ENDOSOMAL CLEAVABLE LINKERS

• US 20190330630A1
• Filed: 01/18/2018
• Published: 10/31/2019
• Est. Priority Date: 01/18/2017
• Status: Active Grant

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Alnylam Pharmaceuticals Incorporated
• Inventors: NAIR, Jayaprakash K., RAJEEV, Kallanthottathil G., LENTINI, Scott, THEILE, Christopher, MAIER, Martin, ZLATEV, Ivan
• Granted Patent: US 11,286,482 B2
• Time in Patent Office: Days
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/04 Centrally acting analgesics, e.g. opioids
• CPC Class Codes: A61P 29/02 without antiinflammatory effect
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2310/314 Phosphoramidates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/318 where the PO2 is completely replaced, e.g. MMI or formacetal
• CPC Class Codes: C12N 2310/3183 Diol linkers, e.g. glycols or propanediols
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 : 2'-R Modification
• CPC Class Codes: C12N 2310/3519 : Fusion with another nucleic acid
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2310/3533 : Halogen
• CPC Class Codes: C12N 2310/51 : in polymeric form, e.g. multimers, concatemers

Abstract

The present disclosure relates generally to cleavable linkers and uses thereof.

Claims

1. A multi-targeted molecule comprising at least two effector molecules, wherein the effector molecules are connected together via an endosomal cleavable linker or a protease cleavable linker, wherein the linker is a carbohydrate linker and wherein the linker is cleaved at least 1.25 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

2. The multi-targeted molecule of claim 1, wherein at least one ligand is conjugated with the multi-targeted molecule.

3. The multi-targeted molecule of claim 1, wherein the effector molecules are selected independently from the group consisting of siRNA, shRNA, antisense oligonucleotide, microRNA, anti-microRNA or antimir, supermir, antagomir, ribozyme, triplex-forming oligonucleotide, decoy oligonucleotide, splice-swithing oligonucleotide, immunostimulatory oligonucleotide, RNA activator, U1 adaptor, CRISPR Cas and any combinations thereof.

4. The multi-targeted molecule of claim 1, wherein said multi-targeted molecule modulates gene expression of at least two target nucleic acids by at least 75% each relative to when said effector molecules are not connected together.

5. The multi-targeted molecule of claim 4, wherein one of said at least two effector molecules modulate gene expression of a first target nucleic acid and another one of said at least two effector molecules modulates gene expression of a second nucleic acid.

6. The multi-targeted molecule of claim 5, wherein the first target nucleic acid and the second target nucleic acid are the same.

7. The multi-targeted molecule of claim 6, wherein the first target nucleic acid and the second target nucleic acid target same nucleotide sequence.

8. The multi-targeted molecule of claim 1, wherein the ligand is conjugated at the 3′

-end of one of said at least two effector molecules.

9. The multi-targeted molecule of claim 1, wherein the ligand is conjugated at the 5′

-end of one of said at least two effector molecules.

10. The multi-targeted molecule of claim 1, wherein the first effector molecule is a first double-stranded siRNA molecule and the second effector molecule is a second double-stranded siRNA molecule.

11. The multi-targeted molecule of claim 10, wherein the sense strand of the first siRNA molecule is covalently linked to the sense strand of the second siRNA molecule.

12. The multi-targeted molecule of claim 10, wherein sense strand of the first siRNA molecule is covalently linked to the antisense strand of the second siRNA molecule.

13. The multi-targeted molecule of claim 10, wherein antisense strand of the first siRNA molecule is covalently linked to the antisense strand of the second siRNA molecule.

14. The multi-targeted molecule of any of claims 10-13, wherein the first and second siRNA independently modulate gene expression of their respective target nucleic acids by at least 70% relative to when the first siRNA and the second siRNA are not part of the multi-targeted molecule.

15. The multi-targeted molecule of any of claims 10-14, wherein the first siRNA modulates gene expression of a first target nucleic acid and the second siRNA modulates gene expression of a second nucleic acid.

16. The multi-targeted molecule of claim 15, wherein the first target nucleic acid and the second target nucleic acid are the same.

17. The multi-targeted molecule of claim 16, wherein the first siRNA and the second siRNA target the same nucleic acid sequence.

18. The multi-targeted molecule of any of claims 10-17, wherein the ligand is conjugated with one of the sense strands.

19. The multi-targeted molecule of claim 18, wherein the ligand is conjugated at the 3′

-end of one of the sense strands.

20. The multi-targeted molecule of claim 19, wherein the ligand is conjugated at the 5′

-end of one of the sense strands.

21. The multi-targeted molecule of claim 19, wherein the ligand is conjugated at the 3′

-end of one of the antisense strands.

22. The multi-targeted molecule of claim 19, wherein the ligand is conjugated at the 5′

-end of one of the antisense strands.

23. The multi-targeted molecule of any of claims 10-22, wherein the multi-targeted molecule comprises at least one modification selected from the group consisting of modified internucleoside linkage, modified nucleobase, modified sugar, and any combinations thereof.

24. The multi-targeted molecule of claim 23, wherein said at least one modification is comprised in a sense strand or an antisense strand.

25. The multi-targeted molecule of any of claims 1-24, wherein the linker is selected from the group consisting of

26. A conjugate comprising an effector molecule linked to a ligand via an endosomal cleavable linker or a protease cleavable linker, wherein the linker is a carbohydrate linker and wherein the linker is cleaved at least 1.25 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions)

27. The conjugate of claim 26, wherein the effector molecule is selected from the group consisting of siRNA, shRNA, antisense oligonucleotide, microRNA, anti-microRNA or antimir, supermir, antagomir, ribozyme, triplex-forming oligonucleotide, decoy oligonucleotide, splice-swithing oligonucleotide, immunostimulatory oligonucleotide, RNA activator, U1 adaptor, and CRISPR Cas.

29. The conjugate of any one of claims 26-28, wherein the linker is selected from the linkers shown in FIGS. 1-6.

28. A conjugate comprising an endosomal agent linked with a ligand via an endosomal cleavable linker or a protease cleavable linker.

30. A prodrug comprising an endosomal cleavable linker or a protease cleavable linker selected from the linkers shown in FIGS. 1-6.