Antisense antiviral compound and method for treating ssRNA viral infection

• US 20060269911A1
• Filed: 05/10/2006
• Published: 11/30/2006
• Est. Priority Date: 09/16/2004
• Status: Active Grant

• Current Assignee: Avi Biopharma, Inc.
• Original Assignee: Avi Biopharma, Inc.
• Inventors: Iversen, Patrick L., Weller, Dwight D., Stein, David A.
• Granted Patent: US 8,084,433 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• 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, Picomoviridae, 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 1240 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 producing an anti-viral compound capable of inhibiting the replication of an RNA virus having a single-stranded, positive-sense genome and selected from one of the Flaviviridae, Picomoviridae, Caliciviridae, Togaviridae, Arteriviridae, Coronaviridae, Astroviridae and Hepeviridae virus families, said method comprising

(a) identifying as a viral target sequence, in the 5′

-terminal 40 bases of the positive strand of the selected virus, a region whose sequence is capable of forming internal stem-loop secondary structure,

(b) constructing, by step-wise solid-phase synthesis, an oligonucleotide analog compound 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 the virus-genome region capable of forming internal duplex structure, and

(v) an ability to form with the viral target sequence, a heteroduplex structure (i) composed of the positive sense strand of the virus and the oligonucleotide compound, and (ii) characterized by a Tm of dissociation of at least 45°

C. and disruption of such stem-loop structure.

2. The method of claim 1, wherein said identifying step includes (i) analyzing the 5′

-terminal 40 bases of the positive strand of the selected virus by a computer program capable of performing secondary structure predictions based on a search for the minimal free energy state of the input RNA sequence, and (ii) identifying as at least a portion of the viral target sequence, a sequence capable of forming internal duplex RNA structure.

3. The method of claim 1, wherein said oligonucleotide is 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.

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

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

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

8. The method of claim 1, wherein the compound to which the cells are exposed is conjugated to an arginine-rich polypeptide effective to promote uptake of the compound into infected host cells.

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

121-126.

10. A method of inhibiting in a mammalian host cell, replication of an infecting RNA virus having a single-stranded, positive-sense genome and selected from one of the Flaviviridae, Picornoviridae, Caliciviridae, Togaviridae, Arteriviridae, Coronaviridae, Astroviridae or Hepeviridae families, comprising

administering to the infected host cells, a virus-inhibitory amount of an oligonucleotide analog compound 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 within the 5′

-terminal 40 bases of the positive-strand viral genome that is capable of forming internal stem-loop secondary structure, and

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

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

11. The method of claim 10, wherein said oligonucleotide 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.

12. The method of claim 10, wherein said intersubunit linkages are phosphorodiamidate linkages.

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

14. The method of claim 13, wherein X═

NR₂, where each R is independently hydrogen or methyl.

15. The method of claim 12, in which at least 2 and no more than half of the total number of intersubunit linkages are positively charged at physiological pH.

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

17. The method of claim 10, wherein said compound is a covalent conjugate of an oligonucleotide analog moiety capable of forming such a heteroduplex structure with the positive sense strand of the virus, and an arginine-rich polypeptide effective to enhance the uptake of the compound into host cells.

18. The method of claim 10, wherein the compound to which the cells are exposed is conjugated to an arginine-rich polypeptide effective to promote uptake of the compound into infected host cells.

19. The method of claim 16, wherein the arginine rich peptide has one of the sequences identified as SEQ ID NOS:

121-126.

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

21. The method of claim 10, wherein the compound administered is a cocktail of such compounds directed against two or more of said positive-strand single-strand RNA viruses.

22. A heteroduplex complex formed between:

(a) a region within the 5′

-terminal 40 bases of the positive strand RNA of an RNA virus having a single-stranded, positive-sense RNA genome and selected from one of the Flaviviridae, Picornoviridae, Caliciviridae, Togaviridae, Arteriviridae, Coronaviridae, Astroviridae or Hepeviridae families, which region is capable of forming internal stem-loop secondary structure, and

(b) an oligonucleotide 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 such stem-loop secondary structure within the 5′

-terminal end 40 bases of the positive-sense RNA strand of the virus,

where said heteroduplex complex has a Tm of dissociation of at least 45°

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

23. The complex of claim 22, wherein said oligonucleotide is 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.

24. The complex of claim 23, wherein said intersubunit linkages are phosphorodiamidate linkages.

25. The complex of claim 24, wherein said morpholino subunits are joined by phosphorodiamidate linkages, in accordance with the structure:

26. The complex of claim 25, wherein X═

NR₂, where each R is independently hydrogen or methyl.

27. The complex of claim 24, in which at least 2 and no more than half of the total number of intersubunit linkages are positively charged at physiological pH.

28. The complex of claim 27, wherein said morpholino subunits are joined by phosphorodiamidate linkages, in accordance with the structure:

29. A method of detecting the presence of a viral infection by an RNA virus having a single-stranded, positive-sense RNA genome and selected from one of the Flaviviridae, Picornoviridae, Caliciviridae, Togaviridae, Arteriviridae, Coronaviridae, Astroviridae or Hepeviridae families a in a mammalian subject, or for confirming the presence of an effective interaction between such a virus infecting a mammalian subject and an antisense oligonucleotide analog compound directed against the virus, comprising

(a) administering to the subject, an uncharged morpholino oligonucleotide analog compound having (a) a sequence of 12-40 subunits, including 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 virus, (b) morpholino subunits linked by uncharged, phosphorus-containing intersubunit linkages, each linkage joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit, and (c) is capable of forming with the positive-strand viral ssRNA genome, a heteroduplex structure characterized by a Tm of dissociation of at least 45°

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

(b) at a selected time after said administering, obtaining a sample of a body fluid from the subject; and

(c) assaying the sample for the presence of a nuclease-resistant heteroduplex comprising the antisense oligonucleotide complexed with a complementary-sequence 5′

-end region of the positive-strand RNA of the virus.

30. The method of claim 29, for use in determining the effectiveness of treating a Flaviviridae, Picornoviridae, Caliciviridae, Togaviridae, Arteriviridae, Coronaviridae, Astroviridae or Hepeviridae infection by administering said compound, wherein said administering, obtaining, and assaying is conducted at periodic intervals throughout a treatment period.

31. An oligonucleotide analog compound for use in inhibiting replication in mammalian host cells of an RNA virus having a single-stranded, positive-sense RNA genome and selected from the Flaviviridae, Picomoviridae, Caliciviridae, Togaviridae, Arteriviridae, Coronaviridae, Astroviridae or Hepeviridae , and 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 virus, and

(v) capable of forming with the positive-strand viral ssRNA genome, a heteroduplex structure having a Tm of dissociation of at least 45°

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

32. The compound of claim 31, 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.

33. The compound of claim 32, wherein said intersubunit linkages are phosphorodiamidate linkages.

34. The compound of claim 33, wherein said morpholino subunits are joined by phosphorodiamidate linkages, in accordance with the structure:

35. The compound of claim 34, wherein X═

NR₂, where each R is independently hydrogen or methyl.

36. The compound of claim 32, in which at least 2 and no more than half of the total number of intersubunit linkages are positively charged at physiological pH.

37. The compound of claim 36, wherein said morpholino subunits are joined by phosphorodiamidate linkages, in accordance with the structure:

38. The compound of claim 31, wherein said oligomer has a T_m, 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.

39. The compound of claim 31, which is a covalent conjugate of an oligonucleotide analog moiety capable of forming such a heteroduplex structure with the positive sense strand of the virus, and an arginine-rich polypeptide effective to enhance the uptake of the compound into host cells.

40. The compound of claim 39, wherein the arginine rich peptide has one of the sequences identified as SEQ ID NOS:

121-126.