Polyconjugates for In Vivo Delivery of Polynucleotides

• US 20080152661A1
• Filed: 08/17/2007
• Published: 06/26/2008
• Est. Priority Date: 08/18/2006
• Status: Active Grant

• Current Assignee: Arrowhead Pharmaceuticals, Inc.
• Original Assignee: Arrowhead Madison Inc (Arrowhead Pharmaceuticals, Inc.)
• Inventors: Klein, Jason, Blokhin, Andrei, Rozema, David B., Lewis, David L., Herweijer, Hans, Hagstrom, James E., Wakefield, Darren H., Wolff, Jon A., Wong, So, Trubetskoy, Vladimir
• Granted Patent: US 8,137,695 B2
• Time in Patent Office: Days
• US Class Current: 424/178.1
• CPC Class Codes: A01K 2217/058 due to expression of inhibitory nucleic acid, e.g. siRNA, antisense
• CPC Class Codes: A01K 2267/0362 Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
• CPC Class Codes: A61K 47/58 obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
• CPC Class Codes: A61K 48/0041 the non-active part being polymeric
• 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 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Abstract

The present invention is directed to compounds, compositions, and methods useful for delivering polynucleotides or other cell-impermeable molecules to mammalian cells. Described are polyconjugates systems that incorporate targeting, anti-opsonization, anti-aggregation, and transfection activities into small biocompatible in vivo delivery vehicles. The use of multiple reversible linkages connecting component parts provides for physiologically responsive activity modulation.

Claims

1. A method for delivering an RNAi polynucleotide to a cell in a mammal in vivo comprising:

a) covalently attaching the RNAi polynucleotide to a polymer via a first physiologically labile bond to form a conjugate; and

,

b) administering said conjugate to said mammal.

2. The method of claim 1 wherein the RNAi polynucleotide is selected from the group consisting of siRNA and miRNA.

3. The method of claim 1 wherein polymer is larger than 5 kDa.

4. The method of claim 3 wherein the polymer is larger than 7.5 kDa.

5. The method of claim 4 wherein the polymer is larger than 10 kDa.

6. The method of claim 1 wherein the conjugate has a zeta potential between +30 and −

30 mV at pH 7.

7. The method of claim 6 wherein the conjugate has a zeta potential between +20 and −

30 mV at pH 7.

8. The method of claim 7 wherein the conjugate has a zeta potential between +10 and +10 mV at pH 7.

9. The method of claim 8 wherein the conjugate has a zeta potential between +5 and −

5 mV at pH 7.

10. The method of claim 1 wherein the conjugate is smaller than 100 nm in diameter.

11. The method of claim 1 wherein the conjugate is smaller than 50 nm in diameter.

12. The method of claim 1 wherein the conjugate is smaller than 20 nm in diameter.

13. The method of claim 1 wherein the conjugate is smaller than 10 nm in diameter.

14. The method of claim 1 wherein the polymer is a membrane active polymer.

15. The method of claim 14 wherein the membrane active polymer is a polyamine.

16. The method of claim 14 wherein the membrane active polymer consists of a polyvinylether random copolymer.

17. The method of claim 14 wherein the membrane active polymer contains two of more different monomers.

20. The method of claim 16 wherein the polyvinylether random copolymer is composed of amine-containing vinyl ether monomers, lower alkyl vinyl ether monomers, and higher alkyl vinyl ether monomers.

21. The method of claim 20 wherein the lower alkyl vinyl ether consists of a butyl vinyl ether and the higher alkyl vinyl ether consists of an octadecyl vinyl ether or dodecyl vinyl ether.

22. The method of claim 21 wherein the polyvinylether random copolymer is soluble in water.

23. The method of claim 21 wherein the polyvinylether random copolymer is surface active.

24. The method of claim 1 wherein the polymer is a biodegradable polymer.

25. The method of claim 1 wherein a masking agent is attached to the polymer via a second physiologically reversible bond.

26. The method of claim 25 wherein a plurality of masking agents are attached to the polymer via a plurality of second physiologically reversible bonds.

27. The method of claim 26 wherein the first physiologically reversible bond and second physiologically reversible bond are orthogonal reversible bonds.

28. The method of claim 27 wherein the first physiologically reversible bond and second physiologically reversible bond are cleaved under distinct conditions.

29. The method of claim 26 wherein the masking agents are the same or different.

30. The method of claim 29 wherein the masking agents are selected from the group consisting of:

steric stabilizers, targeting groups and charge modifying agents.

31. The method of claim 29 wherein the masking agents reversibly alter one or more biophysical characteristics of the polymer.

32. The method of claim 31 wherein the masking agents inhibit non-specific interactions of the conjugate with serum components or non-target cells.

33. The method of claim 30 wherein at least one of the masking agent consists of a targeting group.

34. The method of claim 32 wherein the targeting group is selected from the group consisting of:

compound with affinity to cell surface protein, cell receptor ligand, antibody with affinity to a cell surface molecule, cell receptor ligand, saccharide, galactose, galactose derivative, N-acetylgalactosamine, mannose, mannose derivative, vitamins, folate, biotin, peptide, RGD-containing peptide, insulin, aptamer, and EGF.

35. The method of claim 25 wherein the masking agents are attached to the polymer prior to attachment of the RNAi polynucleotide to the polymer.

36. The method of claim 25 wherein the masking agents are attached to the polymer subsequent to attachment of the RNAi polynucleotide to the polymer.

37. The method of claim 31 wherein cleavage of the second physiologically reversible bonds restores the biophysical characteristics of the polymer altered by the masking agents.

38. The method of claim 27 wherein the first physiologically reversible bond and second physiologically reversible bond are independently selected from the group consisting of:

physiologically labile bond, cellular physiologically labile bond, pH labile bond, very pH labile bond, extremely pH labile bond, a maleamate bond, enzymatically cleavable bond, and disulfide bond.

39. The method of claim 1 wherein the RNAi polynucleotide is attached to the polymer in the presence of an excess of polymer.

40. The method of claim 1 wherein the conjugate is administered to the mammal with an excess of polymer.

41. The method of claim 25 wherein the masking agent is selected from the group consisting of:

dimethylmaleic anhydride, carboxy dimethylmaleic anhydride (CDM), CDM-thioester, CDM derivative, CDM-steric stabilizer, CDM-PEG, CDM-PEG, CDM-ligand, CDM-galactose, CDM-lactobionic acid (LBA), CDM-Pip-LBA, and CDM-NAG.

49. The conjugate of claim 28 wherein the polynucleotide is selected from the groups consisting of:

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

18. The method of claim 18 wherein the membrane active polymer contains three of more different monomers.

19. The method of claim 18 wherein the monomers are selected from the group consisting of:

amine-containing monomer, lower alkyl monomer, butyl monomer, higher alkyl monomer, dodecyl monomer, and octadecyl monomer.

42. The method of claim 19 wherein 1 wherein the conjugate is administered to the mammal in a pharmaceutically acceptable carrier or diluent.

43. A method for delivering an oligonucleotide to a cell in a mammal in vivo comprising:

a) covalently attaching the oligonucleotide to a reversibly masked polymer via a physiologically labile bond to form a conjugate; and

,

b) administering said conjugate to said mammal.

44. A conjugate delivery system for delivering a molecule to a cell comprising:

N-L¹-P-(L²-M)_y,

wherein,

N is a membrane impermeable molecule or molecule with low membrane permeability,

L¹ is a first reversible linkage containing a physiologically labile bond,

P is a polymer,

L² is a second reversible linkage containing an orthogonal physiologically labile bond,

M is a masking agent, and

y is an integer greater than 0.

45. The conjugate of claim 44 wherein the polymer is a membrane active polymer.

46. The conjugate of claim 45 wherein the masking agent modifies a property or interaction of the membrane active polymer.

47. The conjugate of claim 44 wherein y is 2 or greater.

48. The conjugate of claim 44 wherein the molecule is selected from the group consisting of:

polynucleotide, protein, peptide, antibody, and membrane impermeable drug.

50. A conjugate for delivering a molecule to a cell comprising:

a membrane active polymer reversibly linked to the molecule via a physiologically labile bond and further linked to two or more masking agents via physiologically labile bonds wherein the two or more masking agents consist of at least one targeting group and at least one steric stabilizer or charge modifier.