Oligonucleotide conjugates
The present invention relates to oligomeric compounds and conjugates thereof that target Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) PCSK9 mRNA in a cell, leading to reduced expression of PCSK9. Reduction of PCSK9 expression is beneficial for a range of medical disorders, such as hypercholesterolemia and related disorders.
1. An antisense oligonucleotide conjugate comprising:
a. an antisense oligomer (A) 16 nucleotides in length, wherein the sequence of the antisense oligomer is 100% complementary to the sequence of SEQ ID NO: 31, and wherein the antisense oligomer is a gapmer comprising at least one LNA unit; and,
b. at least one asialoglycoprotein receptor targeting conjugate moiety (C) covalently attached to the antisense oligomer (A).
2. The antisense oligonucleotide conjugate of claim 1 , wherein the antisense oligomer (A) targets mRNA encoding PCSK9.
3. The antisense oligonucleotide conjugate of claim 1 , wherein the sequence of the antisense oligomer (A) is SEQ ID NO: 26.
4. The antisense oligonucleotide of claim 1 , wherein the LNA is oxy-LNA, thio-LNA, amino-5 LNA, 5′-methyl-LNA, ENA, cET, cMOE, 5′-Me-LNA, or a combination thereof.
5. The antisense oligonucleotide conjugate of claim 4 , wherein the oxy LNA is beta-D-oxy LNA, alpha-L-oxy-LNA, or a combination thereof.
6. The antisense oligonucleotide conjugate of claim 1 , wherein the antisense oligomer (A) comprises at least one cET unit.
7. The antisense oligonucleotide conjugate of claim 1 , wherein the antisense oligomer (A) comprises six or seven LNA units.
8. The antisense oligonucleotide conjugate of claim 7 , wherein every LNA unit in the antisense oligomer (A) is a beta-D-oxy LNA unit.
9. The antisense oligonucleotide conjugate of claim 8 , wherein the sequence of the antisense oligomer (A) comprises at least one phosphorothioate internucleoside linkage.
10. The antisense oligonucleotide conjugate of claim 9 , wherein the sequence of the antisense oligomer (A) is SEQ ID NO: 2.
11. The antisense oligonucleotide conjugate of claim 9 , wherein the sequence of the antisense oligomer (A) is SEQ ID NO: 3.
12. The antisense oligonucleotide conjugate of claim 11 , wherein the asialoglycoprotein receptor targeting conjugate moiety (C) comprises a trivalent GalNAc moiety.
13. The antisense oligonucleotide conjugate according of claim 12 , wherein the trivalent GalNAc moiety comprises Conj 2a:
14. The antisense oligonucleotide conjugate of claim 1 , wherein the asialoglycoprotein receptor targeting conjugate moiety (C) enhances delivery and/or uptake to liver cells.
15. The antisense oligonucleotide conjugate of claim 1 , wherein the asialoglycoprotein receptor targeting conjugate moiety (C) is covalently attached to the 5′ end of the antisense oligomer (A).
16. The antisense oligonucleotide conjugate of claim 15 , wherein the asialoglycoprotein receptor targeting conjugate moiety (C) comprises a monovalent, divalent, trivalent, or tetravalent N-acetylgalactosamine (GalNAc) moiety.
17. The antisense oligonucleotide conjugate of claim 16 , wherein the asialoglycoprotein receptor targeting conjugate moiety (C) comprises a trivalent GalNAc moiety.
18. The antisense oligonucleotide conjugate of claim 17 , wherein each GalNAc in the trivalent GalNAc moiety is attached to a di-lysine branch point group via a PEG spacer.
19. The antisense oligonucleotide conjugate of claim 18 , wherein the trivalent GalNAc moiety is conjugated to the antisense oligomer (A) via a linker.
20. The antisense oligonucleotide conjugate of claim 19 , wherein the linker is a C6 to C12 amino alkyl group or a biocleavable phosphate nucleotide linker comprising between 1 to 6 nucleotides.
21. The antisense oligonucleotide conjugate according of claim 17 , wherein the trivalent GalNAc moiety comprises Conj 2a:
22. The antisense oligonucleotide conjugate of claim 1 , wherein the conjugate is SEQ ID NO: 18.
23. The antisense oligonucleotide conjugate of claim 1 , wherein the conjugate is SEQ ID NO: 19.
24. An antisense oligonucleotide conjugate consisting of SEQ ID NO: 18
wherein
a superscript L identifies a beta-D-oxy INA unit,
a Me C identifies a 5-methylcytosine unit,
a subscript s identifies a phosphorothioate internucleoside linkage,
and wherein
is a Conj 2a asialoglycoprotein receptor targeting conjugate moiety
and wherein the anti sense oligonucleotide conjugate targets mRNA encoding PCSK9.
25. An antisense oligonucleotide conjugate consisting of SEQ ID NO: 19
wherein
a superscript L identifies a beta-D-oxy LNA unit,
a Me C identifies a 5-methylcytosine unit,
a subscript s identifies a phosphorothioate internucleoside linkage,
and wherein
is a Conj 2a asialoglycoprotein receptor targeting conjugate moiety
and wherein the antisense oligonucleotide conjugate targets mRNA encoding PCSK9.
26. A pharmaceutical composition comprising the antisense oligonucleotide conjugate of claim 1 and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.
27. The pharmaceutical composition of claim 26 , wherein the antisense oligonucleotide conjugate is SEQ ID NO: 18.
28. The pharmaceutical composition of claim 26 , wherein the antisense oligonucleotide conjugate is SEQ ID NO: 19.
29. A method of treating a disorder selected from the group consisting of atherosclerosis, hyperlipidemia, hypercholesterolemia, HDL/LDL cholesterol imbalance, coronary artery disease (CAD), or coronary heart disease (CHD) in a subject in need thereof, said method comprising administering an effective amount of an antisense oligonucleotide conjugate of claim 1 to the subject.
30. The method of claim 29 , wherein the antisense oligonucleotide conjugate is SEQ ID NO: 18.
31. The method of claim 29 , wherein the antisense oligonucleotide conjugate is SEQ ID NO: 19.
32. An in vivo or in vitro method to inhibit PCSK9 expression in a cell expressing PCSK9 comprising contacting the cell with an antisense oligonucleotide conjugate of claim 1 .
33. The method of claim 32 , wherein the antisense oligonucleotide conjugate is SEQ ID NO: 18.
34. The method of claim 32 , wherein the antisense oligonucleotide conjugate is SEQ ID NO: 19.