Antisense antiviral compound and method for treating ssRNA viral infection

• US 8,906,872 B2
• Filed: 12/22/2011
• Issued: 12/09/2014
• Est. Priority Date: 09/16/2004
• Status: Expired due to Fees

• Current Assignee: Sarepta Therapeutics, Inc.
• Original Assignee: Sarepta Therapeutics, Inc.
• Inventors: Iversen, Patrick L., Stein, David A., Weller, Dwight D.
• Primary Examiner(s): Bowman, Amy
• Application Number:
  US13/335,450
• Publication Number:
  US 20120322847A1
• Time in Patent Office: 1,083 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 Coronavirus in a mammalian host cell, comprising

administering to the infected mammalian host cells, a virus-inhibitory amount of an oligonucleotide analog coumpound characterized by:

(i) a nuclease-resistant backbone,

(ii) capable of uptake by mammalian host cells,

(iii) containing between 12-40 nucleotide bases, and

(iv) having a targeting sequence of at least 12 subunits 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 Coronavirus, and

by said administering, forming within the infected mammalian host cell, a heteroduplex structure (i) composed of the positive-sense RNA strand of the Coronavirus and the oligonucleotide analog compound, and (ii) characterized by a Tm of dissociation of at least 45°

C. and disruption of a stem-loop secondary structure.

2. The method of claim 1, wherein said compound is composed of morpholino subunits linked by uncharged, phosphorus-containing intersubunit linkages, joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit.

3. The method of claim 2, wherein said intersubunit linkages are phosphorodiamidate linkages.

4. The method of claim 3, wherein said morpholino subunits are joined by phosphorodiamidate linkages, in accordance with the structure:

5. The method of claim 4, wherein X=NR₂, wherein each R is independently hydrogen or methyl.

6. The method of claim 3, wherein at least 2 and no more than half of the total number of intersubunit linkages are positively charged at physiological pH.

7. The method of claim 6, wherein said morpholino subunits are joined by phosphorodiamidate linkages, in accordance with the structure:

8. The method of claim 1, wherein the compound is conjugated to an arginine-rich.

9. The method of claim 8, wherein the arginine rich peptide has one of the sequences set forth in SEQ ID NOS:

121-126.

10. The method of claim 1, for use in treating a mammalian subject infected with a Coronavirus, or at risk of infection with a Coronavirus, wherein said compound is administered to the mammalian subject, in a therapeutically effective amount, by intravenous or oral administration.

11. The method of claim 1, further comprising administering a second therapeutic agent in combination with the compound.

22. The method of claim 11, wherein the second theraputic agent is a second oligoncleotide analog compound characterized by:

(i) a nuclease-resistant backbone,

(ii) capable of uptake by mammalian host cells,

(iii) containing between 12-40 nucleotide bases,

(iv) having a targeting sequence of at least 12 subunits 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 a second Coronavirus, and

(v) capable of forming with the positive-sense RNA strand of the second Coronavirus, a heteroduplex structure having a Tm of dissociation of at least 45°

C. and disruption of said stem-loop secondary structure.

12. An oligonucleotide analog compound for use in inhibiting replication of a Coronavirus in a mammalian host cell, characterized by:

(i) a nuclease-resistant backbone,

(ii) capable of uptake by mammalian host cells,

(iii) containing between 12-40 nucleotide bases,

(iv) having a targeting sequence of at least 12 subunits 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 Coronavirus, and

(v) capable of forming with the positive-sense RNA strand of the Coronavirus, a heteroduplex structure having a Tm of dissociation of at least 45°

C. and disruption of said stem-loop secondary structure.

13. The compound of claim 12, composed of morpholine subunits linked by uncharged, phosphorus-containing intersubunit linkages, joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit.

14. The compound of claim 13, wherein said intersubunit linkages are phosphorodiamidate linkages.

15. The compound of claim 14, wherein said morpholino subunits are joined by phosphorodiamidate linkages, in accordance with the structure:

16. The compound of claim 15, wherein X=NR₂, wherein each R is independently hydrogen or methyl.

17. The compound of claim 12, wherein the compound is composed of morpholino subunits linked by phosphorus-containing intersubunit linkages, joining a morpnolino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit and at least 2 and no more than half of the total number of intersubunit linkages are positively charged at physiological pH.

18. The compound of claim 17, wherein said morpholino subunits are joined by phosphorodiamidate linkages, in accordance with the structure:

19. The compound of claim 12, wherein said compound has a Tm, with respect to binding to said viral target sequence, of greater than about 50°

C., and said compound is actively taken up by mammalian cells.

20. The compound of claim 12, wherein the compound is covalently conjugated to an arginine-rich peptide.

21. The compound of claim 20, wherein the arginine rich peptide has one of the sequences set forth in SEQ ID NOS:

121-126.