Site-specific delivery of nucleic acids by combining targeting ligands with endosomolytic components

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

wherein

 is the nucleic acid;

E is the endosomolytic component;

L is the targeting ligand;

x represents independently for each occurrence 0 or 1;

y represents independently for each occurrence 1,2,3,4,5, or 6;

z in formula (I) is 1,2,3,4,5, or 6 in one instance, and 0,1,2,3,4,5, or 6 in the other instance; and z in formula (II) represents independently for each occurrence 1,2,3,4,5, or 6;

A is a linking moiety selected from the group consisting of:

wherein

Z 1 represents independently for each occurrence O or S;

Z 2 represents independently for each occurrence —OH, —OM, -Oalkyl, -Oaryl, -Oaralkyl, —SH, —SM, -Salkyl, -Saryl, -aralkyl, —N(R 3 )R 4 , —C(R 11 ) 2 ) m N(R 11 ) 2 , —N(R 11 )(C(R 11 ) 2 ) m N(R 11 ) 2 , or alkyl;

R 3 and R 4 represent independently H or alkyl; or R 3 and R 4 taken together form a 3-, 4-, 5-, 6-, or 7-membered ring;

R 11 represents independently for each occurrence hydrogen or alkyl;

M represents independently for each occurrence an alkali metal or a transition metal with an overall charge of +1;

R 8 represents independently for each occurrence hydrogen, alkyl, aryl, aralkyl, acyl, silyl, a bond to the nucleic acid, or, when combined with B 10 , a bond between linking moieties;

B 10 is a bond between A and the nucleic acid or, when combined with R 8 , is a bond between linking moieties;

A′ is a direct bond or a tether having the formula: —[(P-Q-R) q —X—(P′-Q′-R′) q′ ] q″ -T-, wherein:

P, R, T, P′ and R′ are each independently absent, CO, NH, O, S, OC(O), NHC(O), CH 2 , CH 2 NH, CH 2 O; NHCH(R a )C(O), —C(O)—CH(R a )—NH—, C(O)—(optionally substituted alkyl)-NH—, CH═N—O,

cyclyl, heterocyclyl, aryl or heteroaryl;

R 50 and R 51 are independently alkyl, substitituted alkyl, or R 50 and R 51 taken together form a cyclic ring;

Q and Q′ are each independently for each occurrence absent, —(CH 2 ) n —, —C(R 40 )(R 41 )(CH 2 ) n —, —(CH 2 ) n C(R 40 )(R 41 )—, —(CH 2 CH 2 O) m CH 2 CH 2 —, —(CH 2 CH 2 O) m CH 2 CH 2 NH—, aryl, heteroaryl, cyclyl, or heterocyclyl;

X is absent or a cleavable linking group;

R a is H or an amino acid side chain;

R 40 and R 41 are each independently H, CH 3 , OH, SH or N(R x ) 2 ;

R x is, for each occurrence, H, methyl, ethyl, propyl, isopropyl, butyl or benzyl;

q, q′ and q″ are each independently 0-30;

n is, for each occurrence, an integer from 1-20; and

m is, for each occurrence, an integer from 0-50;

provided that E and L are each present at least once.

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

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

wherein each z is independently 1,2,3,4,5, or 6.

4. The composition of claim 3 , 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:

wherein

m represents independently for each occurrence 1,2,3,4,5,6,7, or 8;

m 1 represents independently for each occurrence 0,1,2,3,4,5,6,7, or 8;

p represents independently for each occurrence 1,2,3, or 4; and

R represents independently for each occurrence hydrogen or alkyl.

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 CHK 6 HC.

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 (Ga1NAc), 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-alactosamine, 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 B 12 , 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 (Ga1NAc), Ga1NAc2, and Ga1NAc3, 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′-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′-O-amine, 2′-SH, 2′-S-alkyl, 2′-O—CH 2 -(4′-C)(LNA), 2′-O—CH 2 CH 2 -(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, N 2 -substituted purines, N 6 -substituted purines and O 6 -substituted purines.

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

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

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

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

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

26. 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.

27. 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.

28. The composition of claim 23 , wherein the endosomolytic agent and the targeting ligand are both on the same strand.

29. The composition of claim 23 , wherein the endosomolytic agent and the targeting ligand are on different strands.

30. The composition of claim 29 , wherein the endosomolytic agent and the targeting ligand are located on the same end of the double-stranded oligonucleotide.

31. The composition of claim 29 , wherein the endosomolytic agent and the targeting ligand are located on the opposite ends of the double-stranded oligonucleotide.

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

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

34. 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 so as to deliver the modular composition to the cell.

35. The method of claim 34 , wherein the targeting ligand provides sufficient permeability and retention to allow the nucleic acid to accumulate in the cell.

36. The method of claim 34 , wherein the method is performed in vitro.

37. The method of claim 34 , wherein the method is performed in vivo.

38. The method of claim 34 , wherein the method is performed ex vivo.

39. 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 so as to inhibit the expression of one or more genes in the one or more cells, wherein the effective amount is an amount that suppresses the expression of the one or more genes.