Antisense antiviral compound and method for treating influenza viral infection
The present invention relates to antisense antiviral compounds and methods of their use and production in inhibition of growth of viruses of the Orthomyxoviridae family and in the treatment of a viral infection. The compounds are particularly useful in the treatment of influenza virus infection in a mammal. Exemplary antisense antiviral compounds are substantially uncharged, or partially positively charged, morpholino oligonucleotides having 1) a nuclease resistant backbone, 2) 12-40 nucleotide bases, and 3) a targeting sequence of at least 12 bases in length that hybridizes to a target region selected from the following: a) the 5′ or 3′ terminal 25 bases of the negative sense viral RNA segment of Influenzavirus A, Influenzavirus B and Influenzavirus C; b) the terminal 30 bases of the 5′ or 3′ terminus of the positive sense vcRNA; c) the 45 bases surrounding the AUG start codon of an influenza viral mRNA and; d) 50 bases surrounding the splice donor or acceptor sites of influenza mRNAs subject to alternative splicing.
1. An isolated antiviral antisense oligonucleotide, comprising
a) a nuclease-resistant backbone;
b) 12-40 nucleotide bases, and
c) a targeting sequence of at least 10 bases in length that hybridizes to a target region selected from the 45 bases surrounding the AUG start codon of an influenza viral M1 or M2 mRNA;
wherein the oligonucleotide (i) comprises morpholino subunits linked by substantially uncharged, phosphorous-containing intersubunit linkages that join a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit; (ii) comprises one or more locked nucleic acid subunits; (iii) comprises 2′OMe residues; or (iv) is a peptide nucleic acid;
wherein the oligonucleotide comprises a base sequence selected from the group consisting of: SEQ ID NOS:12-16, 19-22, 34 and 45-47.
2. The oligonucleotide of claim 1 , wherein the morpholino subunits are joined by phosphorodiamidate linkages in accordance with the structure:
where Y 1 ═O, Z═O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, and X is alkyl, alkoxy, thioalkoxy, or alkyl amino.
3. The oligonucleotide of claim 2 , wherein X is 1-piperazine for at least 2 and no more than half of the total number of intersubunit linkages, and where the uncharged linkages are X≡NR 2 , wherein each R is independently hydrogen or methyl.
4. The oligonucleotide of claim 1 , wherein the oligonucleotide comprises SEQ ID NO:12, 13, 20, 34, 45, 46, or 47.
5. The oligonucleotide of claim 1 , wherein the oligonucleotide is conjugated to an arginine-rich polypeptide that enhances the uptake of the oligonucleotide into host cells.
6. A method of reducing replication of influenza virus, comprising contacting an influenza virus infected cell with an antiviral antisense oligonucleotide of claim 1 .
7. The oligonucleotide of claim 1 , wherein the oligonucleotide is characterized by the ability to form a heteroduplex structure with the viral target region, wherein said heteroduplex structure is:
a) composed of the positive sense strand of the virus and the oligonucleotide, and
b) characterized by a Tm of dissociation of at least 45° C.
8. The oligonucleotide of claim 5 , wherein the arginine-rich polypeptide is selected from the group consisting of SEQ ID NOS:115-128.
9. The oligonucleotide of claim 1 , wherein the oligonucleotide consists essentially of locked nucleic acid subunits.
10. The method of claim 6 , wherein the cell is in a subject, and the method comprises administering the antisense oligonucleotide to the subject.
11. The method of claim 10 , wherein the subject has a secondary bacterial infection, further comprising administering a bacterial antibiotic, separately or concurrently in combination with the antiviral antisense oligonucleotide.
12. The method of claim 11 , wherein the secondary bacterial infection is a Streptococcal pneumonia infection.
13. The method of claim 11 , wherein the antibiotic is a beta-lactam.
14. The method of claim 11 , wherein the antibiotic is selected from penicillin, amoxicillin, cephalosporins, chloramphenicol, and clindamycin.
15. The method of claim 10 , further comprising administering an antisense oligonucleotide targeted against an RNA molecule encoding CD200 or the CD200 receptor, separately or concurrently in combination with the antiviral antisense oligonucleotide.
16. A pharmaceutical composition comprising an antisense oligonucleotide of claim 1 , and a pharmaceutically acceptable carrier.
17. The pharmaceutical composition of claim 16 , further comprising a bacterial antibiotic.
18. The pharmaceutical composition of claim 16 , further comprising an antisense oligonucleotide targeted against an RNA molecule encoding CD200 or the CD200 receptor.