Splice-region antisense composition and method

• US 20060287268A1
• Filed: 05/11/2006
• Published: 12/21/2006
• Est. Priority Date: 05/04/2000
• Status: Active Grant

• Current Assignee: Avi Biopharma, Inc.
• Original Assignee: Avi Biopharma, Inc.
• Inventors: Iversen, Patrick L., Weller, Dwight D., Hudziak, Robert
• Granted Patent: US 8,067,569 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• 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 a phosphorodiamidate-linked morpholino oligonucleotide containing uncharged phosphorodiamidate linkages interspersed with cationic phosphorodiamidate linkages. 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 characterized by:

(a1) a morpholino backbone composed of morpholino subunits that are linked by uncharged phosphorodiamidate linkages interspersed with at least two and up to half positively charged phosphorodiamidate linkages,

(a2);

a base-sequence length of between 12 and 25 nucleotide bases; and

(a3);

a base sequence that 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 a normal splice acceptor site in said preprocessed mRNA, and having the properties that;

(b1);

the compound is taken up by eukaryotic cells;

(b2);

the compound hybridizes to the target region of preprocessed mRNA in such cells, and

(b3);

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

2. The composition of claim 1, wherein said morpholino subunits are linked by phosphorodiamidate linkages, in accordance with the structure:

3. The compound of claim 1, wherein the 5′

end of the target region is 2-20 bases downstream of said normal splice acceptor site.

4. The compound of claim 3, wherein the 5′

end of the target region is 2-15 bases downstream of said normal splice acceptor site.

5. The compound of claim 1, 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.

6. The compound of claim 1, wherein said selected protein has multiple distinct binding regions, and said truncated coding sequence codes for a variant protein in which one such binding region is disabled.

7. The compound of claim 1, wherein said variant protein is a dominant negative protein.

8. The compound of claim 7, wherein said selected protein is human c-myc, and said variant protein is an N-terminal truncated c-myc.

9. The compound of claim 8, having a base sequence selected from the group consisting of SEQ ID NO:

3 and SEQ ID NOs;

13 through 28.

10. The compound of claim 1, wherein the selected protein and corresponding antisense base sequence is selected from the group consisting of

(a) human chorionic gonadotropin, β

subunit;

SEQ ID NO;

6;

(b) human androgen receptor;

SEQ ID NO;

9 or 12;

(c) human c-myc;

SEQ ID NO;

3, 13, or 29;

(d) human p53;

SEQ ID NO;

30;

(e) human abl;

SEQ ID NO;

31; and

(f) HIV-1 rev;

SEQ ID NO;

33.

11. A method of inhibiting normal splicing of mRNA in a eukaryotic cell, comprising contacting the cell with the an antisense compound characterized by:

(a1) a morpholino backbone composed of morpholino subunits that are linked by uncharged phosphorodiamidate linkages interspersed with at least two and up to half positively charged phosphorodiamidate linkages (a2);

a base-sequence length of between 12 and 25 nucleotide bases; and

(a3);

a base sequence that 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;

(b1);

is taken up by the cell;

(b2);

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

(b3);

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

12. The method of claim 11, wherein said morpholino subunits are joined by phosphorodiamidate linkages, in accordance with the structure:

13. The method of claim 11, wherein the 5′

end of the target region is 2-20 bases downstream of said normal splice acceptor site.

14. The compound of claim 13, wherein the 5′

end of the target region is 2-15 bases downstream of said normal splice acceptor site.

15. The method of claim 11, 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.

16. The method of claim 11, 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.

17. The method of claim 16, wherein said variant protein is a dominant negative protein.

18. The method of claim 17, wherein said selected protein is human c-myc, and said variant protein is an N-terminal truncated c-myc.

19. The method of claim 18, wherein the antisense compound has a base sequence selected from the group consisting of SEQ ID NO:

3 and SEQ ID NOs;

13 through 28.

20. The method of claim 11, wherein the selected protein and corresponding antisense base sequence is selected from the group consisting of

(a) human chorionic gonadotropin, β

subunit;

SEQ ID NO;

6;

(b) human androgen receptor;

SEQ ID NO;

9 or 12;

(c) human c-myc;

SEQ ID NO;

3, 13, or 29;

(d) human p53;

SEQ ID NO;

30;

(e) human abl;

SEQ ID NO;

31; and

(f) HIV-1 rev;

SEQ ID NO;

33.