Antisense oligonucleotide directed removal of proteolytic cleavage sites from proteins

• US 10,364,432 B2
• Filed: 02/22/2017
• Issued: 07/30/2019
• Est. Priority Date: 08/05/2010
• Status: Active Grant

• Current Assignee: Academisch Ziekenhuis Leiden H.O.D.N. Lumc
• Original Assignee: Academisch Ziekenhuis Leiden H.O.D.N. Lumc
• Inventors: van Roon-Mom, Wilhelmina M. C., Evers, Melvin Maurice, Pepers, Barry Antonius, Aartsma-Rus, Annemieke, Van Ommen, Garrit-Jan Boudewijn
• Primary Examiner(s): Hill, Kevin K
• Application Number:
  US15/439,776
• Publication Number:
  US 20170175117A1
• Time in Patent Office: 888 Days
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2320/33 Alteration of splicing
• CPC Class Codes: C12Y 304/19012 Ubiquitinyl hydrolase 1 (3.4.19.12)

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.

Claims

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.