COMPOUNDS AND METHODS FOR ALTERING RSV REPLICATION RATE

• US 20150152420A1
• Filed: 11/21/2014
• Published: 06/04/2015
• Est. Priority Date: 11/22/2013
• Status: Active Grant

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: BAO, XIAOYONG, LEE, YONG SUN
• Granted Patent: US 10,219,492 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2760/18511 Pneumovirus, e.g. human respiratory syncytial virus

Abstract

Provided herein are methods for altering respiratory syncytial virus (RSV) replication in a cell using oligonucleotides derived from tRNAs, also referred to as tRFs (tRNA-derived RNA Fragments). The oligonucleotides may be used to decrease or increase replication of RSV. Also provided herein are methods for treating a subject having or at risk of having an RSV infection, and animal models for evaluating viral and host factors in RSV pathogenesis.

Claims

1. A method for decreasing Respiratory Syncytial Virus (RSV) replication in a cell, comprising:

introducing a polynucleotide into a cell, wherein RSV within the cell replicate at a decreased rate compared to RSV within a control cell that does not comprise the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence that is substantially identical to the complement of a reference sequence, wherein the reference sequence is at least 20 consecutive nucleotides selected from first 31 nucleotides at the 5′

end of a mature tRNA, wherein the mature tRNA is selected from a tRNA that specifies glutamine and has the anticodon CTG or TTG, glutamic acid and has the anticodon CTC or TTC, glycine and has the anticodon TCC or CCC or GCC, histidine and has the anticodon GTG, leucine and has the anticodon TAA, lysine and has the anticodon CTT or TTT, methionine and has the anticodon CAT, phenylalanine and has the anticodon GAA, serine and has the anticodon GCT, or valine and has the anticodon AAC or CAC.

2. The method of claim 1 wherein the polynucleotide comprises a nucleotide sequence that is identical to the complement of the reference sequence.

3. The method of claim 1 wherein the mature tRNA is selected from a tRNA that specifies glutamic acid and has the anticodon CTC, glycine and has the anticodon CCC, or lysine and has the anticodon CTT.

4. The method of claim 1 wherein the cell comprises RSV when the polynucleotide is introduced into the cell.

5. The method of claim 1 wherein the method further comprises introducing into the cell an RSV.

6. The method of claim 1 wherein the cell is an ex vivo cell.

7. The method of claim 6 wherein the ex vivo cell is a primary cell.

8. The method of claim 7 wherein the ex vivo cell is a cultured cell.

9. The method of claim 1 wherein the cell is an in vivo cell.

10. The method of claim 9 wherein the in vivo cell is present in an animal selected from a human, a chimpanzee, a cow, a sheep, a cotton rat, and a mouse.

11. The method of claim 9 wherein the in vivo cell is present in a human.

12. The method of claim 10 wherein the human is between 0 and 24 months of age.

13. The method of claim 1 wherein at least 2 polynucleotides are introduced into the cell.

14. The method of claim 1 wherein the polynucleotide comprises RNA.

15. The method of claim 1 wherein the introducing comprises introducing a vector into the cell, wherein the vector encodes the polynucleotide.

16. A method for treating a subject, comprising:

administering to a subject in need thereof an effective amount of a composition comprising a polynucleotide that comprises a nucleotide sequence that is substantially identical to the complement of a reference sequence, wherein the reference sequence is at least 20 consecutive nucleotides selected from first 31 nucleotides at the 5′

end of a mature tRNA, wherein the mature tRNA is selected from a tRNA that specifies glutamine and has the anticodon CTG or TTG, glutamic acid and has the anticodon CTC or TTC, glycine and has the anticodon TCC or CCC or GCC, histidine and has the anticodon GTG, leucine and has the anticodon TAA, lysine and has the anticodon CTT or TTT, methionine and has the anticodon CAT, phenylalanine and has the anticodon GAA, serine and has the anticodon GCT, or valine and has the anticodon AAC or CAC, wherein the subject has or is at risk of having an RSV infection.

17. The method of claim 16 wherein the administering comprises administering the composition to the respiratory tract of the subject.

18. The method of claim 16 wherein the method further comprises administering an antiviral agent.

19. The method of claim 16 wherein the subject is an animal selected from a human, a chimpanzee, a cow, a sheep, a cotton rat, and a mouse.

20. The method of claim 16 wherein the subject is a human between 0 and 24 months of age.

21. The method of claim 19 wherein the human has symptoms of bronchiolitis, pneumonia, or the combination thereof

22. A method for promoting RSV replication in a cell, comprising:

introducing a polynucleotide into a cell, wherein RSV within the cell replicate at an increased rate compared to RSV within a control cell that does not comprise the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence that is substantially identical to a reference sequence, wherein the reference sequence is at least 20 consecutive nucleotides selected from first 31 nucleotides at the 5′

end of a mature tRNA, wherein the mature tRNA is selected from a tRNA that specifies glutamine and has the anticodon CTG or TTG, glutamic acid and has the anticodon CTC or TTC, glycine and has the anticodon TCC, CCC, or GCC, histidine and has the anticodon GTG, leucine and has the anticodon TAA, lysine and has the anticodon CTT or TTT, methionine and has the anticodon CAT, phenylalanine and has the anticodon GAA, serine and has the anticodon GCT, valine and has the anticodon AAC or CAC, or cysteine and has the anticodon GCA.

23. An animal model for evaluating viral and host factors in human RSV pathogenesis, comprising:

an animal comprising a polynucleotide that increases the replication of RSV within the cell compared to RSV within a control cell that does not comprise the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence that is substantially identical to a reference sequence, wherein the reference sequence is at least 20 consecutive nucleotides selected from first 31 nucleotides at the 5′

end of a mature tRNA, wherein the mature tRNA is selected from a tRNA that specifies glutamine and has the anticodon CTG or TTG, glutamic acid and has the anticodon CTC or TTC, glycine and has the anticodon TCC, CCC, or GCC, histidine and has the anticodon GTG, leucine and has the anticodon TAA, lysine and has the anticodon CTT or TTT, methionine and has the anticodon CAT, phenylalanine and has the anticodon GAA, serine and has the anticodon GCT, valine and has the anticodon AAC or CAC, or cysteine and has the anticodon GCA.

24. The animal model of claim 23 wherein the animal model further comprises RSV.

25. A method for using the animal model of claim 23 comprising evaluating viral and host factors in RSV pathogenesis.

26. An animal model for evaluating viral and host factors in human RSV pathogenesis, comprising:

an animal comprising a polynucleotide that decreases the replication of RSV within the cell compared to RSV within a control cell that does not comprise the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence that is substantially identical to the complement of a reference sequence, wherein the reference sequence is at least 20 consecutive nucleotides selected from first 31 nucleotides at the 5′

end of a mature tRNA, wherein the mature tRNA is selected from a tRNA that specifies glutamine and has the anticodon CTG or TTG, glutamic acid and has the anticodon CTC or TTC, glycine and has the anticodon TCC or CCC or GCC, histidine and has the anticodon GTG, leucine and has the anticodon TAA, lysine and has the anticodon CTT or TTT, methionine and has the anticodon CAT, phenylalanine and has the anticodon GAA, serine and has the anticodon GCT, or valine and has the anticodon AAC or CAC.

27. The animal model of claim 26 wherein the animal model further comprises RSV.

28. A kit for treating RSV infection comprising a polynucleotide, wherein the polynucleotide comprises a nucleotide sequence that is substantially identical to the complement of a reference sequence, wherein the reference sequence is at least 20 consecutive nucleotides selected from first 31 nucleotides at the 5′

end of a mature tRNA, wherein the mature tRNA is selected from a tRNA that specifies glutamine and has the anticodon CTG or TTG, glutamic acid and has the anticodon CTC or TTC, glycine and has the anticodon TCC or CCC or GCC, histidine and has the anticodon GTG, leucine and has the anticodon TAA, lysine and has the anticodon CTT or TTT, methionine and has the anticodon CAT, phenylalanine and has the anticodon GAA, serine and has the anticodon GCT, or valine and has the anticodon AAC or CAC.