Antisense oligonucleotide directed removal of proteolytic cleavage sites from proteins
The invention relates to means and methods for removing a proteolytic cleavage site from a protein comprising providing a cell that expresses pre-mRNA encoding the protein with an anti-sense oligonucleotide that induces skipping of the exonic sequence that encodes the proteolytic cleavage site, the method further comprising allowing translation of mRNA produced from the pre-mRNA.
1. A method of promoting production of a human Huntingtin protein lacking a proteolytic caspase-6 cleavage site in a human cell, the method comprising:
a) providing a human cell that expresses a human Huntingtin protein comprising a caspase-6 proteolytic cleavage site from a pre-mRNA encoding the protein, with an antisense oligonucleotide that
is directed toward the interior of exon 12 of the human Huntingtin gene;
binds to the pre-mRNA to form a double-stranded nucleic acid complex; and
induces partial skipping of exon 12,
wherein at least nucleotides 207 to 341 of exon 12 are skipped;
wherein each nucleotide of the antisense oligonucleotide is chemically modified to render the double-stranded nucleic acid complex RNase H resistant; and
b) allowing translation of mRNA produced from the pre-mRNA in the cell to produce an mRNA lacking nucleotides 207 to 341 of exon 12;
wherein the anti-sense oligonucleotide comprises or consists of a sequence selected from SEQ ID NO:178 and 182.
2. The method according to claim 1 , wherein the antisense oligonucleotide is a uniformly 2′-O-methoxyethylribose modified phosphorothioate oligonucleotide.
3. A method for treating Huntington's disease in an individual, the method comprising:
administering to an individual in need thereof an antisense oligonucleotide directed toward the interior of exon 12 of the human Huntingtin gene that binds to a pre-mRNA produced from the human Huntingtin gene to form a double-stranded nucleic acid complex, and induces partial skipping of exon 12 of the human Huntingtin gene wherein at least nucleotides 207 to 341 of exon 12 are skipped;
wherein each nucleotide of the antisense oligonucleotide is chemically modified to render the double-stranded nucleic acid complex RNase H resistant; and
wherein the anti-sense oligonucleotide comprises or consists of a sequence selected from SEQ ID NO:178 and 182.
4. The method according to claim 3 , wherein the antisense oligonucleotide is a uniformly 2′-O-methoxyethylribose modified phosphorothioate oligonucleotide.