Splice-region antisense composition and method

• US 20030166588A1
• Filed: 05/04/2001
• Published: 09/04/2003
• Est. Priority Date: 05/04/2000
• Status: Active Grant

• Current Assignee: Avi Biopharma, Inc.
• Original Assignee: Avi Biopharma, Inc.
• Inventors: Iversen, Patrick L., Hudziak, Robert
• Granted Patent: US 6,784,291 B2
• Time in Patent Office: Days
• US Class Current: 514/44
• 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: A61P 13/08 of the prostate
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 31/20 for DNA viruses
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/06 of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
• CPC Class Codes: A61P 5/28 Antiandrogens
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07F 9/65583 each of the hetero rings containing nitrogen as ring hetero atom
• 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 15/1132 against retroviridae, e.g. HIV
• CPC Class Codes: C12N 15/1135 : against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/1136 : against growth factors, growth regulators, cytokines, lymphokines or hormones
• CPC Class Codes: C12N 15/1138 : against receptors or cell surface proteins
• CPC Class Codes: C12N 2310/11 : Antisense
• CPC Class Codes: C12N 2310/314 : Phosphoramidates
• CPC Class Codes: C12N 2310/3145 : with the nitrogen in 3' or 5'-position
• CPC Class Codes: C12N 2310/3233 : Morpholino-type ring
• CPC Class Codes: C12N 2320/33 : Alteration of splicing

Abstract

Antisense compositions targeted against an mRNA sequence coding for a selected protein, at a region having its 5′ end from 1 to about 25 base pairs downstream of a normal splice acceptor junction in the preprocessed mRNA, are disclosed. The antisense compound is RNase-inactive, and is preferably a phosphorodiamidate-linked morpholino oligonucleotide. Such targeting is effective to inhibit natural mRNA splice processing, produce splice variant mRNAs, and inhibit normal expression of the protein.

Claims

1. An antisense compound having an uncharged morpholino backbone and a base sequence between 12 and 25 nucleotide bases in length which is complementary to a target region of a selected preprocessed mRNA coding for a protein selected from the group consisting of myc, myb, rel, fos, jun, abl, bcl, p53, an integrin, a cathedrin, a telomerase, a cytokine, a kinase, a receptor protein, hCG, HIV rev, human papilloma virus, and human parvovirus B19,

where the 5′

end of the target region is 1-25 bases downstream of a normal splice acceptor site in said preprocessed mRNA.

2. The compound of claim 1, having intersubunit linkages selected from the group consisting of the structures presented in FIGS. 2AA-2EE.

3. The compound of claim 2, wherein the linkage is a phosphorodiamidate linkage as represented at FIG. 2B-B, where X=NH₂, NHR, or NRR′

, Y=O, and Z=O, or where X=OR, Y=NH or NR′

, and Z=O, and R and R′

are groups which do not interfere with target binding.

4. The compound of claim 3, wherein R and R′

are moieties independently selected from alkyl, polyalkyleneoxy, and a combination thereof, which may be substituted with one or more groups selected from hydroxy, alkoxy, amino, alkylamino, thiol, alkanethiol, halogen, oxo, carboxylic acid, carboxylic ester, and inorganic ester.

5. The compound of claim 4, wherein each said moiety R and R′

, independent of substitution, is from 1 to 6 atoms long.

6. The compound of claim 3, wherein NRR′

represents a nitrogen heterocycle having 5-7 ring atoms selected from nitrogen, carbon, oxygen, and sulfur, and having at least as many carbon ring atoms as non-carbon ring atoms.

7. The compound of claim 6, wherein the 5′

end of the target region is 10-15 bases downstream of a normal splice acceptor site.

8. The compound of claim 1, wherein the selected protein is human c-myc.

9. The compound of claim 8, wherein the base sequence is selected from the group consisting of SEQ ID NOs:

16 through 32.

10. The compound of claim 9, wherein the base sequence is SEQ ID NO:

25.

11. The compound of claim 8, wherein the base sequence is a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO:

34.

12. The compound of claim 11, wherein the base sequence is SEQ ID NO:

33.

13. The compound of claim 1, wherein the selected protein is human androgen receptor, and the base sequence is a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO:

9 or SEQ ID NO;

13.

14. The compound of claim 13, wherein the base sequence is SEQ ID NO:

8 or SEQ ID NO;

12.

15. The compound of claim 1, wherein the selected protein is HCG-β

subunit, and the base sequence is a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO;

15.

16. The compound of claim 15, wherein the base sequence is SEQ ID NO:

14.

17. The compound of claim 1, wherein the selected protein is human p53, and the base sequence is a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO:

36.

18. The compound of claim 17, wherein the base sequence is SEQ ID NO:

35.

19. The compound of claim 1, wherein the selected protein is human abl, and the base sequence is a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO:

38.

20. The compound of claim 19, wherein the base sequence is SEQ ID NO:

37.

21. The compound of claim 1, wherein the selected protein is HIV-1 rev, and the base sequence is a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO:

41.

22. The compound of claim 21, wherein the base sequence is SEQ ID NO:

40.

23. A method of inhibiting normal splicing of mRNA in a eukaryotic cell, comprising

contacting the cell with an antisense compound having an uncharged morpholino backbone and a base sequence between 12 and 25 nucleotide bases in length which is complementary to a target region of a selected preprocessed mRNA coding for a selected protein;

where the 5′

end of the target region is 1-25 bases downstream of the a normal splice acceptor site in said preprocessed mRNA,

wherein the compound;

is taken up by the cell;

hybridizes to the target region of preprocessed mRNA in the cell, and

being so hybridized, prevents splicing at said normal acceptor splice site, such that the splice mechanism proceeds to a downstream splice acceptor sequence in the mRNA, producing a splice variant processed mRNA with a truncated coding sequence.

24. The method of claim 23, wherein the protein is selected from the group consisting of myc, myb, rel, fos, jun, abl, bcl, p53, an integrin, a cathedrin, a telomerase, hCG, a receptor protein, a cytokine, a kinase, HIV rev, human papilloma virus, and human parvovirus B 19.

25. The method of claim 24, wherein the compound has intersubunit linkages selected from the group consisting of the structures presented in FIGS. 2AA-2EE.

26. The method of claim 25, wherein the linkage is the phosphorodiamidate linkage represented at FIG. 2B-B, where X=NH₂, NHR, or NRR′

, Y=O, and Z=O, or where X=OR, Y=NH or NR′

, and Z=O, and R and R′

are groups which do not interfere with target binding.

27. The method of claim 26, wherein R and R′

are moieties independently selected from alkyl, polyalkyleneoxy, and a combination thereof, which may be substituted with one or more groups selected from hydroxy, alkoxy, amino, alkylamino, thiol, alkanethiol, halogen, oxo, carboxylic acid, carboxylic ester, and inorganic ester.

28. The method of claim 27, wherein each said moiety R and R′

, independent of substitution, is from 1 to 12 atoms long.

29. The method of claim 26, wherein NRR′

represents a nitrogen heterocycle having 5-7 ring atoms selected from nitrogen, carbon, oxygen, and sulfur, and having at least as many carbon ring atoms as non-carbon ring atoms.

30. The method of claim 23, wherein the 5′

end of the target region is 10-15 bases downstream of a normal splice acceptor site.

31. The method of claim 23, wherein said downstream splice acceptor site is a whole multiple of three bases downstream of the normal splice acceptor site, such that said splice variant mRNA has a coding sequence in frame with that of the processed mRNA when it is normally spliced.

32. The method of claim 23, wherein the selected protein has multiple distinct binding regions, and said truncated coding sequence codes for a variant protein in which a binding region is disabled.

33. The method of claim 32, wherein said variant protein is a dominant negative protein.

34. The method of claim 33, wherein said selected protein is human c-myc, and said variant protein is an N-terminal truncated c-myc protein.

35. The method of claim 34, wherein the antisense compound has a base sequence selected from the group consisting of SEQ ID NOs:

16 through 32.

36. The method of claim 35, wherein the antisense compound has the base sequence SEQ ID NO:

25.

37. The method of claim 23, wherein the selected protein and corresponding antisense base sequence are selected from the group consisting of:

(a) human chorionic gonadotropin, β

subunit;

a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO;

15;

(b) human androgen receptor;

a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO;

9 or SEQ ID NO;

13;

(c) human c-myc;

a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO;

34;

(d) human p53;

a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO;

36;

(e) human abl;

a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO;

38; and

(f) HIV-1 rev;

a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO;

41.

38. The method of claim 37, wherein the selected protein and corresponding antisense base sequence are selected from the group consisting of:

(a) human chorionic gonadotropin, β

subunit;

SEQ ID NO;

14;

(b) human androgen receptor;

SEQ ID NO;

8 or SEQ ID NO;

12;

(c) human c-myc;

SEQ ID NO;

33;

(d) human p53;

SEQ ID NO;

35;

(e) human abl;

SEQ ID NO;

37; and

(f) HIV-1 rev;

SEQ ID NO;

40.