Antisense antiviral compound and method for treating ssRNA viral infection

• US 8,129,352 B2
• Filed: 09/14/2005
• Issued: 03/06/2012
• Est. Priority Date: 09/16/2004
• Status: Active Grant

• Current Assignee: Sarepta Therapeutics, Inc.
• Original Assignee: Avi Biopharma, Inc.
• Inventors: Iversen, Patrick L., Stein, David A.
• Primary Examiner(s): Bowman, Amy
• Application Number:
  US11/226,995
• Publication Number:
  US 20060063150A1
• Time in Patent Office: 2,365 Days
• Field of Search: None
• US Class Current: 514/44
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/1131 against viruses
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/314 Phosphoramidates
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12N 2320/11 for the determination of target sites, i.e. of active nucleic acids
• CPC Class Codes: C12N 2770/00011 Details
• CPC Class Codes: C12N 2770/10011 Arteriviridae
• CPC Class Codes: C12N 2770/12011 Astroviridae
• CPC Class Codes: C12N 2770/16011 Caliciviridae
• CPC Class Codes: C12N 2770/20011 Coronaviridae
• CPC Class Codes: C12N 2770/24011 Flaviviridae
• CPC Class Codes: C12N 2770/28011 Hepeviridae
• CPC Class Codes: C12N 2770/32011 Picornaviridae
• CPC Class Codes: C12N 2770/36011 Togaviridae
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides antisense antiviral compounds and methods of their use and production in inhibition of growth of viruses of the Flaviviridae, Picornoviridae, Caliciviridae, Togaviridae, Arteriviridae, Coronaviridae, Astroviridae and Hepeviridae families in the treatment of a viral infection. The antisense antiviral compounds are substantially uncharged morpholino oligonucleotides having a sequence of 12-40 subunits, including at least 12 subunits having a targeting sequence that is complementary to a region associated with stem-loop secondary structure within the 5′-terminal end 40 bases of the positive-sense RNA strand of the virus.

Claims

1. A method of inhibiting replication of a Flaviviridae positive strand RNA virus selected from yellow fever virus, Dengue virus, tick born encephalitis virus, and West Nile virus, comprising:

administering to mammalian host cells a virus-inhibitory amount of an antisense oligonucleotide analog characterized by (i) a nuclease-resistant backbone, (ii) being capable of uptake by mammalian host cells, (iii) containing up to 25 nucleotide bases, (iv) being composed of morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit, and (v) having a targeting sequence that comprises the sequence set forth in SEQ ID NO;

47, 49, 51, or 55, wherein the antisense oligonucleotide analog disrupts a stem-loop structure in the 5′

-terminal region of the RNA virus and inhibits virus production.

2. The method of claim 1, wherein the heteroduplex has a Tm of dissociation of at least 45°

C.

3. The method of claim 1, wherein the intersubunit linkages are uncharged.

4. The method of claim 1, wherein the morpholino subunits are joined by intersubunit linkages, in accordance with the structure:

5. The method of claim 4, wherein X is —

NR₂, wherein R is independently hydrogen or methyl.

6. The method of claim 1, wherein the antisense oligonucleotide is attached to an arginine-rich polypeptide effective to enhance the uptake of the compound into host cells.

7. The method of claim 6, wherein the arginine-rich polypeptide has the sequence of SEQ ID NO:

122.

8. The method of claim 1, wherein the administration is to a mammalian subject in a therapeutically effective amount to treat a Flaviviridae virus infection.

9. The method of claim 8, wherein the Flaviviridae virus is Dengue virus.

10. The method of claim 1, wherein the administration is by intravenous or oral administration.

11. The method of claim 1, wherein the antisense oligonucleotide analog administered is contained in a cocktail of antisense oligonucleotides directed against two or more Flaviviridae positive-strand RNA viruses.

12. A method of treating an infection by a Flaviviridae positive strand RNA virus selected from yellow fever virus, Dengue virus, tick born encephalitis virus, and West Nile virus, comprising:

administering to a subject a therapeutically effective amount of an antisense morpholino oligonucleotide, wherein the morpholino oligonucleotide has

(i) a targeting sequence that comprises the sequence set forth in SEQ ID NO;

47, 49, 51, or 55,

(ii) a subunit length of up to 25 subunits, wherein morpholino subunits of the oligomer are joined by intersubunit linkages in accordance with the structure;

13. The method of claim 12, wherein the Flaviviridae virus is a Dengue virus and the targeting sequence comprises SEQ ID NO:

51.

14. The method of claim 12, wherein the Flaviviridae is a yellow fever virus and the targeting sequence comprises SEQ ID NO:

49.

15. The method of claim 12, wherein the Flaviviridae is a tick borne encephalitis virus and the targeting sequence comprises SEQ ID NO:

55.

16. The method of claim 12, wherein the Flaviviridae is a West Nile virus and the targeting sequence comprises SEQ ID NO:

47.

17. The method of claim 13, wherein the antisense oligonucleotide has a subunit length of about 22 subunits.

18. The method of claim 17, wherein the antisense oligonucleotide is 22 subunits in length and is 100% complementary to SEQ ID NO:

6.

19. The method of claim 14, wherein the antisense oligonucleotide is attached to an arginine-rich polypeptide effective to enhance the uptake of the compound into host cells.

20. The method of claim 19, wherein the arginine-rich polypeptide has the sequence set forth in SEQ ID NO:

122.