Compositions for Targeted Delivery of siRNA

• US 20110207799A1
• Filed: 02/22/2011
• Published: 08/25/2011
• Est. Priority Date: 02/24/2010
• Status: Active Grant

• Current Assignee: Arrowhead Madison Inc (Arrowhead Pharmaceuticals, Inc.)
• Original Assignee: Roche Madison Inc. (Arrowhead Pharmaceuticals, Inc.)
• Inventors: Hadwiger, Philipp, Rozema, David B., Lewis, David L., Kitas, Erik, Thuer, Wilma, Wakefield, Darren H., Valis, Linda, Mohr, Peter, Hoffmann, Torsten
• Granted Patent: US 8,313,772 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 47/32 Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers , poly(meth)acrylates, or polyvinyl pyrrolidone
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61K 47/65 Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• CPC Class Codes: A61K 48/0041 the non-active part being polymeric
• CPC Class Codes: A61K 9/08 Solutions composition of solutions A61K47/00
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2310/3515 Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific

Abstract

The present invention is directed compositions for targeted delivery of RNA interference (RNAi) polynucleotides to hepatocytes in vivo. Targeted RNAi polynucleotides are administered together with co-targeted delivery polymers. Delivery polymers provide membrane penetration function for movement of the RNAi polynucleotides from outside the cell to inside the cell. Reversible modification provides physiological responsiveness to the delivery polymers.

Claims

1. A conjugate delivery system for delivering an RNA interference polynucleotide to a liver cell in vivo comprising:

2. The composition of claim 1 wherein the RNA interference polynucleotide is selected from the group consisting of:

DNA, RNA, dsRNA, RNA interference polynucleotide, siRNA, and miRNA.

3. The composition of claim 1 wherein the liver cell consists of a hepatocyte.

4. The composition of claim 1 wherein the membrane active polymer contains two or more different monomers.

5. The composition of claim 4 wherein the membrane active polymer is composed of amine-containing monomers, lower hydrophobic group monomers, and higher hydrophobic group monomers.

6. The composition of claim 1 wherein the reversibly masked membrane active polyamine is soluble in water.

7. The composition of claim 1 wherein the membrane active polyamine is a random copolymer

8. The composition of claim 7 wherein the random copolymer is selected from the group consisting of poly(vinyl ether) and poly(acrylate).

9. The composition of claim 5 wherein the amine-containing monomers, lower alkyl monomers, and higher alkyl monomers are present in a ratio of 4-8 amine-containing monomers:

3-5 lower hydrophobic group monomers;

1 higher hydrophobic group monomers.

10. The composition of claim 9 wherein the lower hydrophobic group consists of a butyl group and the higher hydrophobic group consists of an octadecyl or dodecyl group.

11. The composition of claim 1 wherein the masking agents are reversibly linked to at least 70% of the amines on the polyamine.

12. The composition of claim 11 wherein the masking agents are reversibly linked to at least 80% of the amines on the polyamine.

13. The composition of claim 1 wherein the reversibly modified polymer has a zeta potential between +30 and −

30 mV at pH 8.

14. The composition of claim 13 wherein the reversibly modified polymer has a zeta potential between +20 and −

20 mV at pH 8.

15. The composition of claim 14 wherein the reversibly modified polymer has a zeta potential between +10 and −

10 mV at pH 8.

16. The composition of any of claims 1 wherein the reversibly modified polymer has a zeta potential between 0 and −

30 mV at pH 8.

17. The composition of claim 16 wherein the reversibly modified polymer has a zeta potential between 0 and −

20 mV at pH 8.

18. The composition of claim 17 wherein the reversibly modified polymer has a zeta potential between 0 and −

10 mV at pH 8.

19. The composition of claim 1 wherein the composition is provided in a pharmaceutically acceptable carrier or diluent.

26. The composition of claim 1 wherein the ratio of galactose derivative to PEG linked to the polymer is 1 to 0.5-2.

27. The composition of claim 1 wherein the galactose derivative consists of an N-acetylgalactosamine.

20. The composition of 1 wherein L² is a maleamate linkage.

21. The composition of 20 wherein L² is a disubstituted maleamate linkage.

22. The composition of 1 wherein N is linked to A via a physiologically labile linkage L¹.

23. The composition of 22 wherein L¹ is a physiologically labile covalent linkage that is orthogonal to L².

24. The composition of 1 wherein A contains a hydrophobic group having 20 or more carbon atoms.

25. The composition of 1 wherein A contains a galactose trimer.

28. The composition of claim 25 wherein the galactose trimer consists of an N-acetylgalactosamine trimer.

29. A composition for delivering an oligonucleotide to a liver cell in vivo comprising:

a) an oligonucleotide covalently linked to a galactose trimer; and

,

b) a reversibly masked and targeted amphipathic polymer comprising a terpolymer synthesized from amine-containing monomers, lower hydrophobic group-containing monomers, and higher hydrophobic group-containing monomers to which a plurality of galactose derivatives and PEG groups are individually linked to said polymer via pH labile disubstituted maleamic bonds and wherein cleavage of said pH labile disubstituted maleamic bonds yields amine groups thereby generating a membrane active polyamine.

30. A composition for delivering an oligonucleotide to a liver cell in vivo comprising:

a) an oligonucleotide covalently linked to a hydrophobic group having at least 20 carbon atoms; and

,

b) a reversibly masked and targeted amphipathic polymer comprising a terpolymer synthesized from amine-containing monomers, lower hydrophobic group-containing monomers, and higher hydrophobic group-containing monomers to which a plurality of galactose derivatives and PEG groups are individually linked to said polymer via pH labile disubstituted maleamic bonds and wherein cleavage of said pH labile disubstituted maleamic bonds yields amine groups thereby generating a membrane active polyamine.

31. A method of manufacturing an RNA oligonucleotide delivery composition comprising:

a) forming a membrane active polyamine;

b) forming a first masking agent comprising a charge neutral disubstituted maleic anhydride containing a galactose derivative;

c) forming a second masking agent comprising a charge neutral disubstituted maleic anhydride containing a polyethylene glycol;

d) reversibly inhibiting membrane activity of the membrane active polyamine wherein the inhibiting consists of modifying 50% or more of the amines on the polyamine by reacting the polyamine with the first and second masking agents thereby linking a plurality of galactose derivatives and a plurality of polyethylene glycols to the membrane active polymer via physiologically pH-labile disubstituted maleamate linkages; and

,

e) linking the RNA interference polynucleotide to galactose trimer or a hydrophobic group having at least 20 carbon atoms;

f) providing the RNA interference polynucleotide and the reversibly inhibited membrane active polyamine in solution suitable for administration in vivo.