ANTISENSE NUCLEIC ACIDS

• US 20170204410A1
• Filed: 06/16/2015
• Published: 07/20/2017
• Est. Priority Date: 06/17/2014
• Status: Active Grant

• Current Assignee: National Center of Neurology and Psychiatry, Nippon Shinyaku Company Limited
• Original Assignee: National Center of Neurology and Psychiatry, Nippon Shinyaku Company Limited
• Inventors: WATANABE, Naoki, TONE, Yuuichirou, NAGATA, Tetsuya, TAKEDA, Shin'ichi
• Granted Patent: US 9,840,706 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 31/712 Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
• CPC Class Codes: A61K 31/7125 Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• 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 2310/11 Antisense
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2320/10 in screening processes
• CPC Class Codes: C12N 2320/33 Alteration of splicing
• CPC Class Codes: C12N 2330/30 chemically synthesised
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/178 miRNA, siRNA or ncRNA

Abstract

Provided is a drug that allows highly-efficient skipping of exon. The present invention provides an antisense oligomer wherein two or more unit oligomers targeting sequences that are neither consecutive nor overlap with each other in the same exon are connected.

Claims

1. An antisense oligomer having a length of 15 to 30 bases wherein

(a) a first unit oligomer comprising a nucleotide sequence complementary to a first nucleotide sequence of 7 to 15 consecutive bases in a target exon in human dystrophin gene;

and

(b) a second unit oligomer comprising a nucleotide sequence complementary to a second nucleotide sequence of 7 to 15 consecutive bases in the target exon

are connected, wherein

the first nucleotide sequence and the second nucleotide sequence are neither consecutive nor overlap with each other, and

the antisense oligomer induces skipping of the target exon, or a pharmaceutically acceptable salt or hydrate thereof.

2. The antisense oligomer according to claim 1, wherein the first and/or second unit oligomer comprises a nucleotide sequence complementary to a partial nucleotide sequence of an intron adjacent to the target exon, or a pharmaceutically acceptable salt or hydrate thereof.

4. The antisense oligomer according to claim 1, wherein the first nucleotide sequence is a nucleotide sequence of 7 to 15 consecutive bases selected from the nucleotide sequence represented by SEQ ID NO:

1, or a pharmaceutically acceptable salt or hydrate thereof.

5. The antisense oligomer according to claim 1, wherein the second nucleotide sequence is a nucleotide sequence of 7 to 15 consecutive bases selected from the nucleotide sequence represented by SEQ ID NO:

2, or a pharmaceutically acceptable salt or hydrate thereof.

6. The antisense oligomer according to claim 1, wherein two unit oligomers selected from the group consisting of the following (c) to (e) are connected:

(c) an unit oligomer consisting of a nucleotide sequence complementary to a nucleotide sequence of 7 to 15 consecutive bases selected from the nucleotide sequence represented by SEQ ID NO;

3;

(d) an unit oligomer consisting of a nucleotide sequence complementary to a nucleotide sequence of 7 to 15 consecutive bases selected from the nucleotide sequence represented by SEQ ID NO;

4; and

(e) an unit oligomer consisting of a nucleotide sequence complementary to a nucleotide sequence of 7 to 15 consecutive bases selected from the nucleotide sequence represented by SEQ ID NO;

5, or a pharmaceutically acceptable salt or hydrate thereof.

7. The antisense oligomer according to claim 1, which consists of a nucleotide sequence selected from a group consisting of SEQ ID NOs 6 to 9, or a pharmaceutically acceptable salt or hydrate thereof.

8. The antisense oligomer according to claim 1, which is an oligonucleotide, or a pharmaceutically acceptable salt or hydrate thereof.

9. The antisense oligomer according to claim 8, wherein the sugar moiety and/or the phosphate-binding region of at least one nucleotide constituting the oligonucleotide is modified, or a pharmaceutically acceptable salt or hydrate thereof.

10. The antisense oligomer according to claim 8, wherein the sugar moiety of at least one nucleotide constituting the oligonucleotide is a ribose in which the 2′

-OH group is replaced by any one selected from the group consisting of OR, R, R′

OR, SH, SR, NH₂, NHR, NR₂, N₃, CN, F, Cl, Br and I (wherein R is an alkyl or an aryl and R′

is an alkylene), or a pharmaceutically acceptable salt or hydrate thereof.

11. The antisense oligomer according to claim 8, wherein the phosphate-binding region of at least one nucleotide constituting the oligonucleotide is any one selected from the group consisting of a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoramidate bond and a boranophosphate bond, or a pharmaceutically acceptable salt or hydrate thereof.

12. The antisense oligomer according to claim 1, which is a morpholino oligomer, or a pharmaceutically acceptable salt or hydrate thereof.

13. The antisense oligomer according to claim 12, which is a phosphoroamidate morholino oligomer, or a pharmaceutically acceptable salt or hydrate thereof.

14. The antisense oligomer according to claim 12, wherein the 5′

end is any one of chemical formulae (1) to (3) below, or a pharmaceutically acceptable salt or hydrate thereof.

15. A pharmaceutical composition for the treatment of muscular dystrophy, comprising as an active ingredient the antisense oligomer according to claim 1, or a pharmaceutically acceptable salt or hydrate thereof.

16. The pharmaceutical composition according to claim 15, further comprising a pharmaceutically acceptable carrier.

17. A method for treatment of muscular dystrophy, which comprises administering to a patient with muscular dystrophy the antisense oligomer or a pharmaceutically acceptable salt or hydrate thereof according to claim 1-2 or 4-14.

18. The method for treatment according to claim 17, wherein the patient with muscular dystrophy has a mutation(s) which is to be targeted for exon 44 skipping in dystrophin gene.

19. The method for treatment according to claim 17, wherein the patient is a human.

24. A method for manufacturing of the antisense oligomer according to claim 1, which comprises connecting

(a) a first unit oligomer comprising a nucleotide sequence complementary to a first nucleotide sequence of 7 to 15 consecutive bases in a target exon in human dystrophin gene;

and

(b) a second unit oligomer comprising a nucleotide sequence complementary to a second nucleotide sequence of 7 to 15 consecutive bases in the target exon

to produce an antisense oligomer having a length of 15 to 30 bases, wherein

the first nucleotide sequence and the second nucleotide sequence are neither consecutive nor overlap with each other.

25. The method according to claim 24, which further comprises:

measuring the efficiency of skipping by the obtained antisense oligomer; and

selecting an antisense oligomer having the efficiency of skipping that exceeds a reference value.

27. A method for treatment of muscular dystrophy, which comprises administering to a patient with muscular dystrophy the pharmaceutical composition of claim 15 or claim 16.

3. (canceled)

20-23. -23. (canceled)

26. A method for screening of an antisense oligomer, which comprises:

(a) selecting

(i) a first unit oligomer comprising a nucleotide sequence complementary to a first nucleotide sequence of 7 to 15 consecutive bases in a target exon in human dystrophin gene; and

(ii) a second unit oligomer comprising a nucleotide sequence complementary to a second nucleotide sequence of 7 to 15 consecutive bases in the target exon, wherein

the first nucleotide sequence and the second nucleotide sequence are neither consecutive nor overlap with each other;

(b) connecting the first and second unit oligomers to produce an antisense oligomer having a length of 15 to 30 bases;

(c) measuring the efficiency of skipping by the antisense oligomer obtained in the step (b); and

(d) selecting an antisense oligomer having the efficiency of skipping that exceeds a reference value.