Oligonucleotide compound and method for treating nidovirus infections

• US 20050171044A1
• Filed: 12/22/2004
• Published: 08/04/2005
• Est. Priority Date: 12/24/2003
• Status: Abandoned Application

• Current Assignee: Avi Biopharma, Inc., The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Iversen, Patrick L., Stein, David A., Neuman, Benjamin, Buchmeier, Michael, Bestwick, Richard K.
• Application Number:
  US11/022,358
• Publication Number:
  US 20050171044A1
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 15/1131 against viruses
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2770/20022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Abstract

A method and oligonucleotide compound for inhibiting replication of a nidovirus in virus-infected animal cells are disclosed. The compound (i) has a nuclease-resistant backbone, (ii) is capable of uptake by the infected cells, (iii) contains between 8-25 nucleotide bases, and (iv) has a sequence capable of disrupting base pairing between the transcriptional regulatory sequences in the 5′ leader region of the positive-strand viral genome and negative-strand 3′ subgenomic region. In practicing the method, infected cells are exposed to the compound in an amount effective to inhibit viral replication.

Claims

1. A method of inhibiting replication of a nidovirus in virus-infected animal cells, comprising

(a) exposing the cells to an oligonucleotide compound (i) having a nuclease-resistant backbone, (ii) capable of uptake by the cells, (iii) containing between 8-25 nucleotide bases, and (iv) having a sequence complementary to at least 8 bases contained in one of:

(1) a sequence in a 5′

leader sequence of the nidovirus'"'"' positive-strand genomic RNA selected from the group consisting of SEQ ID NOS;

1-9, each sequence of which includes an internal transcription regulatory sequence; and

,

(2) a sequence in a negative-strand 3′

subgenomic region of the virus selected from the group consisting of SEQ ID NOS;

10-18, each sequence of which includes an internal transcriptional regulatory sequence that is substantially complementary to the corresponding transcriptional regulatory sequence contained within SEQ ID NOS;

1-9, respectively; and

(b) by said exposing, forming within said cells a based-paired heteroduplex structure composed of one of (1) the virus positive-strand genomic RNA and the oligonucleotide compound, and (2) the negative-strand 3′

subgenomic region of the virus and the oligonucleotides compound, said structure being characterized by

(1) a Tm of dissociation of at least 45°

C., and

(2) disrupted base pairing between the transcriptional regulatory sequences in the 5′

leader region of the positive-strand viral genome and negative-strand 3′

subgenomic region,

where the amount of compound to which the cells are exposed is sufficient inhibit nidovirus replication in the virus-infected cells.

2. The method of claim 1, wherein said oligonucleotide compound to which the cells are exposed is composed of morpholino subunits and 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 the morpholino subunits in the conjugate to which the cells are exposed are joined by phosphorodiamidate linkages, in accordance with the structure:

4. The method of claim 1, wherein the oligonucleotide analog to which the cells are exposed has a sequence complementary to at least 8 bases contained in the 5′

leader sequence of the nidovirus'"'"' positive-strand genomic RNA selected from the group consisting of SEQ ID NOS;

1-9.

5. The method of claim 4, wherein the oligonucleotide analog to which the cells are exposed has a sequence complementary to at least a portion of the transcriptional regulatory sequence contained within one of the sequences SEQ ID NOS:

1-9.

6. The method of claim 5, wherein the oligonucleotide analog to which the cells are exposed contains a sequence selected from the group consisting of SEQ ID NOS:

19-34.

7. The method of claim 6, for use in inhibiting replication of human SARS virus, wherein the oligonucleotide analog to which the cells are exposed contains a sequence selected from the group consisting of SEQ ID NOS:

25 and 26.

8. The method of claim 6, for use in inhibiting replication of human coronavius-229E or human coronavirus-OC43, wherein the oligonucleotide analog to which the cells are exposed contains the sequence selected from the group consisting of SEQ ID NOS:

21 and 22, for the coronavirus-229E and contains the sequence SEQ ID NOS;

23 for the coronavisus-OC42.

9. The method of claim 6, for use in inhibiting replication of feline coronavirus, wherein the oligonucleotide analog to which the cells are exposed contains the SEQ ID NO:

19.

10. The method of claim 1, wherein the oligonucleotide analog to which the cells are exposed has a sequence complementary to at least 8 bases contained in the negative-strand 3′

subgenomic region of the virus selected from the group consisting of SEQ ID NOS;

10-18.

11. The method of claim 10, wherein the oligonucleotide analog to which the cells are exposed has a sequence complementary to at least a portion of the transcriptional regulatory sequence contained within one of the sequences SEQ ID NOS:

10-18.

12. The method of claim 11, wherein the oligonucleotide analog to which the cells are exposed contains a sequence selected from the group consisting of SEQ ID NOS:

35-50.

13. The method of claim 12, for use in inhibiting replication of human SARS virus, wherein the oligonucleotide analog to which the cells are exposed contains a sequence selected from the group consisting of SEQ ID NOS:

41 and 42.

14. The method of claim 12, for use in inhibiting replication of human coronavius-229E or human coronavirus-OC43, wherein the oligonucleotide analog to which the cells are exposed contains the sequence selected from the group consisting of SEQ ID NOS:

37 and 38, for the coronavirus-229E and contains the sequence SEQ ID NOS;

39 for the coronavisus-OC42.

15. The method of claim 12, for use in inhibiting replication of feline coronavirus, wherein the oligonucleotide analog to which the cells are exposed contains the SEQ ID NO:

35.

16. The method of claim 1, for treating a nidovirus infection in a human or veterinary-animal subject, wherein said exposing includes administering the oligonucotide compound orally to the subject.

17. The method of claim 1, for treating a nidovirus infection in a human or veterinary animal subject, wherein said exposing includes administering the oligonucleotide compound to the subject, and which further includes monitoring a body fluid for the appearance of a heteroduplex composed of the oligonucleotide complementary portion of the viral genome in positive- or negative-strand form.

18. An oligonucleotide compound for use in inhibiting replication of a nidovirus in human cells, characterized by:

(i) a nuclease-resistant backbone,

(ii) capable of uptake by virus-infected human cells,

(iii) containing between 8-25 nucleotide bases,

(iv) having a sequence that is complementary to at least 8 bases contained in one of;

(1) a sequence in a 5′

leader sequence of the nidovirus'"'"' positive-strand genomic RNA selected from the group consisting of SEQ ID NOS;

1-9, each sequence of which includes an internal transcription regulatory sequence; and

,

(2) a sequence in a negative-strand 3′

subgenomic region of the virus selected from the group consisting of SEQ ID NOS;

10-18, each sequence of which includes an internal transcriptional regulatory sequence that is substantially complementary to the corresponding transcriptional regulatory sequence contained within SEQ ID NOS;

1-9, respectively; and

(v) capable of forming with the nidovirus (1) positive-strand genomic RNA or (2) the negative-strand 3′

subgenomic region, a heteroduplex structure characterized by (1) a T_m of dissociation of at least 45°

C., and (2) a disrupted base pairing between the transcriptional regulatory sequences in the 5′

leader region of the positive-strand viral genome and negative-strand 3′

subgenomic region.

19. The compound of claim 18, which is composed of morpholino subunits and uncharged, phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit.

20. The compound of claim 19, wherein the morpholino subunits are joined by phosphorodiamidate linkages, in accordance with the structure:

21. The compound method of claim 18, which has a sequence complementary to at least 8 bases contained in the 5′

leader sequence of the nidovirus'"'"' positive-strand genomic RNA selected from the group consisting of SEQ ID NOS;

1-9.

22. The compound of claim 21, which has a sequence complementary to at least a portion of the transcriptional regulatory sequence contained within one of the sequences SEQ ID NOS:

1-9.

23. The compound of claim 22, which has a sequence selected from the group consisting of SEQ ID NOS:

19-34.

24. The compound of claim 23, for use in inhibiting replication of human SARS virus, which contains a sequence selected from the group consisting of SEQ ID NOS:

25 and 26.

25. The compound of claim 23, for use in inhibiting replication of human coronavius-229E or human coronavirus-OC43, which contains the sequence selected from the group consisting of SEQ ID NOS:

21 and 22, for the coronavirus-229E and contains the sequence SEQ ID NOS;

23 for the coronavisus-OC42.

26. The compound of claim 23, for use in inhibiting replication of feline coronavirus, which contains the SEQ ID NO:

19.

27. The compound of claim 18, which has a sequence complementary to at least 8 bases contained in the negative-strand 3′

subgenomic region of the virus selected from the group consisting of SEQ ID NOS;

10-18.

28. The compound of claim 27, which has a sequence complementary to at least a portion of the transcriptional regulatory sequence contained within one of the sequences SEQ ID NOS:

10-18.

29. The compound of claim 28, which contains a sequence selected from the group consisting of SEQ ID NOS:

35-50.

30. The compound of claim 29, for use in inhibiting replication of human SARS virus, which contains a sequence selected from the group consisting of SEQ ID NOS:

41 and 42.

31. The compound of claim 29, for use in inhibiting replication of human coronavius-229E or human coronavirus-OC43, which contains the sequence selected from the group consisting of SEQ ID NOS:

37 and 38, for the coronavirus-229E and contains the sequence SEQ ID NOS;

39 for the coronavisus-OC42.

32. The compound of claim 29, for use in inhibiting replication of feline coronavirus, which contains the SEQ ID NO:

35.