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

• US 5,665,593 A
• Filed: 01/26/1995
• Issued: 09/09/1997
• Est. Priority Date: 05/11/1993
• Status: Expired due to Term

• Current Assignee: University of South Carolina
• Original Assignee: University of South Carolina
• Inventors: Kole, Ryszard, Dominski, Zbigniew
• Primary Examiner(s): RORIES, CHARLES
• Application Number:
  US08/379,079
• Time in Patent Office: 957 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, 935/3, 935/6, 935/8, 935/9, 935/33, 435/6, 435/240.2, 435/70.1, 435/70.3, 435/70.4
• US Class Current: 435/375
• 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. A method of upregulating expression of a native protein in a cell in vitro, said cell containing a DNA encoding said native protein, which DNA contains a mutation which causes downregulation of said native protein by aberrant splicing in a pre-mRNA,

wherein said DNA encodes said pre-mRNA;

wherein said pre-mRNA contains a native intron having a first set of splice elements, which native intron is removed by splicing when said mutation is absent to produce a first mRNA encoding said native protein;

and wherein said pre-mRNA further contains an aberrant intron different from said native intron having a second set of splice elements, which aberrant intron is removed by splicing when said mutation is present to produce an aberrant second mRNA different from said first mRNA;

said method comprising;

administering to said cell an antisense oligonucleotide which hybridizes to said pre-mRNA in the nucleus of said cell to create a duplex thereof under conditions which permit splicing,

wherein said antisense oligonucleotide does not activate RNase H;

and wherein said antisense oligonucleotide blocks a member of said aberrant second set of splice elements;

so that said native intron is removed by splicing and said native protein is produced.

2. A method according to claim 1, wherein said cell is an eukaryotic cell selected from the group consisting of yeast, insect, and mammalian cells.

3. A method according to claim 1, wherein said antisense oligonucleotide blocks a mutated splice element.

4. A method according to claim 1, wherein said antisense oligonucleotide blocks a native splice element.

5. A method according to claim 1, wherein said antisense oligonucleotide blocks a cryptic splice element.

6. A method according to claim 1, wherein said antisense oligonucleotide blocks a 5'"'"' splice site.

7. A method according to claim 1, wherein said antisense oligonucleotide blocks a 3'"'"' splice site.

8. A method according to claim 1, wherein said antisense oligonucleotide blocks a branch point.

9. A method according to claim 1, wherein said native protein is β

-globin.

10. A method according to claim 1, wherein said native protein is β

-globin.

11. A method according to claim 1, wherein said antisense oligonucleotide is from 8 to 50 nucleotides in length.

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

13. A method according to claim 1, wherein said antisense oligonucleotide contains a nucleotide having a C1 to C4 lower alkyl substituent at the 2'"'"' position thereof.