Antisense oligonucleotides which combat aberrant splicing and methods of using the same

• US 5,627,274 A
• Filed: 05/30/1995
• Issued: 05/06/1997
• Est. Priority Date: 05/11/1993
• Status: Expired due to Term

• Current Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Original Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Inventors: Kole, Ryszard, Dominski, Zbigniew
• Primary Examiner(s): RORIES, CHARLES
• Application Number:
  US08/453,224
• Time in Patent Office: 707 Days
• Field of Search: 514/44, 536/24.5, 536/23.1, 536/24.1, 536/24.3, 536/24.31, 536/24.33, 935/34, 935/44, 935/62, 935/78, 935/33, 935/3, 935/6, 935/8, 935/9, 435/6, 435/240.2, 424/450
• US Class Current: 536/23.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• 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 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3521 Methyl

Abstract

A method of combatting aberrant splicing in a pre-mRNA molecule containing a mutation is disclosed. When present in the pre-mRNA, the mutation causes the pre-mRNA to splice incorrectly and produce an aberrant mRNA or mRNA fragment different from the mRNA ordinarily encoded by the pre-mRNA. The method comprises hybridizing an antisense oligonucleotide to the pre-mRNA molecule to create a duplex molecule under conditions which permit splicing. The antisense oligonucleotide is one which does not activate RNase H, and is selected to block a member of the aberrant set of splice elements created by the mutation so that the native intron is removed by splicing and the first mRNA molecule encoding a native protein is produced. Oligonucleotides useful for carrying out the method are also disclosed.

Claims

1. An oligonucleotide consisting of the nucleotide sequence shown as SEQ ID NO:

1, 3, 4, 5, or 7, which oligonucleotide does not activate RNase H.

2. An antisense oligonucleotide according to claim 1, wherein said oligonucleotide contains a modified internucleotide bridging phosphate residue selected from the group consisting of methyl phosphonates, methyl phosphonothioates, phosphoromorpholidates, phosphoropiperazidates, and phosphoramidates.

3. An antisense oligonucleotide according to claim 1, wherein said antisense oligonucleotide contains a nucleotide having a lower alkyl substituent at the 2'"'"' position thereof.

4. An antisense oligonucleotide according to claim 1 in a lipid vesicle.