Multimeric oligonucleotide compounds having non-nucleotide based 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 compound comprising the general formula: X-L-X,
wherein each X is independently a single-stranded targeting oligonucleotide of 8 to 50 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 each X comprises phosphorothioate internucleotide linkages,
wherein L is a linker that links the two Xs and that is more susceptible to cleavage in a mammalian extract than each X,
wherein L comprises a peptide which includes an endopeptidase cleavage site, wherein the endopeptidase is trypsin, chymotrypsin, elastase, thermolysin, pepsin, endopeptidase V8, cathepsin B, cathepsin D, cathepsin L, cathepsin C, papain, cathepsin S or endosomal acidic insulinase, and
wherein at least one of the different target nucleic acids is an mRNA that encodes a protein having an activity in liver.
2. The compound of claim 1 , wherein the mammalian extract is an extract from kidney, liver, intestinal or tumor tissue.
3. The compound of claim 1 , wherein the targeting oligonucleotide is 8 to 16 nucleotides in length.
4. The compound of claim 1 , wherein each of the different target nucleic acids is an mRNA that encodes a different protein having an activity in liver.
5. The compound of claim 1 , wherein one of the different target nucleic acids is an mRNA that encodes a protein having an activity in liver and the other different target nucleic acid is a long non-coding RNA (lncRNA).
6. The compound of claim 1 , wherein each targeting oligonucleotide comprises phosphorothioate internucleotide linkages between all nucleotides.
7. The compound of claim 1 , wherein at least one targeting oligonucleotide comprises a modified nucleotide.
8. The compound of claim 7 , wherein the modified nucleotide is a bridged nucleotide.
9. The compound of claim 8 , wherein the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.
10. The compound of claim 9 , wherein the bridged nucleotide is an LNA nucleotide.
11. The compound of claim 7 , wherein the modified nucleotide is a 2′-modified nucleotide.
12. The compound of claim 11 , wherein the 2′-modified nucleotide is a 2′-O-methyl or 2′-O-(2-methoxyethyl) nucleotide.
13. The compound of claim 11 , wherein the 2′-modified nucleotide is a 2′-O-methyl nucleotide.
14. The compound of claim 7 , wherein at least one targeting oligonucleotide comprises LNA nucleotides and 2′-O-methyl nucleotides.
15. The compound of claim 7 , wherein at least one targeting oligonucleotide comprises alternating deoxyribonucleotides and 2′-fluoro-deoxyribonucleotides.
16. The compound of claim 7 , wherein at least one targeting oligonucleotide comprises alternating deoxyribonucleotides and 2′-O-methyl nucleotides.
17. The compound of claim 7 , wherein at least one targeting oligonucleotide comprises alternating deoxyribonucleotides and ENA nucleotide analogues.
18. The compound of claim 7 , wherein at least one targeting oligonucleotide comprises alternating LNA nucleotides and 2′-O-methyl nucleotides.
19. The compound of claim 1 , wherein the endopeptidase cleavage site comprises the amino acid sequence ALAL (SEQ ID NO: 125), APISFFELG (SEQ ID NO: 126), FL, GFN, GRWHTVGLRWE (SEQ ID NO: 127), GRWPPMGLPWE (SEQ ID NO.: 137), GRWHPMGAPWE (SEQ ID NO.: 138), YL, GF, FF, or R/KXX, in which X is any amino acid.
20. A method of delivering multiple targeting oligonucleotides to a liver cell, the method comprising contacting the liver cell with a compound of claim 1 under conditions in which the compound enters into the cell.