UBE3A GENES AND EXPRESSION CASSETTES AND THEIR USE
This invention relates to polynucleotides comprising UBE3A open reading frame (ORF) sequences, vectors comprising the same, and methods of using the same for delivery of the ORF to a cell or a subject and to treat disorders associated with aberrant expression of a UBE3A gene or aberrant activity of a UBE3A gene product in the subject, such as Angelman Syndrome.
1 . A method of expressing a UBE3A open reading frame in a subject, comprising delivering to the subject a polynucleotide comprising a human UBE3A open reading frame, wherein the human UBE3A open reading frame encodes human UBE3A short isoform and long isoform, an expression cassette comprising the polynucleotide, a vector comprising the polynucleotide, and/or a transformed cell comprising the polynucleotide, thereby expressing the UBE3A open reading frame in the subject.
2 . A method of treating a disorder associated with aberrant expression of a UBE3A gene or aberrant activity of a UBE3A gene product in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polynucleotide comprising a human UBE3A open reading frame, wherein the human UBE3A open reading frame encodes human UBE3A short isoform and long isoform, an expression cassette comprising the polynucleotide, a vector comprising the polynucleotide, and/or a transformed cell comprising the polynucleotide, such that the UBE3A open reading frame is expressed in the subject.
3 . The method of claim 2 , wherein the disorder associated with expression of the UBE3A gene is Angelman Syndrome.
4 . A method of treating Angelman Syndrome in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polynucleotide comprising a human UBE3A open reading frame, wherein the human UBE3A open reading frame encodes human UBE3A short isoform and long isoform, an expression cassette comprising the polynucleotide, a vector comprising the polynucleotide, and/or a transformed cell comprising the polynucleotide, such that the UBE3A open reading frame is expressed in the subject.
5 . The method of claim 2 , wherein the subject exhibits symptoms of the disease prior to delivery of the polynucleotide, expression cassette, vector, and/or transformed cell.
6 . The method of claim 2 , wherein the polynucleotide, expression cassette, vector, and/or transformed cell is delivered in utero.
7 . The method of claim 2 , wherein the subject is a human.
8 . The method of claim 2 , wherein the polynucleotide, expression cassette, vector, and/or transformed cell is delivered to the nervous system of the subject.
9 . The method of claim 8 , wherein the polynucleotide, expression cassette, vector, and/or transformed cell is delivered intravenously.
10 . The method of claim 8 , wherein the polynucleotide, expression cassette, vector, and/or transformed cell is delivered by intrathecal, intracerebral, intra-cisterna magna , intraparenchymal, intracerebroventricular, intranasal, intra-aural, intra-ocular, or peri-ocular delivery, or any combination thereof.
11 . The method of claim 2 , wherein the AAV vector is administered to the subject at a dose of at least about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 or 10 16 viral genomes/kilogram, optionally from 10 8 -10 14 viral genomes/kilogram.
12 . The method of claim 2 , wherein the human UBE3A open reading frame encodes human UBE3A short isoform (i.e., isoform 1) and long isoform 2.
13 . The method of claim 2 , wherein the human UBE3A open reading frame encodes human UBE3A short isoform (i.e., isoform 1) and long isoform 3.
14 . The method of claim 2 , wherein the human UBE3A open reading frame comprises one or more nucleotide modifications to reduce expression of the long isoform as compared to expression of native ORF which lacks the one or more nucleotide modifications to reduce expression, optionally wherein the expression of the long isoform is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to native ORF not comprising nucleotide modifications to reduce expression.
15 . The method of claim 2 , wherein the human UBE3A open reading frame comprises one or more nucleotide modifications to enhance expression of the short isoform as compared to native ORF not comprising nucleotide modifications to enhance expression, optionally wherein the expression of the short isoform is increased by at 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to native ORF not comprising nucleotide modifications to enhance expression.
16 . The method of claim 2 , wherein the human UBE3A open reading frame comprises one or more nucleotide modifications to encode expression of the short isoform at a ratio of about 1:1 to about 15:1, as compared to expression of the long isoform.
17 . The method of claim 2 , wherein the polynucleotide comprises a first Kozak sequence comprising SEQ ID NO:6 operably linked to coding sequences for the long isoform.
18 . The method of claim 2 , wherein the polynucleotide comprises a second Kozak sequence comprising SEQ ID NO:8 operably linked to coding sequence for the short isoform.
19 . The method of claim 2 , wherein the human UBE3A open reading frame is codon-optimized for expression in a human cell, optionally wherein the human UBE3A open reading frame is codon-optimized for intracellular or non-secretory expression in a human cell.
20 . The method of claim 2 , wherein the human UBE3A open reading frame comprises the nucleotide sequence of SEQ ID NO:9, SEQ ID NO: 26, or SEQ ID NO: 27, or a nucleotide sequence having at least about 70%, 75%, 76%, 77%, 80%, 85%, or 90% identity thereto.