MULTIFUNCTIONAL COPOLYMERS FOR NUCLEIC ACID DELIVERY

• US 20130236968A1
• Filed: 06/20/2011
• Published: 09/12/2013
• Est. Priority Date: 06/21/2010
• Status: Abandoned Application

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Alnylam Pharmaceuticals Incorporated
• Inventors: Manoharan, Muthiah, Rajeev, Kallanthottathil G.
• Application Number:
  US13/805,991
• Publication Number:
  US 20130236968A1
• Time in Patent Office: Days
• US Class Current: 435/375
• 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: C08F 220/58 containing oxygen in addition to the carbonamido oxygen , e.g. N-methylolacrylamide, N-(meth)acryloylmorpholine
• CPC Class Codes: C08F 220/603 and containing oxygen in addition to the carbonamido oxygen and nitrogen
• 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

Abstract

The present invention relates to multifunctional polymers represented by the following formula:

The invention further provides methods for their preparation and methods for site-specific delivery of nucleic acids by combining them with targeting ligands, endosomolytic ligands and/or PK modulator ligands.

Claims

1. A multifunctional copolymer of formula (I):

2. The multifunctional copolymer of claim 1, represented by formula (II):

3. The multifunctional copolymer of claim 2, represented by formula (III):

4. The multifunctional copolymer of claim 3, represented by formula (IV):

5. A multifunctional copolymer of formula (V):

6. The multifunctional copolymer of claim 5, wherein A₁, A₂ and A₃ are absent or independently selected from the group consisting of ester, disulfide, acetal, ketal, and hydrazone.

7. The multifunctional copolymer of claim 5, represented by formula (VI):

8. The multifunctional copolymer of claim 5, wherein the endosomolytic ligand 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.

9. The multifunctional copolymer of claim 5, wherein the endosomolytic ligand is a polyanionic peptide or a polyanionic peptidomimetic.

10. The multifunctional copolymer of claim 5, wherein the endosomolytic ligand is selected from the 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 multifunctional copolymer of claim 5, 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 multifunctional copolymer of claim 11, wherein the targeting ligand is selected from the group consisting of D-galactose, N-acetyl-D-galactose (GalNAc), multivalent N-acetyl-D-galactose, cholesterol, folate, and analogs and derivates thereof.

13. The multifunctional copolymer of claim 5, wherein the nucleic acid is selected from the group consisting of an iRNA agent, an antisense oligonucleotide, an antagomir, an activating RNA, a decoy oligonucleotide, an aptamer, and a ribozyme.

14. The multifunctional copolymer of claim 5, wherein the nucleic acid contains at least one sugar modification.

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

-modification.

16. The multifunctional copolymer of claim 15, wherein said 2′

-modificaiton 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 multifunctional copolymer of claim 5, wherein the nucleic acid contains at least one backbone modification.

18. The multifunctional copolymer 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 multifunctional copolymer of claim 5, wherein the nucleic acid down regulates the expression of a target gene.

20. The multifunctional copolymer of claim 17, wherein the nucleic acid down regulates the expression of a target gene through an RNA interference mechanism.

21. The multifunctional copolymer of claim 5, wherein the nucleic acid is a single-stranded oligonucleotide.

22. The multifunctional copolymer of claim 5, wherein the nucleic acid is a double-stranded oligonucleotide.

23. The multifunctional copolymer of claim 5, wherein Lc is a redox-cleavable linker.

24. The multifunctional copolymer of claim 5, wherein Lc comprises at least one pH-sensitive component.

25. A method of delivering a multifunctional copolymer to a cell, the method comprising (a) contacting a cell with the multifunctional copolymer of claim 5;

and (b) allowing the cell to internalize the multifunctional copolymer.

26. The method of claim 25, wherein at least one of LG₁, LG₂, and LG₃ is a targeting ligand.

27. The method of claim 26, wherein the targeting ligand provides sufficient permeability and retention to allow the nucleic acid to accumulate in the cell.

28. A method of inhibiting the expression of one or more genes, comprising contacting one or more cells with an effective amount of the multifunctional copolymer of claim 5, wherein the effective amount is an amount that suppresses the expression of the one or more genes.