siRNA silencing of genes expressed in cancer

• US 20090149403A1
• Filed: 05/29/2007
• Published: 06/11/2009
• Est. Priority Date: 05/26/2006
• Status: Active Grant

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Protiva Biotherapeutics Inc (Arbutus Biopharma Corporation)
• Inventors: MacLachlan, Ian, Judge, Adam, Heyes, James, Sood, Vandana, Jeffs, Lloyd B., Palmer, Lorne
• Granted Patent: US 8,598,333 B2
• Time in Patent Office: Days
• US Class Current: 514/44.R
• CPC Class Codes: A61K 31/575 substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• 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 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/3515 Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12Y 603/02019 Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme
• CPC Class Codes: Y10T 428/2982 Particulate matter [e.g., sphere, flake, etc.]

Abstract

The present invention provides nucleic acid-lipid particles comprising siRNA molecules that silence genes expressed in cancer (e.g., Eg5, EGFR or XIAP) and methods of using such nucleic acid-lipid particles to silence Eg5, EGFR or XIAP gene expression.

Claims

1. A nucleic acid-lipid particle comprising:

an siRNA molecule that silences expression a gene expressed in cancer, said gene selected from the group consisting of Eg5, EGFR, XIAP, and combinations thereof;

a cationic lipid;

a non-cationic lipid; and

a conjugated lipid that inhibits aggregation of said particle.

2. The nucleic acid-lipid particle of claim 1, wherein said nucleic acid-lipid particle comprises an siRNA molecule comprising at least one sequence set forth in any one of Tables 1-9.

3. The nucleic acid-lipid particle of claim 1, wherein said nucleic acid-lipid particle comprises at least two siRNA molecules, each of which comprises a sequence set forth in any one of Tables 1-9.

4. The nucleic acid-lipid particle of claim 3, wherein the at least two siRNA sequences silence expression of different genes.

5. The nucleic acid-lipid particle of claim 1, wherein said nucleic acid-lipid particle comprises at least one siRNA molecule that silences the XIAP gene, said siRNA molecule comprising a sequence set forth in any of Tables 5-6 and 9.

6. The nucleic acid-lipid particle of claim 1, wherein said cationic lipid is a member selected from the group consisting of N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), and N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLendMA), and a mixture thereof.

7. The nucleic acid-lipid particle of claim 1, wherein said cationic lipid is DLinDMA.

8. The nucleic acid-lipid particle of claim 1, wherein said non-cationic lipid is a member selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), palmitoyloleyolphosphatidylglycerol (POPG), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE),16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, palmitoyloleoyl-phosphatidylethanolamine (POPE),1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, and a mixture thereof.

9. The nucleic acid-lipid particle of claim 1, wherein said non-cationic lipid is DSPC.

10. The nucleic acid-lipid particle of claim 1, wherein the conjugated lipid that inhibits aggregation of particles is a polyethyleneglycol (PEG)-dialkyloxypropyl (PEG-DAA) conjugate selected from the group consisting of a PEG-dilauryloxypropyl (C_1-2), a PEG-dimyristyloxypropyl (C₁₄), a PEG-dipalmityloxypropyl (C₁₆), and a PEG-distearyloxypropyl (C₁₈).

11. The nucleic acid-lipid particle of claim 10, wherein the PEG-DAA conjugate is a PEG-dimyristyloxypropyl (C₁₄).

12. The nucleic acid-lipid particle of claim 1, wherein said cationic lipid comprises from about 30 mol % to about 50 mol % of the total lipid present in said particle.

13. The nucleic acid-lipid particle of claim 1, wherein said non-cationic lipid comprises from about 5 mol % to about 90 mol % of the total lipid present in said particle.

14. The nucleic acid-lipid particle of claim 10, wherein said PEG-DAA conjugate comprises from 1 mol % to about 20 mol % of the total lipid present in said particle.

15. The nucleic acid-lipid particle of claim 1, further comprising cholesterol.

16. The nucleic acid-lipid particle of claim 15, wherein the cholesterol comprises from about 10 mol % to about 60 mol % of the total lipid present in said particle.

17. The nucleic acid-lipid particle of claim 1, wherein

said cationic lipid is DLinDMA;

said non-cationic lipid is DSPC; and

said conjugated lipid that inhibits aggregation of said particles is PEG-cDMA.

18. The nucleic acid-lipid particle of claim 17, further comprising cholesterol.

19. The nucleic acid-lipid particle of claim 18, comprisingmol % DLinDMA;

10 mol % DSPC;

2 mol % PEG-cDMA; and

48 mol % cholesterol.

20. The nucleic acid-lipid particle of claim 1, wherein the nucleic acid is fully encapsulated in said nucleic acid-lipid particle.

21. The nucleic acid-lipid particle of claim 1, wherein said particle has a nucleic acid:

lipid ratio of from about 0.01 to about 0.2.

22. The nucleic acid-lipid particle of claim 1, wherein said particle has a median diameter of less than about 150 nm.

23. A composition comprising the nucleic acid-lipid particle of claim 2 and a pharmaceutically acceptable carrier.

24. A method of introducing an siRNA that silences expression of a Eg5, EGFR or XIAP gene into a cell, said method comprising:

contacting said cell with a nucleic acid-lipid particle comprising

a cationic lipid;

a non-cationic lipid;

a conjugated lipid that inhibits aggregation of particles; and

said siRNA.

25. The method of claim 24, wherein said siRNA comprises at least one of the sequences set forth in Tables 1-9.

26. The method of claim 24, wherein said cationic lipid is a member selected from the group consisting of N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), and N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA)), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA) and 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), and a mixture thereof.

27. The method of claim 24, wherein said non-cationic lipid is a member selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), palmitoyloleyolphosphatidylglycerol (POPG), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE),16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, palmitoyloleoyl-phosphatidylethanolamine (POPE),1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, and a mixture thereof.

28. The method of claim 24, wherein the conjugated lipid that inhibits aggregation of particles is a polyethyleneglycol (PEG)-dialkyloxypropyl (PEG-DAA) conjugate selected from the group consisting of a PEG-dilauryloxypropyl (C₁₂), a PEG-dimyristyloxypropyl (C₁₄), a PEG-dipalmityloxypropyl (C₁₆), and a PEG-distearyloxypropyl (C₁₈).

29. The method of claim 24, wherein the nucleic acid-lipid particle further comprises cholesterol.

30. The method of claim 24, wherein said cell is in a mammal.

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

32. The method of claim 31, wherein the mammal has been diagnosed with cancer.

33. An isolated siRNA molecule comprising a sequence set forth in any one of Tables 1-9.