Compositions comprising reversibly modified oligonucleotides and uses thereof

1. A glutathione-sensitive oligonucleotide comprising at least one nucleotide comprising a glutathione-sensitive bond, wherein the at least one nucleotide comprising the glutathione-sensitive bond is represented by Formula I:

wherein X is O, S, Se or NR′, wherein R′ is hydrogen, halogen, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms or a heterocyclic having 3-14 carbon atoms;

wherein R 1 , R 2 , R 3 and R 4 are each independently selected from hydrogen, halogen, OH, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl or wherein two of R 1 , R 2 , R 3 and R 4 are taken together to form a 5-8 membered ring, wherein the ring optionally contains a heteroatom;

wherein J is O, S, NR′, or CR′R″, wherein each of R′ and R″ is independently hydrogen, halogen, an aliphatic, aryl or heteroaryl having 5-19 carbon atoms;

wherein B is hydrogen, a natural nucleobase, a modified nucleobase or a universal nucleobase;

wherein U 2 is absent or O, S, NR′, or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a heterocyclic having 3-14 carbon atoms or a cycloalkyl;

wherein W is hydrogen, a phosphate group, an internucleotide linking group attaching the at least one nucleotide represented by Formula I to a nucleotide or an oligonucleotide, a halogen, OR′, SR′, NR′R″, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a cycloalkyl, a heterocyclic having 3-14 carbon atoms, wherein R′ and R″ are each independently hydrogen, halogen, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a heterocyclic having 3-14 carbon atoms, or are taken together to form a heterocyclic having 3-14 carbon atoms;

wherein I is absent or is selected from O, S, NR′, or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a heterocyclic having 3-14 carbon atoms and a cycloalkyl;

wherein U 1 is absent, hydrogen, an internucleotide linking group attaching the at least one nucleotide represented by Formula I to a nucleotide or an oligonucleotide, or O, S, NR′ or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a heterocyclic having 3-14 carbon atoms and a cycloalkyl and wherein at least one of U 1 or W is an internucleotide linking group attaching the at least one nucleotide represented by Formula I to a nucleotide or an oligonucleotide and provided that if U 1 is an internucleotide linking group, A is absent;

wherein I and U 1 can be combined to form CR′-CR″ alkyl, CR′-CR″ alkenyl, CR′-CR″ alkynyl, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a heterocyclic having 3-14 carbon atoms, or taken together to form a cycloalkyl or heterocyclic having 3-14 carbon atoms;

wherein A is absent, a hydrogen, a phosphate group, a chemical moiety that mimics electrostatic and steric properties of a phosphate group, or a phosphoramidate; and

wherein L is a glutathione-sensitive moiety comprising the glutathione-sensitive bond, wherein the glutathione-sensitive bond is a disulfide bridge or a sulfonyl group, and wherein L is represented by:

wherein, in Formula IVb(i) and Formula IVb(ii), R is hydrogen, CH 3 , an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a cycloalkyl or a heterocyclic having 3-14 carbon atoms, or R is a targeting ligand optionally connected via a spacer;

wherein, in Formula IVc(i), R is hydrogen, CH 3 , an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a cycloalkyl or a heterocyclic having 3-14 carbon atoms, or R is a targeting ligand optionally connected via a spacer;

wherein, in Formulae IVe(iii), IVe(iv), and IVe(x), R is hydrogen, CH 3 , an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a cycloalkyl or a heterocyclic having 3-14 carbon atoms, or R is a targeting ligand optionally connected via a spacer;

wherein, in Formulae IVb(i), IVb(ii), IVc(i), IVe(iii), IVe(iv), and IVe(x), the targeting ligand directs delivery of the glutathione-sensitive oligonucleotide to a tissue of interest, and wherein the spacer is a molecule that couples the targeting ligand to the glutathione-sensitive oligonucleotide.

2. The glutathione-sensitive oligonucleotide of claim 1 , wherein the glutathione-sensitive moiety is represented by Formula IVb(ii), IVd(i), IVe(ii) or IVe(ix).

3. The glutathione-sensitive oligonucleotide according to claim 1 , wherein the internucleotide linking group contains a phosphorous atom.

4. The glutathione-sensitive oligonucleotide according to claim 1 , wherein the oligonucleotide is a double-stranded oligonucleotide comprising a first strand and a second strand.

5. The glutathione-sensitive oligonucleotide according to claim 4 , wherein the double stranded oligonucleotide is a double-stranded RNAi inhibitor molecule and the first strand comprises a sense strand and the second strand comprises an antisense strand, wherein the antisense strand or part of the antisense strand is used by an Argonaute 2 (Ago2) endonuclease in the cleavage of a target mRNA.

6. The glutathione-sensitive oligonucleotide according to claim 5 , wherein the at least one nucleotide represented by Formula I is located at nucleotide position 1 of the antisense strand; at nucleotide position 14 of the antisense strand; or at a nucleotide position at or adjacent to the Ago2 cleavage site of the sense strand.

7. The glutathione-sensitive oligonucleotide according to claim 5 , wherein the double stranded RNAi inhibitor molecule contains a tetraloop.

8. The glutathione-sensitive oligonucleotide according to claim 1 , wherein the oligonucleotide is a single stranded oligonucleotide.

9. The glutathione-sensitive oligonucleotide according to claim 8 , wherein the single stranded oligonucleotide is a single stranded RNAi nucleic acid inhibitor molecule.

10. The glutathione-sensitive oligonucleotide according to claim 8 , wherein the single-stranded oligonucleotide is a conventional antisense oligonucleotide, a ribozyme, a microRNA, an antagomir, or an aptamer.

11. The glutathione-sensitive oligonucleotide according to claim 1 , wherein the glutathione-sensitive oligonucleotide contains 1-5 nucleotides represented by Formula I.

12. The glutathione-sensitive oligonucleotide according to claim 1 , wherein every nucleotide of the glutathione-sensitive oligonucleotide is modified and wherein every nucleotide that is not modified with the glutathione-sensitive moiety is modified with an irreversible covalent chemical modification.

13. A pharmaceutical composition comprising the glutathione-sensitive oligonucleotide according to claim 1 and a pharmaceutically acceptable carrier.

14. A pharmaceutical composition comprising the glutathione-sensitive oligonucleotide according to claim 4 and a pharmaceutically acceptable carrier.

15. A method for reducing expression of a target gene in a subject comprising administering the pharmaceutical composition of claim 14 to a subject in need thereof in an amount sufficient to reduce expression of the target gene.

16. A nucleoside phosphoramidite, wherein the nucleoside phosphoramidite is represented by Formula VIII:

wherein L 1 is a glutathione-sensitive moiety comprising a glutathione-sensitive bond, wherein the glutathione-sensitive bond is a disulfide bridge or a sulfonyl group, and wherein L 1 is represented by:

wherein, in Formula IVb(i) and Formula IVb(ii), R is hydrogen, CH 3 , an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a cycloalkyl or a heterocyclic having 3-14 carbon atoms, or R is a targeting ligand optionally connected via a spacer;

wherein, in Formula IVc(i), R is hydrogen, CH 3 , an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a cycloalkyl or a heterocyclic having 3-14 carbon atoms, or R is a targeting ligand optionally connected via a spacer;

wherein, in Formulae IVe(iii), IVe(iv), and IVe(x), R is hydrogen, CH 3 , an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a cycloalkyl or a heterocycle heterocyclic having 3-14 carbon atoms, or R is a targeting ligand optionally connected via a spacer;

wherein, in Formulae IVb(i), IVb(ii), IVc(i), IVe(iii), IVe(iv), and IVe(x), the targeting ligand directs delivery of the glutathione-sensitive oligonucleotide to a tissue of interest, and wherein the spacer is a molecule that couples the targeting ligand to the glutathione-sensitive oligonucleotide;

wherein A 1 is absent, hydrogen, a phosphate group, a chemical moiety that mimics electrostatic and steric properties of a phosphate group, a phosphoramidate, a phosphoramidite, a protecting group, or a solid support;

wherein W 1 is a phosphoramidite, a protecting group, a solid support, hydrogen, halogen, OR′, SR′, NR′R″, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a cycloalkyl, a heterocyclic having 3-14 carbon atoms, wherein R′ and R″ are each independently hydrogen, halogen, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a heterocyclic having 3-14 carbon atoms, or are taken together to form a heterocyclic having 3-14 carbon atoms;

wherein U 3 is a hydrogen or O, S, NR′ or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a heterocycle heterocyclic having 3-14 carbon atoms and a cycloalkyl;

wherein at least A 1 is a phosphoramidite and U 3 is O or at least W 1 is a phosphoramidite and U 2 is O;

wherein X is O, S, Se or NR′, wherein R′ is hydrogen, halogen, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms or a heterocyclic having 3-14 carbon atoms;

wherein R 1 , R 2 , R 3 and R 4 are each independently hydrogen, halogen, OH, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl or wherein two of R 1 , R 2 , R 3 and R 4 are taken together to form a 5-8 membered ring, wherein the ring optionally contains a heteroatom;

wherein J is O, S, NR′, CR′R″, wherein each of R′ and R″ is independently hydrogen, halogen, an aliphatic, an aryl or a heteroaryl having 5-19 carbon atoms;

wherein B is hydrogen, an aliphatic, a natural nucleobase, a modified nucleobase or a universal nucleobase;

wherein U 2 is absent or O, S, NR′, or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a heterocyclic having 3-14 carbon atoms or a cycloalkyl;

wherein I is absent or is O, S, NR′, or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a heterocyclic having 3-14 carbon atoms, or and a cycloalkyl; and

wherein I and U 3 can be combined to form CR′-CR″ alkyl, CR′-CR″ alkenyl, CR′-CR″ alkynyl, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms a heterocyclic having 3-14 carbon atoms, or taken together to form a cycloalkyl or heterocyclic having 3-14 carbon atoms.

17. The nucleoside phosphoramidite of claim 16 , wherein the glutathione-sensitive moiety (L 1 ) is represented by Formula IVb(ii), IVd(i), IVe(ii) or IVe(ix).

18. The nucleoside phosphoramidite of claim 16 , wherein:

a) J is O; B is a natural nucleobase; U 2 is O; I is CH 2 ; W 1 is a phosphoramidite; A 1 is a protecting group, hydrogen, or solid support; and U 3 is O;

b) J is O; B is a natural nucleobase; U 2 is O; I is CH 2 ; W 1 is a phosphoramidite; A 1 is a protecting group, hydrogen, or solid support; U 3 is O; X is O; and R 1 , R 2 , R 3 and R 4 are hydrogen;

c) J is O; B is a natural nucleobase; U 2 is O; I is CH 2 ; W 1 is a protecting group, hydrogen or solid support; A 1 is a phosphoramidite, and U 3 is O;

d) J is O; B is a natural nucleobase; U 2 is O; I is CH 2 ; W 1 is a protecting group, hydrogen or solid support; A 1 is a phosphoramidite, U 3 is O; X is O; and R 1 , R 2 , R 3 and R 4 are hydrogen;

e) J is O; B is a natural nucleobase; U 2 is O; I is CH 2 ; W 1 is a phosphoramidite; A 1 is hydrogen or a protecting group; U 3 is O; X is O; R 1 , R 2 , R 3 and R 4 are hydrogen; and L 1 is represented by Formula IVe(ix);

f) J is O; B is a natural nucleobase; U 2 is O; I is CH 2 ; W 1 is a phosphoramidite; A 1 is hydrogen or a protecting group; U 3 is O; X is O; R 1 , R 2 , R 3 and R 4 are hydrogen; and L 1 is represented by Formula IVe(xi); or

g) U 2 is O; W 1 is a phosphoramidite; and A 1 is a is absent, hydrogen, a phosphate group, a phosphate mimic a chemical moiety that mimics electrostatic and steric properties of a phosphate group, a phosphoramidate, a protecting group, or a solid support.

19. The nucleoside phosphoramidite of claim 16 , wherein the phosphoramidite has the formula —P(OR x )N(R y ) 2 , wherein R x is selected from the group consisting of methyl, 2-cyanoethyl and benzyl, wherein each of R y is selected from the group consisting of ethyl and isopropyl.

20. A method for preparing a glutathione-sensitive oligonucleotide comprising:

(a) attaching a nucleoside to a solid support via a covalent linkage;

(b) coupling the nucleoside phosphoramidite according to claim 16 to a hydroxyl group on the nucleoside of step (a) to form a phosphorus nucleoside linkage therebetween, wherein any uncoupled nucleoside on the solid support is capped with a capping reagent;

(c) oxidizing said phosphorus nucleoside linkage with an oxidizing reagent; and

(d) repeating steps (b) to (d) iteratively with one or more subsequent nucleoside phosphoramidites according to claim 16 or one or more subsequent nucleoside phosphoramidites that do not contain a glutathione-sensitive moiety, to form the glutathione-sensitive oligonucleotide; and

(f) optionally removing said glutathione-sensitive oligonucleotide from said solid support.

21. A glutathione-sensitive oligonucleotide made by the method of claim 20 .

22. A glutathione-sensitive nucleoside or nucleotide comprising a glutathione-sensitive bond, wherein the glutathione-sensitive nucleoside or nucleotide is represented by Formula XI:

wherein L 2 is a glutathione-sensitive moiety comprising the glutathione-sensitive bond, wherein the glutathione-sensitive bond is a disulfide bridge or a sulfonyl group, and wherein L 2 is represented by:

wherein, in Formula IVb(i) and Formula IVb(ii), R is hydrogen, CH 3 , an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a cycloalkyl or a heterocyclic having 3-14 carbon atoms, or R is a targeting ligand optionally connected via a spacer;

wherein, in Formula IVc(i), R is hydrogen, CH 3 , an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a cycloalkyl or a heterocyclic having 3-14 carbon atoms, or R is a targeting ligand optionally connected via a spacer;

wherein, in Formulae IVe(iii), IVe(iv), and IVe(x), R is hydrogen, CH 3 , an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a cycloalkyl or a heterocyclic having 3-14 carbon atoms, or R is a targeting ligand optionally connected via a spacer, or is absent if one of A 2 or W 2 is the glutathione-sensitive moiety represented by Formula IIa, IVb(i), IVb(ii), IVc(i), IVd(i), IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(v), IVe(ix), IVe(x) or IVe(xi);

wherein, in Formulae IVb(i), IVb(ii), IVc(i), IVe(iii), IVe(iv), and IVe(x), the targeting ligand directs delivery of the glutathione-sensitive oligonucleotide to a tissue of interest, and wherein the spacer is a molecule that couples the targeting ligand to the glutathione-sensitive oligonucleotide;

wherein if L 2 is a glutathione-sensitive moiety, X is O, S, Se, or NR′, wherein R′ is hydrogen, halogen, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms or a heterocyclic having 3-14 carbon atoms, or if L 2 is absent, X is H, OH, SH, NH 2 , halogen, alkoxy, alkyl, alkenyl, alkynyl, alkylthio, alkylamino or dialkylamino wherein one or more methylenes in the alkyl, alkenyl, and alkynyl may be interrupted with one or more of O, S, S(O), SO 2 , N(R′), C(O), N(R′)C(O)O, OC(O)N(R′) aryl, heteroaryl, heterocyclic or cycloalkyl, O, S, Se or NHR′, wherein R′ is hydrogen, halogen, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms or a heterocyclic having 3-14 carbon atoms;

wherein R 1 , R 2 , R 3 and R 4 are each independently hydrogen, halogen, OH, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl or wherein two of R 1 , R 2 , R 3 and R 4 are taken together to form a 5-8 membered ring, wherein the ring optionally contains a heteroatom;

wherein J is O, S, NR′, CR′R″, wherein each of R′ and R″ is independently hydrogen, halogen, an aliphatic, an aryl or a heteroaryl having 5-19 carbon atoms;

wherein B is hydrogen, a natural nucleobase, a modified nucleobase or a universal nucleobase;

wherein U 2 is absent or O, S, NR′, or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a heterocyclic having 3-14 carbon atoms or a cycloalkyl;

wherein W 2 is a glutathione-sensitive moiety represented by Formula IIa, IVb(i), IVb(ii), IVc(i), IVd(i), IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(v), IVe(ix), IVe(x) or IVe(xi); hydrogen, halogen, OR′, SR′, NR′R″, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a cycloalkyl, a heterocyclic having 3-14 carbon atoms, wherein R′ and R″ are each independently hydrogen, halogen, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a heterocyclic having 3-14 carbon atoms, or are taken together to form a heterocyclic having 3-14 carbon atoms;

wherein I is absent or is O, S, NR′, or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a heterocyclic having 3-14 carbon atoms or a cycloalkyl;

wherein U 3 is hydrogen, or O, S, NR′ or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a heterocyclic having 3-14 carbon atoms or a cycloalkyl;

wherein I and U 3 can be combined to form CR′-CR″ alkyl, CR′-CR″ alkenyl, CR′-CR″ alkynyl, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a heterocyclic having 3-14 carbon atoms, or taken together to form a cycloalkyl or heterocyclic having 3-14 carbon atoms; and

wherein A 2 is absent, hydrogen, a phosphate group, a chemical moiety that mimics electrostatic and steric properties of a phosphate group, a phosphoramidate, or a glutathione-sensitive moiety represented by Formula IIa, IVb(i), IVb(ii), IVc(i), IVd(i), IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(v), IVe(ix), IVe(x) or IVe(xi).

23. The glutathione-sensitive nucleoside or nucleotide of claim 22 , wherein:

a) J is O; X is O; L 2 is a glutathione-sensitive moiety represented by IIa, IVb(i), IVb(ii), IVc(i), IVd(i), IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(v), IVe(ix), IVe(x) or IVe(xi); W 2 is hydrogen, halogen, OR′, SR′, NR′R″, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a cycloalkyl, a heterocyclic having 3-14 carbon atoms, wherein R′ and R″ are each independently hydrogen, halogen, an aliphatic, an aryl, a heteroaryl having 5-19 carbon atoms, a heterocyclic having 3-14 carbon atoms, or are taken together to form a heterocyclic having 3-14 carbon atoms; and A 2 is absent, hydrogen, a phosphate group, a chemical moiety that mimics electrostatic and steric properties of a phosphate group, or a phosphoramidate;

b) R 1 , R 2 , R 3 , and R 4 are hydrogen; U 2 is oxygen; W 2 is hydrogen; I is CH 2 ; U 3 is O; and A 2 is hydrogen or a phosphate group;

c) J is O; R 1 , R 2 , R 3 , and R 4 are hydrogen; B is a natural nucleobase; U 2 is O; X is O; I is CH 2 , A 2 is absent, a hydrogen, a phosphate group, or a a chemical moiety that mimics electrostatic and steric properties of a phosphate group; U 3 is O or an internucleotide linking group attaching the glutathione-sensitive nucleoside or nucleotide to a nucleotide or an oligonucleotide; L 2 is a represented by Formula IVe(ix); and W 2 is hydrogen or an internucleotide linking group attaching the glutathione-sensitive nucleoside or nucleotide to a nucleotide or an oligonucleotide, wherein at least one of U 3 or W 2 is an internucleotide linking group attaching the glutathione-sensitive nucleoside or nucleotide to an oligonucleotide and provided that if U 3 is an internucleotide linking group, A 2 is absent;

d) J is O; R 1 , R 2 , R 3 , and R 4 are hydrogen; B is a natural nucleobase; U 3 is O; U 2 is O; X is O; I is CH 2 , U 3 is O or an internucleotide linking group attaching the glutathione-sensitive nucleoside or nucleotide to a nucleotide or an oligonucleotide; L 2 is a represented by Formula IVe(ix); A 2 is hydrogen and W 2 is an internucleotide linking group attaching the attaching the glutathione-sensitive nucleoside or nucleotide to an oligonucleotide and wherein the glutathione-sensitive nucleoside or nucleotide is located at nucleotide position 1 of an antisense strand of a double-stranded RNAi inhibitor molecule; or

e) J is O; R 1 , R 2 , R 3 , and R 4 are hydrogen; B is a natural nucleobase; U 2 is O; X is O; I is CH 2 ; L 2 is a represented by Formula IVe(ix); A 2 is absent; W 2 is a internucleotide linking group attaching the glutathione-sensitive nucleoside or nucleotide to a first oligonucleotide; and U 3 is a internucleotide linking group attaching the glutathione-sensitive nucleoside or nucleotide to a second oligonucleotide; and wherein the glutathione-sensitive nucleoside or nucleotide is located at nucleotide position 14 of an antisense strand of a double-stranded RNAi inhibitor molecule.