Multimeric oligonucleotide compounds
The disclosure provides multimeric oligonucleotide compounds, comprising two or more target-specific oligonucleotides (e.g., antisense oligonucleotides (ASOs)), each being resistant to cleavage, and linked together by a cleavable linker. In particular, two or more linked target-specific oligonucleotides, each to a different target, allows concomitant inhibition of multiple genes' expression levels, while exhibiting favorable pharmacokinetic and pharmacodynamic properties. Methods of making and uses of the described compounds are also provided.
1. A single-stranded nucleic acid compound comprising the general formula: 5′X3′-L-5′X3′,
wherein each X is independently a single-stranded targeting oligonucleotide of 8 to 16 nucleotides in length having a region of complementarity comprising at least 7 contiguous nucleotides complementary to a target region of an mRNA, wherein adjacent nucleotides of the region of complementarity of each X comprise phosphorothioate linkages, and
wherein L is a linker consisting of 1 to 10 pyrimidine nucleotides linked through phosphodiester linkages that links at least two Xs and that is i) more susceptible to cleavage in a liver mammalian extract than each X and ii) more susceptible to cleavage in liver mammalian extract than mammalian serum or plasma, and
wherein when the target regions complementary to the first X and second X do not overlap in the mRNA, the 5′-end of the target region complementary to the first X and the 3′-end of the target region complementary to the second X are not within a distance of 0 to 4 nucleotides in the mRNA, and
wherein at least one targeting oligonucleotide is a mixmer or gapmer.
2. The single-stranded nucleic acid compound of claim 1 , wherein the pyrimidine nucleotides are thymidines or uridines.
3. A composition comprising a single-stranded nucleic acid compound of claim 1 and a carrier.
4. A composition comprising a single-stranded nucleic acid compound of claim 1 in a buffered solution.
5. A pharmaceutical composition comprising a single-stranded nucleic acid compound of claim 1 and a pharmaceutically acceptable carrier.
6. A kit comprising a container housing the composition of claim 3 .
7. The single-stranded nucleic acid compound of claim 2 , wherein the pyrimidine nucleotides are thymidines.
8. The single-stranded nucleic acid compound of claim 2 , wherein the pyrimidine nucleotides are uridines.
9. The single-stranded nucleic acid compound of claim 1 , wherein at least one targeting oligonucleotide is a gapmer.
10. The single-stranded nucleic acid compound of claim 1 , wherein at least one targeting oligonucleotide is a mixmer.
11. The single-stranded nucleic acid compound of claim 1 , wherein each targeting oligonucleotide is a gapmer.
12. The single-stranded nucleic acid compound of claim 1 , wherein each targeting oligonucleotide is a mixmer.
13. The single-stranded nucleic acid compound of claim 1 , wherein at least one targeting oligonucleotide is a mixmer that comprises alternating deoxyribonucleotides and 2′-fluoro-deoxyribonucleotides, alternating deoxyribonucleotides and 2′-O-methyl nucleotides, alternating deoxyribonucleotides and ENA nucleotide analogues, alternating deoxyribonucleotides and LNA nucleotides, or alternating LNA nucleotides and 2′-O-methyl nucleotides.
14. The single-stranded nucleic acid compound of claim 13 , wherein each targeting oligonucleotide is a mixmer that comprises alternating deoxyribonucleotides and 2′-fluoro-deoxyribonucleotides, alternating deoxyribonucleotides and 2′-O-methyl nucleotides, alternating deoxyribonucleotides and ENA nucleotide analogues, alternating deoxyribonucleotides and LNA nucleotides, or alternating LNA nucleotides and 2′-O-methyl nucleotides.
15. The single-stranded nucleic acid compound of claim 1 , wherein at least one targeting oligonucleotide is an X—N-Y gapmer, wherein X and Y each independently contain 1, 2, 3, 4, 5 or more modified nucleotides and N is 3, 4, 5, 6, 7, 8, 9, or 10 deoxynucleotides with non-modified sugars.
16. The single-stranded nucleic acid compound of claim 15 , wherein each targeting oligonucleotide is an X—N-Y gapmer, wherein X and Y each independently contain 1, 2, 3, 4, 5 or more modified nucleotides and N is 3, 4, 5, 6, 7, 8, 9, or 10 deoxynucleotides with non-modified sugars.
17. The single-stranded nucleic acid compound of claim 1 , wherein each targeting oligonucleotide comprises phosphorothioate internucleotide linkages between all nucleotides.
18. The single-stranded nucleic acid compound of claim 1 , wherein each targeting oligonucleotide comprises a locked nucleic acid (LNA) nucleotide, ethylene bridged nucleic acid (ENA) nucleotide, 2′-O-methyl nucleotide, or 2′-fluoro-deoxyribonucleotide.
19. The single-stranded nucleic acid compound of claim 1 , wherein the linker consists of 2 to 4 pyrimidine nucleotides linked through phosphodiester linkages.
20. A method of delivering multiple targeting oligonucleotides to a cell, the method comprising:
contacting a cell with a single-stranded nucleic acid compound of claim 1 under conditions in which the compound enters into the cell.
21. The method of claim 20 , wherein the cell is in vitro.
22. The method of claim 20 , wherein the cell is in vivo.
23. The method of claim 20 , wherein the single-stranded nucleic acid compound is delivered systemically to a subject comprising the cell.
24. A method of delivering multiple targeting oligonucleotides to a cell, the method comprising:
contacting a cell with a single-stranded nucleic acid compound of claim 11 under conditions in which the compound enters into the cell.
25. The method of claim 24 , wherein the cell is in vitro.
26. The method of claim 24 , wherein the cell is in vivo.
27. The method of claim 24 , wherein the single-stranded nucleic acid compound is delivered systemically to a subject comprising the cell.
28. A method of delivering multiple targeting oligonucleotides to a cell, the method comprising:
contacting a cell with a single-stranded nucleic acid compound of claim 12 under conditions in which the compound enters into the cell.
29. The method of claim 28 , wherein the cell is in vitro.
30. The method of claim 28 , wherein the cell is in vivo.
31. The method of claim 28 , wherein the single-stranded nucleic acid compound is delivered systemically to a subject comprising the cell.