Antisense antiviral agent and method for treating ssRNA viral infection

• US 20030224353A1
• Filed: 04/24/2003
• Published: 12/04/2003
• Est. Priority Date: 10/16/2001
• Status: Abandoned Application

• Current Assignee: Alvin W. Smith, David A. Stein, Douglas E. Skilling, Patrick L. Iversen
• Original Assignee: Alvin W. Smith, David A. Stein, Douglas E. Skilling, Patrick L. Iversen
• Inventors: Iversen, Patrick L., Stein, David A., Smith, Alvin W., Skilling, Douglas E.
• Application Number:
  US10/422,671
• Publication Number:
  US 20030224353A1
• Time in Patent Office: Days
• US Class Current: 435/5
• 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 31/14 for RNA viruses
• CPC Class Codes: C12N 15/1131 against viruses
• CPC Class Codes: C12N 2310/11 Antisense
• 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 2770/20011 Coronaviridae
• CPC Class Codes: C12N 2770/24111 Flavivirus, e.g. yellow fever virus, dengue, JEV
• 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 in inhibition of growth of viruses of the picornavirus, calicivirus, togavirus, coronavirus, and flavivirus families, as in treatment of a viral infection. The antisense antiviral compounds are substantially uncharged oligomers having a targeting base sequence that is substantially complementary to a viral target sequence which spans the AUG start site of the first open reading frame of the viral genome.

Claims

1. An antiviral compound directed against an RNA virus from the picornavirus, calicivirus, togavirus, coronavirus, or flavivirus families, said virus having a single-stranded, positive sense genome of less than 12 kb and a first open reading frame that encodes a polyprotein containing multiple functional proteins, said compound comprising

a morpholino oligomer having (a) a sequence of 12 to 40 morpholino subunits, supporting a targeting base sequence that is substantially complementary to a viral target sequence which spans the translation initiation region of said first open reading frame, and (b) a substantially uncharged backbone.

2. The compound of claim 1, wherein the subunits are connected by uncharged, phosphorus-containing intersubunit linkages, joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit.

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

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

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

6. The compound of claim 1, 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.

7. The compound of claim 1, directed against a picornavirus, and having a targeting sequence having at least 90% homology to a sequence selected from the group consisting of:

(i) SEQ ID NO. 16, for a polio virus of the Mahoney and Sabin strains,

(ii) SEQ ID NO. 17, for a hepatitis A virus,

(iii) SEQ ID NO. 18, for a rhinovirus 14,

(iv) SEQ ID NO. 19, for a rhinovirus 16,

(v) SEQ ID NO. 20, for a rhinovirus 1 B,

(vi) SEQ ID NOs. 21 and 22, for an Aphthovirus, and

(vii) SEQ ID NOs 23, 24 and 25, for a coxsackie virus.

8. The compound of claim 7, directed against human rhinovirus 16, and having a targeting sequence represented by SEQ ID NO. 18 or 19.

9. The compound of claim 1, directed against a calicivirus, and having a targeting sequence having at least 90% homology to a sequence selected from the group consisting of:

(i) SEQ ID NOs. 27, 28, and 29, for a serotype Pan-1 vesivirus,

(ii) SEQ ID NO. 30, for a porcine vesivirus,

(iii) SEQ ID NO. 31, for a Norwalk virus, and

(iv) SEQ ID NO. 32, for a feline vesivirus.

10. The compound of claim 9, directed against a flavivirus, wherein the oligomer is a phosphorodiamidate-linked morpholino oligomer having a targeting sequence having at least 90% homology to a sequence selected from the group consisting of SEQ ID NOs. 35, 37, 38, and 39.

11. The compound of claim 9, directed against a corona virus, wherein the oligomer is a phosphorodiamidate-linked morpholino oligomer having a targeting sequence having at least 90% homology to a sequence selected from the group consisting of SEQ ID NO. 40 and 41.

12. The compound of claim 1, directed against hepatitis E virus, and having a targeting sequence having at least 90% homology to a sequence selected from the group consisting of SEQ ID NOs. 33 and 34.

13. The compound of claim 12, wherein the oligomer is a phosphorodiamidate-linked morpholino oligomer having a sequence selected from the group consisting of SEQ ID NOs:

33 and 34.

14. The compound of claim 1, directed against a hepatitis C flavivirus, and having a targeting sequence having at least 90% homology to the sequence SEQ ID NO. 35.

15. The compound of claim 14, wherein the oligomer is a phosphorodiamidate-linked morpholino oligomer having the targeting sequence SEQ ID NO:

35.

16. The compound of claim 1, wherein said oligomer can be recovered, in a heteroduplex form consisting of the oligomer and a complementary portion of the viral genome of said RNA virus, from the serum or urine of a mammalian subject, several hours after being administered to said subject.

17. A method of inhibiting replication of an RNA virus from the picornavirus, calicivirus, togavirus, coronavirus, or flavivirus families which has a single-stranded, positive sense genome of less than 12 kb, and a first open reading frame that encodes a polyprotein containing multiple functional proteins, comprising

exposing said virus to a morpholino oligomer having (a) a sequence of 12 to 40 morpholino subunits, supporting a targeting base sequence that is substantially complementary to a viral target sequence which spans the translation initiation region of said first open reading frame, and (b) a substantially uncharged backbone.

18. The method of claim 17, wherein said oligomer is administered to a mammalian subject infected with said virus.

19. The method of claim 17, wherein said oligomer can be recovered from the serum or urine of said subject, several hours after said administering, in a heteroduplex form consisting of the oligomer and a complementary portion of the viral genome.

20. The method of claim 17, wherein the oligomer has uncharged, phosphorus-containing intersubunit linkages, joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit.

21. The method of claim 20, wherein said intersubunit linkages are phosphorodiamidate linkages.

22. The method of claim 21, wherein said morpholino subunits are joined by phosphorodiamidate linkages in accordance with the structure:

23. The method of claim 22, wherein X=NR₂, where each R is independently hydrogen or methyl. 24.

24. The method of claim 17, for inhibition of replication of a picornavirus, wherein said targeting sequence has at least 90% homology to a sequence selected from the group consisting of:

(i) SEQ ID NO. 16 for a polio virus of the Mahoney and Sabin strains,

(ii) SEQ ID NO. 17, for a hepatitis A virus,

(iii) SEQ ID NO. 18, for a rhinovirus 14,

(iv) SEQ ID NO. 19, for a rhinovirus 16,

(v) SEQ ID NO. 20, for a rhinovirus 1B,

(vi) SEQ ID NOs. 21 and 22, for an Aphthovirus,

(vii) SEQ ID NOs 23, 24 and 25, for a coxsackie virus.

25. The method of claim 24, for inhibition of replication of human rhinovirus 16, wherein the targeting sequence is SEQ ID NO. 18 or 19, and the oligomer is a phosphorodiamidate-linked morpholino oligomer.

26. The method of claim 17, for inhibition of replication of a calicivirus, wherein said targeting sequence has at least 90% homology to a sequence selected from the group consisting of:

(i) SEQ ID NOs. 27, 28 and 29, for a serotype Pan-1 vesivirus,

(ii) SEQ ID NO. 30, for a porcine vesivirus,

(iii) SEQ ID NO. 31, for a Norwalk virus, and

(iv) SEQ ID NO. 32, for a feline vesivirus.

27. The method of claim 17, for inhibition of a flavivirus, wherein the oligomer is a phosphorodiamidate-linked morpholino oligomer having a targeting sequence having at least 90% homology to a sequence selected from the group consisting of SEQ ID NOs. 35, 37, 38, and 39.

28. The method of claim 17, directed against a corona virus, wherein the oligomer is a phosphorodiamidate-linked morpholino oligomer having a targeting sequence having at least 90% homology to a sequence selected from the group consisting of SEQ ID NO. 40 and 41.

29. The method of claim 17, for inhibition of replication of a hepatitis E virus, wherein the targeting sequence has at least 90% homology to a sequence selected from the group consisting of SEQ ID NOs. 33 and 34, and the oligomer is a phosphorodiamidate-linked morpholino oligomer.

30. The method of claim 17, for inhibition of replication of a hepatitis C flavivirus, wherein the targeting sequence has at least 90% homology to SEQ ID NO:

35, and the oligomer is a phosphorodiamidate-linked morpholino oligomer.

31. The method of claim 17, wherein said oligomer has a T_m, with respect to binding to said viral target sequence, of greater than about 50°

C., and is able to be actively taken up by mammalian cells.

32. A method of confirming the presence of an effective interaction between a picornavirus, calicivirus, togavirus, coronavirus, or flavivirus infecting a mammalian subject, and an antisense oligomer targeted against the infecting virus, comprising

(a) administering said oligomer to the subject, wherein said oligomer has a sequence of 12 to 40 morpholino subunits, supporting a targeting base sequence that is substantially complementary to a viral target sequence which spans the translation initiation region of the first open reading frame of the infecting virus,

(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 oligomer and a complementary portion of said viral target sequence.

33. The method of claim 32, wherein the oligomer has uncharged, phosphorus-containing intersubunit linkages, joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit.

34. The method of claim 33, wherein the linkages are phosphorodiamidate linkages.

35. The method of claim 32, for use in determining the effectiveness of treating a picornavirus, calicivirus, togavirus, coronavirus, or flavivirus infection by administering said oligomer, wherein said administering, obtaining, and assaying is conducted at periodic intervals throughout a treatment period.

36. A method of determining the family or genus of an infecting picornavirus, calicivirus, togavirus, coronavirus, or flavivirus, the method comprising

(a) providing a plurality of antisense oligomers, each said oligomer having a base sequence that is substantially complementary to a viral target sequence of a plurality of known viruses selected from picornaviruses, caliciviruses, togaviruses or flaviviruses, wherein each said viral target sequence is (i) common to a virus family or genus, and (ii) not found in humans;

(b) administering at least one oligomer of the plurality to the subject,

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

(d) assaying the sample for the presence of a nuclease-resistant heteroduplex comprising the antisense oligomer and a complementary portion of the viral target sequence, and

(e) identifying the family or genus of the infecting virus, based on the presence or absence of a heteroduplex comprising an administered antisense oligomer and a complementary portion of said viral target base sequence.

37. The method of claim 36, for use in identifying a specific infecting picornavirus, calicivirus, togavirus, coronavirus, or flavivirus, further comprising

providing a second plurality of antisense oligomers, each said oligomer having a base sequence that is substantially complementary to a viral target sequence of one of a plurality of known viruses from the family or genus identified in step (e), wherein each said viral target sequence is (i) specific to one of said known viruses, and (ii) not found in humans;

(b) administering at least one oligomer of the plurality to the subject,

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

(d) assaying the sample for the presence of a nuclease-resistant heteroduplex comprising the antisense oligomer and a complementary portion of the viral target sequence, and

(e) identifying the infecting virus, based on the presence or absence of a heteroduplex comprising an administered antisense oligomer and a complementary portion of said viral target base sequence.