Multimeric oligonucleotides 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 different target nucleic acid than the other X, 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 the 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 in mammalian serum or plasma.
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 pharmaceutical composition comprising a single-stranded nucleic acid compound of claim 1 and a pharmaceutically acceptable carrier.
5. A kit comprising a container housing the composition of claim 3 .
6. The single-stranded nucleic acid compound of claim 2 , wherein the pyrimidine nucleotides are thymidines.
7. The single-stranded nucleic acid compound of claim 2 , wherein the pyrimidine nucleotides are uridines.
8. The single-stranded nucleic acid compound of claim 1 , wherein one of the different target nucleic acids is an mRNA.
9. The single-stranded nucleic acid compound of claim 1 , wherein one of the different target nucleic acids is a long non-coding RNA (lncRNA).
10. The single-stranded nucleic acid compound of claim 1 , wherein each targeting oligonucleotide comprises phosphorothioate internucleotide linkages between all nucleotides.
11. 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.
12. 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.
13. The method of claim 12 , wherein the cell is in vitro.
14. The method of claim 12 , wherein the cell is in vivo.
15. The method of claim 14 , wherein the single-stranded nucleic acid compound is delivered systemically to a subject comprising the cell.
16. The single-stranded nucleic acid compound of claim 1 , wherein the linker consists of 2 to 4 pyrimidine nucleotides linked through phosphodiester linkages.
17. A 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 different target nucleic acid than the other X, wherein adjacent nucleotides of the region of complementarity of each X comprise phosphorothioate linkages,
wherein L is a linker consisting of 1 to 10 pyrimidine nucleotides linked through phosphodiester linkages that links the 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 in mammalian serum or plasma, and
wherein the compound does not mediate degradation of the target nucleic acids by an RNAi pathway.
18. The compound of claim 17 , wherein the pyrimidine nucleotides are thymidines or uridines.
19. The compound of claim 18 , wherein the pyrimidine nucleotides are thymidines.
20. The compound of claim 18 , wherein the pyrimidine nucleotides are uridines.
21. The compound of claim 17 , wherein one of the different target nucleic acids is an mRNA.
22. The compound of claim 17 , wherein one of the different target nucleic acids is a long non-coding RNA (lncRNA).
23. The compound of claim 17 , wherein each targeting oligonucleotide comprises phosphorothioate internucleotide linkages between all nucleotides.
24. The compound of claim 17 , 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.
25. The compound of claim 17 , wherein the linker consists of 2 to 4 pyrimidine nucleotides linked through phosphodiester linkages.