Methods of delivering multiple targeting oligonucleotides to a cell using cleavable linkers
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 method of delivering multiple single stranded targeting oligonucleotides to a liver or kidney cell, the method comprising:
contacting a liver or kidney cell with a compound under conditions in which the compound enters into the cell, wherein the compound comprises the general formula: X-L-[X-L] i -X,
wherein i is an integer from 0 to 9, the value of which indicates the number of units of [X-L] i present in the compound,
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 a genomic target sequence, wherein adjacent nucleotides of the region of complementarity of each X comprise phosphorothioate linkages, and each L is an single stranded oligonucleotide linker consisting of 2 to 4 contiguous thymidine or uridine nucleotide residues linked through phosphodiester linkages, that links at least two Xs and that is more susceptible to cleavage in a mammalian liver or kidney cell extract than each X,
wherein when i=0, and the general formula is 5′X3′-L-5′X3′ and when the target regions complementary to the first X and second X do not overlap in the genomic target sequence, 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 genomic target sequence,
wherein at least one L does not comprise an oligonucleotide having a self-complementary nucleotide sequence, and
wherein at least one L does not comprise an oligonucleotide having a nucleotide sequence that is complementary to a region of the genomic target sequence that is contiguous with the target regions complementary to two immediately flanking Xs.
2. The method of claim 1 , wherein i is an integer from 1 to 9.
3. The method of claim 1 , wherein each L consists of 3 or 4 contiguous thymidine or uridine nucleotide residues linked through phosphodiester linkages.
4. The method of claim 1 , wherein the linker is cleaved after the compound enters the cell.
5. The method of claim 1 , wherein i is 0.
6. The method of claim 1 , wherein each X of the compound has a different region of complementarity than each other X of the compound, such that the Xs of the compound target different genomic target sequences.
7. The method of claim 1 , wherein each X is independently complementary to an mRNA or a long non-coding RNA.
8. The method of claim 5 , wherein L consists of 3 or 4 contiguous thymidine or uridine nucleotide residues linked through phosphodiester linkages.
9. The method of claim 3 , wherein each L consists of 3 or 4 contiguous thymidine nucleotide residues linked through phosphodiester linkages.
10. The method of claim 3 , wherein each L consists of 4 contiguous thymidine nucleotide residues linked through phosphodiester linkages.
11. The method of claim 3 , wherein each L consists of 3 or 4 contiguous uridine nucleotide residues linked through phosphodiester linkages.
12. The method of claim 3 , wherein each L consists of 4 contiguous uridine nucleotide residues linked through phosphodiester linkages.
13. The method of claim 8 , wherein L consists of 3 or 4 contiguous thymidine nucleotide residues linked through phosphodiester linkages.
14. The method of claim 8 , wherein L consists of 4 contiguous thymidine nucleotide residues linked through phosphodiester linkages.
15. The method of claim 8 , wherein L consists of 3 or 4 contiguous uridine nucleotide residues linked through phosphodiester linkages.
16. The method of claim 8 , wherein L consists of 4 contiguous uridine nucleotide residues linked through phosphodiester linkages.
17. The method of claim 1 , wherein the cell is a liver cell.
18. The method of claim 5 , wherein the cell is a liver cell.