Enhancement of influenza gene expression
View Patent ↗The invention relates to compositions and methods for modulating the expression of influenza viral genes, and more particularly to the downregulation of influenza viral genes by chemically modified oligonucleotides.
1. A method of enhancing gene expression of an influenza gene in cell culture comprising,
(a) contacting a cell culture selected from MDCK cells and Vero cells with an siRNA molecule, wherein the cells of said cell culture have been engineered to express a recombinant influenza target gene;
(b) measuring the level of gene expression of said recombinant influenza target gene; and
(c) comparing the level of expression determined in step (b) to the level of expression in mock-treated cells, wherein an increased level of expression over mock-treated cells is evidence that enhanced gene expression of an influenza gene has occurred.
2. The method of claim 1 wherein recombinant influenza target gene is at least 80% homologous to a consensus sequence of the MP gene (SEQ ID NO: 1453).
3. The method of claim 2 wherein recombinant influenza target gene is the MP gene (SEQ ID NO: 1453).
4. The method of claim 3 wherein the siRNA molecule targets the first 500 nucleotides of said recombinant influenza target gene.
5. the method of claim 4 wherein the siRNA molecule comprises a sense strand 15 to 30 nucleotides in length and an antisense strand 15 to 30 nucleotides in length.
6. The method of claim 1 wherein the cell culture is selected from MDCK cells and Vero cells.
7. The method of claim 1 wherein measuring is performed via an ELISA assay.
8. The method of claim 5 wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the sense strand sequence of SEQ ID NO. 23, and wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the antisense sequences of SEQ ID NO 24.
9. The method of claim 8 wherein the siRNA molecule increases the expression of said recombinant influenza target gene by more than 10%, 20%, 30%, 40%, 50%, or 60%, in either MDCK cells or Vero cells compared to mock-treated cells.
10. The method of claim 8 wherein the siRNA molecule increases the expression of said recombinant influenza target gene by more than 5%, 10%, 15%, 20%, or 30%, in both MDCK cells and Vero cells compared to mock-treated cells.
11. The method of claim 8 wherein the antisense siRNA strand is 15 to 30 nucleotides in length and the duplex region of the siRNA molecule is 15 to 30 nucleotide pairs in length.
12. The method of claim 11 wherein the siRNA molecule comprises a modification that causes the siRNA molecule to have increased stability in a biological sample.
13. The method of claim 12 wherein the siRNA molecule comprises a phosphorothioate or a 2′-modified nucleotide.
14. The method of claim 11 wherein the siRNA molecule comprises a 5′ phosphate group.
15. The method of claim 12 wherein the siRNA molecule comprises a nucleotide overhang having 1 to 4 unpaired nucleotides.
16. The method of claim 15 wherein the siRNA molecule has 2 or 3 unpaired nucleotides.
17. The method of claim 11 wherein the siRNA molecule comprises at least one 1 non-natural nucleobase.
18. The method of claim 17 wherein said non-natural nucleobase is a universal base.
19. The method of claim 18 wherein the siRNA molecule consists of at least 3 universal base modifications.