neurodegenerative disease.

[0065] The pharmaceutical compositions of the subject invention can be formulated according to known methods for preparing pharmaceutically useful compositions. Furthermore, as used herein, the phrase pharmaceutically acceptable carrier means any of the standard pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier can include diluents, adjuvants, and vehicles, as well as implant carriers, and inert, non-toxic solid or liquid fillers, diluents, or encapsulating material that does not react with the active ingredients of the invention. Examples include, but are not limited to, phosphate buffered saline, physiological saline, water, and emulsions, such as oil/water emulsions. In some embodiments, the pharmaceutically acceptable carrier can be a blood brain permeabilizer including, but not limited to, mannitol. The carrier can be a solvent or dispersing medium containing, for example, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Formulations are described in a number of sources that are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Sciences (Martin E W [1995] Easton Pa., Mack Publishing Company, 19.sup.th ed.) describes formulations which can be used in connection with the subject invention.

[0066] As used herein animal means a multicellular, eukaryotic organism classified in the kingdom Animalia or Metazoa. The term includes, but is not limited to, mammals. Nonlimiting examples include rodents, mammals, aquatic mammals, domestic animals such as dogs and cats, farm animals such as sheep, pigs, cows and horses, and humans. Wherein the terms animal or the plural animals are used, it is contemplated that it also applies to any animals.

[0067] As used herein the phrase conservative substitution refers to substitution of amino acids with other amino acids having similar properties (e.g. acidic, basic, positively or negatively charged, polar or non-polar). The following six groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0068] As used herein conservative mutation, refers to a substitution of a nucleotide for one which results in no alteration in the encoding for an amino acid, i.e. a change to a redundant sequence in the degenerate codons, or a substitution that results in a conservative substitution. An example of codon redundancy is seen in Tables 1 and 2.

TABLE-US-00001 TABLE1 AminoAcids(Category-Based)and TripletCodeandRedundant CorrespondingEncodedAminoAcids (FunctionalGroupCategory-Based) Nonpolar,aliphatic Gly G GGT GGC GGA GGG Ala A GCT GCC GCA GCG Val V GTT GTC GTA GTG Leu L TTA TTG CTT CTC CTA CTG Met M ATG Ile I ATT ATC ATA Aromatic Phe F TTT TTC Tyr Y TAT TAC Trp W TGG Negativecharge Asp D GAT GAC Glu E GAA GAG Polar,uncharged Ser S AGT AGC TCT TCC TCA TCG Thr T ACT ACC ACA ACG Cys C TGT TGC Pro P CCT CCC CCA CCG Asn N AAT AAC Gln Q CAA CAG Positivecharge Lys K AAA AAG His H CAT CAC Arg R CGT CGC CGA CGG AGA AGG OTHER stop TTA TAG TGA

TABLE-US-00002 TABLE2 RedundantTripletCodeandCorresponding EncodedAminoAcids. U C A G U UUU Phe UCU Ser UAU Tyr UGU Cys UUC Phe UCC Ser UAC Tyr UGC Cys UUA Leu UCA Ser UAA END UGA END UUG Leu UCG Ser UAG END UGG Trp C CUU Leu CCU Pro CAU His CGU Arg CUC Leu CCC Pro CAC His CGC Arg CUA Leu CCA Pro CAA Gln CGA Arg CUG Leu CCG Pro CAG Gln CGG Arg A AUU Ile ACU Thr AAU Asn AGU Ser AUC Ile ACC Thr AAC Asn AGC Ser AUA Ile ACA Thr AAA Lys AGA Arg AUG Met ACG The AAG Lys AGG Arg G GUU Val GCU Ala GAU Asp GGU Gly GUC Val GCC Ala GAC Asp GGC Gly GUA Val GCA Ala GAA Glu GGA Gly GUG Val GCG Ala GAG Glu GGG Gly
Thus, according to Table 2, conservative mutations to the codon UUA include UUG, CUU, CUC, CUA, and CUG.

[0069] As used herein, the term homologous means a nucleotide sequence possessing at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, and even more preferably at least 98% sequence identity to the target sequence. Variations in the nucleotide sequence can be conservative mutations in the nucleotide sequence, i.e. mutations in the triplet code that encode for the same amino acid as seen in the Table 2.

[0070] As used herein, the term therapeutically effective amount refers to that amount of a therapy (e.g., a therapeutic agent or vector) sufficient to result in the amelioration of Angelman syndrome or other UBE3A-related disorder or one or more symptoms thereof, prevent advancement of Angelman syndrome or other UBE3A-related disorder, or cause regression of Angelman syndrome or other UBE3A-related disorder. In accordance with the present invention, a suitable single dose size is a dose that is capable of preventing or alleviating (reducing or eliminating) a symptom in a patient when administered one or more times over a suitable time period. One of skill in the art can readily determine appropriate single dose sizes for systemic administration based on the size of a mammal and the route of administration.

[0071] The dosing of compounds and compositions of the present invention to obtain a therapeutic or prophylactic effect is determined by the circumstances of the patient, as known in the art. The dosing of a patient herein may be accomplished through individual or unit doses of the compounds or compositions herein or by a combined or prepackaged or pre-formulated dose of a compounds or compositions. An average 40 g mouse has a brain weighing 0.416 g, and a 160 g mouse has a brain weighing 1.02 g, a 250 g mouse has a brain weighing 1.802 g. An average human brain weighs 1508 g, which can be used to direct the amount of therapeutic needed or useful to accomplish the treatment described herein.

[0072] Nonlimiting examples of dosages include, but are not limited to: 5.5510.sup.11 genomes/g brain mass, 5.7510.sup.11 genomes/g brain mass, 5.810.sup.11 genomes/g brain mass, 5.910.sup.11 genomes/g brain mass, 6.010.sup.11 genomes/g brain mass, 6.110.sup.11 genomes/g brain mass, 6.210.sup.11 genomes/g brain mass, 6.310.sup.11 genomes/g brain mass, 6.410.sup.11 genomes/g brain mass, 6.510.sup.11 genomes/g brain mass, 6.6.10.sup.11 genomes/g brain mass, 6.710.sup.11 genomes/g brain mass, 6.810.sup.11 genomes/g brain mass, 6.9.10.sup.11 genomes/g brain mass, 7.010.sup.11 genomes/g brain mass, 7.110.sup.11 genomes/g brain mass, 7.210.sup.11 genomes/g brain mass, 7.310.sup.11 genomes/g brain mass, 7.410.sup.11 genomes/g brain mass, 7.510.sup.11 genomes/g brain mass, 7.610.sup.11 genomes/g brain mass, 7.710.sup.11 genomes/g brain mass, 7.810.sup.11 genomes/g brain mass, 7.910.sup.11 genomes/g brain mass, 8.010.sup.11 genomes/g brain mass, 8.110.sup.11 genomes/g brain mass, 8.210.sup.11 genomes/g brain mass, 8.310.sup.11 genomes/g brain mass, 8.410.sup.11 genomes/g brain mass, 8.510.sup.11 genomes/g brain mass, 8.610.sup.11 genomes/g brain mass, 8.710.sup.11 genomes/g brain mass, 8.810.sup.11 genomes/g brain mass, 8.910.sup.11 genomes/g brain mass, 9.010.sup.11 genomes/g brain mass, 9.110.sup.11 genomes/g brain mass, 9.210.sup.11 genomes/g brain mass, 9.310.sup.11 genomes/g brain mass, 9.410.sup.11 genomes/g brain mass, 9.510.sup.11 genomes/g brain mass, 9.610.sup.11 genomes/g brain mass, 9.710.sup.11 genomes/g brain mass, 9.8010.sup.11 genomes/g brain mass, 1.010.sup.12 genomes/g brain mass, 1.110.sup.12 genomes/g brain mass, 1.210.sup.12 genomes/g brain mass, 1.310.sup.12 genomes/g brain mass, 1.410.sup.12 genomes/g brain mass, 1.510.sup.12 genomes/g brain mass, 1.610.sup.12 genomes/g brain mass, 1.710.sup.12 genomes/g brain mass, 1.810.sup.12 genomes/g brain mass, 1.910.sup.12 genomes/g brain mass, 2.010.sup.12 genomes/g brain mass, 2.110.sup.12 genomes/g brain mass, 2.210.sup.12 genomes/g brain mass, 2.310.sup.12 genomes/g brain mass, 2.4010.sup.12 genomes/g brain mass, 2.510.sup.12 genomes/g brain mass, 2.610.sup.12 genomes/g brain mass, 2.710.sup.12 genomes/g brain mass, 2.7510.sup.12 genomes/g brain mass, 2.810.sup.12 genomes/g brain mass, or 2.8610.sup.12 genomes/g brain mass.

[0073] The compositions used in the present invention may be administered individually, or in combination with or concurrently with one or more other therapeutics for neurodegenerative disorders, specifically UBE3A deficient disorders.

[0074] As used herein patient is used to describe an animal, preferably a human, to whom treatment is administered, including prophylactic treatment with the compositions of the present invention.

[0075] Neurodegenerative disorder or neurodegenerative disease as used herein refers to any abnormal physical or mental behavior or experience where the death or dysfunction of neuronal cells is involved in the etiology of the disorder. Further, the term neurodegenerative disease as used herein describes neurodegenerative diseases which are associated with UBE3A deficiencies. Exemplary neurodegenerative diseases include Angelman's Syndrome, Huntington's disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, autistic spectrum disorders, epilepsy, multiple sclerosis, Prader-Willi syndrome, Fragile X syndrome, Rett syndrome and Pick's Disease.

[0076] UBE3A deficiency as used herein refers to a mutation or deletion in the UBE3A gene.

[0077] The term normal or control as used herein refers to a sample or cells or patient which are assessed as not having Angelman syndrome or any other neurodegenerative disease or any other UBE3A deficient neurological disorder.

[0078] Generally, a UBE3A vector was formed using a transcription initiation sequence, and a UBE construct disposed downstream of the transcription initiation sequence. The UBE construct is formed of a UBE3A sequence, a secretion sequence, and a cell uptake sequence. Nonlimiting examples of the UBE3A sequence are SEQ ID No: 4, SEQ ID No: 9, SEQ ID No: 14, SEQ ID No:15, SEQ ID NO: 17, a cDNA of SEQ ID No: 10, a cDNA of SEQ ID No: 16, or a homologous sequence. Variations of the DNA sequence include conservative mutations in the DNA triplet code, as seen in Tables 1 and 2. In specific variations, the UBE3A sequence is Mus musculus UBE3A, Homo sapiens UBE3A variant 1, variant 2, or variant 3.

[0079] Nonlimiting examples of the secretion sequence are SEQ ID No: 2, SEQ ID No: 5, SEQ ID No: 11, SEQ ID No: 12, a cDNA of SEQ ID No: 3, a cDNA of SEQ ID NO: 7, a cDNA of SEQ ID NO: 18. A cDNA of SEQ ID NO: 19, or a homologous sequence, with variations of the DNA sequence that include the aforementioned conservative mutations.

[0080] Nonlimiting examples of the cell uptake sequence are SEQ ID No: 6, a cDNA of SEQ ID No. 8, a cDNA of SEQ ID No: 13, a cDNA of SEQ ID No: 20, a cDNA of SEQ ID No: 21, a cDNA of SEQ ID No: 22, or a homologous sequence. Variations of the DNA sequence include the aforementioned conservative mutations.

[0081] In specific variations of the invention, the secretion sequence is disposed upstream of the UBE3A sequence, and more specifically is optionally is disposed upstream of the UBE3A sequence and downstream of the secretion sequence. Other possible uptake proteins include penetratin, TATk, pVEC, transportan, MPG, Pep-1, polyarginines, MAP, and R6W3.

[0082] In some variations of the invention, the transcription initiation sequence is a cytomegalovirus chicken-beta actin hybrid promoter, or human ubiquitin c promoter. The invention optionally includes an enhancer sequence. A nonlimiting example of the enhancer sequence is a cytomegalovirus immediate-early enhancer sequence disposed upstream of the transcription initiation sequence. The vector optionally also includes a woodchuck hepatitis post-transcriptional regulatory element. The listed promotors, enhancer sequence and post-transcriptional regulatory element are well known in the art. (Garg S. et al., The hybrid cytomegalovirus enhancer/chicken beta-actin promotor along with woodchuck hepatitis virus posttranscriptional regulatory element enhances the protective efficacy of DNA vaccines, J. Immunol., Jul. 1, 2004; 173(1):550-558; Higashimoto, T. et al., The woodchuck hepatitis virus post-transcriptional regulatory element reduces readthrough transcription from retroviral vectors, September 2007; 14(17): 1298-304; Cooper, A. R. et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter, Nucleic Acids Res., January 2015; 43(1):682-90).

[0083] In variations, the vector is inserted into a plasmid, such as a recombinant adeno-associated virus serotype 2-based plasmid. In specific variations, the recombinant adeno-associated virus serotype 2-based plasmid lacks DNA integration elements. A nonlimiting example of the recombinant adeno-associated virus serotype 2-based plasmid is a pTR plasmid.

[0084] A method of synthesizing the UBE3A vector includes inserting a UBE3A construct into a backbone plasmid having a transcription initiation sequence. The TBE3A construct is formed of a UBE3A sequence, a secretion sequence, and a cell uptake sequence as described above. For example, Ube3a gene was cloned and fused in frame to the 3 DNA sequence (N-terminus with two other peptide sequences), signal peptide and HIV TAT sequences, which were cloned into a recombinant adeno-associated viral vector for expression of the secreted E6-AP protein in the brain and spinal cord of AS patients. The UBE construct is optionally inserted by cleaving the backbone plasmid with at least one endonuclease, and the UBE3A construct ligated to the cleaved ends of the backbone plasmid.

[0085] The vector was then optionally inserted into an amplification host, possessing an antibiotic resistance gene, and subjected to an antibiotic selection corresponding to the antibiotic resistance gene. The amplification host was then expanded in a medium containing the antibiotic selection and the expanded amplification host collected. The vector was then isolated from the amplification host. In specific variations of the invention, the antibiotic resistance gene is an ampicillin resistance gene, with the corresponding antibiotic selection, ampicillin.

[0086] In a preferred embodiment, a UBE3A vector is formed from cDNA cloned from a Homo sapiens UBE3A gene to form the UBE3A, version 1 gene (SEQ ID No: 9) which is fused to a gene encoding a secretion signaling peptide, such as GDNF, insulin or IgK. In a preferred embodiment, GDNF is used. The construct is inserted into the hSTUb vector, under a CMV chicken-beta actin hybrid promoter (preferred) or a human ubiquitin c promoter. Woodchuck hepatitis post-transcriptional regulatory element (WPRE) is present to increase expression levels.

[0087] The UBE3A-seretion signal construct is then attached to a cellular uptake peptide (cell penetrating peptide or CPP) such as HIV TAT or HIV TATk (preferred). The human UBE3A vector is then transformed into an amplification host such as E. coli using the heat shock method described in Example 2. The transformed E. coli were expanded in broth containing ampicillin to select for the vector and collect large amounts of vector. Therapeutically effective doses of vector can then the administered to a patient as a gene therapy for treating Angelman syndrome or another neurological disorder having UBE3A deficiency. The vector may be administered via injection into the hippocampus or ventricles, in some cases, bilaterally. Dosages of the therapeutic can range between about 5.5510.sup.11 to 2.8610.sup.12 genomes/g brain mass.

Example 1Efficiency of the Secretion Signal

[0088] To test the efficacy of the secretion signal, GFP (SEQ ID No: 1) (XM 013480425.1) was cloned in frame with human insulin, GDNF (SEQ ID No: 2) (AB675653.1) or IgK signal peptides.

TABLE-US-00003 (SEQIDNo:1) ATGGCTCGTCTTTCTTTTGTTTCTCTTCTTTCTCTGTCAC TGCTCTTCGGGCAGCAAGCAGTCAGAGCTCAGAATTACAC CATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTG CCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACA AGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTA CGGCAAGGACTGCCTGAAGTTCATCTGCACCACCGGCAAG CTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCT ACGGCCTGATGTGCTTCGCCCGCTACCCCGACCACATGAA GCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTAC GTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACT ACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCT GGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAG GACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACA ACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAA CGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAG GACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACA CCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA CTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAAC GAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCG CCGCCGGGATCACTCTCGGCATGGACGAGCTATACAAGTG GGCGCGCCACTCGAGACGAATCACTAGTGAATTCGCGGCC GCCTGCAGGTCGAGGTTTGCAGCAGAGTAG,

[0089] fused with a secretion protein based on GDNF;

TABLE-US-00004 (SEQIDNo:2) ATGAAGTTATGGGATGTCGTGGCTGTCTGCCTGGTGCTGCTCCACACC GCGTCCGCC (XM017009337.2),whichencodes (SEQIDNO:3) MKLWDVVAVCLVLLHTASA (AAC98782.1)

[0090] The construct was inserted into a pTR plasmid and transfected into HEK293 cells (American Type Culture Collection, Manassas, Va.). HEK293 cells were grown at 37 C. 5% CO.sub.2 in Dulbecco's Modified Essential Medium (DMEM) with 10% FBS and 1% Pen/Strep and subcultured at 80% confluence.

[0091] The vector (2 g/well in a 6-well plate) was transfected into the cells using PEI transfection method. The cells were subcultured at 0.510.sup.6 cells per well in a 6-well plate with DMEM medium two days before the transfection. Medium was replaced the night before transfection. Endotoxin-free dH.sub.2O was heated to at around 80 C., and polyethylenimine (Sigma-Aldrich Co. LLC, St. Louis, Mo.) dissolved. The solution was cooled to around 25 C., and the solution neutralized using sodium hydroxide. AAV4-STUb vector or negative control (medium only) was added to serum-free DMEM at 2 g to every 200 L for each well transfected, and 9 L of 1 g/L polyethylenimine added to the mix for each well. The transfection mix was incubated at room temperature for 15 minutes, then added to each well of cells at 210 L per well and incubated for 48 hours.

[0092] Media was collected from each culture well and 2 L spotted onto a nitrocellulose membrane using a narrow-tipped pipette. After the samples dried, the membrane was blocked applying 5% BSA in TBS-T to the membrane and incubating at room temperature for 30 minutes to 1 hour, followed by incubating the membrane with chicken anti-GFP (5 g/mL, Abcam PLC, Cambridge, UK; # ab13970) in BSA/TBS-T for 30 min at room temperature. The membrane was washed with TBS-T 3 times, 5 minutes for each wash. The membrane was incubated with anti-chicken HRP conjugate secondary antibody (Southern Biotechnology, Thermo Fisher Scientific, Inc., Waltham, Mass.; #6100-05, 1/3000) conjugated with HRP for 30 minutes at room temperature, followed by washing the membrane three times with TBS-T, once for 15 minutes, and subsequent washed at 5 minutes each. The membrane was washed with TBS for 5 minutes at room temperature, and incubated with luminescence reagent for 1 minute (Millipore, Merck KGaA, Darmstadt, DE; # WBKLS0100). The membrane was recorded on a GE Amersham Imager 600 (General Electric, Fairfield, Calif.), shown in FIG. 1.

[0093] As seen from FIG. 1, all three secretion signals resulted in release of GFP-tagged protein from cells as observed by comparison to untransfected control cells. Of the three secretion constructs, the IgK construct showed the highest level of secretion, though clone 2 of the GDNF construct did display similarly high secretion of GFP-tagged protein.

Example 2Mouse-UBE3A Vector Construct

[0094] A mouse-UBE3A vector construct was generated using a pTR plasmid. The mouse (Mus musculus) UBE3A gene was formed from cDNA (U82122.1);

TABLE-US-00005 (SEQIDNo:4) ATGAAGCGAGCAGCTGCAAAGCATCTAATAGAACGCTACT ACCATCAGTTAACTGAGGGCTGTGGAAATGAGGCCTGCAC GAATGAGTTTTGTGCTTCCTGTCCAACTTTTCTTCGTATG GATAACAATGCAGCAGCTATTAAAGCCCTTGAGCTTTATA AAATTAATGCAAAACTCTGTGATCCTCATCCCTCCAAGAA AGGAGCAAGCTCAGCTTACCTTGAGAACTCAAAAGGTGCA TCTAACAACTCAGAGATAAAAATGAACAAGAAGGAAGGAA AAGATTTTAAAGATGTGATTTACCTAACTGAAGAGAAAGT ATATGAAATTTATGAATTTTGTAGAGAGAGTGAGGATTAT TCCCCTTTAATTCGTGTAATTGGAAGAATATTTTCTAGTG CTGAGGCACTGGTTCTGAGCTTTCGGAAAGTCAAACAGCA CACAAAGGAGGAATTGAAATCTCTTCAAGAAAAGGATGAA GACAAGGATGAAGATGAAAAGGAAAAAGCTGCATGTTCTG CTGCTGCTATGGAAGAAGACTCAGAAGCATCTTCTTCAAG GATGGGTGATAGTTCACAGGGAGACAACAATGTACAAAAA TTAGGTCCTGATGATGTGACTGTGGATATTGATGCTATTA GAAGGGTCTACAGCAGTTTGCTCGCTAATGAAAAATTAGA AACTGCCTTCCTGAATGCACTTGTATATCTGTCACCTAAC GTGGAATGTGATTTGACATATCATAATGTGTATACTCGAG ATCCTAATTATCTCAATTTGTTCATTATTGTAATGGAGAA TAGTAATCTCCACAGTCCTGAATATCTGGAAATGGCGTTG CCATTATTTTGCAAAGCTATGTGTAAGCTACCCCTTGAAG CTCAAGGAAAACTGATTAGGCTGTGGTCTAAATACAGTGC TGACCAGATTCGGAGAATGATGGAAACATTTCAGCAACTT ATTACCTACAAAGTCATAAGCAATGAATTTAATAGCCGAA ATCTAGTGAATGATGATGATGCCATTGTTGCTGCTTCAAA GTGTTTGAAAATGGTTTACTATGCAAATGTAGTGGGAGGG GATGTGGACACAAATCATAATGAGGAAGATGATGAAGAAC CCATACCTGAGTCCAGCGAATTAACACTTCAGGAGCTTCT GGGAGATGAAAGAAGAAATAAGAAAGGTCCTCGAGTGGAT CCACTAGAAACCGAACTTGGCGTTAAAACTCTAGACTGTC GAAAACCACTTATCTCCTTTGAAGAATTCATTAATGAACC ACTGAATGATGTTCTAGAAATGGACAAAGATTATACCTTT TTCAAAGTTGAAACAGAGAACAAATTCTCTTTTATGACAT GTCCCTTTATATTGAATGCTGTCACAAAGAATCTGGGATT ATATTATGACAATAGAATTCGCATGTACAGTGAAAGAAGA ATCACTGTTCTTTACAGCCTAGTTCAAGGACAGCAGTTGA ATCCGTATTTGAGACTCAAAGTCAGACGTGACCATATTAT AGATGATGCACTGGTCCGGCTAGAGATGATTGCTATGGAA AATCCTGCAGACTTGAAGAAGCAGTTGTATGTGGAATTTG AAGGAGAACAAGGAGTAATGAGGGAGGCGTTTCCAAAGAG TTTTTTCAGTTGGGTTGTGGAGGAAATTTTTAATCCAAAT ATTGGTATGTTCACATATGATGAAGCTACGAAATTATTTT GGTTTAATCCATCTTCTTTTGAAACTGAGGGTCAGGTTTA CTCTGATTGGCATATCCTGGGTCTGGCTATTTACAATAAT TGTATACTGGATGTCCATTTTCCCATGGTTGTATACAGGA AGCTAATGGGGAAAAAAGGAACCTTTCGTGACTTGGGAGA CTCTCACCCAGTTTTATATCAGAGTTTAAAGGATTTATTG GAATATGAAGGGAGTGTGGAAGATGATATGATGATCACTT TCCAGATATCACAGACAGATCTTTTTGGTAACCCAATGAT GTATGATCTAAAAGAAAATGGTGATAAAATTCCAATTACA AATGAAAACAGGAAGGAATTTGTCAATCTCTATTCAGACT ACATTCTCAATAAATCTGTAGAAAAACAATTCAAGGCATT TCGCAGAGGTTTTCATATGGTGACTAATGAATCGCCCTTA AAATACTTATTCAGACCAGAAGAAATTGAATTGCTTATAT GTGGAAGCCGGAATCTAGATTTCCAGGCACTAGAAGAAAC TACAGAGTATGACGGTGGCTATACGAGGGAATCTGTTGTG ATTAGGGAGTTCTGGGAAATTGTTCATTCGTTTACAGATG AACAGAAAAGACTCTTTCTGCAGTTTACAACAGGCACAGA CAGAGCACCTGTTGGAGGACTAGGAAAATTGAAGATGATT ATAGCCAAAAATGGCCCAGACACAGAAAGGTTACCTACAT CTCATACTTGCTTTAATGTCCTTTTACTTCCGGAATATTC AAGCAAAGAAAAACTTAAAGAGAGATTGTTGAAGGCCATC ACATATGCCAAAGGATTTGGCATGCTGTAA (U82122.1).

[0095] The cDNA was subcloned and sequenced. The mouse UBE3A gene (SEQ ID No. 4) was fused to DNA sequences encoding the secretion signaling peptide GDNF (SEQ ID No. 5) and cell uptake peptide HIV TAT sequence (SEQ ID No: 6). The secretion signaling peptide has the DNA sequence;

TABLE-US-00006 (SEQIDNo:5) ATGGCCCTGTTGGTGCACTTCCTACCCCTGCTGGCC CTGCTTGCCCTCTGGGAGCCCAAACCCACCCAGGCT TTTGTC (NM008386.4),encodingtoproteinsequence; (SEQIDNo:7) MALLVHFLPLLALLALWEPKPTQAFV (NP032412.3);

[0096] while HIV TAT sequence is;

TABLE-US-00007 (SEQIDNo:6) TACGGCAGAAAGAAGAGGAGGCAGAGAAGGAGA, encodingtoproteinsequence; (SEQIDNo:8) YGRKKRRQRRR (AIW51918.1).

[0097] The construct sequence of SEQ ID No: 4 fused with SEQ ID No: 5 and SEQ ID No: 6 was inserted into a pTR plasmid. The plasmid was cleaved using Age I and Xho I endonucleases and the construct sequence ligated using ligase. The vector contains AAV serotype 2 terminal repeats, CMV-chicken-beta actin hybrid promoter and a WPRE, seen in FIG. 2. The recombinant plasmid lacks the Rep and Cap elements, limiting integration of the plasmid into host DNA.

[0098] The vector (AAV4-STUb vector) was then transformed into Escherichia coli (E. coli, Invitrogen, Thermo Fisher Scientific, Inc., Waltham, Mass.; SURE2 cells). Briefly, cells were equilibrated on ice and 1 pg to 500 ng of the vector were added to the E. coli and allowed to incubate for about 1 minute. The cells were electroporated with a BioRad Gene Pulser in a 0.1 cm cuvette (1.7V, 200 Ohms). The E. Coli were then grown in media for 60 min prior to being plated onto agar, such as ATCC medium 1065 (American Type Culture Collection, Manassas, Va.), with ampicillin (50 g/mL). E. coli was expanded in broth containing ampicillin to collect large amounts of vector.

Example 3In Vitro Testing of Mouse-UBE3A Vector Construct

[0099] The mouse vector properties of the construct generated in Example 2 were tested in HEK293 cells (American Type Culture Collection, Manassas, Va.). HEK293 cells were grown at 37 C. 5% CO.sub.2 in Dulbecco's Modified Essential Medium (DMEM) with 10% FBS and 1% Pen/Strep and subcultured at 80% confluence.

[0100] The vector (2 g/well in a 6-well plate) was transfected into the cells using PEI transfection method. The cells were subcultured at 0.510.sup.6 cells per well in a 6-well plate with DMEM medium two days before the transfection. Medium was replaced the night before transfection. Endotoxin-free dH.sub.2O was heated to at around 80 C., and polyethylenimine (Sigma-Aldrich Co. LLC, St. Louis, Mo.) dissolved. The solution was allowed to cool to around 25 C., and the solution neutralized using sodium hydroxide. AAV4-STUb vector or negative control (medium only) was added to serum-free DMEM at 2 g to every 200 l for each well transfected, and 9p of 1 g/l polyethylenimine added to the mix for each well. The transfection mix was incubated at room temperature for 15 minutes, then added to each well of cells at 210 l per well and incubated for 48 hours.

[0101] Media was collected from AAV4-STUb vector transfected cells, medium-only transfected control cells, and untransfected control cells. The medium was run on Western blot and stained with rabbit anti-E6-AP antibody (A300-351A, Bethyl Labs, Montgomery, Tex.), which is reactive against human and mouse E6-AP, at 0.4 g/ml. Secondary conjugation was performed with rabbit-conjugated horseradish peroxidase (Southern Biotechnology, Thermo Fisher Scientific, Inc., Waltham, Mass.). The results were determined densiometrically, and show the HEK293 cells transfected with AAV4-STUb secrete E6-AP protein into the medium, as seen in FIG. 3.

Example 4In Vivo Testing of Mouse-UBE3A Vector Construct

[0102] Transgenic mice were formed by crossbreeding mice having a deletion in the maternal UBE3A (Jiang, et al., Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation. Neuron. 1998 October; 21(4):799-811; Gustin, et al., Tissue-specific variation of Ube3a protein expression in rodents and in a mouse model of Angelman syndrome. Neurobiol Dis. 2010 September; 39(3):283-91; Heck, et al., Analysis of cerebellar function in Ube3a-deficient mice reveals novel genotype-specific behaviors. Hum Mol Genet. 2008 Jul. 15; 17(14):2181-9) and GABARB3. Mice were housed in a 12-hour day-light cycle and fed food and water ad libitum. Three month old mice were treated with the vector.

[0103] Mice were anesthetized with isoflurane and placed in the stereotaxic apparatus (51725D Digital Just for Mice Stereotaxic Instrument, Stoelting, Wood Dale, Ill.). An incision was made sagittally over the middle of the cranium and the surrounding skin pushed back to enlarge the opening. The following coordinates were used to locate the left and right hippocampus: AP 22.7 mm, L 62.7 mm, and V 23.0 mm. Mice received bilateral intrahippocampal injections of either AAV4-STUb particles at a concentration of 110.sup.12 genomes/mL (N=2) in 10 L of 20% mannitol or vehicle (10 L of 20% mannitol) using a 10 mL Hamilton syringe in each hemisphere. The wound was cleaned with saline and closed using Vetbond (NC9286393 Fisher Scientific, Pittsburgh, Pa.). Control animals included uninjected AS mice and littermate wild type mice (n=2). Mice recovered in a clean, empty cage on a warm heating pad and were then singly housed until sacrificed. The mice were monitored over the course of the experiment.

[0104] At day 30 after treatment, the mice were euthanized by injecting a commercial euthanasia solution, Somnasol, (0.22 ml/kg) intraperitoneally. After euthanizing the animals, CSF was collected and the animals were perfused with PBS and the brain removed. The brain was fixed in 4% paraformaldehyde solution overnight prior to cryoprotection in sucrose solutions. Brains were sectioned at 25 m using a microtome.

[0105] Most recombinant adeno-associated virus vector studies inject the vector directly into the parenchymal, which typically results in limited cellular transduction (Li, et al., Intra-ventricular infusion of rAAV-1-EGFP resulted in transduction in multiple regions of adult rat brain: a comparative study with rAAV2 and rAAV5 vectors. Brain Res. 2006 Nov. 29; 1122(1):1-9). However, appending a secretion signaling sequence and TAT sequence to the Ube3A protein allows for secretion of the HECT protein (i.e., UBE3A) from transfected cells and uptake of the peptide by adjacent neurons, allowing injection into a discrete site to serve as a supply of protein for other sites throughout the brain.

[0106] Brains from sacrificed mice were sliced using a microtome and stained for E6-AP protein using anti-E6-AP antibody (A300-351A, Bethyl Labs, Montgomery, Tex.) with a biotinylated anti-rabbit secondary antibody (Vector Labs # AB-1000). Staining was completed with ABC (Vector Labs) and DAB reaction. Sections were mounted and scanned using Zeiss Axio Scan microscope. Percentage area staining was quantified using IAE-NearCYTE image analysis software (University of Pittsburgh Starzl Transplant Institute, Pittsburgh, Pa.).

[0107] Nontransgenic (Ntg) control mice shows the level of UBE3a expression in a normal mouse brain, which was about 40%, as seen in FIG. 4. By comparison, Angelman syndrome mice (AS) show Ube3a protein staining levels of about 25%. Insertion of the AAV4-STUb vector into the lateral ventricles of an AS mouse shows the vector increased the level of E6-AP to around 30-35%.

[0108] Immunohistochemical analysis of brain slices indicate nontransgenic mice possess relatively high levels of E6-AP, with region-specific staining, seen in FIGS. 5 and 6. In Angelman syndrome-model mice, staining patterns of E6-AP are similar, but the levels of E6-AP are drastically reduced, seen in FIGS. 7 and 8, as expected. Administration of the mouse UBE3A vector to Angelman syndrome model mice did increase levels of E6-AP, though not to the level of nontransgenic mice, as seen in FIGS. 9 and 10. A detailed analysis of the lateral ventricle shows that the injection of UBE3A vector resulted in uptake of the vector by ependymal cells, as seen in FIG. 11. However, in addition to the uptake of UBE3A vector and expression of E6-AP by ependymal cells, adjacent cells in the parenchyma also stained positive for E6-AP, as seen by arrows in the Figure. Moreover, staining was seen in more distal locations, such as the 3d ventricle, seen in FIG. 12. This indicates that E6-AP was being secreted by the transfected cells and successfully uptaken by adjacent cells, confirming that the construct can be used to introduce E6-AP and that the E6-AP construct can be used as a therapeutic to treat global cerebral deficiency in E6-AP expression, such as Angelman syndrome. Control treatment using AAV4-GFP vector did not exhibit uptake of the control protein, as seen in FIG. 13, as only transduction of the ependymal and choroid plexus cells.

[0109] Detailed analysis of the coronal cross sections of Angelman syndrome-model mice confirmed that administration of the UBE3A construct increased levels of E6-AP in and around the lateral ventricle, as seen in FIGS. 14 through 20.

Example 5Human UBE3A Vector Construct

[0110] A human vector construct was generated using a pTR plasmid. A Homo sapiens UBE3A gene was formed from cDNA (AH005553.1);

TABLE-US-00008 (SEQIDNo:9) GGAGTAGTTTACTGAGCCACTAATCTAAAGTTTAATACTG TGAGTGAATACCAGTGAGTACCTTTGTTAATGTGGATAACCAATACTTGG CTATAGGAAGTTTTTTAGTTGTGTGTTTTATNACACGTATTTGACTTTGT GAATAATTATGGCTTATAATGGCTTGTCTGTTGGTATCTATGTATAGCGT TTACAGTTTCCTTTAAAAAACATGCATTGAGTTTTTTAATAGTCCAACCC TTAAAATAAATGTGTTGTATGGCCACCTGATCTGACCACTTTCTTTCATG TTGACATCTTTAATTTTAAAACTGTTTTATTTAGTGCTTAAATCTTGTTN ACAAAATTGTCTTCCTAAGTAATATGTCTACCTTTTTTTTTGGAATATGG AATATTTTGCTAACTGTTTCTCAATTGCATTTTACAGATCAGGAGAACCT CAGTCTGACGACATTGAAGCTAGCCGAATGTAAGTGTAACTTGGTTGAGA CTGTGGTTCTTATTTTGAGTTGCCCTAGACTGCTTTAAATTACGTCACAT TATTTGGAAATAATTTCTGGTTAAAAGAAAGGAATCATTTAGCAGTAAAT GGGAGATAGGAACATACCTACTTTTTTTCCTATCAGATAACTCTAAACCT CGGTAACAGTTTACTAGGTTTCTACTACTAGATAGATAAATGCACACGCC TAAATTCTTAGTCTTTTTGCTTCCCTGGTAGCAGTTGTAGGGAAATAGGG AGGTTGAGGAAAGAGTTTAACAGTCTCAACGCCTACCATATTTAAGGCAT CAAGTACTATGTTATAGATACAGAGATGCGTAATAATTAGTTTTCACCCT ACAGAAATTTATATTATACTCAAGAGTGAAAGATGCAGAAGCAAATAATT TCAGTCACTGAGGTAGAATGGTATCCAAAATACAATAGTAACATGAAGGA GTACTGGAGTACCAGGTATGCAATAGGAATCTAGTGTAGATGGCAGGGAA GTAAGAGTGGCCAGGAAATGCTAAGTTCAGTCTTGAAATGTGACTGGGAA TCAGGCAGCTATCAACTATAAGTCAAATGTTTACAAGCTGTTAAAAATGA AATACTGATTATGTAAAAGAAAACCGGATTGATGCTTTAAATAGACTCAT TTTCNTAATGCTAATTTTTAAAATGATAGAATCCTACAANTCTTAGCTGT AAACCTTGTGATTTTTCAGCTGTTGTACTAAACAACTTAAGCACATATAC CATCAGACAAGCCCCCNTCCCCCCTTTTAAACCAAAGGAATGTATACTCT GTTAATACAGTCAGTAAGCATTGACATTCTTTATCATAATATCCTAGAAA ATATTTATTAACTATTTCACTAGTCAGGAGTTGTGGTAAATAGTGCATCT CCATTTTCTACTTCTCATCTTCATACACAGGTTAATCACTTCAGTGCTTG ACTAACTTTTGCCTTGATGATATGTTGAGCTTTGTACTTGAGAGCTGTAC TAATCACTGTGCTTATTGTTTGAATGTTTGGTACAGGAAGCGAGCAGCTG CAAAGCATCTAATAGAACGCTACTACCACCAGTTAACTGAGGGCTGTGGA AATGAAGCCTGCACGAATGAGTTTTGTGCTTCCTGTCCAACTTTTCTTCG TATGGATAATAATGCAGCAGCTATTAAAGCCCTCGAGCTTTATAAGATTA ATGCAAAACTCTGTGATCCTCATCCCTCCAAGAAAGGAGCAAGCTCAGCT TACCTTGAGAACTCGAAAGGTGCCCCCAACAACTCCTGCTCTGAGATAAA AATGAACAAGAAAGGCGCTAGAATTGATTTTAAAGGTAAGATGTTTTATT TTCAATTGAGAATTGTTGCCTGAAAACCATGTGGGAGATTTAAATGTATT AGTTTTTATTTGTTTTTTCTTCTGTGACATAAAGACATTTTGATATCGTA GAACCAATTTTTTATTGTGGTAACGGACAGGAATAATAACTACATTTTAC AGGTCTAATCATTGCTAATTAGAAGCAGATCATATGCCAAAAGTTCATTT GTTAATAGATTGATTTGAACTTTTTAAAATTCTTAGGAAAAATGTATTAA GTGGTAGTGAATCTCCAAAACTATTTAAGAGCTGTATTATGATTAATCAG TACATGACATATTGGTTCATATTTATAATTAAAGCTATACATTAATAGAT ATCTTGATTATAAAGAAAGTTTAAACTCATGATCTTATTAAGAGTTATAC ATTGTTGAAAGAATGTAAAAGCATGGGTGAGGTCATTGGTATAGGTAGGT AGTTCATTGAAAAAAATAGGTAAGCATTAAATTTTGTTTGCTGAATCTAA GTATTAGATACTTTAAGAGTTGTATATCATAAATGATATTGAGCCTAGAA TGTTTGGCTGTTTTACTTTTAGAACTTTTTGCAACAGAGTAAACATACAT ATTATGAAAATAAATGTTCTCTTTTTTCCTCTGATTTTCTAGATGTGACT TACTTAACAGAAGAGAAGGTATATGAAATTCTTGAATTATGTAGAGAAAG AGAGGATTATTCCCCTTTAATCCGTGTTATTGGAAGAGTTTTTTCTAGTG CTGAGGCATTGGTACAGAGCTTCCGGAAAGTTAAACAACACACCAAGGAA GAACTGAAATCTCTTCAAGCAAAAGATGAAGACAAAGATG AAGATGAAAAGGAAAAAGCTGCATGTTCTGCTGCTGCTATGGAAGAAGAC TCAGAAGCATCTTCCTCAAGGATAGGTGATAGCTCACAGGGAGACAACAA TTTGCAAAAATTAGGCCCTGATGATGTGTCTGTGGATATTGATGCCATTA GAAGGGTCTACACCAGATTGCTCTCTAATGAAAAAATTGAAACTGCCTTT CTCAATGCACTTGTATATTTGTCACCTAACGTGGAATGTGACTTGACGTA TCACAATGTATACTCTCGAGATCCTAATTATCTGAATTTGTTCATTATCG TAATGGAGAATAGAAATCTCCACAGTCCTGAATATCTGGAAATGGCTTTG CCATTATTTTGCAAAGCGATGAGCAAGCTACCCCTTGCAGCCCAAGGAAA ACTGATCAGACTGTGGTCTAAATACAATGCAGACCAGATTCGGAGAATGA TGGAGACATTTCAGCAACTTATTACTTATAAAGTCATAAGCAATGAATTT AACAGTCGAAATCTAGTGAATGATGATGATGCCATTGTTGCTGCTTCGAA GTGCTTGAAAATGGTTTACTATGCAAATGTAGTGGGAGGGGAAGTGGACA CAAATCACAATGAAGAAGATGATGAAGAGCCCATCCCTGA GTCCAGCGAGCTGACACTTCAGGAACTTTTGGGAGAAGAA AGAAGAAACAAGAAAGGTCCTCGAGTGGACCCCCTGGAAA CTGAACTTGGTGTTAAAACCCTGGATTGTCGAAAACCACTTATCCCTTTT GAAGAGTTTATTAATGAACCACTGAATGAGGTTCTAGAAATGGATAAAGA TTATACTTTTTTCAAAGTAGAAACAGAGAACAAATTCTCTTTTATGACAT GTCCCTTTATATTGAATGCTGTCACAAAGAATTTGGGATTATATTATGAC AATAGAATTCGCATGTACAGTGAACGAAGAATCACTGTTCTCTACAGCTT AGTTCAAGGACAGCAGTTGAATCCATATTTGAGACTCAAAGTTAGACGTG ACCATATCATAGATGATGCACTTGTCCGGGTAAGTTGGGCTGCTAGATTA AAAACCTAATAATGGGGATATCATGATACAGTTCAGTGAATTCATTTTAA AAGTGACTGAAAAAAATGATACCATATAGCATAGGAACACATGGACATTT CTGATCTTATATAAGTATTATACTTTTGTTGTTCCTGTGCAAGTTTATAG ATGTGTTCTACAAAGTATCGGTTGTATTATATAATGGTCATGCTATCTTT GAAAAAGAATGGGTTTTCTAAATCTTGAAAACTAAATCCAAAGTTTCTTT CATTCAGAAGAGAATAGAGTGTTGGACAAAGACCAGAACA AGAGAAATGTGGAGATACCCAATAATAAGTGTGGATGTGCAGTCTTGAAC TGGGAGTAATGGTACAGTAAAACCATACCATAAAATTATAGGTAGTGTCC AAAAAATTCCATCGTGTAAAATTCAGAGTTGCATTATTGTGGACTTGAAG AAGCAGTTGTATGTGGGACGGTATCGATAAGCTTGATATCGAATTCCTGC AGCCCGGGGGATCCACTAGTGTGGTAATTAATACTAAGTCTTACTGTGAG AGACCATAAACTGCTTTAGTATTCAGTGTATTTTTCTTAATTGAAATATT TAACTTATGACTTAGTAGATACTAAGACTTAACCCTTGAGTTTCTATTCT AATAAAGGACTACTAATGAACAATTTTGAGGTTAGACCTCTACTCCATTG TTTTTGCTGAAATGATTTAGCTGCTTTTCCATGTCCTGTGTAGTCCAGAC TTAACACACAAGTAATAAAATCTTAATTAATTGTATGTTAATTTCATAAC AAATCAGTAAAGTTAGCTTTTTACTATGCTAGTGTCTGTTTTGTGTCTGT CTTTTTGATTATCTTTAAGACTGAATCTTTGTCTTCACTGGCTTTTTATC AGTTTGCTTTCTGTTTCCATTTACATACAAAAAGTCAAAAATTTGTATTT GTTTCCTAATCCTACTCCTTGTTTTTATTTTGTTTTTTTCCTGATACTAG CAATCATCTTCTTTTCATGTTTATCTTTTCAATCACTAGCTAGAGATGAT CGCTATGGAAAATCCTGCAGACTTGAAGAAGCAGTTGTATGTGGAATTTG AAGGAGAACAAGGAGTTGATGAGGGAGGTGTTTCCAAAGAATTTTTTCAG CTGGTTGTGGAGGAAATCTTCAATCCAGATATTGGTAAATACATTAGTAA TGTGATTATGGTGTCGTATCATCTTTTGAGTTAGTTATTTGTTTATCTTA CTTTGTAAATATTTTCAGCTATGAAGAGCAGCAAAAGAAGGATTTGGTAT GGATTACCCAGAATCACACATCATGACTGAATTTGTAGGTTTTAGGAACT GATTTGTATCACTAATTTATTCAAATTCTTTTATTTCTTAGAAGGAATAT TCTAATGAAGGAAATTATCTCTTTGGTAAACTGAATTGAAAGCACTTTAG AATGGTATATTGGAACAGTTGGAGGGATTTCTTTGCTTTTTGTTGTCTAA AACCATCATCAAACTCACGGTTTTCCTGACCTGTGAACTTCAAAGAACAA TGGTTTGAAGAGTATTGAGAGACTGTCTCACAAGTATGTCATGCTCAAAG TTCAGAAACACTAGCTGATATCACATTAATTAGGTTTATTTGCTATAAGA TTTCTTGGGGCTTAATATANGTAGTGTTCCCCCAAACTTTTTGAACTCCA GAACTCTTTTCTGCCCTAACAGTAGCTACTCAGGAGCTGAGGCAGGAGAA TTGTTTGAACCTAGGAGGCAGAGGTTGCAGTGAGCTGAGATCGTGCCACT CCAGCCCACCCCTGGGTAACAGAGCGAGACTCCATCTCAAAGAAAAAAAT GAAAAATTGTTTTCAAAAATAGTACGTGTGGTACAGATATAAGTAATTAT ATTTTTATAAATGAAACACTTTGGAAATGTAGCCATTTTTTGTTTTTTTA TGTTTATTTTTCAGCTATGGGTGGATAAAGCATGAATATAACTTTTCTTA TGTGTTAGTAGAAAATTAGAAAGCTTGAATTTAATTAACGTATTTTTCTA CCCGATGCCACCAAATTACTTACTACTTTATTCCTTTGGCTTCATAAAAT TACATATCACCATTCACCCCAATTTATAGCAGATATATGTGGACATTGTT TTCTCAAGTGCTAATATAATAGAAATCAATGTTGCATGCCTAATTACATA TATTTTAAATGTTTTATATGCATAATTATTTTAAGTTTATATTTGTATTA TTCATCAGTCCTTAATAAAATACAAAAGTAATGTATTTTTAAAAATCATT TCTTATAGGTATGTTCACATACGATGAATCTACAAAATTGTTTTGGTTTA ATCCATCTTCTTTTGAAACTGAGGGTCAGTTTACTCTGATTGGCATAGTA CTGGGTCTGGCTATTTACAATAACTGTATACTGGATGTACATTTTCCCAT GGTTGTCTACAGGAAGCTAATGGGGAAAAAAGGAACTTTTCGTGACTTGG GAGACTCTCACCCAGTAAGTTCTTTGTCATTTTTTTAATTCAGTCTCTTA GATTTTATTTAAATGCAAAAATTTAATTTATGTCAAAATTTTAAAGTTTT TGTTTAGAATCTTTGTTGATACTCTTATCAATAAGATAAAAATGTTTTAA TCTGACCGAAGTACCAGAAACACTTAAAAACTCAAAGGGGGACATTTTTA TATATTGCTGTCAGCACGAAGCTTTCGTAAGATTGATTTCATAGAGAAGT GTTTCTAAACATTTTGTTTGTGTTTTAGTGAAATCTTAAGAGATAGGTAA AAATCAGAGTAGCCCTGGCTAAGGGTCTTGGTAGTTACAACGAGTGTGCC TGCTCCTACCACCCCCACCCCCACCTTGAGACACCACAGAATTTCTCATA GAGCACAGTGTGAATTCTATTGCTAAATTGGTGGTATGGGGTTTCTCAGC AGAGAATGGGACATCACAGTGACTGACAATCTTTCTTTTATAGGTTGGAA ACTATTTGGGGGACTGGAGGGATACTGTCTACACTTTTTACAATTTTTAT TGATAAGATTTTTGTTGTCTTCTAAGAAGAGTGATATAAATTATTTGTTG TATTTTGTAGTTCTATGGTGGCCTCAATTTACCATTTCTGGTTGCTAGGT TCTATATCAGAGTTTAAAAGATTTATTGGAGTATGAAGGGAATGTGGAAG ATGACATGATGATCACTTTCCAGATATCACAGACAGATCTTTTTGGTAAC CCAATGATGTATGATCTAAAGGAAAATGGTGATAAAATTCCAATTACAAA TGAAAACAGGAAGGTAATAAATGTTTTTATGTCACATTTTGTCTCTTCAT TAACACTTTCAAAGCATGTATGCTTATAATTTTTAAAGAAGTATCTAATA TAGTCTGTACAAAAAAAAAACAAGTAACTAAGTTTATGTAAATGCTAGAG TCCACTTTTCTAAATCTTGGATATAAGTTGGTATGAAAGCACACAGTTGG GCACTAAAGCCCCTTTTAGAGAAAGAGGACATGAAGCAGG AGATAGTTAATAGCTAAGTGTGGTTGTAGTATAAAGCAAGAAGCAGGGTG TTTCTTGTATTAAGCTGTAAGCAGGAACCTCATGATTAAGGTCTTTATCA CAGAACAAATAAAAATTACATTTAATTTACACATGTATATCCTGTTTGTG ATAAAAATACATTTCTGAAAAGTATACTTTACGTCAGATTTGGGTTCTAT TGACTAAAATGTGTTCATCGGGAATGGGAATAACCCAGAACATAACAAGC AAAAAATTATGACAAATATATAGTATACCTTTAAGAAACATGTTTATATT GATATAATTTTTTGATTAAATATTATACACACTAAGGGTACAANGCACAT TTTCCTTTTATGANTTNGATACAGTAGTTTATGTGTCAGTCAGATACTTC CACATTTTTGCTGAACTGGATACAGTAAGCAGCTTACCAAATATTCTATG GTAGAAAACTNGGACTTCCTGGTTTGCTTAAATCAAATATATTGTACTCT CTTAAAACGGTTGGCATTTATAAATAGATGGATACATGGTTTAAATGTGT CTGTTNACATACCTAGTTGAGAGAACCTAAAGAATTTTCTGCGTCTCCAG CATTTATATTCAGTTCTGTTTAATACATTATCGAAATTGACATTTATAAG TATGACAGTTTTGTGTATATGGCCTTTTCATAGCTTAATATTGGCTGTAA CAGAGAATTGTGAAATTGTAAGAAGTAGTTTTCTTTGTAGGTGTAAAATT GAATTTTTAAGAATATTCTTGACAGTTTTATGTATATGGCCTTTTCATAG CTTAATATTGGCTATAACAGAGAATTGTGAAATTGTTAAGAAGTAGGTGT AAAATTGAATTTTTAAGAATATTCTTGAATGTTTTTTTCTTGGAAAAATT AAAAAGCTATGCAGCCCAATAACTTGTGTTTTGTTTGCATAGCATATTAT AAGAAGTTCTTGTGATTAATGTTTTCTACAGGAATTTGTCAATCTTTATT CTGACTACATTCTCAATAAATCAGTAGAAAAACAGTTCAAGGCTTTTCGG AGAGGTTTTCATATGGTGACCAATGAATCTCCCTTAAAGTACTTATTCAG ACCAGAAGAAATTGAATTGCTTATATGTGGAAGCCGGGTA AGAAAGCAGGTGTCTGCAAAAAGTCATGTATCGATTTATTGTTTGTAATG ATACAGTAGTATAGCAGATAACTAAGACATATTTTCTTGAATTTGCAGAA TCTAGATTTCCAAGCACTAGAAGAAACTACAGAATATGACGGTGGCTATA CCAGGGACTCTGTTCTGATTAGGTGAGGTACTTAGTTCTTCAGAGGAAGA TTTGATTCACCAAAGGGGTGTGTGATTTTGCTTCAGACCTTTATCTCTAG GTACTAATTCCCAAATAAGCAAACTCACAAATTGTCATCTATATACTTAG ATTTGTATTTGTAATATAATCACCATTTTTCAGAGCTAATCTTGTGATTT ATTTCATGAATGAAGTGTTGTTATATATAAGTCTCATGTAATCTCCTGCA TTTGGCGTATGGATTATCTAGTATTCCTCACTGGTTAGAGTATGCTTACT GCTGGTTAGAAGATAATTAAAATAAGGCTACCATGTCTGCAATTTTTCCT TTCTTTTGAACTCTGCATTTGTGAACTGTTACATGGCTTCCCAGGATCAA GCACTTTTTGAGTGAAATGGTAGTCTTTTATTTAATTCTTAAGATAATAT GTCCAGATACATACTAGTATTTCCATTTTACACCCTAAAAAACTAAGCCC TGAATTCTCACAGAAAGATGTAGAGGTTCCCAGTTCTATCTGCTTTTAAA CAAATGCCCTTACTACTCTACTGTCTACTTCTGTGTACTACATCATCGTA TGTAGTTGTTTGCATTTGGGCCAGTTGGTTGGGGCAGGGGTCTTTTTTTC TTTTGTCCCTTAATCTGTATCACTTTTTCCTCCCAAAGTTGAGTTAAAGG ATGAGTAGACCAGGAGAATAAAGGAGAAAGGATAAATAAA ATATATACCCAAAGGCACCTGGAGTTAATTTTTCCAAATATTCATTTCAG TCTTTTTCAATTCATAGGATTTTGTCTTTTGCTCATTACTGACTGCATAA TGTGATTATACCATAGTTTAAATAGTCACTTCCTGTTACTACACACTTGG GTTTTCTCAATTTTTTACTATTGTAGTACTAATATTTTACTATATTGTAA TCTAATCCAAATTTTTACGTATTCAGAGCTGTTCAGGATAAATTTGCTTG GAAATTTTTAAATCACCAGAAGTGATACTATCCTGATAATTAACTTCCAA GTTGTCTCTTAATATAGTTTTAATGCAAATCATAAGCTTATGTTAGTACC AGTCATAATGAATGCCAAACTGAAACCAGTATTGTATTTTTTCTCATTAG GGAGTTCTGGGAAATCGTTCATTCATTTACAGATGAACAGAAAAGACTCT TCTTGCAGTTTACAACGGGCACAGACAGAGCACCTGTGGGAGGACTAGGA AAATTAAAGATGATTATAGCCAAAAATGGCCCAGACACAG AAAGGTAGGTAATTATTAACTTGTGACTGTATACCTACCGAAAACCTTGC ATTCCTCGTCACATACATATGAACTGTCTTTATAGTTTCTGAGCACATTC GTGATTTTATATACAAATCCCCAAATCATATTAGACAATTGAGAAAATAC TTTGCTGTCATTGTGTGAGGAAACTTTTAAGAAATTGCCCTAGTTAAAAA TTATTATGGGGCTCACATTGGTTTGGAATCAAATTAGTGTGATTCATTTA CTTTTTTGATTCCCAGCTTGTTAATTGAAAGCCATATAACATGATCATCT ATTTAGAATGGTTACATTGAGGCTCGGAAGATTATCATTTGATTGTGCTA GAATCCTGTTATCAAATCATTTTCTTAGTCATATTGCCAGCAGTGTTTCT AATAAGCATTTAAGAGCACACACTTTGCAGTCTTGTAAAACAGGTTTGAG TATTTTCTCCACCTTAGAGGAAGTTACTTGACTTCTCAGTGACCTAACCT CTAAAGTGCATTTACTGATGTCCTCTCTGTGGTTTTGTTGTGGAAAGATT TAGTTAAATGAACTGTAAGAATTCAGTACCTAAAATGGTATCTGTTATGT AGTAAAAACTCAATGGATACAGTATCTTATCATCGTCACTAGCTTTGAGT AATTTATAGGATAAAGGCAACTTGGTAGTTACACAACAAAAAGTTTATGA TTTGCATTAATGTATAGTTTGCATTGCAGACCGTCTCAACTATATACAAT CTAAAAATAGGAGCATTTAATTCTAAGTGTATTTCCCATGACTTACAGTT TTCCTGTTTTTTTCCCCTTTTCTCTATTTAGGTTACCTACATCTCATACT TGCTTTAATGTGCTTTTACTTCCGGAATACTCAAGCAAAGAAAAACTTAA AGAGAGATTGTTGAAGGCCATCACGTATGCCAAAGGATTTGGCATGCTGT AAAACAAAACAAAACAAAATAAAACAAAAAAAAGGAAGGA AAAAAAAAGAAAAAATTTAAAAAATTTTAAAAATATAACG AGGGATAAATTTT(AH005553.1),whichencodesfor; (SEQIDNo:10) MKRAAAKHLIERYYHQLTEGCGNEACTNEFCASCPTFLRMDNNAAAIKA LELYKINAKLCDPHPSKKGASSAYLENSKGAPNNSCSEIKMNKKGARIDFKDVT YLTEEKVYEILELCREREDYSPLIRVIGRVFSSAEALVQSFRKVKQHTKEELKSL QAKDEDKDEDEKEKAACSAAAMEEDSEASSSRIGDSSQGDNNLQKLGPDDVS VDIDAIRRVYTRLLSNEKIETAFLNALVYLSPNVECDLTYHNVYSRDPNYLNLFI IVMENRNLHSPEYLEMALPLFCKAMSKLPLAAQGKLIRLWSKYNADQIRRMME TFQQLITYKVISNEFNSRNLVNDDDAIVAASKCLKMVYYANVVGGEVDTNHNE EDDEEPIPESSELTLQELLGEERRNKKGPRVDPLETELGVKTLDCRKPLIPFEEFI NEPLNEVLEMDKDYTFFKVETENKFSFMTCPFILNAVTKNLGLYYDNRIRMYSE RRITVLYSLVQGQQLNPYLRLKVRRDHIIDDALVRLEMIAMENPADLKKQLYV EFEGEQGVDEGGVSKEFFQLVVEEIFNPDIGMFTYDESTKLFWFNPSSFETEGQF TLIGIVLGLAIYNNCILDVHFPMVVYRKLMGKKGTFRDLGDSHPVLYQSLKDLL EYEGNVEDDMMITFQISQTDLFGNPMMYDLKENGDKIPITNENRKEFVNLYSD YILNKSVEKQFKAFRRGFHMVTNESPLKYLFRPEEIELLICGSRNLDFQALEETT EYDGGYTRDSVLIREFWEIVHSFTDEQKRLFLQFTTGTDRAPVGGLGKLKMIIA KNGPDTERLPTSHTCFNVLLLPEYSSKEKLKERLLKAITYAKGFGML(NP570853.1).

[0111] The cDNA was subcloned and sequenced. The UBE3A, version 1 gene (hUBEv1) (SEQ ID No: 9) was fused to one of three genes encoding a secretion signaling peptide, based on GDNF;

TABLE-US-00009 (SEQIDNo:2) ATGAAGTTATGGGATGTCGTGGCTGTCTGCCTGGTGCTGCTCCACACC GCGTCCGCC,

[0112] from insulin protein;

TABLE-US-00010 (SEQIDNo:11) ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCT GGGGACCTGACCCAGCCGCAGCC (AH002844.2),

[0113] or from IgK;

TABLE-US-00011 (SEQIDNo:12) ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCA GGTTCCACTGGT (NG000834.1).

[0114] The construct was inserted into the hSTUb vector, under a CMV chicken-beta actin hybrid promoter or human ubiquitin c promoter. Woodchuck hepatitis post-transcriptional regulatory element (WPRE) is present to increase expression levels.

[0115] The UBE3A-seretion signal construct was then attached to a cellular uptake peptide (cell penetrating peptide); either a

TABLE-US-00012 HIVTATsequence YGRKKRRQRRR; (SEQIDNo.8) or HIVTATksequence YARKAARQARA. (SEQIDNo.13)

[0116] The human UBE3A vector, seen in FIG. 21, is then transformed into E. coli using the heat shock method described in Example 2. The transformed E. coli were expanded in broth containing ampicillin to select for the vector and collect large amounts of vector.

[0117] Other sequences of UBE3A include variants 1, 2, or 3, seen below;

[0118] H sapiens UBE3A variant 1:

TABLE-US-00013 (SEQIDNo:14) ACAGTATGACATCTGATGCTGGAGGGTCGCACTTTCACAA ATGAGTCAGCTGGTACATGGGGTTATCATCAATTTTTAGC TCTTCTGTCTGGGAGATACAAGTTTGGAAGCAATCTTGGG GTACTTACCCACAAGGCTGGTGGAGACCAGATCAGGAGAA CCTCAGTCTGACGACATTGAAGCTAGCCGAATGAAGCGAG CAGCTGCAAAGCATCTAATAGAACGCTACTACCACCAGTT AACTGAGGGCTGTGGAAATGAAGCCTGCACGAATGAGTTT TGTGCTTCCTGTCCAACTTTTCTTCGTATGGATAATAATG CAGCAGCTATTAAAGCCCTCGAGCTTTATAAGATTAATGC AAAACTCTGTGATCCTCATCCCTCCAAGAAAGGAGCAAGC TCAGCTTACCTTGAGAACTCGAAAGGTGCCCCCAACAACT CCTGCTCTGAGATAAAAATGAACAAGAAAGGCGCTAGAAT TGATTTTAAAGATGTGACTTACTTAACAGAAGAGAAGGTA TATGAAATTCTTGAATTATGTAGAGAAAGAGAGGATTATT CCCCTTTAATCCGTGTTATTGGAAGAGTTTTTTCTAGTGC TGAGGCATTGGTACAGAGCTTCCGGAAAGTTAAACAACAC ACCAAGGAAGAACTGAAATCTCTTCAAGCAAAAGATGAAG ACAAAGATGAGGATGAAAAGGAAAAAGCTGCATGTTCTGC TGCTGCTATGGAAGAAGACTCAGAAGCATCTTCCTCAAGG ATAGGTGATAGCTCACAGGGAGACAACAATTTGCAAAAAT TAGGCCCTGATGATGTGTCTGTGGATATTGATGCCATTAG AAGGGTCTACACCAGATTGCTCTCTAATGAAAAAATTGAA ACTGCCTTTCTCAATGCACTTGTATATTTGTCACCTAACG TGGAATGTGACTTGACGTATCACAATGTATACTCTCGAGA TCCTAATTATCTGAATTTGTTCATTATCGTAATGGAGAAT AGAAATCTCCACAGTCCTGAATATCTGGAAATGGCTTTGC CATTATTTTGCAAAGCGATGAGCAAGCTACCCCTTGCAGC CCAAGGAAAACTGATCAGACTGTGGTCTAAATACAATGCA GACCAGATTCGGAGAATGATGGAGACATTTCAGCAACTTA TTACTTATAAAGTCATAAGCAATGAATTTAACAGTCGAAA TCTAGTGAATGATGATGATGCCATTGTTGCTGCTTCGAAG TGCTTGAAAATGGTTTACTATGCAAATGTAGTGGGAGGGG AAGTGGACACAAATCACAATGAAGAAGATGATGAAGAGCC CATCCCTGAGTCCAGCGAGCTGACACTTCAGGAACTTTTG GGAGAAGAAAGAAGAAACAAGAAAGGTCCTCGAGTGGACC CCCTGGAAACTGAACTTGGTGTTAAAACCCTGGATTGTCG AAAACCACTTATCCCTTTTGAAGAGTTTATTAATGAACCA CTGAATGAGGTTCTAGAAATGGATAAAGATTATACTTTTT TCAAAGTAGAAACAGAGAACAAATTCTCTTTTATGACATG TCCCTTTATATTGAATGCTGTCACAAAGAATTTGGGATTA TATTATGACAATAGAATTCGCATGTACAGTGAACGAAGAA TCACTGTTCTCTACAGCTTAGTTCAAGGACAGCAGTTGAA TCCATATTTGAGACTCAAAGTTAGACGTGACCATATCATA GATGATGCACTTGTCCGGCTAGAGATGATCGCTATGGAAA ATCCTGCAGACTTGAAGAAGCAGTTGTATGTGGAATTTGA AGGAGAACAAGGAGTTGATGAGGGAGGTGTTTCCAAAGAA TTTTTTCAGCTGGTTGTGGAGGAAATCTTCAATCCAGATA TTGGTATGTTCACATACGATGAATCTACAAAATTGTTTTG GTTTAATCCATCTTCTTTTGAAACTGAGGGTCAGTTTACT CTGATTGGCATAGTACTGGGTCTGGCTATTTACAATAACT GTATACTGGATGTACATTTTCCCATGGTTGTCTACAGGAA GCTAATGGGGAAAAAAGGAACTTTTCGTGACTTGGGAGAC TCTCACCCAGTTCTATATCAGAGTTTAAAAGATTTATTGG AGTATGAAGGGAATGTGGAAGATGACATGATGATCACTTT CCAGATATCACAGACAGATCTTTTTGGTAACCCAATGATG TATGATCTAAAGGAAAATGGTGATAAAATTCCAATTACAA ATGAAAACAGGAAGGAATTTGTCAATCTTTATTCTGACTA CATTCTCAATAAATCAGTAGAAAAACAGTTCAAGGCTTTT CGGAGAGGTTTTCATATGGTGACCAATGAATCTCCCTTAA AGTACTTATTCAGACCAGAAGAAATTGAATTGCTTATATG TGGAAGCCGGAATCTAGATTTCCAAGCACTAGAAGAAACT ACAGAATATGACGGTGGCTATACCAGGGACTCTGTTCTGA TTAGGGAGTTCTGGGAAATCGTTCATTCATTTACAGATGA ACAGAAAAGACTCTTCTTGCAGTTTACAACGGGCACAGAC AGAGCACCTGTGGGAGGACTAGGAAAATTAAAGATGATTA TAGCCAAAAATGGCCCAGACACAGAAAGGTTACCTACATC TCATACTTGCTTTAATGTGCTTTTACTTCCGGAATACTCA AGCAAAGAAAAACTTAAAGAGAGATTGTTGAAGGCCATCA CGTATGCCAAAGGATTTGGCATGCTGTAAAACAAAACAAA ACAAAAT (AK291405.1);

[0119] H sapiens UBE3A variant 2;

TABLE-US-00014 (SEQIDNo:15) AGCCAGTCCTCCCGTCTTGCGCCGCGGCCGCGAGATCCGT GTGTCTCCCAAGATGGTGGCGCTGGGCTCGGGGTGACTAC AGGAGACGACGGGGCCTTTTCCCTTCGCCAGGACCCGACA CACCAGGCTTCGCTCGCTCGCGCACCCCTCCGCCGCGTAG CCATCCGCCAGCGCGGGCGCCCGCCATCCGCCGCCTACTT ACGCTTCACCTCTGCCGACCCGGCGCGCTCGGCTGCGGGC GGCGGCGCCTCCTTCGGCTCCTCCTCGGAATAGCTCGCGG CCTGTAGCCCCTGGCAGGAGGGCCCCTCAGCCCCCCGGTG TGGACAGGCAGCGGCGGCTGGCGACGAACGCCGGGATTTC GGCGGCCCCGGCGCTCCCTTTCCCGGCCTCGTTTTCCGGA TAAGGAAGCGCGGGTCCCGCATGAGCCCCGGCGGTGGCGG CAGCGAAAGAGAACGAGGCGGTGGCGGGCGGAGGCGGCGG GCGAGGGCGACTACGACCAGTGAGGCGGCCGCCGCAGCCC AGGCGCGGGGGCGACGACAGGTTAAAAATCTGTAAGAGCC TGATTTTAGAATTCACCAGCTCCTCAGAAGTTTGGCGAAA TATGAGTTATTAAGCCTACGCTCAGATCAAGGTAGCAGCT AGACTGGTGTGACAACCTGTTTTTAATCAGTGACTCAAAG CTGTGATCACCCTGATGTCACCGAATGGCCACAGCTTGTA AAAGAGAGTTACAGTGGAGGTAAAAGGAGTGGCTTGCAGG ATGGAGAAGCTGCACCAGTGTTATTGGAAATCAGGAGAAC CTCAGTCTGACGACATTGAAGCTAGCCGAATGAAGCGAGC AGCTGCAAAGCATCTAATAGAACGCTACTACCACCAGTTA ACTGAGGGCTGTGGAAATGAAGCCTGCACGAATGAGTTTT GTGCTTCCTGTCCAACTTTTCTTCGTATGGATAATAATGC AGCAGCTATTAAAGCCCTCGAGCTTTATAAGATTAATGCA AAACTCTGTGATCCTCATCCCTCCAAGAAAGGAGCAAGCT CAGCTTACCTTGAGAACTCGAAAGGTGCCCCCAACAACTC CTGCTCTGAGATAAAAATGAACAAGAAAGGCGCTAGAATT GATTTTAAAGATGTGACTTACTTAACAGAAGAGAAGGTAT ATGAAATTCTTGAATTATGTAGAGAAAGAGAGGATTATTC CCCTTTAATCCGTGTTATTGGAAGAGTTTTTTCTAGTGCT GAGGCATTGGTACAGAGCTTCCGGAAAGTTAAACAACACA CCAAGGAAGAACTGAAATCTCTTCAAGCAAAAGATGAAGA CAAAGATGAAGATGAAAAGGAAAAAGCTGCATGTTCTGCT GCTGCTATGGAAGAAGACTCAGAAGCATCTTCCTCAAGGA TAGGTGATAGCTCACAGGGAGACAACAATTTGCAAAAATT AGGCCCTGATGATGTGTCTGTGGATATTGATGCCATTAGA AGGGTCTACACCAGATTGCTCTCTAATGAAAAAATTGAAA CTGCCTTTCTCAATGCACTTGTATATTTGTCACCTAACGT GGAATGTGACTTGACGTATCACAATGTATACTCTCGAGAT CCTAATTATCTGAATTTGTTCATTATCGTAATGGAGAATA GAAATCTCCACAGTCCTGAATATCTGGAAATGGCTTTGCC ATTATTTTGCAAAGCGATGAGCAAGCTACCCCTTGCAGCC CAAGGAAAACTGATCAGACTGTGGTCTAAATACAATGCAG ACCAGATTCGGAGAATGATGGAGACATTTCAGCAACTTAT TACTTATAAAGTCATAAGCAATGAATTTAACAGTCGAAAT CTAGTGAATGATGATGATGCCATTGTTGCTGCTTCGAAGT GCTTGAAAATGGTTTACTATGCAAATGTAGTGGGAGGGGA AGTGGACACAAATCACAATGAAGAAGATGATGAAGAGCCC ATCCCTGAGTCCAGCGAGCTGACACTTCAGGAACTTTTGG GAGAAGAAAGAAGAAACAAGAAAGGTCCTCGAGTGGACCC CCTGGAAACTGAACTTGGTGTTAAAACCCTGGATTGTCGA AAACCACTTATCCCTTTTGAAGAGTTTATTAATGAACCAC TGAATGAGGTTCTAGAAATGGATAAAGATTATACTTTTTT CAAAGTAGAAACAGAGAACAAATTCTCTTTTATGACATGT CCCTTTATATTGAATGCTGTCACAAAGAATTTGGGATTAT ATTATGACAATAGAATTCGCATGTACAGTGAACGAAGAAT CACTGTTCTCTACAGCTTAGTTCAAGGACAGCAGTTGAAT CCATATTTGAGACTCAAAGTTAGACGTGACCATATCATAG ATGATGCACTTGTCCGGCTAGAGATGATCGCTATGGAAAA TCCTGCAGACTTGAAGAAGCAGTTGTATGTGGAATTTGAA GGAGAACAAGGAGTTGATGAGGGAGGTGTTTCCAAAGAAT TTTTTCAGCTGGTTGTGGAGGAAATCTTCAATCCAGATAT TGGTATGTTCACATACGATGAATCTACAAAATTGTTTTGG TTTAATCCATCTTCTTTTGAAACTGAGGGTCAGTTTACTC TGATTGGCATAGTACTGGGTCTGGCTATTTACAATAACTG TATACTGGATGTACATTTTCCCATGGTTGTCTACAGGAAG CTAATGGGGAAAAAAGGAACTTTTCGTGACTTGGGAGACT CTCACCCAGTTCTATATCAGAGTTTAAAAGATTTATTGGA GTATGAAGGGAATGTGGAAGATGACATGATGATCACTTTC CAGATATCACAGACAGATCTTTTTGGTAACCCAATGATGT ATGATCTAAAGGAAAATGGTGATAAAATTCCAATTACAAA TGAAAACAGGAAGGAATTTGTCAATCTTTATTCTGACTAC ATTCTCAATAAATCAGTAGAAAAACAGTTCAAGGCTTTTC GGAGAGGTTTTCATATGGTGACCAATGAATCTCCCTTAAA GTACTTATTCAGACCAGAAGAAATTGAATTGCTTATATGT GGAAGCCGGAATCTAGATTTCCAAGCACTAGAAGAAACTA CAGAATATGACGGTGGCTATACCAGGGACTCTGTTCTGAT TAGGGAGTTCTGGGAAATCGTTCATTCATTTACAGATGAA CAGAAAAGACTCTTCTTGCAGTTTACAACGGGCACAGACA GAGCACCTGTGGGAGGACTAGGAAAATTAAAGATGATTAT AGCCAAAAATGGCCCAGACACAGAAAGGTTACCTACATCT CATACTTGCTTTAATGTGCTTTTACTTCCGGAATACTCAA GCAAAGAAAAACTTAAAGAGAGATTGTTGAAGGCCATCAC GTATGCCAAAGGATTTGGCATGCTGTAAAACAAAACAAAA CAAAATAAAACAAAAAAAAGGAAGGAAAAAAAAAGAAAAA ATTTAAAAAATTTTAAAAATATAACGAGGGATAAATTTTT GGTGGTGATAGTGTCCCAGTACAAAAAGGCTGTAAGATAG TCAACCACAGTAGTCACCTATGTCTGTGCCTCCCTTCTTT ATTGGGGACATGTGGGCTGGAACAGCAGATTTCAGCTACA TATATGAACAAATCCTTTATTATTATTATAATTATTTTTT TGCGTGAAAGTGTTACATATTCTTTCACTTGTATGTACAG AGAGGTTTTTCTGAATATTTATTTTAAGGGTTAAATCACT TTTGCTTGTGTTTATTACTGCTTGAGGTTGAGCCTTTTGA GTATTTAAAAAATATATACCAACAGAACTACTCTCCCAAG GAAAATATTGCCACCATTTGTAGACCACGTAACCTTCAAG TATGTGCTACTTTTTTGTCCCTGTATCTAACTCAAATCAG GAACTGTATTTTTTTTAATGATTTGCTTTTGAAACTTGAA GTCTTGAAAACAGTGTGATGCAATTACTGCTGTTCTAGCC CCCAAAGAGTTTTCTGTGCAAAATCTTGAGAATCAATCAA TAAAGAAAGATGGAAGGAAGGGAGAAATTGGAATGTTTTA ACTGCAGCCCTCAGAACTTTAGTAACAGCACAACAAATTA AAAACAAAAACAACTCATGCCACAGTATGTCGTCTTCATG TGTCTTGCAATGAACTGTTTCAGTAGCCAATCCTCTTTCT TAGTATATGAAAGGACAGGGATTTTTGTTCTTGTTGTTCT CGTTGTTGTTTTAAGTTTACTGGGGAAAGTGCATTTGGCC AAATGAAATGGTAGTCAAGCCTATTGCAACAAAGTTAGGA AGTTTGTTGTTTGTTTATTATAAACAAAAAGCATGTGAAA GTGCACTTAAGATAGAGTTTTTATTAATTACTTACTTATT ACCTAGATTTTAAATAGACAATCCAAAGTCTCCCCTTCGT GTTGCCATCATCTTGTTGAATCAGCCATTTTATCGAGGCA CGTGATCAGTGTTGCAACATAATGAAAAAGATGGCTACTG TGCCTTGTGTTACTTAATCATACAGTAAGCTGACCTGGAA ATGAATGAAACTATTACTCCTAAGAATTACATTGTATAGC CCCACAGATTAAATTTAATTAATTAATTCAAAACATGTTA AACGTTACTTTCATGTACTATGGAAAAGTACAAGTAGGTT TACATTACTGATTTCCAGAAGTAAGTAGTTTCCCCTTTCC TAGTCTTCTGTGTATGTGATGTTGTTAATTTCTTTTATTG CATTATAAAATAAAAGGATTATGTATTTTTAACTAAGGTG AGACATTGATATATCCTTTTGCTACAAGCTATAGCTAATG TGCTGAGCTTGTGCCTTGGTGATTGATTGATTGATTGACT GATTGTTTTAACTGATTACTGTAGATCAACCTGATGATTT GTTTGTTTGAAATTGGCAGGAAAAATGCAGCTTTCAAATC ATTGGGGGGAGAAAAAGGATGTCTTTCAGGATTATTTTAA TTAATTTTTTTCATAATTGAGACAGAACTGTTTGTTATGT ACCATAATGCTAAATAAAACTGTGGCACTTTTCACCATAA TTTAATTTAGTGGAAAAAGAAGACAATGCTTTCCATATTG TGATAAGGTAACATGGGGTTTTTCTGGGCCAGCCTTTAGA ACACTGTTAGGGTACATACGCTACCTTGATGAAAGGGACC TTCGTGCAACTGTAGTCATCTTAAAGGCTTCTCATCCACT GTGCTTCTTAATGTGTAATTAAAGTGAGGAGAAATTAAAT ACTCTGAGGGCGTTTTATATAATAAATTCGTGAAGA (NM000462.4),whichencodestheprotein: (SEQIDNo:16) MEKLHQCYWKSGEPQSDDIEASRMKRAAAKHLIERYYHQL TEGCGNEACTNEFCASCPTFLRMDNNAAAIKALELYKINA KLCDPHPSKKGASSAYLENSKGAPNNSCSEIKMNKKGARI DFKDVTYLTEEKVYEILELCREREDYSPLIRVIGRVFSSA EALVQSFRKVKQHTKEELKSLQAKDEDKDEDEKEKAACSA AAMEEDSEASSSRIGDSSQGDNNLQKLGPDDVSVDIDAIR RVYTRLLSNEKIETAFLNALVYLSPNVECDLTYHNVYSRD PNYLNLFIIVMENRNLHSPEYLEMALPLFCKAMSKLPLAA QGKLIRLWSKYNADQIRRMMETFQQLITYKVISNEFNSRN LVNDDDAIVAASKCLKMVYYANVVGGEVDTNHNEEDDEEP IPESSELTLQELLGEERRNKKGPRVDPLETELGVKTLDCR KPLIPFEEFINEPLNEVLEMDKDYTFFKVETENKFSFMTC PFILNAVTKNLGLYYDNRIRMYSERRITVLYSLVQGQQLN PYLRLKVRRDHIIDDALVRLEMIAMENPADLKKQLYVEFE GEQGVDEGGVSKEFFQLVVEEIFNPDIGMFTYDESTKLFW FNPSSFETEGQFTLIGIVLGLAIYNNCILDVHFPMVVYRK LMGKKGTFRDLGDSHPVLYQSLKDLLEYEGNVEDDMMITF QISQTDLFGNPMMYDLKENGDKIPITNENRKEFVNLYSDY ILNKSVEKQFKAFRRGFHMVTNESPLKYLFRPEEIELLIC GSRNLDFQALEETTEYDGGYTRDSVLIREFWEIVHSFTDE QKRLFLQFTTGTDRAPVGGLGKLKMIIAKNGPDTERLPTS HTCFNVLLLPEYSSKEKLKERLLKAITYAKGFGML (NP000453.2);

[0120] H sapiens UBE3A variant 3

TABLE-US-00015 (SEQIDNo:17) TTTTTCCGGATAAGGAAGCGCGGGTCCCGCATGAGCCCCG GCGGTGGCGGCAGCGAAAGAGAACGAGGCGGTGGCGGGCG GAGGCGGCGGGCGAGGGCGACTACGACCAGTGAGGCGGCC GCCGCAGCCCAGGCGCGGGGGCGACGACAGGTTAAAAATC TGTAAGAGCCTGATTTTAGAATTCACCAGCTCCTCAGAAG TTTGGCGAAATATGAGTTATTAAGCCTACGCTCAGATCAA GGTAGCAGCTAGACTGGTGTGACAACCTGTTTTTAATCAG TGACTCAAAGCTGTGATCACCCTGATGTCACCGAATGGCC ACAGCTTGTAAAAGATCAGGAGAACCTCAGTCTGACGACA TTGAAGCTAGCCGAATGAAGCGAGCAGCTGCAAAGCATCT AATAGAACGCTACTACCACCAGTTAACTGAGGGCTGTGGA AATGAAGCCTGCACGAATGAGTTTTGTGCTTCCTGTCCAA CTTTTCTTCGTATGGATAATAATGCAGCAGCTATTAAAGC CCTCGAGCTTTATAAGATTAATGCAAAACTCTGTGATCCT CATCCCTCCAAGAAAGGAGCAAGCTCAGCTTACCTTGAGA ACTCGAAAGGTGCCCCCAACAACTCCTGCTCTGAGATAAA AATGAACAAGAAAGGCGCTAGAATTGATTTTAAAGATGTG ACTTACTTAACAGAAGAGAAGGTATATGAAATTCTTGAAT TATGTAGAGAAAGAGAGGATTATTCCCCTTTAATCCGTGT TATTGGAAGAGTTTTTTCTAGTGCTGAGGCATTGGTACAG AGCTTCCGGAAAGTTAAACAACACACCAAGGAAGAACTGA AATCTCTTCAAGCAAAAGATGAAGACAAAGATGAAGATGA AAAGGAAAAAGCTGCATGTTCTGCTGCTGCTATGGAAGAA GACTCAGAGGCATCTTCCTCAAGGATAGGTGATAGCTCAC AGGGAGACAACAATTTGCAAAAATTAGGCCCTGATGATGT GTCTGTGGATATTGATGCCATTAGAAGGGTCTACACCAGA TTGCTCTCTAATGAAAAAATTGAAACTGCCTTTCTCAATG CACTTGTATATTTGTCACCTAACGTGGAATGTGACTTGAC GTATCACAATGTATACTCTCGAGATCCTAATTATCTGAAT TTGTTCATTATCGTAATGGAGAATAGAAATCTCCACAGTC CTGAATATCTGGAAATGGCTTTGCCATTATTTTGCAAAGC GATGAGCAAGCTACCCCTTGCAGCCCAAGGAAAACTGATC AGACTGTGGTCTAAATACAATGCAGACCAGATTCGGAGAA TGATGGAGACATTTCAGCAACTTATTACTTATAAAGTCAT AAGCAATGAATTTAACAGTCGAAATCTAGTGAATGATGAT GATGCCATTGTTGCTGCTTCGAAGTGCTTGAAAATGGTTT ACTATGCAAATGTAGTGGGAGGGGAAGTGGACACAAATCA CAATGAAGAAGATGATGAAGAGCCCATCCCTGAGTCCAGC GAGCTGACACTTCAGGAACTTTTGGGAGAAGAAAGAAGAA ACAAGAAAGGTCCTCGAGTGGACCCCCTGGAAACTGAACT TGGTGTTAAAACCCTGGATTGTCGAAAACCACTTATCCCT TTTGAAGAGTTTATTAATGAACCACTGAATGAGGTTCTAG AAATGGATAAAGATTATACTTTTTTCAAAGTAGAAACAGA GAACAAATTCTCTTTTATGACATGTCCCTTTATATTGAAT GCTGTCACAAAGAATTTGGGATTATATTATGACAATAGAA TTCGCATGTACAGTGAACGAAGAATCACTGTTCTCTACAG CTTAGTTCAAGGACAGCAGTTGAATCCATATTTGAGACTC AAAGTTAGACGTGACCATATCATAGATGATGCACTTGTCC GGCTAGAGATGATCGCTATGGAAAATCCTGCAGACTTGAA GAAGCAGTTGTATGTGGAATTTGAAGGAGAACAAGGAGTT GATGAGGGAGGTGTTTCCAAAGAATTTTTTCAGCTGGTTG TGGAGGAAATCTTCAATCCAGATATTGGTATGTTCACATA CGATGAATCTACAAAATTGTTTTGGTTTAATCCATCTTCT TTTGAAACTGAGGGTCAGTTTACTCTGATTGGCATAGTAC TGGGTCTGGCTATTTACAATAACTGTATACTGGATGTACA TTTTCCCATGGTTGTCTACAGGAAGCTAATGGGGAAAAAA GGAACTTTTCGTGACTTGGGAGACTCTCACCCAGTTCTAT ATCAGAGTTTAAAAGATTTATTGGAGTATGAAGGGAATGT GGAAGATGACATGATGATCACTTTCCAGATATCACAGACA GATCTTTTTGGTAACCCAATGATGTATGATCTAAAGGAAA ATGGTGATAAAATTCCAATTACAAATGAAAACAGGAAGGA ATTTGTCAATCTTTATTCTGACTACATTCTCAATAAATCA GTAGAAAAACAGTTCAAGGCTTTTCGGAGAGGTTTTCATA TGGTGACCAATGAATCTCCCTTAAAGTACTTATTCAGACC AGAAGAAATTGAATTGCTTATATGTGGAAGCCGGAATCTA GATTTCCAAGCACTAGAAGAAACTACAGAATATGACGGTG GCTATACCAGGGACTCTGTTCTGATTAGGGAGTTCTGGGA AATCGTTCATTCATTTACAGATGAACAGAAAAGACTCTTC TTGCAGTTTACAACGGGCACAGACAGAGCACCTGTGGGAG GACTAGGAAAATTAAAGATGATTATAGCCAAAAATGGCCC AGACACAGAAAGGTTACCTACATCTCATACTTGCTTTAAT GTGCTTTTACTTCCGGAATACTCAAGCAAAGAAAAACTTA AAGAGAGATTGTTGAAGGCCATCACGTATGCCAAAGGATT TGGCATGCTGTAAAACAAAACAAAACAAAATAAAACAAAA AAAAGGAAGG (AK292514.1).

Example 6In Vitro Testing of Human UBE3A Vector Construct

[0121] Human vector properties were tested in HEK293 cells (American Type Culture Collection, Manassas, Va.), grown at 37 C. 5% CO.sub.2 in DMEM with 10% FBS and 1% Pen/Strep and subcultured at 80% confluence.

[0122] The vector (2 g/well in a 6-well plate) was transfected into the cells using PEI transfection method. The cells were subcultured at 0.510.sup.6 cells per well in a 6-well plate with DMEM medium two days before the transfection. Medium was replaced the night before transfection. Endotoxin-free dH.sub.2O was heated to at around 80 C., and polyethylenimine (Sigma-Aldrich Co. LLC, St. Louis, Mo.) dissolved. The solution was allowed to cool to around 25 C., and the solution neutralized using sodium hydroxide. AAV4-STUb vector or negative control (medium only) was added to serum-free DMEM at 2 g to every 200 l for each well transfected, and 9p of 1 g/l polyethylenimine added to the mix for each well. The transfection mix was incubated at room temperature for 15 minutes, then added to each well of cells at 210 l per well and incubated for 48 hours. Cells and media were harvested by scraping the cells from the plates. The medium and cells were then centrifuged at 5000g for 5 minutes.

[0123] For Western blotting of the extracts, cell pellets were resuspended in 50 L of hypo-osmotic buffer and the cells lysed by three repeated freeze/thaws. 15 L of lysate was heated with Lamelli sample buffer and run on a BioRad 4-20% acrylamide gel. Transferred to nitrocellulose membrane using a TransBlot. The blot was blocked with 5% milk and protein detected using an anti-E6AP antibody.

[0124] As seen in FIG. 22, cells transfected with the construct express the UBE3A gene, i.e. E6-AP. Furthermore, appending the gene to the various secretion signals exhibited mixed results, based on the secretion signal peptide. For example, transfection using constructs based on the GDNF secretion signal exhibited less expression and no detectable secretion from the transfected cells, as seen in FIG. 23. Use of the insulin secretion signal resulted in moderate secretion of E6AP from transfected cells, along with high expression of the construct within the cell. The results of insulin-signal secretion were confirmed using an HA-tagged construct, as seen in FIG. 24.

Example 7Efficacy of Secretion Peptides

[0125] The efficacy of secretion peptides in promoting extracellular secretion of the protein by neurons was measured by creating plasmid constructs containing the various secretion signals, GFP or a human Ube3A version 1 (hUbev1) gene, and the CPP TATk, as seen in FIGS. 25(A) and 26(A). GFP was generated to use as a reporter gene for in vivo testing and to act as a control to hUbev1 in future AS studies. The secretion signals tested in this experiment were GDNF secretion signal, human insulin secretion signal, and IgK secretion signal. The amino acid sequences for the secretion signals are as follows;

TABLE-US-00016 forinsulin: (SEQIDNO:18) MALWMRLLPLLALLALWGPDPAAA (CAA08766.1); forGDNF: (SEQIDNO:3) MKLWDVVAVCLVLLHTASA; forIgK: (SEQIDNO:19) METDTLLLWVLLLWVPGSTG (AAH80787.1).

[0126] The plasmid constructs containing the various secretion signals were generated and gel electrophoresis run to confirm successful gene insertion for each plasmid. As seen in FIGS. 25(B) and 26(B), both GFP and hUbev1 were successfully integrated into the plasmids. The efficacy of the selected secretion signals in inducing secretion of peptide by neurons was measured by transfecting the plasmid constructs into HEK293 cells and measuring the concentration of GFP in the media via dot blot. Extracts from the media were collected and X l were placed onto nitrocellulose paper, followed by immunostaining. The results indicate that insulin signal resulted in moderate extracellular protein levels, and strong to high extracellular protein levels with IgK and GDNF signals, as seen in FIGS. 25(C) and 26(C). Thus, each signal is effective at inducing secretion of peptide in neurons, and that the hUbev1/GDNF signal-containing plasmid was particularly effective at inducing secretion of E6-AP.

Example 8Efficacy of Cell Penetrating Peptide

[0127] The efficacy of the select CPP signals in inducing reuptake of the protein by neurons was measured by creating plasmid constructs containing the secretion signal (GDNF), the hUbev1 gene, and the various CPP signals, outlined below, and transfecting them into HEK293 cells.

TABLE-US-00017 (SEQIDNO:20) forpenetratin: RQIKIWFQNRRMKWKK; (SEQIDNO:12) forTATk: YARKAARQARA; (SEQIDNO:21) forR6W3: RRWWRRWRR; (SEQIDNO:22) forpVEC LLIILRRRIRKQAHAHSK.

[0128] The cell lyses from these cells was then taken and added to new cell cultures of HEK293 cells and the concentration of E6-AP in these cells after incubation measured via Western blot. Results of the uptake for the CPP signals penetratin, TATk, R6RW, and pVEC are seen in FIG. 27.

Example 9In Vivo Testing of Human UBE3A Vector Construct in Mouse Model

[0129] To ensure that the Ube3A gene modified to include secretion and reuptake signals maintained its ability to improve cognitive deficits associated with AS, a plasmid construct (hSTUb) containing human Ube3A version 1 (hUbev1), a secretion signal, and the CPP TATk was transduced via an rAAV vector into mouse models of AS. Long-term potentiation of the murine brain was measured via electrophysiology post-mortem and compared to GFP-transfected AS model control mice and wild-type control mice. The results indicate that the hSTUb plasmid successfully rescued LTP deficits, as seen in FIGS. 28(A) and (B).

Example 10Human UBE3A Vector Construct as Gene Therapy in Mouse Model

[0130] The potential of secretion and CPP signal peptides were analyzed for their ability to promote greater global distribution of E6-AP in neurons for use in a gene therapy for AS. Rescue of LTP by the hSTUb plasmid in the mouse model suggests that the UBE3A gene retains its efficacy in treating cognitive deficits in AS following the addition of secretion and CPP signals, supporting the potential of the construct in a gene therapy. The GDNF signal presents as the optimal signal for utilization in this proposed therapy as indicated by its plasmid construct showing the most secretion of E6-AP into media following transduction. Failure of the CPP signals to induce measurable reuptake of E6-AP after the application of cell lyses to the cells may be due to several factors, including insufficient concentration of E6-AP in the lyses.

Example 11Prophetic Human Gene Therapy

[0131] A human child presents with severe developmental delay that becomes apparent around the age of 12 months. The child later presents with absent speech, seizures, hypotonia, ataxia and mricrocephaly. The child moves with a jerky, puppet like gait and displays an unusually happy demeanor that is accompanied by laughing spells. The child has dysmorphic facial features characterized by a prominent chin, an unusually wide smile and deep-set eyes. The child diagnoses with Angelman's Syndrome. The child is treated with a therapeutically effective amount of UBE3A vector which is injected bilaterally into the left and right hippocampal hemispheres of the brain. Improvement is seen in the symptoms after treatment with a decrease in seizures, increased muscle tone, increased coordination of muscle movement and improvement in speech.

[0132] The UBE3A vector is formed from cDNA cloned from a Homo sapiens UBE3A gene. The UBE3A, version 1 gene (SEQ ID No: 9) is fused to a gene encoding a secretion signaling peptide, in this case GDNF, although insulin or IgK may also be used. The construct is inserted into the hSTUb vector, under a CMV chicken-beta actin hybrid promoter or human ubiquitin c promoter. Woodchuck hepatitis post-transcriptional regulatory element (WPRE) is present to increase expression levels.

[0133] The UBE3A-seretion signal construct is attached to a cellular uptake peptide (cell penetrating peptide or CPP) such as HIV TAT or HIV TATk. The human UBE3A vector is then transformed into E. coli using the heat shock method described in Example 2. The transformed E. coli were expanded in broth containing ampicillin to select for the vector and collect large amounts of vector.

[0134] In the preceding specification, all documents, acts, or information disclosed does not constitute an admission that the document, act, or information of any combination thereof was publicly available, known to the public, part of the general knowledge in the art, or was known to be relevant to solve any problem at the time of priority.

[0135] The disclosures of all publications cited above are expressly incorporated herein by reference, each in its entirety, to the same extent as if each were incorporated by reference individually.

[0136] While there has been described and illustrated specific embodiments of a method of treating UBE3A deficiencies, it will be apparent to those skilled in the art that variations and modifications are possible without deviating from the broad spirit and principle of the present invention. It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween.