Compositions and methods for mediating RNAi in vivo

• US 8,148,344 B2
• Filed: 03/26/2009
• Issued: 04/03/2012
• Est. Priority Date: 03/27/2008
• Status: Active Grant

• Current Assignee: Alnylam Pharmaceuticals Incorporated, ETH Zurich
• Original Assignee: Alnylam Pharmaceuticals Incorporated, ETH Zurich
• Inventors: Akinc, Akin, Nakayama, Tomoko, Konys, David, Stoffel, Markus, Wolfrum, Christian
• Primary Examiner(s): McGarry, Sean
• Application Number:
  US12/412,206
• Publication Number:
  US 20100003317A1
• Time in Patent Office: 1,104 Days
• Field of Search: 435/6, 435/91.1, 435/91.31, 435/455, 536/23.1, 536/24.5
• US Class Current: 514/44.A
• CPC Class Codes: A61K 47/554 the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
• CPC Class Codes: A61K 47/69 the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Abstract

This invention relates to new formulated lipid particles (FLiPs) comprising at least one of a single or double stranded oligonucleotide, where the oligonucleotide has been conjugated to a lipophile and at least one of an emulsion or liposome to which the conjugated oligonucleotide has been aggregated, admixed or associated. These particles have surprisingly been shown to effectively deliver oligonucleotides to heart, lung and muscle where they effect gene silencing.

Claims

1. A formulated lipid particle (FLiP) comprising:

(a) a double stranded oligonucleotide conjugated to a lipophile, and

(b) a 20% intravenous fat emulsion to which the lipophile-conjugated double stranded oligonucleotide is associated.

2. The FLiP of claim 1, wherein the double stranded oligonucleotide is a double stranded RNA (dsRNA) comprising at least two strands that are complementary to each other and wherein each of the two strands are at least 15 nucleotides long, and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is complementary to at least a part of a target gene, and wherein said region of complementarity is less than 30 nucleotides in length.

3. The FLiP of claim 2, wherein at least one nucleotide of the dsRNA is a modified nucleotide.

4. The FLiP of claim 3, wherein the modified nucleotide is chosen from the group of:

a 2′

-O-methyl modified nucleotide, a nucleotide comprising a 5′

-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.

5. The FLiP of claim 3, wherein said modified nucleotide is chosen from the group of:

a 2′

-deoxy-2′

-fluoro modified nucleotide, a 2′

-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′

-amino-modified nucleotide, 2′

-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

6. The FLiP of claim 2, wherein the region of complementarity is between 19 and 21 nucleotides in length.

7. The FLiP of claim 2, wherein the dsRNA comprises a single strand overhang at one or both ends of the dsRNA.

8. The FLiP of claim 2, wherein the sense and antisense strands are each 21 nucleotides in length with 19 base pair complementarity, and wherein the dsRNA has a single strand overhang of 2 nucleotides at the 3′

end of each strand.

9. The FLiP of claim 1, wherein the lipophile conjugate is a cholesterol moiety.

10. A method for selectively targeting or delivering a double stranded oligonucleotide comprising two strands to a mammalian tissue comprising contacting a mammal with the FLiP of claim 1.

11. The method of claim 10, wherein said mammalian tissue is heart, lung and/or muscle tissue.

12. The method of claim 10, each strand being 21 nucleotides in length with 19 base pair complementarity and each strand having a single strand overhang of 2 nucleotides at the 3′

end.

13. The method of claim 12, wherein the double stranded oligonucleotide is modified with a cholesterol moiety.

14. The method of claim 10, wherein the double stranded oligonucleotide targets ApoB or RhoA.

15. A method of reducing expression of a gene in mammalian tissue comprising contacting said tissue with the FLiP of claim 1 or claim 2 or claim 8.

16. The method of claim 15, wherein said mammalian tissue is heart, lung and/or muscle tissue.

17. The method of claim 15, wherein expression of the gene in the tissue is reduced in vivo in a mammal and said contacting is via systemic delivery to the mammal.

18. A lyophilisat comprising the FLiP of claim 1 or claim 2 or claim 8.

19. The FLiP of claim 1 or claim 2 or claim 8, wherein the oligonucleotide targets ApoB or RhoA.

20. The FLiP of claim 1 or claim 2 or claim 8, wherein the 20% intravenous fat emulsion comprises triacylglycerol, phospholipids, glycerol, and one or several lipid-binding proteins.

21. The FLiP of claim 20, wherein the 20% intravenous fat emulsion comprises 20% triacylglycerol, 1.2% phospholipids, 2.25% glycerol, and one or several lipid-binding proteins.

22. The FLiP of claim 20, wherein the 20% intravenous fat emulsion comprises 20% soybean oil, 1.2% egg yolk phospholipids, 2.25% glycerin, and water.

23. The FLiP of claim 1 or claim 2 or claim 8 having a particle size of 20-50 nm.

24. The FLiP of claim 1 or claim 2 or claim 8 having a particle size of 35 nm.

25. The FLiP of claim 1 or claim 2 or claim 8, wherein the lipophile conjugate comprises cholesterol and the 20% intravenous fat emulsion comprises 20% soybean oil, 1.2% egg yolk phospholipids, 2.25% glycerin, and water, and the particle size is 35 nm.