Compositions and methods for silencing SMAD4

• US 9,035,039 B2
• Filed: 12/21/2012
• Issued: 05/19/2015
• Est. Priority Date: 12/22/2011
• Status: Active Grant

• Current Assignee: Protiva Biotherapeutics Inc (Arbutus Biopharma Corporation)
• Original Assignee: Protiva Biotherapeutics Inc (Arbutus Biopharma Corporation)
• Inventors: Dhillon, Ammen P., Lee, Amy C. H., MacLachlan, Ian
• Primary Examiner(s): WHITEMAN, BRIAN A
• Application Number:
  US13/725,796
• Publication Number:
  US 20130172405A1
• Time in Patent Office: 879 Days
• Field of Search: 536/24.5
• US Class Current: 536/24.5
• CPC Class Codes: A61K 31/7105 Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical

Abstract

The present invention provides compositions comprising therapeutic nucleic acids such as interfering RNA (e.g., dsRNA such as siRNA) that target SMAD4 gene expression, lipid particles comprising one or more (e.g., a cocktail) of the therapeutic nucleic acids, methods of making the lipid particles, and methods of delivering and/or administering the lipid particles (e.g., for treating anemia of inflammation in humans).

Claims

1. A composition comprising an interfering RNA that silences SMAD4 gene expression, wherein the interfering RNA is an siRNA that comprises a sense strand and a complementary antisense strand, wherein the siRNA is Identifier 3 (SEQ ID NO:

7 and SEQ ID NO;

8).

2. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.

3. A nucleic acid-lipid particle comprising:

(a) an interfering RNA that silences SMAD4 gene expression, wherein the interfering RNA is an siRNA that comprises a sense strand and a complementary antisense strand, wherein the siRNA is Identifier 3 (SEQ ID NO;

7 and SEQ ID NO;

8);

(b) a cationic lipid; and

(c) a non-cationic lipid.

4. The nucleic acid-lipid particle of claim 3, wherein the cationic lipid comprises 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2- dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-y-linolenyloxy-N,Ndimethylaminopropane (y-DLenDMA), a salt thereof, or a mixture thereof.

5. The nucleic acid-lipid particle of claim 3, wherein the cationic lipid comprises 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), a salt thereof, or a mixture thereof.

6. The nucleic acid-lipid particle of claim 3, wherein the cationic lipid comprises (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-M-C3-DMA), dilinoleylmethyl-3-dimethylaminopropionate (DLin-MC2-DMA), a salt thereof, or a mixture thereof.

7. The nucleic acid-lipid particle of claim 6, wherein the cationic lipid is DLin-M-C3-DMA, a salt thereof.

8. The nucleic acid-lipid particle of claim 3, wherein the non-cationic lipid is a phospholipid.

9. The nucleic acid-lipid particle of claim 3, wherein the non-cationic lipid is cholesterol or a derivative thereof.

10. The nucleic acid-lipid particle of claim 9, wherein the non-cationic lipid is cholesterol.

11. The nucleic acid-lipid particle of claim 3, wherein the non-cationic lipid is a mixture of a phospholipid and cholesterol or a derivative thereof.

12. The nucleic acid-lipid particle of claim 3, wherein the phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), or a mixture thereof.

13. The nucleic acid-lipid particle of claim 3, wherein the non-cationic lipid is a mixture of DPPC and cholesterol.

14. The nucleic acid-lipid particle of claim 3, further comprising a conjugated lipid that inhibits aggregation of particles.

15. The nucleic acid-lipid particle of claim 14, wherein the conjugated lipid that inhibits aggregation of particles is a polyethyleneglycol (PEG)-lipid conjugate.

16. The nucleic acid-lipid particle of claim 15, wherein the PEG-lipid conjugate is selected from the group consisting of a PEG-diacylglycerol (PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, a PEG-phospholipid conjugate, a PEGceramide (PEG-Cer) conjugate, and a mixture thereof.

17. The nucleic acid-lipid particle of claim 15, wherein the PEG-lipid conjugate is a PEG-DAA conjugate.

18. The nucleic acid-lipid particle of claim 17, wherein the PEG-DAA conjugate is selected from the group consisting of a PEG-didecyloxypropyl (C10) conjugate, a PEG-dilauryloxypropyl (C12) conjugate, a PEG-dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (C16) conjugate, a PEG-distearyloxypropyl (C18) conjugate, and a mixture thereof.

19. The nucleic acid-lipid particle of claim 14, wherein the conjugated lipid that inhibits aggregation of particles is a polyoxazoline (POZ)-lipid conjugate.

20. The nucleic acid-lipid particle of claim 19, wherein the POZ-lipid conjugate is a POZ-DAA conjugate.

21. The nucleic acid-lipid particle of claim 14, wherein the cationic lipid is (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-M-C3-DMA), the non-cationic lipid is cholesterol, and the conjugated lipid is a a polyethyleneglycol (PEG)-lipid conjugate.

22. The nucleic acid-lipid particle of claim 3, wherein the interfering RNA is fully encapsulated in the particle.

23. The nucleic acid-lipid particle of claim 3, wherein the particle has a lipid:

interfering RNA mass ratio of from about 5;

1 to about 15;

1.

24. The nucleic acid-lipid particle of claim 3, wherein the particle has a median diameter of from about 30 nm to about 150 nm.

25. The nucleic acid-lipid particle of claim 3, wherein the cationic lipid comprises from about 50 mol % to about 65 mol % of the total lipid present in the particle.

26. The nucleic acid-lipid particle of claim 3, wherein the non-cationic lipid comprises a mixture of a phospholipid and cholesterol or a derivative thereof, wherein the phospholipid comprises from about 4 mol % to about 10 mol % of the total lipid present in the particle and the cholesterol or derivative thereof comprises from about 30 mol % to about 40 mol % of the total lipid present in the particle.

27. The nucleic acid-lipid particle of claim 26, wherein the phospholipid comprises from about 5 mol % to about 9 mol % of the total lipid present in the particle and the cholesterol or derivative thereof comprises from about 32 mol % to about 37 mol % of the total lipid present in the particle.

28. The nucleic acid-lipid particle of claim 14, wherein the conjugated lipid that inhibits aggregation of particles comprises from about 0.5 mol % to about 2 mol % of the total lipid present in the particle.

29. A pharmaceutical composition comprising a nucleic acid-lipid particle of claim 3 and a pharmaceutically acceptable carrier.

30. A method for introducing an interfering RNA that silences SMAD4 gene expression into a cell, the method comprising:

(a) contacting the cell with a nucleic acid-lipid particle of claim 3.

31. The method of claim 30, wherein the cell is in a mammal.

32. The method of claim 30, wherein the cell is contacted by administering the particle to the mammal via a systemic route.

33. The method of claim 30, wherein the mammal is a human.

34. The method of claim 30, wherein the mammal has been diagnosed with anemia of inflammation.

35. A method for treating and/or ameriorating one or more symptoms associated with anemia of inflammation in a human, the method comprising:

(a) administering to the human a therapeutically effective amount of a nucleic acid-lipid particle of claim 3.

36. The method of claim 35, wherein the particle is administered via a systemic route.

37. The method of claim 35, wherein the human has anemia of inflammation.