SITE-SPECIFIC DELIVERY OF NUCLEIC ACIDS BY COMBINING TARGETING LIGANDS WITH ENDOSOMOLYTIC COMPONENTS

• US 20110123520A1
• Filed: 04/10/2009
• Published: 05/26/2011
• Est. Priority Date: 04/11/2008
• Status: Active Grant

• Current Assignee: Arbutus Biopharma Corporation
• Original Assignee: Alnylam Pharmaceuticals Incorporated
• Inventors: Butler, David, Jayaraman, Muthusamy, Manoharan, Muthiah, Rajeev, Kallanthottathil G.
• Granted Patent: US 8,575,123 B2
• Time in Patent Office: Days
• US Class Current: 424/130.1
• CPC Class Codes: A61K 47/545 Heterocyclic compounds A61K47/558 takes precedence
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61K 47/551 one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
• CPC Class Codes: A61K 47/554 the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
• CPC Class Codes: A61K 48/0008 characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/04 Centrally acting analgesics, e.g. opioids
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/06 : for tuberculosis
• CPC Class Codes: A61P 31/08 : for leprosy
• CPC Class Codes: A61P 31/10 : Antimycotics
• CPC Class Codes: A61P 31/12 : Antivirals
• CPC Class Codes: A61P 31/14 : for RNA viruses
• CPC Class Codes: A61P 31/18 : for HIV
• CPC Class Codes: A61P 31/20 : for DNA viruses
• CPC Class Codes: A61P 31/22 : for herpes viruses
• CPC Class Codes: A61P 33/00 : Antiparasitic agents
• CPC Class Codes: A61P 33/06 : Antimalarials
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 37/00 : Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• 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 15/111 : General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/1137 : against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• 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 2310/11 : Antisense
• CPC Class Codes: C12N 2310/14 : interfering N.A.
• CPC Class Codes: C12N 2310/315 : Phosphorothioates
• CPC Class Codes: C12N 2310/322 : 2'-R Modification
• CPC Class Codes: C12N 2310/346 : having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/351 : Conjugate
• CPC Class Codes: C12N 2310/3513 : Protein; Peptide
• CPC Class Codes: C12N 2320/32 : Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12Y 304/21021 : Coagulation factor VIIa (3.4.21.21)

Abstract

The invention relates to compositions and methods for site-specific delivery of nucleic acids by combining them with targeting ligands and endosomolytic components.

Claims

1. A modular composition, comprising a nucleic acid, at least one endosomolytic component, and at least one targeting ligand, the composition having the formula:

2. The composition of claim 1, wherein the composition has the formula:

3. The composition of claim 1, wherein the composition has the formula:

4. The composition of claim 1, wherein the composition has the formula:

5. The composition of claim 1, wherein the A′

tether is an alkyl diradical, heteroalkyl diradical, alkenyl diradical, alkynyl diradical, alkylalkynyl diradical, aminoalkyl diradical, thioether, —

C(O)—

, —

S(O)—

, —

S(O)₂—

, —

C(R)₂—

, or has the formula selected from the group consisting of;

6. The composition of claim 1, wherein the A′

tether is selected from the group consisting of;

7. The composition of claim 1, wherein the endosomolytic component is selected from the group consisting of imidazoles, poly or oligoimidazoles, linear or brached polyethyleneimines (PEIs), linear and branched polyamines, cationic linear and branched polyamines, polycarboxylates, polycations, masked oligo or poly cations or anions, acetals, polyacetals, ketals, polyketals, orthoesters, linear or branched polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, and natural and synthetic fusogenic lipids.

8. The composition of claim 1, wherein the endosomolytic component is a polyanionic peptide or a polyanionic peptidomimetic.

9. The composition of claim 7, wherein the fusogenic lipid is selected from the group consisting of 1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine (DLin-k-DMA) and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine (XTC).

10. The composition of claim 1, wherein the endosomolytic component is selected from a group consisting of GALA, EALA, INF-7, Inf HA-2, diINF-7, diINF3, GLF, GALA-INF3, INF-5, JTS-1, ppTG1, ppTG20, KALA, HA, melittin, and histinde-rich peptide CHK₆HC.

11. The composition of claim 1, wherein the targeting ligand is selected from the group consisting of an antibody, a ligand-binding portion of a receptor, a ligand for a receptor, an aptamer, D-galactose, N-acetyl-D-galactose (GalNAc), multivalent N-acetyl-D-galactose, D-mannose, cholesterol, a fatty acid, a lipoprotein, folate, thyrotropin, melanotropin, surfactant protein A, mucin, carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine, multivalent mannose, multivalent fucose, glycosylated polyaminoacids, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipophilic moiety that enhance plasma protein binding, a steroid, bile acid, vitamin B₁₂, biotin, an RGD peptide, an RGD peptide mimetic, ibuprofen, naproxen, aspirin, folate, and analogs and derivatives thereof.

12. The composition of claim 11, wherein the targeting ligand is selected from the group consisting of D-galactose, N-acetyl-D-galactose (GalNAc), GalNAc2, and GalNAc3, cholesterol, folate, and analogs and derivates thereof.

13. The composition of claim 1, wherein the nucleic acid is selected from a group consisting of an iRNA agent, an antisense oligonucleotide, an antagomir, an activating RNA, a decoy oligonucleotide, an aptamer, a microRNA (miRNA), miRNA mimics, antimir, activating RNA (RNAa), a supermir, a U1 adaptor and a ribozyme.

14. The composition of claim 1, wherein the nucleic acid contains at least one sugar modification.

15. The composition of claim 14, wherein said sugar modification is a 2′

-modification.

16. The composition of claim 15, wherein said 2′

-modification is selected from the group consisting of 2′

-O-Me (2′

-O-methyl), 2′

-O-MOE (2′

-O-methoxyethyl), 2′

-F, 2′

-O-[2-(methylamino)-2-oxoethyl] (2′

-O-NMA), 2′

—

NH₂, 2′

-O-amine, 2′

-SH, 2′

-S-alkyl, 2′

-O—

CH₂-(4′

-C) (LNA), 2′

-O—

CH₂CH₂-(4′

-C) (ENA), 2′

-O-aminopropyl (2′

-O-AP), 2′

-O-dimethylaminoethyl (2′

-O-DMAOE), 2′

-O-dimethylaminopropyl (2′

-O-DMAP), and 2′

-O-dimethylaminoethyloxyethyl (2′

-O-DMAEOE).

17. The composition of claim 1, wherein the nucleic acid contains at least one backbone modification.

18. The composition of claim 17, wherein said backbone modification is selected from the group consisting of phosophorothioate, phosphorodithioate, phosphoramidate, phosphonate, alkylphosphonate, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methyleneaminocarbonyl, methylenemethylimino (MMI), methylenehydrazo, methylenedimethylhydrazo (MDH), and methyleneoxymethylimino.

19. The composition of claim 1, where in the nucleic acid comprises at least one nucleobase modification.

20. The composition of claim 19, wherein the nucleobase modification is selected from the group consisting of difluorotolyl, 5-nitroindole, 3-nitropyrrole, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidines, N²-substituted purines, N⁶-substituted purines and O⁶-substituted purines.

21. The composition of claim 1, wherein the nucleic acid down regulates the expression of a target gene.

22. The composition of claim 21, wherein the nucleic acid down regulates the expression of a target gene through an RNA interference mechanism.

23. The composition of claim 1, wherein the nucleic acid is a single-stranded oligonucleotide.

24. The composition of claim 1, wherein the nucleic acid is a hairpin oligonucleotide.

25. The composition of claim 1, wherein the nucleic acid is a double-stranded oligonucleotide.

26. The composition of claim 1, wherein the endosomolytic agent and the targeting ligand are on the same terminal end of the nucleic acid.

27. The composition of claim 1, wherein the endosomolytic agent and the targeting ligands are on the opposite terminal ends of the nucleic acid.

28. The composition of claim 1, wherein one of the endosomolytic agent and the targeting ligand is at the terminal end of the nucleic acid and the other is at an internal, non-terminal position on the nucleic acid.

29. The composition of claim 1, wherein both the endosomolytic agent and the targeting ligand are located at internal, non-terminal positions on the nucleic acid.

30. The composition of claim 25, wherein the endosomolytic agent and the targeting ligand are both on the same strand.

31. The composition of claim 25, wherein the endosomolytic agent and the targeting ligand are on different strands.

32. The composition of claim 31, wherein the endosomolytic agent and the targeting ligand are located on the same end of the double-stranded oligonucleotide.

33. The composition of claim 31, wherein the endosomolytic agent and the targeting ligand are located on the opposite ends of the double-stranded oligonucleotide.

34. The composition of claim 1, wherein the tether comprises at least on redox cleavable linking group.

35. The composition of claim 1, wherein the tether comprises at least one pH sensitive component.

36. A method of delivering a modular composition to a cell, the method comprising (a) contacting a cell with a modular composition of claim 1;

and (b) allowing the cell to internalize the modular composition.

37. The method of claim 36, wherein the targeting ligand provides sufficient permeability and retention to allow the nucleic acid to accumulate in the cell.

38. The method of claim 36, wherein the method is preformed in vitro.

39. The method of claim 36, wherein the method is performed in vivo.

40. The method of claim 36, wherein the method is performed ex vivo.

41. A method of inhibiting the expression of one or more genes, comprising contacting one or more cells with an effective amount of the modular composition of claim 1, wherein the effective amount is an amount that suppresses the expression of the one or more genes.