Antisense oligonucleotide directed removal of proteolytic cleavage sites from proteins
Described are means and methods for removing a proteolytic cleavage site from a protein, the method 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, and allowing translation of mRNA produced from the pre-mRNA.
1. A method of promoting the production of a human Huntingtin protein lacking a proteolytic caspase-6 cleavage site in a human cell, the method comprising:
providing a human cell that expresses a human Huntingtin protein comprising a caspase-6 proteolytic cleavage site from a pre-mRNA encoding said protein with an anti-sense 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;
induces partial skipping of exon 12, wherein at least nucleotides 207 to 341 of exon 12 are skipped; and
wherein each nucleotide of the anti-sense oligonucleotide is chemically modified to render the double-stranded nucleic acid complex RNase H resistant, and
allowing translation of mRNA produced from said pre-mRNA in the cell to produce an mRNA lacking nucleotides 207 to 341 of exon 12.
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 anti-sense 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; and
wherein each nucleotide of the anti-sense oligonucleotide is chemically modified to render the double-stranded nucleic acid complex RNase H resistant.
4. The method according to claim 3 , wherein the antisense oligonucleotide is a uniformly 2′-O-methoxyethylribose modified phosphorothioate oligonucleotide.
5. The method according to claim 1 , wherein the antisense nucleotide comprises a sequence according to SEQ ID NO:1.
6. The method according to claim 4 , wherein the antisense nucleotide comprises a sequence according to SEQ ID NO:1.