Multimetric 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.
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 each targeting oligonucleotide comprises phosphorothioate internucleotide linkages between all nucleotides.
10. 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.
11. 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.
12. The method of claim 11 , wherein the cell is in vitro.
13. The method of claim 11 , wherein the cell is in vivo.
14. The method of claim 11 , wherein the single-stranded nucleic acid compound is delivered systemically to a subject comprising the cell.
15. The single-stranded nucleic acid compound of claim 1 , wherein the linker consists of 2 to 4 pyrimidine nucleotides linked through phosphodiester linkages.
16. 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 target region of an mRNA, 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,
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 the compound does not mediate degradation of the target nucleic acids by an RNAi pathway.
17. The compound of claim 16 , wherein the pyrimidine nucleotides are thymidines or uridines.
18. The compound of claim 17 , wherein the pyrimidine nucleotides are thymidines.
19. The compound of claim 17 , wherein the pyrimidine nucleotides are uridines.
20. The compound of claim 16 , wherein each targeting oligonucleotide comprises phosphorothioate internucleotide linkages between all nucleotides.
21. The compound of claim 16 , 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.
22. The compound of claim 16 , wherein the linker consists of 2 to 4 pyrimidine nucleotides linked through phosphodiester linkages.