MODIFIED UBE3A GENE FOR A GENE THERAPY APPROACH FOR ANGELMAN SYNDROME

Abstract

Angelman Syndrome (AS) is a genetic disorder occurring in approximately one in every 15,000 births. It is characterized by severe mental retardation, seizures, difficulty speaking and ataxia. The gene responsible for AS was discovered to be UBE3A and encodes for E6-AP, an ubiquitin ligase. 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. While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. We have generated a Ube3a protein with additional sequences that should allow the secretion from cells and uptake by neighboring neuronal cells. This would 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, wherein the UBE3A sequence is SEQ ID No. 1, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, a cDNA of SEQ ID No. 7, or a homologous sequence; a secretion sequence disposed downstream of the transcription initiation sequence, wherein the secretion sequence is SEQ ID No. 2, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, a cDNA of SEQ ID No. 3, or a homologous sequence; and a cell uptake sequence disposed downstream of the transcription initiation sequence, wherein the cell uptake sequence is SEQ ID No. 4, SEQ ID No. 11, a cDNA of SEQ ID No. 5, 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 5, wherein the recombinant adeno-associated virus serotype 2-based plasmid is a pTR plasmid.

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

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

9. A method of synthesizing a UBE3A vector, comprising: providing a backbone plasmid; wherein backbone plasmid has a transcription initiation sequence; forming a UBE3A construct, further comprising: providing a UBE3A sequence, wherein the UBE3A sequence is SEQ ID No. 1, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, a cDNA of SEQ ID No. 7, or a homologous sequence; appending a secretion sequence to the UBE3A sequence, wherein the secretion sequence is SEQ ID No. 2, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, a cDNA of SEQ ID No. 3, or a homologous sequence; and appending a cell uptake sequence to the XXX, wherein the cell uptake sequence is SEQ ID No. 4, SEQ ID No. 11, a cDNA of SEQ ID No. 5, or a homologous sequence; inserting the UBE3A construct downstream of the transcription initiation sequence.

10. The method of claim 9, further comprising: inserting the vector into an amplifaction host; subjecting the amplifaction host to an antibiotic selection; where the backbone plasmid has an antibiotic resistance gene; expanding the amplifaction host in a medium containing the antibiotic selection; collecting the expanded amplifaction host; and isolating the vector from the amplifaction host.

11. The method of claim 10, wherein the antibiotic resistance gene is an ampicillin resistance gene, and wherein the antibiotic selection is ampicillin selection.

12. The method of claim 9, further comprising: cleaving the backbone plasmid with at least one endonuclease; and ligating the UBE3A construct to the cleaved ends of the backbone plasmid.

13. The method of claim 9, 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.

14. A method of treating a UBE3A deficiency disease, comprising: administering a vector to the brain of a patient suffering from the UBE3A deficiency disease, where the vector comprises: a transcription initiation sequence; a UBE3A sequence disposed downstream of the transcription initiation sequence, wherein the UBE3A sequence is SEQ ID No. 1, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, a cDNA of SEQ ID No. 7, or a homologous sequence; a secretion sequence disposed downstream of the transcription initiation sequence, wherein the secretion sequence is SEQ ID No. 2, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, a cDNA of SEQ ID No. 3, or a homologous sequence; a cell uptake sequence disposed downstream of the transcription initiation sequence, wherein the cell uptake sequence is SEQ ID No. 4, SEQ ID No. 11, a cDNA of SEQ ID No. 5, or a homologous sequence; and wherein the UBE3A deficiency disease is Angelman syndrome, Prader-Willi syndrome, or Huntington's disease.

15. The method of claim 14, wherein the administering a vector to the brain comprises injecting the vector into the brain.

16. The method of claim 15, wherein the vector is injected into the hippocampus or ventricle.

17. The method of claim 16, wherein the vector is injected bilaterally.

18. The method of claim 14, wherein the vector is administered at about 5.55?10.sup.11 to about 2.86?10.sup.12 genomes/g brain mass.

19. The method of claim 14, wherein the vector is administered at 5.55?10.sup.11 to 2.86?10.sup.12 genomes/g brain mass, 2.86?10.sup.12 genomes/g brain mass, 2.40?10.sup.12 genomes/g brain mass, 9.80?10.sup.11 genomes/g brain mass, or 5.55?10.sup.11 genomes/g brain mass.

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] 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:

[0023] FIG. 1 is a dot blot of anti-GFP on media from HEK293 cells transfected with GFP clones containing signal peptides as indicated.

[0024] FIG. 2 is a map of the mouse UBE3A vector construct used in the present invention. Major genes are noted.

[0025] 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.

[0026] 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

[0027] 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.

[0028] 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), 3rd 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.

[0029] FIG. 7 is a microscopic image of anti-E6-AP staining in an uninjected AS mouse.

[0030] 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), 3rd ventricle).

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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 (3rd 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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 3rd ventricle (3V) in the brain showing Ube3a expression after AAV4-STUb delivery.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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) 3rd ventricle (3V) of Ube3a expression after AAV4-STUb delivery.

[0043] FIG. 21 is a map of the human UBE3A vector construct used in the present invention. Major genes are noted.

[0044] FIG. 22 is a Western blot of HEK293 cell lysate transfected with hSTUb construct. The proteins were stained with anti-E6AP.

[0045] 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.

[0046] FIG. 24 is a dot blot confirming insulin signal secretion using anti-HA tag antibody.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0047] 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.

[0048] 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

[0049] 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 oral, parenteral (referring to intravenous and intraarterial and other appropriate parenteral routes), intratheceally, intramuscularly, subcutaneously, colonically, rectally, and nasally, 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. 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 thereapeutic needed or useful to accomplish the treatment described herein.

[0050] 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. 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.

[0051] 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. Non-limiting 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.

[0052] 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.

[0053] 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.

[0054] 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.

Example 1

[0055] To test the efficacy of the secretion signal, GFP was cloned in frame with human insulin, GDNF or IgK signal peptides. 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.

[0056] The vector (2 ?g/well in a 6-well plate) was transfected into the cells using PEI transfection method. The cells were subcultured at 0.5?10.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 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 then added to each well of cells at 210 ?L per well and incubated for 48 hours.

[0057] 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 temperatire, and incubated with luminescence reagent for 1 minute (Millipore, Merck KGaA, Darmstadt, Del.; #WBKLS0100). The membrane was recorded on a GE Amersham Imager 600 (General Electric, Fairfield, Calif.), shown in FIG. 1.

[0058] 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 2

[0059] 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-00002 (SEQIDNo.1) 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.

[0060] The cDNA was subcloned and sequenced. The mouse UBE3A gene (SEQ ID No. 1) was fused to DNA sequences encoding a section signaling peptide (SEQ ID No. 2) and HIV TAT sequence (SEQ ID No. 4). The section signaling peptide has the DNA sequence;

TABLE-US-00003 (SEQIDNo.2) atggccctgttggtgcacttcctacccctgctggcc ctgcttgccctctgggagcccaaacccacccaggct tttgtc, encodingtoproteinsequence; (SEQIDNo.3) MALLVHFLPLLALLALWEPKPTQAFV;
while HIV TAT sequence is;

TABLE-US-00004 (SEQIDNo.4) tacggcagaaagaagaggaggcagagaaggaga, encodingtoproteinsequence; (SEQIDNo.5) YGRKKRRQRRR.

[0061] The construct sequence of SEQ ID No. 1 fused with SEQ ID No. 2 and SEQ ID No. 4 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.

[0062] 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 preior to being plated onto agar, such as ATCC medium 1065 (American Type Culture Collection, Manassas, Va.), with ampicillin (50 ?g/mL).

[0063] E. coli was expanded in broth containing ampicillin to collect large amounts of vector.

Example 3

[0064] 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.

[0065] The vector (2 ?g/well in a 6-well plate) was transfected into the cells using PEI transfection method. The cells were subcultured at 0.5?10.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 9 ?l of 1 ?g/?; polyethylenimine added to the mix for each well. The transfection mix was incubated at room temperature for 15 minutes, then then added to each well of cells at 210 ?l per well and incubated for 48 hours.

[0066] 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 4

[0067] 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.

[0068] 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 sagitally 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 1?10.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.

[0069] 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.

[0070] 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 service as a supply of protein for other sites throughout the brain.

[0071] 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.).

[0072] 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%.

[0073] 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 epxression, 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.

[0074] Detailed analysis of the cornocal cross sections of Anegelman syndrome-model mice confirmed that administration of the UBE3A construct increased levels of E6-AP in and around the the lateral ventricle, as seen in FIGS. 14 through 20.

Example 5

[0075] A human vector construct was generated using a pTR plasmid. A Homo sapien UBE3A gene was formed from cDNA (AH005553.1);

TABLE-US-00005 (SEQIDNo.6) ggagtagtttactgagccactaatctaaagtttaatactg tgagtgaataccagtgagtacctttgttaatgtggataac caatacttggctataggaagttttttagttgtgtgtttta tnacacgtatttgactttgtgaataattatggcttataat ggcttgtctgttggtatctatgtatagcgtttacagtttc ctttaaaaaacatgcattgagttttttaatagtccaaccc ttaaaataaatgtgttgtatggccacctgatctgaccact ttctttcatgttgacatctttaattttaaaactgttttat ttagtgcttaaatcttgttnacaaaattgtcttcctaagt aatatgtctacctttttttttggaatatggaatattttgc taactgtttctcaattgcattttacagatcaggagaacct cagtctgacgacattgaagctagccgaatgtaagtgtaac ttggttgagactgtggttcttattttgagttgccctagac tgctttaaattacgtcacattatttggaaataatttctgg ttaaaagaaaggaatcatttagcagtaaatgggagatagg aacatacctactttttttcctatcagataactctaaacct cggtaacagtttactaggtttctactactagatagataaa tgcacacgcctaaattcttagtctttttgcttccctggta gcagttgtagggaaatagggaggttgaggaaagagtttaa cagtctcaacgcctaccatatttaaggcatcaagtactat gttatagatacagagatgcgtaataattagttttcaccct acagaaatttatattatactcaagagtgaaagatgcagaa gcaaataatttcagtcactgaggtagaatggtatccaaaa tacaatagtaacatgaaggagtactggagtaccaggtatg caataggaatctagtgtagatggcagggaagtaagagtgg ccaggaaatgctaagttcagtcttgaaatgtgactgggaa tcaggcagctatcaactataagtcaaatgtttacaagctg ttaaaaatgaaatactgattatgtaaaagaaaaccggatt gatgctttaaatagactcattttcntaatgctaattttta aaatgatagaatcctacaantcttagctgtaaaccttgtg atttttcagctgttgtactaaacaacttaagcacatatac catcagacaagcccccntccccccttttaaaccaaaggaa tgtatactctgttaatacagtcagtaagcattgacattct ttatcataatatcctagaaaatatttattaactatttcac tagtcaggagttgtggtaaatagtgcatctccattttcta cttctcatcttcatacacaggttaatcacttcagtgcttg actaacttttgccttgatgatatgttgagctttgtacttg agagctgtactaatcactgtgcttattgtttgaatgtttg gtacaggaagcgagcagctgcaaagcatctaatagaacgc tactaccaccagttaactgagggctgtggaaatgaagcct gcacgaatgagttttgtgcttcctgtccaacttttcttcg tatggataataatgcagcagctattaaagccctcgagctt tataagattaatgcaaaactctgtgatcctcatccctcca agaaaggagcaagctcagcttaccttgagaactcgaaagg tgcccccaacaactcctgctctgagataaaaatgaacaag aaaggcgctagaattgattttaaaggtaagatgttttatt ttcaattgagaattgttgcctgaaaaccatgtgggagatt taaatgtattagtttttatttgttttttcttctgtgacat aaagacattttgatatcgtagaaccaattttttattgtgg taacggacaggaataataactacattttacaggtctaatc attgctaattagaagcagatcatatgccaaaagttcattt gttaatagattgatttgaactttttaaaattcttaggaaa aatgtattaagtggtagtgaatctccaaaactatttaaga gctgtattatgattaatcagtacatgacatattggttcat atttataattaaagctatacattaatagatatcttgatta taaagaaagtttaaactcatgatcttattaagagttatac attgttgaaagaatgtaaaagcatgggtgaggtcattggt ataggtaggtagttcattgaaaaaaataggtaagcattaa attttgtttgctgaatctaagtattagatactttaagagt tgtatatcataaatgatattgagcctagaatgtttggctg ttttacttttagaactttttgcaacagagtaaacatacat attatgaaaataaatgttctcttttttcctctgattttct agatgtgacttacttaacagaagagaaggtatatgaaatt cttgaattatgtagagaaagagaggattattcccctttaa tccgtgttattggaagagttttttctagtgctgaggcatt ggtacagagcttccggaaagttaaacaacacaccaaggaa gaactgaaatctcttcaagcaaaagatgaagacaaagatg aagatgaaaaggaaaaagctgcatgttctgctgctgctat ggaagaagactcagaagcatcttcctcaaggataggtgat agctcacagggagacaacaatttgcaaaaattaggccctg atgatgtgtctgtggatattgatgccattagaagggtcta caccagattgctctctaatgaaaaaattgaaactgccttt ctcaatgcacttgtatatttgtcacctaacgtggaatgtg acttgacgtatcacaatgtatactctcgagatcctaatta tctgaatttgttcattatcgtaatggagaatagaaatctc cacagtcctgaatatctggaaatggctttgccattatttt gcaaagcgatgagcaagctaccccttgcagcccaaggaaa actgatcagactgtggtctaaatacaatgcagaccagatt cggagaatgatggagacatttcagcaacttattacttata aagtcataagcaatgaatttaacagtcgaaatctagtgaa tgatgatgatgccattgttgctgcttcgaagtgcttgaaa atggtttactatgcaaatgtagtgggaggggaagtggaca caaatcacaatgaagaagatgatgaagagcccatccctga gtccagcgagctgacacttcaggaacttttgggagaagaa agaagaaacaagaaaggtcctcgagtggaccccctggaaa ctgaacttggtgttaaaaccctggattgtcgaaaaccact tatcccttttgaagagtttattaatgaaccactgaatgag gttctagaaatggataaagattatacttttttcaaagtag aaacagagaacaaattctcttttatgacatgtccctttat attgaatgctgtcacaaagaatttgggattatattatgac aatagaattcgcatgtacagtgaacgaagaatcactgttc tctacagcttagttcaaggacagcagttgaatccatattt gagactcaaagttagacgtgaccatatcatagatgatgca cttgtccgggtaagttgggctgctagattaaaaacctaat aatggggatatcatgatacagttcagtgaattcattttaa aagtgactgaaaaaaatgataccatatagcataggaacac atggacatttctgatcttatataagtattatacttttgtt gttcctgtgcaagtttatagatgtgttctacaaagtatcg gttgtattatataatggtcatgctatctttgaaaaagaat gggttttctaaatcttgaaaactaaatccaaagtttcttt cattcagaagagaatagagtgttggacaaagaccagaaca agagaaatgtggagatacccaataataagtgtggatgtgc agtcttgaactgggagtaatggtacagtaaaaccatacca taaaattataggtagtgtccaaaaaattccatcgtgtaaa attcagagttgcattattgtggacttgaagaagcagttgt atgtgggacggtatcgataagcttgatatcgaattcctgc agcccgggggatccactagtgtggtaattaatactaagtc ttactgtgagagaccataaactgctttagtattcagtgta tttttcttaattgaaatatttaacttatgacttagtagat actaagacttaacccttgagtttctattctaataaaggac tactaatgaacaattttgaggttagacctctactccattg tttttgctgaaatgatttagctgcttttccatgtcctgtg tagtccagacttaacacacaagtaataaaatcttaattaa ttgtatgttaatttcataacaaatcagtaaagttagcttt ttactatgctagtgtctgttttgtgtctgtctttttgatt atctttaagactgaatctttgtcttcactggctttttatc agtttgctttctgtttccatttacatacaaaaagtcaaaa atttgtatttgtttcctaatcctactccttgtttttattt tgtttttttcctgatactagcaatcatcttcttttcatgt ttatcttttcaatcactagctagagatgatcgctatggaa aatcctgcagacttgaagaagcagttgtatgtggaatttg aaggagaacaaggagttgatgagggaggtgtttccaaaga attttttcagctggttgtggaggaaatcttcaatccagat attggtaaatacattagtaatgtgattatggtgtcgtatc atcttttgagttagttatttgtttatcttactttgtaaat attttcagctatgaagagcagcaaaagaaggatttggtat ggattacccagaatcacacatcatgactgaatttgtaggt tttaggaactgatttgtatcactaatttattcaaattctt ttatttcttagaaggaatattctaatgaaggaaattatct ctttggtaaactgaattgaaagcactttagaatggtatat tggaacagttggagggatttctttgctttttgttgtctaa aaccatcatcaaactcacggttttcctgacctgtgaactt caaagaacaatggtttgaagagtattgagagactgtctca caagtatgtcatgctcaaagttcagaaacactagctgata tcacattaattaggtttatttgctataagatttcttgggg cttaatatangtagtgttcccccaaactttttgaactcca gaactcttttctgccctaacagtagctactcaggagctga ggcaggagaattgtttgaacctaggaggcagaggttgcag tgagctgagatcgtgccactccagcccacccctgggtaac agagcgagactccatctcaaagaaaaaaatgaaaaattgt tttcaaaaatagtacgtgtggtacagatataagtaattat atttttataaatgaaacactttggaaatgtagccattttt tgtttttttatgtttatttttcagctatgggtggataaag catgaatataacttttcttatgtgttagtagaaaattaga aagcttgaatttaattaacgtatttttctacccgatgcca ccaaattacttactactttattcctttggcttcataaaat tacatatcaccattcaccccaatttatagcagatatatgt ggacattgttttctcaagtgctaatataatagaaatcaat gttgcatgcctaattacatatattttaaatgttttatatg cataattattttaagtttatatttgtattattcatcagtc cttaataaaatacaaaagtaatgtatttttaaaaatcatt tcttataggtatgttcacatacgatgaatctacaaaattg ttttggtttaatccatcttcttttgaaactgagggtcagt ttactctgattggcatagtactgggtctggctatttacaa taactgtatactggatgtacattttcccatggttgtctac aggaagctaatggggaaaaaaggaacttttcgtgacttgg gagactctcacccagtaagttctttgtcatttttttaatt cagtctcttagattttatttaaatgcaaaaatttaattta tgtcaaaattttaaagtttttgtttagaatctttgttgat actcttatcaataagataaaaatgttttaatctgaccgaa gtaccagaaacacttaaaaactcaaagggggacattttta tatattgctgtcagcacgaagctttcgtaagattgatttc atagagaagtgtttctaaacattttgtttgtgttttagtg aaatcttaagagataggtaaaaatcagagtagccctggct aagggtcttggtagttacaacgagtgtgcctgctcctacc acccccacccccaccttgagacaccacagaatttctcata gagcacagtgtgaattctattgctaaattggtggtatggg gtttctcagcagagaatgggacatcacagtgactgacaat ctttcttttataggttggaaactatttgggggactggagg gatactgtctacactttttacaatttttattgataagatt tttgttgtcttctaagaagagtgatataaattatttgttg tattttgtagttctatggtggcctcaatttaccatttctg gttgctaggttctatatcagagtttaaaagatttattgga gtatgaagggaatgtggaagatgacatgatgatcactttc cagatatcacagacagatctttttggtaacccaatgatgt atgatctaaaggaaaatggtgataaaattccaattacaaa tgaaaacaggaaggtaataaatgtttttatgtcacatttt gtctcttcattaacactttcaaagcatgtatgcttataat ttttaaagaagtatctaatatagtctgtacaaaaaaaaaa caagtaactaagtttatgtaaatgctagagtccacttttc taaatcttggatataagttggtatgaaagcacacagttgg gcactaaagccccttttagagaaagaggacatgaagcagg agatagttaatagctaagtgtggttgtagtataaagcaag aagcagggtgtttcttgtattaagctgtaagcaggaacct catgattaaggtctttatcacagaacaaataaaaattaca tttaatttacacatgtatatcctgtttgtgataaaaatac atttctgaaaagtatactttacgtcagatttgggttctat tgactaaaatgtgttcatcgggaatgggaataacccagaa cataacaagcaaaaaattatgacaaatatatagtatacct ttaagaaacatgtttatattgatataattttttgattaaa tattatacacactaagggtacaangcacattttcctttta tganttngatacagtagtttatgtgtcagtcagatacttc cacatttttgctgaactggatacagtaagcagcttaccaa atattctatggtagaaaactnggacttcctggtttgctta aatcaaatatattgtactctcttaaaacggttggcattta taaatagatggatacatggtttaaatgtgtctgttnacat acctagttgagagaacctaaagaattttctgcgtctccag catttatattcagttctgtttaatacattatcgaaattga catttataagtatgacagttttgtgtatatggccttttca tagcttaatattggctgtaacagagaattgtgaaattgta agaagtagttttctttgtaggtgtaaaattgaatttttaa gaatattcttgacagttttatgtatatggccttttcatag cttaatattggctataacagagaattgtgaaattgttaag aagtaggtgtaaaattgaatttttaagaatattcttgaat gtttttttcttggaaaaattaaaaagctatgcagcccaat aacttgtgttttgtttgcatagcatattataagaagttct tgtgattaatgttttctacaggaatttgtcaatctttatt ctgactacattctcaataaatcagtagaaaaacagttcaa ggcttttcggagaggttttcatatggtgaccaatgaatct cccttaaagtacttattcagaccagaagaaattgaattgc ttatatgtggaagccgggtaagaaagcaggtgtctgcaaa aagtcatgtatcgatttattgtttgtaatgatacagtagt atagcagataactaagacatattttcttgaatttgcagaa tctagatttccaagcactagaagaaactacagaatatgac ggtggctataccagggactctgttctgattaggtgaggta cttagttcttcagaggaagatttgattcaccaaaggggtg tgtgattttgcttcagacctttatctctaggtactaattc ccaaataagcaaactcacaaattgtcatctatatacttag atttgtatttgtaatataatcaccatttttcagagctaat cttgtgatttatttcatgaatgaagtgttgttatatataa gtctcatgtaatctcctgcatttggcgtatggattatcta gtattcctcactggttagagtatgcttactgctggttaga agataattaaaataaggctaccatgtctgcaatttttcct ttcttttgaactctgcatttgtgaactgttacatggcttc ccaggatcaagcactttttgagtgaaatggtagtctttta tttaattcttaagataatatgtccagatacatactagtat ttccattttacaccctaaaaaactaagccctgaattctca cagaaagatgtagaggttcccagttctatctgcttttaaa caaatgcccttactactctactgtctacttctgtgtacta catcatcgtatgtagttgtttgcatttgggccagttggtt ggggcaggggtctttttttcttttgtcccttaatctgtat cactttttcctcccaaagttgagttaaaggatgagtagac caggagaataaaggagaaaggataaataaaatatataccc aaaggcacctggagttaatttttccaaatattcatttcag tctttttcaattcataggattttgtcttttgctcattact gactgcataatgtgattataccatagtttaaatagtcact tcctgttactacacacttgggttttctcaattttttacta ttgtagtactaatattttactatattgtaatctaatccaa atttttacgtattcagagctgttcaggataaatttgcttg gaaatttttaaatcaccagaagtgatactatcctgataat taacttccaagttgtctcttaatatagttttaatgcaaat cataagcttatgttagtaccagtcataatgaatgccaaac tgaaaccagtattgtattttttctcattagggagttctgg gaaatcgttcattcatttacagatgaacagaaaagactct tcttgcagtttacaacgggcacagacagagcacctgtggg aggactaggaaaattaaagatgattatagccaaaaatggc ccagacacagaaaggtaggtaattattaacttgtgactgt atacctaccgaaaaccttgcattcctcgtcacatacatat gaactgtctttatagtttctgagcacattcgtgattttat atacaaatccccaaatcatattagacaattgagaaaatac tttgctgtcattgtgtgaggaaacttttaagaaattgccc tagttaaaaattattatggggctcacattggtttggaatc aaattagtgtgattcatttacttttttgattcccagcttg ttaattgaaagccatataacatgatcatctatttagaatg gttacattgaggctcggaagattatcatttgattgtgcta gaatcctgttatcaaatcattttcttagtcatattgccag cagtgtttctaataagcatttaagagcacacactttgcag tcttgtaaaacaggtttgagtattttctccaccttagagg aagttacttgacttctcagtgacctaacctctaaagtgca tttactgatgtcctctctgtggttttgttgtggaaagatt tagttaaatgaactgtaagaattcagtacctaaaatggta tctgttatgtagtaaaaactcaatggatacagtatcttat catcgtcactagctttgagtaatttataggataaaggcaa cttggtagttacacaacaaaaagtttatgatttgcattaa tgtatagtttgcattgcagaccgtctcaactatatacaat ctaaaaataggagcatttaattctaagtgtatttcccatg acttacagttttcctgtttttttccccttttctctattta ggttacctacatctcatacttgctttaatgtgcttttact tccggaatactcaagcaaagaaaaacttaaagagagattg ttgaaggccatcacgtatgccaaaggatttggcatgctgt aaaacaaaacaaaacaaaataaaacaaaaaaaaggaagga aaaaaaaagaaaaaatttaaaaaattttaaaaatataacg agggataaatttt, whichencodesfor; (SEQIDNo.7) MKRAAAKHLIERYYHQLTEGCGNEACTNEFCASCPTFLRMDNNAAAI KALELYKINAKLCDPHPSKKGASSAYLENSKGAPNNSCSEIKMNKKG ARIDFKDVTYLTEEKVYEILELCREREDYSPLIRVIGRVFSSAEALV QSFRKVKQHTKEELKSLQAKDEDKDEDEKEKAACSAAAMEEDSEASS SRIGDSSQGDNNLQKLGPDDVSVDIDAIRRVYTRLLSNEKIETAFLN ALVYLSPNVECDLTYHNVYSRDPNYLNLFIIVMENRNLHSPEYLEMA LPLFCKAMSKLPLAAQGKLIRLWSKYNADQIRRMMETFQQLITYKVI SNEFNSRNLVNDDDAIVAASKCLKMVYYANVVGGEVDTNHNEEDDEE PIPESSELTLQELLGEERRNKKGPRVDPLETELGVKTLDCRKPLIPF EEFINEPLNEVLEMDKDYTFFKVETENKFSFMTCPFILNAVTKNLGL YYDNRIRMYSERRITVLYSLVQGQQLNPYLRLKVRRDHIIDDALVRL EMIAMENPADLKKQLYVEFEGEQGVDEGGVSKEFFQLVVEEIFNPDI GMFTYDESTKLFWFNPSSFETEGQFTLIGIVLGLAIYNNCILDVHFP MVVYRKLMGKKGTFRDLGDSHPVLYQSLKDLLEYEGNVEDDMMITFQ ISQTDLFGNPMMYDLKENGDKIPITNENRKEFVNLYSDYILNKSVEK QFKAFRRGFHMVTNESPLKYLFRPEEIELLICGSRNLDFQALEETTE YDGGYTRDSVLIREFWEIVHSFTDEQKRLFLQFTTGTDRAPVGGLGK LKMIIAKNGPDTERLPTSHTCFNVLLLPEYSSKEKLKERLLKAITYA KGFGML.

[0076] The cDNA was subcloned and sequenced. The UBE3A, variant 1 gene (SEQ ID No. 6) was fused to one of three genes encoding a section signaling peptide, based on GDNF;

TABLE-US-00006 (SEQIDNo.8) ATGAAGTTATGGGATGTCGTGGCTGTCTGCCTGGTGCTGCTCCACAC CGCGTCCGCC, frominsulinprotein; (SEQIDNo.9) ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCT CTGGGGACCTGACCCAGCCGCAGCC, orfromIgK; (SEQIDNo.10) ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCC AGGTTCCACTGGT.

[0077] 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.

[0078] The UBE3A-seretion signal construct was then attached to a cellular uptake peptide (cell penetrating peptide); either a HIV TAT sequence

TABLE-US-00007 (SEQIDNo.5) YGRKKRRQRRR; or HIVTATksequence (SEQIDNo.11) YARKAARQARA.

[0079] The human UBE3A vector, seen in FIG. 21, is then 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.

Example 6

[0080] 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.

[0081] The vector (2 ?g/well in a 6-well plate) was transfected into the cells using PEI transfection method. The cells were subcultured at 0.5?10.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 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 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 5000?g for 5 minutes.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.