Antisense oligonucleotide directed removal of proteolytic cleavage sites from proteins

Abstract

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 .- 20 . (canceled) 
     
     
         21 . An oligonucleotide of between fourteen (14) and forty (40) nucleotides that induces skipping of an exon or a part thereof that encodes a proteolytic cleavage site in a protein involved in a disease that is associated with a proteolytic cleavage product of the protein, wherein the oligonucleotide binds to pre-mRNA of the protein to form a double-stranded nucleic acid complex, wherein the oligonucleotide is chemically modified to render the double-stranded nucleic acid complex RNase H resistant, and wherein the disease is a polyglutamine disorder. 
     
     
         22 . The oligonucleotide of  claim 21 , wherein the polyglutamine disorder is Huntington's disease (“HD”) or Alzheimer's disease (“AD”). 
     
     
         23 . The oligonucleotide of  claim 21 , wherein the polyglutamine disorder is Huntington's disease (“HD”), and wherein part of exon 12 of human huntingtin pre-mRNA is skipped. 
     
     
         24 . The oligonucleotide of  claim 21 , wherein the polyglutamine disorder is Huntington's disease (“HD”), and wherein nucleotides 207 to 341 of exon 12 of human huntingtin pre-mRNA is skipped. 
     
     
         25 . The oligonucleotide of  claim 21 , wherein the polyglutamine disorder is Huntington's disease (“HD”), and wherein the oligonucleotide comprises a polynucleotide selected from the group consisting of SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, and SEQ ID NO: 188. 
     
     
         26 . The oligonucleotide of  claim 21 , wherein the polyglutamine disorder is Huntington's disease (“HD”), and wherein the oligonucleotide consists of a polynucleotide selected from the group consisting of SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, and SEQ ID NO: 188. 
     
     
         27 . The oligonucleotide of  claim 21 , wherein the proteolytic cleavage site is a caspase-3 cleavage site or a caspase-6 cleavage site. 
     
     
         28 . The oligonucleotide of  claim 21 , wherein at least one nucleotide of the oligonucleotide is chemically modified by a 2′-O-methyl substitution. 
     
     
         29 . The oligonucleotide of  claim 27 , wherein each nucleotide of the oligonucleotide is chemically modified by a 2′-O-methyl substitution. 
     
     
         30 . The oligonucleotide of  claim 21 , wherein at least one nucleotide of the oligonucleotide is chemically modified by a 2′-O-methoxyethyl substitution. 
     
     
         31 . The oligonucleotide of  claim 29 , wherein each nucleotide of the oligonucleotide is chemically modified by a 2′-O-methoxyethyl substitution. 
     
     
         32 . The oligonucleotide of  claim 21 , wherein all internucleoside linkages of the oligonucleotide are phosphorothioated. 
     
     
         33 . The oligonucleotide of  claim 21 , wherein the oligonucleotide is a uniformly 2′-O-methoxyethylribose modified phosphorothioate oligonucleotide.