COMPOSITIONS AND METHODS FOR MEDIATING RNAI IN VIVO

• US 20100003317A1
• Filed: 03/26/2009
• Published: 01/07/2010
• Est. Priority Date: 03/27/2008
• Status: Active Grant

• Current Assignee: Alnylam Pharmaceuticals Incorporated, ETH Zurich
• Original Assignee: ETH Zurich
• Inventors: Wolfrum, Christian, Konys, David, Stoffel, Markus, Akinc, Akin, Nakayama, Tomoko
• Granted Patent: US 8,148,344 B2
• Time in Patent Office: Days
• US Class Current: 424/450
• 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 composition comprising:

(a) at least one of a single or double stranded oligonucleotide, wherein the oligonucleotide has been conjugated to a lipophile, and

(b) an emulsion to which the lipophile-conjugated oligonucleotide has been aggregated, admixed or associated, wherein said composition has a size of at least 100 nm.

2. The composition of claim 1, wherein the lipophile-conjugated oligonucleotide comprises a double stranded oligonucleotide.

3. The composition of claim 2, 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 substantially complementary to at least a part of a target gene, and wherein said region of complementarity is less than 30 nucleotides in length.

4. The composition of claim 3, wherein the dsRNA comprises at least one modified nucleotide.

5. The composition of claim 3, wherein at least one of said modified nucleotides 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.

6. The composition 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.

7. The composition of claim 3, wherein the region of complementarity is between 19 and 21 nucleotides in length.

8. The composition of claim 3, wherein the dsRNA comprises a single strand overhang at one or both ends of the dsRNA.

9. The composition of claim 3, 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.

10. The composition of claim 1, wherein the lipophile conjugate is a cholesterol moiety.

11. The composition of claim 1, wherein the lipophile-conjugated oligonucleotide comprises a single stranded oligonucleotide, and wherein said single stranded oligonucleotide is a microRNA or microRNA targeting compound.

18. A method of reducing expression of a gene in mammalian tissue comprising contacting said tissue with the composition of claim 1.

19. The method of claim 18, wherein said mammalian tissue is heart, lung and/or muscle tissue.

20. The method of claim 18, 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.

12. A method for selectively targeting or delivering an oligonucleotide to a mammalian tissue comprising contacting a mammal with said oligonucleotide, wherein said oligonucleotide has been formulated in a Formulated Lipid Particle (FLiP) emulsion, and wherein said emulsion is at least 100 nm in size.

13. The method of claim 12, wherein said mammalian tissue is heart, lung and/or muscle tissue.

14. The method of claim 12, wherein the oligonucleotide comprises a double stranded RNA (dsRNA) having two strands, 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.

15. The method of claim 14, wherein the oligonucleotide is modified with a cholesterol moiety.

16. The method of claim 12, wherein the oligonucleotide targets ApoB or RhoA.

17. The method of claim 12, wherein the oligonucleotide comprises an miRNA antagonist or antagomir.

21. A liposome comprising triacylglycerol, phospholipids, glycerol and one or several lipid-binding proteins aggregated via a lipophilic linker molecule with a single- or double-stranded oligonucleotide, wherein said liposome has an affinity to heart, lung and/or muscle tissue.

22. The liposome of claim 21, wherein the oligonucleotide is stabilized by one or more oligonucleotide modifications.

23. The liposome of claim 21, comprising about 20% triacylglycerol, about 1.2% phospholipids and about 2.25% glycerol and one or several lipid-binding proteins.

24. The liposome of claim 21 having a particle size of 20-50 nm, preferably of about 35 nm.

25. The liposome of claim 21, wherein the oligonucleotide is an oligonucleotide mediating RNA interference, an oligonucleotide targeting RNAs or an oligonucleotide targeting proteins.

26. The liposome of claim 21, wherein the oligonucleotide is an RNA.

27. The liposome of claim 21 obtainable by fractionating an emulsion of lipid particles and selecting for particles of 20-50 nm, preferably of about 35 nm in size.

28. A process for making a liposome aggregate according to claim 21 comprising:

a. mixing a lipid emulsion with one or several single- or double-stranded oligonucleotides;

b. fractionating the mixture of step a);

c. selecting the fraction with particles of 20-50 nm, preferably of about 35 nm in size.

29. A process for making a liposome aggregate according to claim 21, wherein the lipid particle comprising triacylglycerol, phospholipids, glycerol and one or several lipid-binding proteins is first isolated from a lipid emulsion and then mixed with >

2-fold molar excess of one or several lipophile conjugated single- or double-stranded oligonucleotide.

30. The liposome of claim 21 for the use as a medicament.

31. The liposome of claim 21 for the treatment of heart, lung and/or muscle diseases.

32. Use of the liposome of claim 21 for the manufacture of a medication for the treatment of heart, lung and/or muscle diseases.

33. A lyophilisat comprising the liposome of claim 21.