NZ750307B2 - Compositions comprising reversibly modified oligonucleotides and uses thereof - Google Patents

Abstract

Disclosed herein are glutathione-sensitive oligonucleotides and methods of using the same. Any oligonucleotide of interest may be modified with a glutathione-sensitive moiety, including oligonucleotides used for in vivo delivery, such as nucleic acid inhibitor molecules. Typically, the glutathione-sensitive moiety is used to reversibly modify the 2'-carbon of a sugar moiety in one or more nucleotides in the oligonucleotide, although other carbon positions may also be modified with the glutathione-sensitive moiety. Also disclosed are glutathione- sensitive nucleotide and nucleoside monomers, including glutathione-sensitive nucleoside phosphoramidites that can be used, for example, in standard oligonucleotide synthesis methods. In addition, glutathione-sensitive nucleotide and nucleoside monomers without a phosphoramidite can be used therapeutically, for example, as anti-viral agents. ensitive moiety is used to reversibly modify the 2'-carbon of a sugar moiety in one or more nucleotides in the oligonucleotide, although other carbon positions may also be modified with the glutathione-sensitive moiety. Also disclosed are glutathione- sensitive nucleotide and nucleoside monomers, including glutathione-sensitive nucleoside phosphoramidites that can be used, for example, in standard oligonucleotide synthesis methods. In addition, glutathione-sensitive nucleotide and nucleoside monomers without a phosphoramidite can be used therapeutically, for example, as anti-viral agents.

Description

COMPOSITIONS COMPRISING REVERSIBLY MODIFIED OLIGONUCLEOTIDES AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of, and relies on the filing date of, US. provisional patent application number 62/378,635, filed 23 August 2016, the entire disclosure of which is incorporated herein by reference.

BACKGROUND Oligonucleotides have various uses in molecular biology, including, for e, use as probes, primers, or linkers. Oligonucleotides can also be used therapeutically, for example, to edit genomic DNA sequences (e.g., Clustered Regularly Interspaced Short Palindromic Repeats “CRISPR”), to e defective or missing genes using gene therapy techniques, or as nucleic acid inhibitor les to modulate intracellular RNA levels through a diverse set of mechanisms. Small interfering RNA (“siRNA”), antisense oligonucleotides, ribozymes, microRNA, antagomirs, and aptamers are all examples of nucleic acid molecules that have demonstrated early promise in the treatment of cancers, viral ions, and genetic disorders.

Nucleoside and nucleotide analogs are also ly used therapeutically, ularly as antiviral or anticancer agents.

Like other drugs, therapeutic oligonucleotides need, among other , stability in biological systems and sufficient potency at the ed site of action. The in viva environment presents challenges to the stability of therapeutic oligonucleotides because of the conditions that these les experience as they navigate their way through the body and into the cytosol of a target cell. For example, oligonucleotides are susceptible to degradation by nucleases in the serum, including 3’-exonucleases. See Behlke, M.A., Oligonucleotides, 2008,18z305-320. c acid inhibitor molecules having a single-stranded 3’-overhang, such as certain canonical 2l-mer siRNA and other siRNA s known in the art and described herein may, consequently, be particularly susceptible to degradation by such 3’-exonucleases.

, M.A., Oligonucleotides, 2008,18z305-320. In addition, an RNase A-like ty has been implicated in the degradation of siRNAs in serum.

Furthermore, even if an oligonucleotide makes it through the environment of the serum and enters a target cell of interest, it may still be exposed to enzymes or conditions (e.g., pH) that impair the stability of the oligonucleotide. For example, endent ribonucleases and deoxyribonucleases are present in the vesicles of cells, e.g., lysosomes, endosomes and fused endosomal/lysosomal vesicles.

Over the years, different approaches have been pursued in an attempt to protect therapeutic oligonucleotides from these environmental conditions. A major approach to addressing problems associated within viva administration of nucleic acid inhibitor les has been to introduce certain irreversible, covalent chemical ation to one or more nucleotides in the nucleic acid molecule. Many types of irreversible nucleotide chemical modification have been reported over the years. See e. g., Bramsen et al., Nucleic Acids Res.,2009, 37 :2867-2881. Such irreversible chemical modifications often involve changes to the sugar moiety of a nucleotide.

Commonly, the 2’-carbon (C2’) ofthe sugar moiety of a nucleotide has been irreversibly modified because the 2’-hydroxyl (2’-OH) group makes the ribonucleotide more susceptible to certain ribonucleases. For e, many groups have modified the 2’ on of the sugar moiety from ahydroxyl group to a 2’-fluoro (2’-F) or a2’-O-methyl (2’-OMe), and such changes have effectively increased nuclease resistance of RNA oligonucleotides. See Behlke, M.A., Oligonucleoiides, 2008,18:305-320.

The 5’-end of the oligonucleotide is another position that has been commonly modified in an irreversible manner. Typical irreversible modifications at the 5’-end of the nucleic acid tor molecule include a oramidate or a chemical moiety that mimics the electrostatic and steric properties of a phosphate group (“phosphate ). See Prakash et al., 2015,43(6):2993-3011. Typically, these 5’-phosphate mimics contain atase- resistant linkages.

It is also possible to irreversibly modify the backbone of an oligonucleotide. For example, the phosphorothioate (PS) backbone modification replaces a non-bridging oxygen atom with a sulfur atom and may extend the ife of oligonucleotides in plasma from minutes to days. See Shen ei dl. Nucleic Acids Res., 2015,43z4569—4578, Eckstein, F. Nucleic Acid Therd., 2014,24(6):374-387.

Often it is desirable to rsibly modify one or more nucleotide positions in the same nucleic acid inhibitor molecule with more than one type of irreversible ation. For example, it is common to modify a siRNA molecule with multiple 2’-F, , and phosphorothioate modifications. See Podbevsek et al., Nucleic Acid Res, 2010,38(20):7298- 7307.

While these irreversible modifications may help to e the stability of a nucleic acid and/or protect it from s in the serum or in a cell, depending on the position of the modified nucleotide and/or the number of modifications, these irreversible modifications can also reduce the potency or activity of the c acid inhibitor molecule once it reaches the cytosol of the cell. See Behlke, M.A., ucleotides, 8z305-320. Furthermore, because these modifications are irreversible under intracellular conditions, they are not removed from the nucleic acid inhibitor molecule before it exerts its biological activity in the cytosol of the cell. If the irreversible modifications result in d potency or ty, they can limit the therapeutic y of nucleic acid tor molecules containing them.

While research and drug pment efforts have focused on irreversible modifications to protect nucleic acid inhibitor molecules, there have also been, on a smaller scale, reports of oligonucleotides containing a chemical modification that is reversible and can be removed after an oligonucleotide enters a cell. The reversible modifications can be removed, for example, by the action of an intracellular enzyme or by the chemical conditions inside a cell (e.g., through reduction by intracellular glutathione). Typically, nucleic acid les have been chemically modified with cyclic disulfide moieties to mask the negative charge created by the intemucleotide diphosphate linkages and improve cellular uptake and nuclease resistance. See US. Published ation No. 2011/0294869 originally ed to Traversa Therapeutics, Inc. (“Traversa”), PCT Publication No. to Solstice Biologics, Ltd. (“Solstice”), Meade et al., Nature Biotechnology, 2014,32:1256-1263 (“Meade”), PCT Publication No. to Merck Sharp & Dohme Corp. This reversible modification of the intemucleotide diphosphate linkages is designed to be cleaved intracellularly by the reducing environment of the cytosol (e. g. glutathione). Earlier es include neutralizing otriester modifications that were reported to be cleavable inside cells (Dellinger et al. J. Am. Chem. Soc. 2003,125z940-950).

There has been less effort in the art to reversibly modify other ons in the sugar moiety of nucleotides, such as the 2’-carbon (also referred to as C2’). C2’ has been ibly modified using modifications that are sensitive to enzymatic cleavage (Lavergne et al., J. Org.

Chem. and light-stimulated cleavage son et al., Bioorgemic &Med. , 2011,76:5719-5731) Chem. Letters, 2011,21z3721-25). Very recently, a reversible disulfide modification was applied to RNA molecules at the 2’ carbon. More particularly, a specific, 2’-O- methyldithiomethyl (2’-O MDTM) RNA was designed with a disulfide bridge cleavable intracellularly by glutathione and was shown in vitro to be able to inhibit the expression of an exogenously added luciferase gene in isolated A549 cells. See Ochi et al., Bioorganic Medicinal Chemistry Letters, 2016,26z845-848. However, the authors in Ochi found that nucleoside phosphoramidites ning the 2’-O-MDTM group are incompatible with standard oligonucleotide solid phase synthesis. See Ochi 2016, see also, Ochi et al., Curr.

Protoc. Nucleic Acid Chem, 2015,(62):4.63.1-4.63.20. Thus, Ochi had to use a post-synthetic approach to synthesize their 2’-O-MDTM-modified RNA molecules. See Ochi 2016, see also s et al., Org. Biomol. Chem, 2016,14z7010-17, recognizing Ochi’s post-synthetic approach to prepare 2’-O-MDTM-modified RNA molecules as a way to avoid the instability of the disulfide bond in side phosphoramidites containing the 2’-O-MDTM group and proposing alternative post-synthetic approaches for preparing RNA containing various 2’- alkyldithiomethyl groups.

Notwithstanding the advances that have been made in the art to e the stability of oligonucleotides and/or protect them from enzymes in the serum or in a cell, there remains a need in the art for improved strategies for the reversible modification ofnucleic acid molecules, ularly reversible modifications that are compatible with standard, oramidite oligonucleotide sis.

SUMMARY This application discloses various new glutathione-sensitive, reversibly modified nucleotides and nucleosides that can be incorporated into any oligonucleotide of interest, including nucleic acid inhibitor molecules, such as siRNA, antisense ucleotides, microRNA, ribozymes, antagomirs, and aptamers. They can also be incorporated into other oligonucleotides, such as, Clustered Regularly Interspaced Short romic Repeats R) nucleic acids, nucleic acids for gene therapy, nucleic acids for DNA editing, probes, or any other oligonucleotide that is susceptible to degradation by nucleases and/or harsh environmental conditions (e.g., pH), ing other oligonucleotides that are to be administered in viva.

The glutathione-sensitive reversible modifications of the invention can also be used to reversibly modify nucleotide and nucleoside monomers, including glutathione-sensitive nucleoside phosphoramidites that can be used, for example, in standard oligonucleotide synthesis s. In addition, glutathione-sensitive nucleotide and nucleoside monomers t a phosphoramidite can be used therapeutically, for example as anti-viral agents.

Typically, the glutathione-sensitive moiety is used to reversibly modify the bon of a sugar moiety in the nucleotide, although other carbon positions may also be modified with the glutathione-sensitive moiety. One or more glutathione-sensitive nucleotides can be incorporated into an oligonucleotide to help protect the oligonucleotide during in viva 2017/048239 administration (e.g., transit through the blood and/or the lysosomal/endosomal compartments of a cell) where the oligonucleotide will be exposed to ses and other harsh environmental conditions (e.g., pH). When the reversibly d oligonucleotide is released into the cytosol, the intracellular conditions, including a high level of hione, cause the glutathione-sensitive moiety to be removed from the oligonucleotide. In certain embodiments, the l of the hione-sensitive moiety yields a yl group at the 2’-carbon position, which is the natural substituent for a ribonucleotide at that position (see, 6. g. , Scheme 7 in Example 3).

Using reversible, glutathione-sensitive es according to the teachings of the instant application, it is possible to introduce sterically larger chemical groups into the oligonucleotide of interest as compared to the options available using irreversible chemical modifications. This is because these larger chemical groups will be removed in the cytosol and, therefore, should not interfere with the biological activity of the oligonucleotides inside the cytosol of a cell. As a result, these larger chemical groups can be engineered to confer various advantages to the nucleotide or oligonucleotide, such as nuclease resistance, lipophilicity, charge, thermal stability, specificity, and reduced immunogenicity. In some embodiments, the structure of the hione-sensitive moiety can be engineered to modify the kinetics of its release.

Moreover, the present reversibly modified, glutathione-sensitive ucleotides, can be synthesized using conventional solid-phase synthesis. Accordingly, these reversibly modified, glutathione-sensitive oligonucleotides are readily prepared and are suitable for use in therapeutic applications. In addition, because the nucleic acids can be synthesized using conventional phase synthesis, the glutathione-sensitive nucleotides can be incorporated into a nucleic acid molecule at selected positions in the oligonucleotide depending on the desired effect. The incorporation of the hione-sensitive moiety at specific positions of an oligonucleotide, such as a nucleic acid inhibitor molecule, can affect the properties of the oligonucleotide. For example, the glutathione-sensitive moiety can be incorporated at nucleotide position 1 (i.e., the 5’-terminal nucleotide) of a nucleic acid inhibitor molecule, which increases the stability of the molecule as compared to a molecule that has a 2’-F at nucleotide position 1.

With this technology, it is now le to readily synthesize therapeutically useful, glutathione-sensitive oligonucleotides having the glutathione-sensitive moiety incorporated at one or more nucleotide positions of interest. Thus, in one aspect, the glutathione-sensitive ucleotides described herein can be used as pharmaceuticals and formulated with a pharmaceutically acceptable excipient as a pharmaceutical composition and used, for example, to edit genomic DNA or to modulate the expression of target genes and to treat patients in need thereof.

In certain aspects, the present disclosure is directed to oligonucleotides that contain one or more reversibly modified nucleotides, where the reversibly modified nucleotide comprises a glutathione-sensitive moiety ed to the 2’-carbon of the sugar ring (or analog thereof).

In certain embodiments, the glutathione-sensitive moiety is represented by Formula II, III, or IV, or any of the sub genera f, as described herein, including, for example, Formula IIa, IIIa, ), IIIb, and IIIb(i), Formula IVa, IVb, IVc, IVd, or IVe, Formula IVa(i), IVb(i), IVb(ii), IVc(i), or IVd(i), or Formula IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(v), IVe(vi), IVe(vii), ii), IVe(ix), IVe(x), or IVe(xi). In embodiments where the oligonucleotide ns more than one reversibly modified nucleotide, each reversibly ed nucleotide may comprise the same glutathione-sensitive moiety or at least one of the reversibly modified tides may n a glutathione-sensitive moiety that is different from the at least one glutathione-sensitive moiety in the other reversibly modified nucleotides of the oligonucleotide. In certain embodiments, each reversibly modified nucleotide of the oligonucleotide comprises a different glutathione-sensitive .

In n aspects, the present disclosure is directed to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a glutathione-sensitive oligonucleotide comprising at least one nucleotide comprising a glutathione-sensitive moiety attached to the 2’-carbon of the sugar moiety (or analog f).

In certain embodiments, the glutathione-sensitive oligonucleotide comprises at least one tide ented by Formula I, as described , wherein L is a glutathione- sensitive moiety selected from Formula II, III, or IV, as described herein, or any of the sub genera thereof, as described herein, including, for example, Formula IIa, IIIa, IIIa(i), IIIb, and IIIb(i), Formula IVa, IVb, IVc, IVd, or IVe, Formula IVa(i), IVb(i), ), IVc(i), or IVd(i), or Formula IVe(i), IVe(ii), IVe(iii), IVe(iv), , IVe(vi), IVe(vii), IVe(viii), IVe(ix), IVe(x), or IVe(xi).

In certain embodiments of the glutathione-sensitive oligonucleotide, L is represented by Formula II, as described herein, wherein Y is 0, wherein Z is NR’, wherein R’ is hydrogen or substituted or tituted aliphatic, and wherein V is C and optionally wherein X2 and X3 are independently selected from hydrogen, halogen, nitro or amino.

In certain embodiments, L is represented by Formula IIa; as described herein.

In certain embodiments of the hione-sensitive oligonucleotide; L is ented by Formula 111, as described herein; wherein Y is O; S or NH; wherein Z1 is N or CH; wherein V is C; and optionally; wherein M1 and M2 are substituted or unsubstituted C2 to C6 alkyl or are taken together with P1 to Q1 to form a 5-8 membered ring; wherein the ring is substituted or unsubstituted cycloalkyl.

In n embodiments; L is represented by Formula IIIa; as described herein; wherein Y is O; S or NH; and Z1 is N or CR’; wherein R’ is ed from en; halogen; substituted or unsubstituted aliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted heterocycle. In one embodiment; Y is O and Z1 is N (see Formula IIIa(i)).

In certain ments; L is represented by Formula IIIb; as described ; wherein Y is O; S or NH; Z1 is N or CR’; wherein R’ is selected from hydrogen; n; substituted or unsubstituted aliphatic; substituted or unsubstituted aryl; substituted or unsubstituted aryl; substituted or unsubstituted heterocycle; and Ta and Tb are each independently absent or ed from CH3; substituted or substituted aliphatic; substituted or unsubstituted aryl; substituted or tituted heteroaryl; substituted or unsubstituted heterocycle or a ligand optionally connected Via spacer to a sulfur atom. In one embodiment; L is represented by Formula IIIb(i); as described .

In certain embodiments of the glutathione-sensitive oligonucleotide; L is ented by Formula IV; as described ; wherein Y is O; S or NH; wherein Z is NH or NCH3; wherein V is C; wherein G is CH2 and E is absent or G is absent and E is CH2; and optionally wherein M3 and M4 are independently substituted or unsubstituted C2 to C6 alkyl or taken together to form a 5-8 membered ring; wherein the ring is substituted or unsubstituted cycloalkyl.

In certain embodiments; L is represented by Formula IVa; as described herein; wherein Y is O; S; NH; wherein Z is O; S or NH; wherein R5; R6; and R7 are each independently selected from OAcyl; NHR’; NR’; CR’R”; wherein R’ and R” are each independently selected from en; halogen; CH2; CH; substituted aliphatic or unsubstituted aliphatic; aryl; heteroaryl; heterocyclic; or can be taken together to form a heterocyclic ring; and wherein T is a branched or unbranched C2-C6 alkyl or a ligand optionally connected Via a spacer to a sulfur atom. In one embodiment; L is represented by Formula IVa(i); as described herein.

In certain embodiments, L is represented by Formula IVb wherein Y is O; 8, NH; Z is O; S or NH; V is C; M3 and M4 are hydrogen; K is CH or a substituted or unsubstituted aliphatic; E is NH or NR’; wherein R’ is substituted or unsubstituted aliphatic; n is 0-5; T is substituted or unsubstituted C2 to C6 alkyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl or T is a ligand optionally ted Via a spacer to a sulfur atom. In certain embodiments; L is represented by Formula IVb(i) or IVb(ii); as described herein; wherein R is selected from hydrogen; CH3; substituted or tituted tic; aryl; heteroaryl; cycloalkyl or a heterocycle or R is a targeting ligand optionally ted Via a .

In certain embodiments; L is represented by Formula IVc; as described herein; wherein Y is O; 8, NH; Z is selected from O; S; or NR’; wherein R’ is selected from hydrogen; halogen; CH3; substituted or unsubstituted aliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted heterocycle; V is C; M3 and M4 are taken together to form a 5-8 membered ring; wherein the ring is a substituted or unsubstituted cycloalkyl; optionally substituted with a heteroatom; K is a branched or unbranched substituted or unsubstituted C2 to C6 alkyl; 11 is 0-5; T is substituted or unsubstituted C2 to C6 alkyl; tuted or unsubstituted aryl; substituted or unsubstituted heteroaryl or T is a ligand optionally connected Via a spacer; wherein R is selected from en; CH3; substituted or unsubstituted aliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted lkyl or a substituted or unsubstituted heterocycle or R is a targeting ligand optionally connected Via a spacer. In one embodiment; L is represented by a IVc(i); as described herein; wherein R is ed from hydrogen; CH3; substituted or tituted aliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted lkyl or a substituted or unsubstituted heterocycle or R is a targeting ligand optionally connected Via a spacer.

In certain embodiments; L is represented by Formula IVd; as described herein; n Y is O; 8, NH; Z is selected from O; 8, NH; or NCH3; T is substituted or unsubstituted C2 to C6 alkyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl or T is a ligand optionally connected Via a spacer to a sulfur atom; and R is selected from hydrogen; CH3 or a substituted or unsubstituted C2 to C6 alkyl. In one embodiment; L is represented by Formula ; as described herein.

In certain embodiments of the glutathione-sensitive oligonucleotide; L is represented by Formula IVe; as described herein; n Y is O; 8, NH; Z is selected from O; S; or NR’; wherein R’ is selected from hydrogen; halogen; CH3; substituted or unsubstituted aliphatic; WO 39364 substituted or unsubstituted aryl, tuted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, V is C or SO, G and E can be each ndently absent, or selected from CH2, CHR’, CR’R”, NH, NR’, wherein R’ and R” are each independently selected from hydrogen, halogen, a substituted or unsubstituted aliphatic, a substituted or unsubstituted aryl, a substituted or unsubstituted aryl, a substituted or unsubstituted heterocycle or R’ and R” are taken together to form a heterocyclic ring, K is C or CH, 11 is 0-5, T is substituted or unsubstituted C2 to C6 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or T is a ligand optionally connected Via a spacer, wherein R is selected from hydrogen, CH3, substituted or unsubstituted tic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or tituted cycloalkyl or a tuted or unsubstituted heterocycle or R is a targeting ligand optionally connected Via a spacer. In certain ments, Z is NH or NCH3 and one or both of G and E are absent, CH2, or CR’R”, NH, NR’, wherein R’ and R” are each ndently selected from hydrogen or substituted or unsubstituted aliphatic. In certain embodiments, L is represented by Formula IVe(i), IVe(ii), IVe(iii), IVe(iV), IVe(V), IVe(Vi), IVe(Vii), IVe(Viii), IVe(iX), IVe(X), or IVe(Xi), as described herein, wherein R is selected from hydrogen, CH3, substituted or unsubstituted aliphatic, tuted or tituted aryl, substituted or unsubstituted heteroaryl, substituted or tituted cycloalkyl or a substituted or unsubstituted heterocycle or R is a targeting ligand optionally connected Via a spacer.

In certain embodiments, the glutathione-sensitive oligonucleotide comprises at least one nucleotide represented by Formula VIIe(iX), wherein A is absent, a hydrogen, a phosphate group, or a phosphate mimic, wherein U1 is O or an intemucleotide linking group attaching the at least one nucleotide represented by Formula VIIe(iX) to a nucleotide or an oligonucleotide, wherein B is a natural nucleobase, wherein U2 is 0, wherein W is hydrogen or an intemucleotide linking group ing the at least one nucleotide represented by a VIIe(iX) to a nucleotide or an oligonucleotide, wherein at least one of U1 or W is an intemucleotide linking group attaching the at least one nucleotide represented by Formula VIIe(iX) to an oligonucleotide and provided that if U1 is an cleotide linking group, A is absent, and wherein the glutathione-sensitive oligonucleotide is a double-stranded RNAi inhibitor molecule comprising a sense strand and an antisense strand.

In n embodiments, A is hydrogen and W is an intemucleotide linking group attaching the at least one nucleotide represented by Formula VIIe(iX) to an oligonucleotide and the at least one nucleotide ented by Formula X) is located at nucleotide position 1 of the antisense strand.

In certain embodiments, A is absent; W is a intemucleotide linking group attaching the at least one nucleotide represented by Formula VIIe(iX) to a first oligonucleotide, and U1 is a cleotide linking group attaching the at least one nucleotide represented by Formula VIIe(iX) to a second oligonucleotide, and the at least one nucleotide represented by Formula VIIe(iX) is located at tide position 14 of the antisense strand.

In certain embodiments, the glutathione-sensitive oligonucleotide comprises at least one tide represented by Formula VIIe(Xi), n A is absent, a hydrogen, a phosphate group, or a ate mimic, wherein U1 is O or an intemucleotide linking group attaching the at least one nucleotide represented by Formula VIIe(Xi) to a nucleotide or an ucleotide, wherein B is a natural nucleobase, wherein U2 is 0, wherein W is hydrogen or an intemucleotide linking group attaching the at least one nucleotide represented by Formula VIIe(Xi) to a nucleotide or an oligonucleotide, wherein at least one of U1 or W is an intemucleotide linking group attaching the at least one nucleotide ented by a VIIe(Xi) to an oligonucleotide and provided that if U1 is an intemucleotide linking group, A is absent, and wherein the glutathione-sensitive oligonucleotide is a double-stranded RNAi inhibitor molecule sing a sense strand and an antisense strand. [03 8] In certain embodiments, the cleotide linking group contains a phosphorous atom.

In certain embodiments, the oligonucleotide is a double-stranded oligonucleotide comprising a first strand and a second strand.

In certain embodiments, the double stranded oligonucleotide is a double-stranded RNAi inhibitor molecule and the first strand and comprises a sense strand and the second strand comprises an antisense . In certain embodiments, the double ed RNAi inhibitor molecule comprises a region of mentarity between the sense strand and the antisense strand of about 15 to 45, 20 to 30, 21 to 26, 19 to 24, or 19 to 21 tides.

In certain embodiments, the at least one nucleotide represented by Formula I is located on the antisense strand. In certain embodiments, the at least one nucleotide represented by Formula I is located on the sense strand.

In certain embodiments, the at least one nucleotide represented by Formula I is located at nucleotide position 1 of the antisense strand. In certain ments, the at least one nucleotide represented by FormulaI is located at nucleotide position 14 of the antisense strand.

In certain embodiments, the at least one nucleotide represented by FormulaI is located at one or more nucleotide positions at or adjacent to the Ag02 cleavage site of the sense strand. In certain embodiments, the at least one nucleotide represented by a I is located at one, two, or three nucleotides that are immediately 5’ or 3’ of the Ag02 cleavage site. In certain embodiments, the at least one nucleotide represented by Formula I is located on both sides of the Ag02 cleavage site, e.g., at one or more nucleotides that are immediately 5’ of the Ag02 cleavage site and at one or more nucleotides that are immediately 3’ of the Ag02 cleavage site.

In certain embodiments, the double stranded RNAi tor le contains a tetraloop.

In certain embodiments, the hione-sensitive oligonucleotide is a single stranded ucleotide. In certain embodiments, the single stranded oligonucleotide is a single stranded RNAi inhibitor molecule. In certain embodiments, the single-stranded oligonucleotide is a conventional antisense oligonucleotide, a ribozyme, microRNA, antagomir, or an aptamer. In certain embodiments, the single stranded RNAi inhibitor molecule is about 14-50, 16-30, 18-22, or 20-22 nucleotides in length.

In certain embodiments, the glutathione-sensitive oligonucleotide contains 1-5 nucleotides represented by Formula I. In certain ments, 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 modification.

In certain embodiments, the glutathione-sensitive oligonucleotide further comprises a delivery agent, wherein the delivery agent facilitates ort of the glutathione-sensitive oligonucleotide across an outer membrane of a cell. In certain embodiments, the delivery agent is selected from the group ting of carbohydrates, peptides, lipids, vitamins and antibodies. In certain embodiments, the delivery agent is selected from N—Acetylgalactosamine (GalNAc), mannosephosphate, galactose, oligosaccharide, polysaccharide, cholesterol, polyethylene glycol, folate, vitamin A, n E, lithocholic acid and a ic lipid.

In certain embodiment, the glutathione-sensitive oligonucleotide is ned in a lipid nanoparticle. In certain embodiments, the glutathione-sensitive oligonucleotide is a naked, glutathione-sensitive oligonucleotide.

In certain embodiments, the hione-sensitive oligonucleotide comprises at least one nucleotide having a hione-sensitive moiety bound to an oxygen atom that is covalently bound to a 2’-carbon of a sugar moiety of the nucleotide, n the glutathione- sensitive oligonucleotide is prepared by a phosphoramidite-based oligonucleotide synthesis method using a nucleoside phosphoramidite having a glutathione-sensitive moiety.

In certain embodiments, the glutathione-sensitive oligonucleotide is a Clustered Regularly Interspaced Short Palindromic Repeats “CRISPR” nucleic acid sequence having a chNA sequence having a first portion capable of hybridizing to a target sequence in a cell and/or a trachNA sequence that hybridizes with a second portion of the chNA sequence to form a guide sequence. In certain embodiments, the guide sequence is a chimeric guide sequence, wherein the chNA sequence is fused to the trachNA sequence.

In certain aspects, the present disclosure is directed to a pharmaceutical composition comprising a glutathione-sensitive oligonucleotide as described herein and a pharmaceutically acceptable excipient and methods of using the same. In certain embodiments, the hione- sensitive oligonucleotide comprises at least one glutathione-sensitive tide, wherein the at least one glutathione-sensitive nucleotide comprises a substitution of a hydroxyl group at the bon of a ribose or analog thereof with a glutathione-sensitive moiety. In certain embodiments, the glutathione-sensitive oligonucleotide is a double stranded RNAi inhibitor molecule. In certain aspects, the present disclosure is ed to a method for reducing sion of a target gene in a subject comprising administering a pharmaceutical composition comprising a hione-sensitive double-stranded RNAi inhibitor molecule to a subject in need thereof in an amount sufficient to reduce expression of the target gene. In certain embodiments, the stering ses systemic administration.

In certain aspects, the present disclosure is directed to a side comprising a phosphoramidite and a glutathione-sensitive moiety, wherein the nucleoside is compatible with phosphoramidite-based oligonucleotide synthesis. In certain embodiments, the phosphoramidite is bound to the 5’-or 3’-carbon of the sugar moiety of the nucleoside and the glutathione-sensitive moiety is bound to an oxygen atom that is covalently bound to the 2’- carbon of the sugar moiety of the side. In certain embodiments of the nucleoside phosphoramidite, the glutathione-sensitive moiety is represented by Formula II, Formula III, or Formula IV or any subgenera thereof, including Formula IIa, IIIa, IIIb, IIIa(i), IIIb(i), IVa, IVb, IVc, IVd, IVe, IVa(i), IVb(i), ), IVc(i), IVd(i), IVe(i), ), IVe(iii), IVe(iv), IVe(v), IVe(vi), IVe(vii), ii), IVe(ix), IVe(x), or IVe(xi), as described herein.

In certain s, the t disclosure is ed to a glutathione-sensitive nucleoside phosphoramidite, wherein the side phosphoramidite is represented by Formula VIII, as described herein. In certain embodiments, the nucleoside phosphoramidite is represented by Formula IX. In certain embodiments, the hione-sensitive moiety (Li) comprises a disulfide bridge or a sulfonyl group. 2017/048239 In certain embodiments, the nucleoside phosphoramidite is represented by Formula VIII, n J is O, B is a natural nucleobase, U2 is O, I is CH2, W1 is a phosphoramidite, A1 is a ting group, hydrogen, or solid support, and U3 is O and optionally, wherein X is O and R1, R2, R3 and R4 are en.

In certain embodiments, the nucleoside phosphoramidite is represented by Formula VIII, wherein J is O, B is a natural nucleobase, U2 is O, I is CH2, W1 is a protecting group, hydrogen or solid support, A1 is a phosphoramidite, and U3 is O and optionally, n X is O and R1, R2, R3 and R4 are hydrogen.

In certain embodiments, the nucleoside phosphoramidite is represented by Formula X, wherein R8 is H or a protecting group, R7 is a phosphoramidite, B is a natural nucleobase, X is O, and wherein L1 is represented by Formula IVe(ix).

In certain embodiments, the nucleoside phosphoramidite is represented by Formula X, wherein R8 is H or a protecting group, R7 is a phosphoramidite, B is a natural base, and X is O, and wherein L1 is represented by Formula IVe(xi).

In n ments, the phosphoramidite has the formula —P(ORX)—N(Ry)2, wherein RX is selected from the group consisting of an optionally substituted methyl, 2- cyanoethyl and benzyl, wherein each of Ry is selected from the group consisting of an optionally substituted ethyl and isopropyl.

In certain aspects, the present disclosure is directed to a method for preparing a glutathione-sensitive oligonucleotide comprising: (a) ing a side to a solid t via a covalent linkage, (b) coupling the glutathione-sensitive nucleoside phosphoramidite, as described herein, to a hydroxyl group on the nucleoside of step (a) to form a phosphorus nucleoside linkage etween, 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 glutathione-sensitive side phosphoramidites, as described herein, 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. In another aspect, the present disclosure is directed to an oligonucleotide made by the method. In certain embodiments, the hione- sensitive moiety comprises a disulfide bridge or sulfonyl group, including, for example, the glutathione-sensitive moiety represented by Formula II, III, or IV, as described herein, or any of the era thereof, including Formula IIa, IIIa, IIIb, IIIa(i), IIIb(i), IVa, IVb, IVc, IVd, IVe, IVa(i), IVb(i), IVb(ii), IVc(i), IVd(i), , IVe(ii), IVe(iii), IVe(iv), IVe(v), IVe(vi), IVe(vii), IVe(viii), IVe(ix), IVe(x), or ), as described herein.In certain aspects, the present disclosure is directed to a glutathione-sensitive nucleoside or tide that does not contain a phosphoramidite, wherein the hione-sensitive nucleoside or nucleotide comprises a glutathione-sensitive moiety that is bound to an oxygen atom that is covalently bound to the 2′-carbon of the sugar moiety of the nucleotide or nucleoside; and wherein the glutathione-sensitive moiety is represented by Formula II, Formula III, or Formula IV, as described herein, or any subgenera thereof.

In certain embodiments, the glutathione-sensitive nucleoside or nucleotide is represented by Formula XI, as described . In certain embodiments, J is O; X is O; L2 is a glutathione-sensitive moiety represented by Formula II, III, or IV; W2 is hydrogen, halogen, OR′, SR′, NR′R″, a substituted or unsubstituted aliphatic, a substituted or tituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycle, wherein R′ and R″ are each independently selected from hydrogen, halogen, a substituted or unsubstituted aliphatic, an aryl, a aryl, a heterocycle or are taken together to form a heterocyclic ring; and A2 is absent, hydrogen, a phosphate group, a ate mimic, or a phosphoramidate; and optionally wherein R1, R2, R3, and R4 are hydrogen; U2 is oxygen; W2 is en; I is CH2; U3 is O; and A2 is hydrogen or a phosphate group. [059A] In a particular aspect, the present invention provides a glutathione-sensitive oligonucleotide, wherein the glutathione-sensitive ucleotide ses at least one nucleotide ented by Formula I: A U1 I B R4 R1 R3 R2 U2 X wherein X is O, S, Se or NR′, wherein R′ is selected from hydrogen, halogen, an aliphatic, an aryl, a heteroaryl or a heterocycle; wherein R1, R2, R3 and R4 are each independently selected from hydrogen, halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl or wherein two of R1, R2, R3 and R4 are taken together to form a 5-8 ed ring, wherein the ring optionally contains a heteroatom; (followed by page 14a) wherein J is O, S, NR′, CR′R″, n each of R′ and R″ is independently selected from hydrogen, halogen, an aliphatic, aryl or heteroaryl; wherein B is selected from hydrogen, a l nucleobase, a modified nucleobase or a universal nucleobase; wherein U2 is absent or selected from O, S, NR′, or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl, a heterocycle 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 tic, an aryl, a heteroaryl, a cycloalkyl, a heterocycle, wherein R′ and R″ are each independently selected from hydrogen, halogen, an aliphatic, an aryl, a heteroaryl, a heterocycle or are taken together to form a heterocyclic ring; wherein I is absent or is selected from O, S, NR′, CR′R″, n R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl, a heterocycle and a cycloalkyl; wherein U1 is absent, hydrogen, an internucleotide linking group attaching the at least one nucleotide ented by Formula I to a nucleotide or an oligonucleotide, or selected from O, S, NR′ or CR′R″, wherein R′ and R″ are each independently en, an aliphatic, an aryl, a heteroaryl, a heterocycle and a cycloalkyl and wherein at least one of U1 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 U1 is an internucleotide linking group, A is absent; wherein I and U1 can be combined to form ″ alkyl, CR′-CR″ alkenyl, CR′- CR″ alkynyl, an aliphatic, an aryl, a heteroaryl a heterocycle or taken together to form cycloalkyl or heterocyclic ring; n A is absent, a en, a phosphate group, a phosphate mimic or a phosphoramidate; and wherein L is a hione-sensitive moiety represented by: Formula IIa: IIa; (followed by page 14b) Formula IIIa(i): IIIa(i); Formula IIIb(i): IIIb(i); Formula IVa(i): IVa(i); Formula : IVb(i); (followed by page 14c) Formula IVb(ii): IVb(ii); wherein, in Formula IVb(i) and a IVb(ii), R is selected from hydrogen, CH3, tic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected via a spacer; Formula IVc(i): IVc(i); n, in Formula IVc(i), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected via a spacer; Formula IVd(i): IVd(i); (followed by page 14d) Formula IVe(i): IVe(i); Formula IVe(ii): Formula IVe(iii): IVe(iii); (followed by page 14e) Formula IVe(iv): IVe(iv); Formula IVe(ix): IVe(ix); Formula IVe(x): IVe(x); Formula IVe(xi): (followed by page 14f) wherein, in Formulae i), IVe(iv), and IVe(x), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected via a spacer. [059B] In another particular aspect, the present invention provides a nucleoside phosphoramidite, wherein the nucleoside phosphoramidite is represented by Formula VIII: VIII wherein L1 is a glutathione-sensitive moiety represented by a IIa, IIIa(i), IIIb(i), IVa(i), IVb(i), ), IVc(i), IVd(i), IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(ix), IVe(x) or IVe(xi); IIa; IIIa(i); (followed by page 14g) IVa(i); IVb(i); IVb(ii); (followed by page 14h) n, in Formula IVb(i) and Formula IVb(ii), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand ally connected via a spacer; IVc(i); wherein, in Formula IVc(i), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a ing ligand optionally connected via a spacer; IVd(i); IVe(i); (followed by page 14i) IVe(ii); IVe(iv); IVe(ix); (followed by page 14j) IVe(x); IVe(xi); wherein, in ae i), IVe(iv), and IVe(x), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected via a spacer; wherein A1 is absent, hydrogen, a phosphate group, a phosphate mimic, a phosphoramidate, a protecting group, or a solid support; wherein W1 is a phosphoramidite, a protecting group, a solid support, hydrogen, n, OR′, SR′, NR′R″, an aliphatic, an aryl, a heteroaryl, a cycloalkyl, a heterocycle, wherein R′ and R″ are each independently selected from hydrogen, halogen, an aliphatic, an aryl, a heteroaryl, a heterocycle or are taken together to form a heterocyclic ring; wherein U3 is a hydrogen or selected from O, S, NR′ or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl, a heterocycle and a cycloalkyl; wherein at least A1 is a phosphoramidite and U3 is O or at least W1 is a oramidite and U2 is O; wherein X is O, S, Se or NR′, wherein R′ is selected from hydrogen, n, an aliphatic, an aryl, a heteroaryl or a heterocycle; n R1, R2, R3 and R4 are each independently selected from hydrogen, halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl or wherein two of R1, R2, R3 and R4 are taken together to form a -8 membered ring, wherein the ring ally contains a heteroatom; (followed by page 14k) wherein J is O, S, NR′, CR′R″, wherein each of R′ and R″ is independently selected from en, halogen, an aliphatic, aryl or heteroaryl; wherein B is selected from hydrogen, an aliphatic, a l nucleobase, a ed nucleobase or a universal nucleobase; wherein U2 is absent or selected from O, S, NR′, or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a aryl, a heterocycle or a cycloalkyl; wherein I is absent or is selected from O, S, NR′, CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl, a heterocycle and a cycloalkyl; and wherein I and U3 can be combined to form CR′-CR″ alkyl, CR′-CR″ alkenyl, CR′-CR″ alkynyl, an tic, an aryl, a heteroaryl a heterocycle or taken together to form cycloalkyl or heterocyclic ring. [059C] In another particular aspect, the t invention provides a glutathionesensitive nucleoside or nucleotide, wherein the glutathione-sensitive nucleoside or nucleotide comprises a glutathionesensitive moiety; wherein the glutathione-sensitive moiety is bound to an oxygen atom that is covalently bound to the bon of the sugar moiety of the nucleotide or nucleoside; and n the glutathione-sensitive moiety is represented by Formula IIa, IIIa(i), IIIb(i), IVa(i), IVb(i), IVb(ii), IVc(i), IVd(i), IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(ix), IVe(x) or IVe(xi): IIa; (followed by page 14l) IIIa(i); IVa(i); IVb(i); (followed by page 14m) IVb(ii); wherein, in Formula IVb(i) and Formula ), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected via a spacer; IVc(i); wherein, in Formula IVc(i), R is selected from hydrogen, CH3, aliphatic, aryl, aryl, cycloalkyl or a cycle or R is a targeting ligand optionally connected via a spacer; IVd(i); (followed by page 14n) IVe(ii); IVe(iii); IVe(iv); (followed by page 14o) IVe(ix); IVe(x); IVe(xi); wherein, in Formulae IVe(iii), IVe(iv), and IVe(x), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a cycle or R is a targeting ligand optionally connected via [059D] In a further particular aspect, the present invention provides a glutathionesensitive side or nucleotide, wherein the glutathione-sensitive nucleoside or nucleotide is represented by Formula XI: (followed by page 14p) wherein L2 is a glutathione-sensitive moiety represented by Formula IIa, IIIa(i), IIIb(i), IVa(i), IVb(i), ), IVc(i), IVd(i), IVe(i), IVe(ii), IVe(iii), IVe(iv), ), IVe(x) or IVe(xi): IIa; IIIa(i); IIIb(i); (followed by page 14q) IVa(i); IVb(i); IVb(ii); wherein, in Formula IVb(i) and Formula IVb(ii), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a cycle or R is a targeting ligand optionally connected via a spacer; (followed by page 14r) IVc(i); n, in Formula IVc(i), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected via a spacer; IVd(i); IVe(i); IVe(ii); (followed by page 14s) IVe(iii); IVe(ix); IVe(x); (followed by page 14t) IVe(xi); wherein, in Formulae IVe(iii), ), and IVe(x), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected via a spacer; or wherein L2 is absent if one of A2 or W2 is the glutathione-sensitive moiety represented by a IIa, IIIa(i), IIIb(i), IVa(i), IVb(i), IVb(ii), IVc(i), IVd(i), IVe(i), IVe(ii), i), IVe(iv), IVe(ix), IVe(x) or IVe(xi); wherein if L2 is a glutathione-sensitive moiety, X is O, S, Se, or NR′, wherein R′ is selected from hydrogen, halogen, an aliphatic, an aryl, a heteroaryl or a heterocycle or if L2 is absent, X is H, OH, SH, NH2, halogen, alkoxy, alkyl, alkenyl, alkynyl, hio, alkylamino or dialkylamino wherein one or more methylenes in the alkyl, alkenyl, and alkynyl may be upted with one or more of O, S, S(O), SO2, N(R′), C(O), N(R′)C(O)O, OC(O)N(R′), aryl, heteroaryl, heterocyclic or lkyl, O, S, Se or NHR′, wherein R′ is selected from hydrogen, halogen, an aliphatic, an aryl, a heteroaryl or a cycle; wherein R1, R2, R3 and R4 are each independently selected from en, n, OH, C1-C6 alkyl, C1-C6 haloalkyl or wherein two of R1, R2, R3 and R4 are taken together to form a -8 ed ring, wherein the ring optionally contains a heteroatom; wherein J is O, S, NR′, CR′R″, wherein each of R′ and R″ is independently selected from hydrogen, halogen, an aliphatic, aryl or heteroaryl; wherein B is selected from hydrogen, a natural nucleobase, a modified nucleobase or a sal nucleobase; wherein U2 is absent or ed from O, S, NR′, or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl, a heterocycle or a cycloalkyl; wherein W2 is a glutathione-sensitive moiety represented by Formula IIa, IIIa(i), ), IVa(i), IVb(i), IVb(ii), IVc(i), IVd(i), IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(ix), IVe(x) or IVe(xi); hydrogen, halogen, OR′, SR′, NR′R″, an aliphatic, an aryl, a heteroaryl, a cycloalkyl, (followed by page 14u) a heterocycle, wherein R′ and R″ are each ndently selected from en, halogen, an aliphatic, an aryl, a heteroaryl, a heterocycle or are taken together to form a heterocyclic ring; wherein I is absent or is selected from O, S, NR′, CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl, a heterocycle and a cycloalkyl; wherein U3 is hydrogen, or selected from O, S, NR′ or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl, a heterocycle and a cycloalkyl; wherein I and U3 can be combined to form CR′-CR ″ alkyl, CR′-CR ″ alkenyl, CR′-CR ″ alkynyl, an aliphatic, an aryl, a heteroaryl, a heterocycle or taken together to form cycloalkyl or heterocyclic ring; and wherein A2 is absent, hydrogen, a phosphate group, a phosphate mimic, a phosphoramidate, or a glutathione-sensitive moiety represented by Formula IIa, ), IIIb(i), , IVb(i), IVb(ii), IVc(i), IVd(i), IVe(i), ), IVe(iii), IVe(iv), IVe(ix), IVe(x) or IVe(xi).

BRIEF DESCRIPTION OF THE DRAWINGS -1B depict examples of four representative double ed RNAi inhibitor molecules as described in the Examples: Control Compound A and Control Compound B (Fig. 1A) and Test Compound 1 and Test Compound 2 (Fig. 1B). Test Compounds 1 and 2 contain the indicated hione-sensitive moiety at the bon at nucleotide positions 1 (“Guide position 1”) and 14 (“Guide position 14”), respectively, of the guide strand of the double stranded RNAi inhibitor molecules, according to the present disclosure. Except for the glutathione-sensitive nucleotide at nucleotide position 1 and 14 of the guide strands of Test Compounds 1 and 2, respectively, the remaining nucleotides in Test Compounds 1 and 2 were irreversibly modified with either 2′-F or 2′-OMe. Control Compounds A and B are identical to Test Compounds 1 and 2 except for the nucleotides at positions 1 and 14 of the guide s. l Compounds A and B contain a 2′-F at tide position 1 of the guide strand (“Guide position 1”). l Compound A differs from Control Compound B because it contains [FOLLOWED BY PAGE 15] natural phosphate (5’-PO42') at the 5’-carbon of the 5’-terminal nucleotide of the guide strand, whereas l Compound B contains a free hydroxyl group (5’-OH) at the 5’-carbon of the ’-terminal nucleotide of the guide strand. The guide strands of Control Compounds A and B contain the same nucleotide sequence and, therefore, recognize the same target mRNA sequence as Test Compounds 1 and 2. depicts the release rate of uridine from a reversibly-modified e having a glutathione-sensitive moiety at the 2’-carbon in accordance with the present disclosure, ing incubation with glutathione, as described in Example 3. depicts the rate of disappearance of Test Compound 2 ing incubation with glutathione, as described in Example 3. depicts the potency (including IC50) of Test Compound 1 in comparison to the control compounds (Compound A and Compound B), as measured by the knockdown of target mRNA 48 hours after transfection of the compounds into murine hepatocytes, as described in Example 4. depicts the y (including IC50) of Test Compound 1 in monkey hepatocytes, as measured by the knockdown of target mRNA 24 hours after transfection, as described in Example 4. depicts the potency in mice, as measured by the knockdown of target mRNA, and duration of effect following the in viva administration of Test Compound 1 in comparison to a control PBS injection, as described in Example 5. depicts the potency in mice, as measured by the knockdown of target mRNA, and duration of effect following the in viva administration of Test nd 2 in comparison to a control PBS injection, as bed in Example 5.

DETAILED DESCRIPTION Definitions In order for the present sure to be more y understood, certain terms are first defined below. onal definitions for the following terms and other terms may be set forth through the ication. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.

WO 39364 As used in this cation and the appended claims, the singular forms cc 7) (C a, an,” and “the” include plural references unless the context clearly dictates otherwise. Thus for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

Acyl: As used herein, the term “acyl” refers to an alkylcarbonyl, lkylcarbonyl and arylcarbonyl moiety.

Aliphatic group: As used herein, the term “aliphatic group” refers to both saturated and unsaturated, straight chain (i.e., unbranched), or branched, hydrocarbons, which are optionally substituted with one or more functional groups. The term “substituted tic” refers to aliphatic moieties bearing substituents.

: As used herein, the term “alkoxy” refers to an alkyl group attached to a molecular moiety through an oxygen atom. l: As used herein, the term “alkenyl” refers to straight or branched chain hydrocarbyl groups having at least one carbon-carbon double bond, and having in the range of about 2 to about 20 carbon atoms. “Substituted alkenyl” refers to alkenyl groups further bearing one or more substituents. As used , “lower alkenyl” refers to alkenyl moieties having from 2 to about 6 carbon atoms.

Alkyl: As used herein, the term “alkyl” refers to straight or branched chain hydrocarbyl groups having from 1 up to about 20 carbon atoms. er it appears herein, a numerical range, such as “C1-C6 alkyl” means that an alkyl group may comprise only 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 6 carbon atoms, although the term “alkyl” also includes instances where no numerical range of carbon atoms is designated. For example, the term “alkyl” can refer to a sub-range between C1-C10 (e. g. C1-C6). “Substituted alkyl” refers to alkyl moieties bearing substituents. As used herein, “lower alkyl” refers to alkyl moieties having from 1 to about 6 carbon atoms.

Alkylamino: As used herein, the term “alkylamino” refers to an alkyl radical bearing an amine functionality. Alkylaminos may be tuted or tituted.

Alkynyl: As used herein, “alkynyl” refers to straight or branched chain hydrocarbyl groups having at least one carbon-carbon triple bond, and having in the range of about 2 to about 20 carbon atoms. “Substituted alkynyl” refers to alkynyl groups r bearing one or more substituents. As used herein, “lower l” refers to alkynyl moieties having from about 2 to about 6 carbon atoms.

Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, %, 4%, 3%, 2%, 1%, or less in either ion (greater than or less than) ofthe stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

Aptamer: As used herein, the term “aptamer” refers to an oligonucleotide that has binding affinity for a specific target including a nucleic acid, a protein, a specific whole cell or a particular tissue. Aptamers may be ed using methods known in the art, for example, by in vitro selection from a large random sequence pool of nucleic acids. Lee et al., Nucleic Acid Res. , 2004,32:D95-D100. mir: As used herein, the term “antagomir” refers to an ucleotide that has binding affinity for a specific target including the guide strand of an exogenous RNAi inhibitor molecule or natural miRNA (Krutzfeldt et al. Nature 2005,438(7068):685-689).

Antisense : A double stranded RNAi inhibitor molecule comprises two oligonucleotide s: an antisense strand and a sense . The antisense strand or a region f is partially, substantially or fully complementary to a corresponding region of a target nucleic acid. In on, the antisense strand of the double stranded RNAi inhibitor molecule or a region thereof is partially, substantially or fully complementary to the sense strand of the double stranded RNAi inhibitor molecule or a region thereof. In certain embodiments, the antisense strand may also contain nucleotides that are non-complementary to the target nucleic acid sequence. The non-complementary nucleotides may be on either side of the complementary sequence or may be on both sides of the complementary sequence. In certain embodiments, where the antisense strand or a region thereof is partially or substantially complementary to the sense strand or a region thereof, the non-complementary nucleotides may be located between one or more s of complementarity (e.g., one or more mismatches).

The nse strand of a double stranded RNAi inhibitor le is also referred to as the guide .

Aromatic Group: The term “aromatic group” as used herein refers to a planar ring having a delocalized 7t-electron system containing 4n+27t electrons, where n is an integer.

Aromatic rings can be formed from five, six, seven, eight, nine, or more than nine atoms. The term “aromatic” is intended to encompass both carbocyclic aryl (e.g., phenyl) and heterocyclic aryl (or “heteroaryl” or “heteroaromatic”) groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic rings, i.e., rings which share adjacent pairs of carbon atoms. “Substituted aromatic” refers to an aromatic group further bearing one or more substituents.

Araliphatic: As used herein, the terms “araliphatic,77 (Caryl aliphatic,” or “aromatic aliphatic” are used hangeably and refer to compounds that contain one or more aromatic moieties and one or more aliphatic es.

Aryl: As used herein, the term “aryl” refers to an aromatic monocyclic or multicyclic groups having in the range of 5 up to 19 carbon atoms. “Substituted aryl” refers to aryl groups further bearing one or more substituents.

Carboxylic: As used herein, “carboxylic77 (C , carboxy” or “carboxyl” generally refers to the radical C(O)OH.

Canonical RNA tor molecule: As used herein, the term “canonical RNA inhibitor molecule” refers to two strands of nucleic acids, each 21 nucleotides long with a central region of complementarity that is 19 base-pairs long for the formation of a double ed c acid and two nucleotide nds at each of the 3’-ends.

Complementary: As used herein, the term “complementary” refers to a structural relationship between two nucleotides (e.g., on two opposing c acids or on opposing regions of a single nucleic acid strand) that permits the two nucleotides to form base pairs with one another. For example, a purine nucleotide of one nucleic acid that is mentary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another. In some embodiments, complementary nucleotides can base pair in the Watson-Crick manner or in any other manner that allows for the formation of stable duplexes. “Fully complementarity” or 100% complementarity refers to the situation in which each nucleotide monomer of a first oligonucleotide strand or of a segment of a first oligonucleotide strand can form a base pair with each nucleotide monomer of a second oligonucleotide strand or of a segment of a second oligonucleotide strand. Less than 100% complementarity refers to the situation in which some, but not all, nucleotide monomers oftwo oligonucleotide strands (or two segments of two ucleotide strands) can form base pairs with each other. “Substantial complementarity” refers to two ucleotide strands (or segments of two oligonucleotide s) exhibiting 90% or greater mentarity to each other. “Sufficiently complementary” refers to complementarity between a target mRNA and a nucleic acid inhibitor molecule, such that there is a reduction in the amount ofprotein d by a target mRNA.

Complementary strand: As used herein, the term ementary ” refers to a strand of a double stranded nucleic acid tor molecule that is partially, substantially or fully complementary to the other strand.

Conventional antisense oligonucleotide: As used , the term “conventional antisense oligonucleotide” refers to single stranded ucleotides that inhibit the expression of a targeted gene by one of the following mechanisms: (1) Steric hindrance, e.g., the antisense oligonucleotide interferes with some step in the sequence of events involved in gene expression and/or production ofthe encoded protein by directly interfering with, for example, transcription ofthe gene, splicing ofthe pre-mRNA and translation ofthe mRNA, (2) Induction of enzymatic ion of the RNA transcripts of the targeted gene by RNase H, (3) Induction of enzymatic digestion of the RNA transcripts of the targeted gene by RNase L, (4) Induction of enzymatic digestion of the RNA transcripts of the targeted gene by RNase P: (5) Induction of enzymatic digestion of the RNA transcripts of the targeted gene by double stranded RNase, and (6) Combined steric hindrance and induction of enzymatic digestion activity in the same antisense oligo. Conventional nse oligonucleotides do not have an RNAi mechanism of action like RNAi tor les. RNAi inhibitor molecules can be distinguished from conventional antisense oligonucleotides in several ways including the requirement for Ag02 that combines with an RNAi nse strand such that the antisense strand directs the Ag02 protein to the intended target(s) and where Ag02 is required for silencing of the target.

CRISPR RNA: Clustered rly Interspaced Short Palindromic Repeats (“CRISPR”) is a microbial nuclease system involved in defense against invading phages and plasmids. Wright et al., Cell, 2016,164:29-44. This prokaryotic system has been adapted for use in editing target nucleic acid sequences of interest in the genome of eukaryotic cells. Cong et al., Science, 2013,339:8l9-23, Mali et al., Science, 2013,339:823-26, Woo Cho et al., Nat.

Biotechnology, 2013,3l(3):230-232. As used herein, the term “CRISPR RNA” refers to a nucleic acid comprising a “CRISPR” RNA (chNA) n and/or a trans activating chNA (trachNA) portion, wherein the CRISPR portion has a first sequence that is partially, substantially or fully mentary to a target nucleic acid and a second sequence (also called the tracer mate sequence) that is sufficiently complementary to the trachNA portion, such that the tracer mate sequence and trachNA portion hybridize to form a guide RNA. The guide RNA forms a complex with an endonuclease, such as a Cas endonuclease (e.g., Cas9) and directs the nuclease to mediate cleavage of the target nucleic acid. In certain embodiments, the chNA portion is fused to the trachNA portion to form a chimeric guide RNA. Jinek et al., Science, 2012,337z816-21. In certain embodiments, the first sequence of the chNA portion includes between about 16 to about 24 nucleotides, preferably about 20 nucleotides, which hybridize to the target nucleic acid. In certain embodiments, the guide RNA is about 10-500 nucleotides. In other embodiments, the guide RNA is about 20-100 nucleotides.

Cycloalkyl: As used herein, the term “cycloalkyl” refers to cyclic (i.e., ringcontaining ) hydrocarbon groups ning 3 to 12 carbons, for example, 3 to 8 carbons and, for example, 3 to 6 carbons. “Substituted cycloalkyl” refers to cycloalkyl groups further bearing one or more substituents.

Delivery agent: As used herein, the term “delivery agent” refers to a transfection agent or a ligand that is complexed with or bound to an ucleotide and which mediates its entry into cells. The term encompasses ic liposomes, for example, which have a net positive charge that binds to the oligonucleotide’s negative charge. This term also encompasses the conjugates as described , such as GalNAc and cholesterol, which can be covalently attached to an oligonucleotide to direct delivery to certain tissues. r specific suitable delivery agents are also described herein.

Deoxyribonucleotide: As used herein, the term “deoxyribonucleotide” refers to a natural or modified nucleotide which has a hydrogen group at the ition of the sugar moiety.

Disulfide: As used herein, the term “disulfide” refers to a al compound %s—s containing the group i. Typically, each sulfur atom is ntly bound to a hydrocarbon group. In certain ments, at least one sulfur atom is covalently bound to a group other than a hydrocarbon. The e is also called an SS-bond or a disulfide bridge.

Duplex: As used herein, the term “duplex” in reference to nucleic acids (e.g., oligonucleotides), refers to a double helical structure formed through complementary base pairing of two antiparallel sequences of nucleotides.

Excipient: As used herein, the term “excipient” refers to a non-therapeutic agent that may be included in a composition, for example to provide or contribute to a desired consistency or izing effect.

Glutathione: As used , the term “glutathione” (GSH) refers to a tripeptide having the structure of a XIII, below. GSH is present in cells at a concentration of approximately 1-10 mM. GSH reduces glutathione-sensitive bonds, including disulfide bonds.

In the s, glutathione is converted to its oxidized form, glutathione disulfide (GSSG).

Once ed, glutathione can be reduced back by glutathione reductase, using NADPH as an electron donor. 0 O O HO IZ OH ““2 0 (X111) Glutathione—sensitive compound or glutathione—sensitive moiety: As used herein, the terms “glutathione-sensitive compound”, or “glutathione-sensitive moiety”, are used hangeably and refers to any chemical nd (e.g., oligonucleotide, nucleotide, or nucleoside) or moiety containing at least one glutathione-sensitive bond, such as a disulfide bridge or a sulfonyl group. As used herein, a “glutathione—sensitive oligonucleotide” is an oligonucleotide containing at least one nucleotide containing a glutathione-sensitive bond.

Half-life: As used herein, the terms “serum half-life”, “plasma half-life” and “vesicle half-life” refer to the amount of time by which half of an amount of a molecule, such as a reversibly modified oligonucleotide, is degraded or removed under a specific condition, e. g. in the presence of serum, plasma or in endosomal or lysosomal es.

Halo: As used herein, the terms “halo” and “halogen” are interchangeable and refer to an atom selected from fluorine, chlorine, e and iodine.

Haloalkyl: As used herein, the term “haloalkyl” refers to an alkyl group having one or more halogen atoms attached o and is exemplified by such groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.

Heteroaryl: As used herein, the term “heteroaryl” refers to an aromatic ring system ning at least one heteroatom selected from nitrogen, oxygen and sulfur. The heteroaryl ring can be fused or ise ed to one or more heteroaryl rings, aromatic or non- aromatic hydrocarbon rings or heterocycloalkyl rings.

Heterocycle: As used herein, the terms “heterocycle” or “heterocyclic” refer to non- aromatic cyclic (i.e., ontaining) groups containing one or more heteroatoms (e.g., N, O, S, or the like) as part of the ring structure, and having in the range of 3 up to 14 carbon atoms. ituted heterocyclic” or “substituted heterocycle” refer to cyclic groups further bearing one or more substituents.

IC50: As used herein, the term “ICso” refers to a quantitative measure that indicates how much of a particular drug or other nce (inhibitor) is needed to inhibit a given biological process (e. g. sion of an mRNA) by half.

Intemucleotide linking group: As used herein, the term “intemucleotide linking group” or “intemucleotide linkage” refers to a chemical group e of covalently linking two nucleoside moieties. Typically, the chemical group is a phosphorus-containing linkage group containing a phospho or phosphite group. Phospho linking groups are meant to include a phosphodiester linkage, a phosphorodithioate linkage, a phosphorothioate linkage, a phosphotriester linkage, a thionoalkylphosphonate linkage, a thionalkylphosphotriester linkage, a phosphoramidite linkage, a phosphonate e and/or a boranophosphate linkage.

Many phosphorus-containing linkages are well known in the art, as disclosed, for example, in US. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 196, 5,188,897, 5,264,423, ,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 233, ,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,306, 5,550,111, 5,563,253, 5,571,799, ,587,361, 599, 5,565,555, 5,527,899, 5,721,218, 5,672,697 and 5,625,050. In other embodiments, the oligonucleotide ns one or more intemucleotide linking groups that do not contain a phosphorous atom, such short chain alkyl or cycloalkyl intemucleotide linkages, mixed heteroatom and alkyl or lkyl intemucleotide linkages, or one or more short chain heteroatomic or heterocyclic intemucleotide linkages, including, but not limited to, those having siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl nes, methylene formacetyl and thioformacetyl backbones, etyl backbones, alkene ning backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, and amide backbones.

Non-phosphorous containing linkages are well known in the art, as disclosed, for example, in US. Pat. Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 562, ,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, 5,541,307, 5,561,225, ,596,086, 240, 5,610,289, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, ,663,312, 5,633,360, 5,677,437, 5,792,608, 5,646,269 and 5,677,439.

Loop: As used herein, the term “loop” refers to a structure formed by a single strand of a nucleic acid, in which mentary regions that flank a particular single stranded nucleotide region hybridize in a way that the single stranded nucleotide region between the 2017/048239 complementary regions is excluded from duplex ion or Watson-Crick base pairing. A loop is a single stranded nucleotide region of any length. Examples of loops include the unpaired nucleotides present in such structures as hairpins and tetraloops.

MicroRNA: As used , the terms “microRNA” “mature microRNA” “miRNA” and “miR” are interchangeable and refer to non-coding RNA molecules encoded in the genomes of plants and animals. Typically, mature microRNA are about 18-25 nucleotides in length. In certain instances, highly conserved, endogenously expressed microRNAs regulate the expression of genes by binding to the 3’-untranslated regions (3’-UTR) of specific mRNAs.

Certain mature microRNAs appear to originate from long endogenous primary NA ripts (also known as pre-microRNAs, pri-microRNAs, pri-mirs, pri-miRs or pri-pre- microRNAs) that are often hundreds of nucleotides in length (Lee, et al., EMBO J., 2002, 21(17), 4663-4670).

Modified nucleoside: As used herein, the term “modified nucleoside” refers to a side ning one or more of a modified or universal nucleobase or a modified sugar.

The modified or universal nucleobases (also ed to herein as base analogs) are generally located at the 1’-position of a nucleoside sugar moiety and refer to nucleobases other than adenine, guanine, cytosine, thymine and uracil at the 1’-position. In certain ments, the modified or universal nucleobase is a nitrogenous base. In certain embodiments, the modified base does not n nitrogen atom. See e. g, U.S. Published Patent Application No. 20080274462. In certain embodiments, the modified nucleotide does not contain a nucleobase c). A modified sugar (also referred herein to a sugar ) includes modified deoxyribose or ribose moieties, e.g., where the modification occurs at the 2’-, 3’-, 4’-, or 5’- carbon on of the sugar. The modified sugar may also include non-natural alternative carbon structures such as those present in locked nucleic acids (“LNA”) (see, e.g., n et al. (1998), Tetrahedron, 54,3607-3630), bridged c acids (“BNA”) (see, e.g., US. Patent No. 7,427,672 and Mitsuoka et al. (2009), Nucleic Acids Res., 37(4):1225-38) and unlocked nucleic acids (“UNA”) (see, e.g., Snead et al. (2013), Molecular Therapy 4 Nucleic Acids, 2,e103(doi: 10.1038/mtna.2013.36)). Suitable modified or universal nucleobases or modified sugars in the context of the present disclosure are described herein.

Modified nucleotide: As used herein, the term “modified nucleotide” refers to a nucleotide containing one or more of a modified or universal base, a modified sugar, or a modified phosphate group. The modified or universal nucleobases (also referred to herein as base s) are generally located at the 1’-position of a nucleoside sugar moiety and refer to nucleobases other than adenine, guanine, cytosine, thymine and uracil at the 1’-position. In certain embodiments, the modified or universal nucleobase is a enous base. In certain embodiments, the modified nucleobase does not contain nitrogen atom. See e. g., U.S.

Published Patent Application No. 20080274462. In certain embodiments, the d nucleotide does not contain a nucleobase (abasic). A d sugar (also referred herein to a sugar analog) includes d deoxyribose or ribose moieties, e.g., where the modification occurs at the 2’-, 3’-, 4’-, or 5’-carbon on of the sugar. The d sugar may also include non-natural alternative carbon structures such as those present in locked c acids (“LNA”) (see, e.g., Koshkin et al. (1998), Tetrahedron, 54,3607-3630), bridged nucleic acids (“BNA”) (see, e.g., US. Patent No. 7,427,672 and Mitsuoka et al. (2009), Nucleic Acids Res., 37(4): 1225-38) and unlocked nucleic acids (“UNA”) (see, e.g., Snead et al. (2013), Molecular Therapy 4 Nucleic Acids, 2,e103(doi: 10.1038/mtna.2013.36)). Modified phosphate groups refer to a modification of the phosphate group that does not occur in natural nucleotides and includes turally occurring phosphate mimics as described herein, including phosphate mimics that include a phosphorous atom and anionic phosphate mimics that do not include phosphate (e.g. e). Modified phosphate groups also include non-naturally occurring cleotide linking groups, including both phosphorous-containing cleotide linking groups and non-phosphorous containing linking , as bed herein. Suitable modified or universal nucleobases, modified sugars, or modified phosphates in the context of the present disclosure are described herein.

Naked glutathione-sensitive oligonucleotide: As used herein, the term “naked glutathione-sensitive oligonucleotide” refers to a glutathione-sensitive oligonucleotide as described herein, which is not formulated in a tive lipid nanoparticle or other protective formulation and is thus exposed to the blood and endosomal/lysosomal compartments when administered in viva.

Natural nucleoside: As used herein, the term al nucleoside” refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar (e.g., deoxyribose or ribose or analog thereof). The natural heterocyclic nitrogenous bases include adenine, guanine, cytosine, uracil and thymine.

Natural nucleotide: As used herein, the term al nucleotide” refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar (e.g., ribose or deoxyribose or analog thereof) that is linked to a phosphate group. The natural heterocyclic nitrogenous bases include adenine, guanine, ne, uracil and thymine. 2017/048239 Nucleic acid tor molecule: As used , the term “nucleic acid tor molecule” refers to an oligonucleotide molecule that reduces or eliminates the expression of a target gene wherein the ucleotide molecule contains a region that specifically targets a sequence in the target gene mRNA. Typically, the targeting region of the nucleic acid inhibitor molecule comprises a sequence that is iently complementary to a sequence on the target gene mRNA to direct the effect of the nucleic acid inhibitor molecule to the specified target gene. The nucleic acid inhibitor molecule may include ribonucleotides, deoxyribonucleotides, and/or modified nucleotides.

Nucleoside: As used herein, the term oside” refers to a natural nucleotide or a modified nucleoside.

Nucleotide: As used herein, the term “nucleotide” refers to a natural nucleotide or a modified nucleotide.

Nucleotide position: As used herein, the term otide position” refers to a position of a nucleotide in an oligonucleotide as counted from the nucleotide at the 5’-terminus.

Oligonucleotide: As used herein, the term “oligonucleotide” as used herein refers to a polymeric form of nucleotides ranging from 2 to 2500 tides. Oligonucleotides may be single-stranded or double-stranded. In certain embodiments, the oligonucleotide has 500-1500 nucleotides, typically, for example, where the oligonucleotide is used in gene therapy. In n embodiments, the oligonucleotide is single or double stranded and has 7-100 nucleotides. In another embodiment, the oligonucleotide is single or double stranded and has -50 nucleotides, typically, for example, where the oligonucleotide is a nucleic acid inhibitor molecule. In yet another embodiment, the oligonucleotide is single or double stranded and has 19-40 or 19-25 nucleotides, typically, for example, where the oligonucleotide is a double- stranded nucleic acid inhibitor molecule and forms a duplex of at least 18-26 base pairs. In other embodiments, the oligonucleotide is single ed and has 15-25 nucleotides, typically, for example, where the oligonucleotide nucleotide is a single stranded RNAi tor molecule. Typically, the oligonucleotide contains one or more orous-containing intemucleotide linking groups, as described herein. In other embodiments, the intemucleotide linking group is a non-phosphorus containing linkage, as described herein. ng: As used herein, the term “overhang” refers to terminal non-base pairing nucleotide(s) at either end of either strand of a double-stranded nucleic acid inhibitor molecule.

In certain ments, the overhang results from one strand or region extending beyond the terminus of the complementary strand to which the first strand or region forms a duplex. One or both of two ucleotide regions that are capable of forming a duplex through hydrogen bonding of base pairs may have a 5’- and/or 3’-end that extends beyond the 3’- and/or 5’-end of complementarity shared by the two polynucleotides or regions. The single-stranded region extending beyond the 3’- and/or 5’-end of the duplex is ed to as an overhang.

Pharmaceutical ition: As used herein, the term “pharmaceutical composition” comprises a pharmacologically effective amount of an instant ibly-modified oligonucleotide or other bioactive agent and a pharmaceutically acceptable excipient. As used , “pharmacologically effective amount77 cctherapeutically effective amount” or “effective amount” refers to that amount of a ibly modified oligonucleotide ofthe present disclosure or other active agent effective to produce the intended pharmacological, therapeutic or preventive result.

Pharmaceutically acceptable excipient: As used herein, the term “pharmaceutically acceptable excipient” means that the excipient is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.

Phosphate mimic: As used herein, the term “phosphate mimic” refers to a al moiety that mimics the electrostatic and steric properties of a phosphate group. Typically, a ate analog is positioned at the 5’ terminal nucleotide of an oligonucleotide in place of a ’-phosphate, which is often susceptible to enzymatic removal. In some ments, these ’-phosphate mimics contain phosphatase-resistant linkages. Suitable phosphate mimics include 5’-phosphonates, such as 5’-methylenephosphonate ) and — vinylphosphonate (5’-VP) and 4’-phosphate analogs that are bound to the 4’-carbon ofthe sugar moiety (e.g., a ribose or deoxyribose or analog thereof) of the 5’-terminal nucleotide of an oligonucleotide, such as 4’-oxymethylphosphonate, 4’-thiomethylphosphonate, or 4’- ethylphosphonate, as described in US. Provisional Application No. 62/393,401, which is hereby incorporated by reference in its entirety. Other modifications have been developed for the 5’-end of oligonucleotides (see, e.g., US. Patent No. 8,927,513, Prakash et al. Nucleic Acids Res., 2015,43(6):2993-3011, ). oramidite: As used herein, the term “phosphoramidite” refers to a nitrogen containing a ent phosphorus derivative. Examples of suitable phosphoramidites are described herein. y: As used herein, “potency” refers to the amount of an oligonucleotide or other drug that must be administered in vivo or in vitro to obtain a particular level of activity against an intended target in cells. For example, an oligonucleotide that suppresses the expression of its target by 90% in a subject at a dosage of 1 mg/kg has a greater potency than an oligonucleotide that suppresses the expression of its target by 90% in a subject at a dosage of 100 mg/kg.

Protecting group: As used herein, the term “protecting group” is used in the conventional chemical sense as a group which reversibly renders unreactive a functional group under certain conditions of a desired reaction. After the desired on, protecting groups may be removed to deprotect the protected functional group. All protecting groups should be removable under conditions which do not degrade a substantial proportion of the molecules being synthesized.

Ribonucleotide: As used herein, the term ucleotide” refers to a natural or modified tide which has a hydroxyl group at the 2’-position of the sugar moiety.

Ribozyme: As used herein, the term “ribozyme” refers to a catalytic c acid molecule that specifically recognizes and cleaves a distinct target nucleic acid sequence, which can be either DNA or RNA. Each ribozyme has a catalytic component (also referred to as a “catalytic domain”) and a target sequence-binding component consisting of two binding s, one on either side of the tic domain.

RNAi inhibitor molecule: As used herein, the term “RNAi inhibitor molecule” refers to either (a) a double stranded nucleic acid inhibitor molecule (“dsRNAi inhibitor molecule”) having a sense strand (passenger) and antisense strand (guide), where the antisense strand or part of the antisense strand is used by the Argonaute 2 (Ago2) clease in the cleavage of a target mRNA or (b) a single stranded nucleic acid inhibitor molecule (“ssRNAi inhibitor le”) having a single antisense strand, where that antisense strand (or part of that antisense strand) is used by the Ago2 endonuclease in the cleavage of a target mRNA.

Sense strand: A double stranded RNAi inhibitor molecule comprises two ucleotide strands: an antisense strand and a sense strand. The sense strand or a region f is lly, substantially or fully complementary to the antisense strand of the double stranded RNAi inhibitor molecule or a region thereof. In certain embodiments, the sense strand may also contain nucleotides that are non-complementary to the antisense strand. The non- complementary nucleotides may be on either side of the complementary sequence or may be on both sides of the complementary sequence. In n embodiments, where the sense strand or a region f is partially or ntially complementary to the antisense strand or a region thereof, the non-complementary nucleotides may be located between one or more regions of complementarity (e.g., one or more mismatches). The sense strand is also called the passenger strand.

Solid support: As used herein, “solid support” refers to a non-liquid and non-gaseous substance to which chemical compounds such as oligonucleotides can attach. The term asses a variety of als including but not limited to gels, resins, beads, c, glass, n, metal and cellulose.

Spacer: As used herein, the term “spacer” refers to a molecule that couples a ligand to an oligonucleotide, nucleotide, or nucleoside. s include, but are not limited to, '(CH2)n' n=3 or 6), carbohydrates, , '(CH2)nN', 'CH2)nO', '(CH2)nS', O(CH2CH20)nCH2CH20H (e.g., a peptide, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, amide, morpholino or biotin.

Substituent or tuted: The terms “substituent” or ituted” as used herein refer to the replacement of hydrogen radicals in a given structure with the l of a substituent. When more than one position in any given structure may be substituted with more than one substituent, the substituent may be either the same or different at every position unless otherwise indicated. As used herein, the term “substituted” is contemplated to include all permissible substituents that are compatible with organic compounds. The permissible tuents include acyclic and cyclic, branched and unbranched, carbocyclic and cyclic, aromatic and nonaromatic substituents of c compounds. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds.

Sulfonyl group: As used herein, the term “sulfonyl group” refers to a al compound containing the bivalent group, —S02 —. In certain embodiments, the sulfur atom is covalently bound to two carbon atoms and two oxygen atoms. In other embodiments, the sulfur atom is covalently bound to a carbon atom, a nitrogen atom, and two oxygen atoms.

Systemic administration: As used herein, the term “systemic administration” refers to in viva systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body.

Target site: As used herein, the term “target sitea: Cctarget sequence, 77 cctarget nucleic acid”, “target region, 77 cctarget gene” are used interchangeably and refer to a RNA or DNA sequence that is “targeted,” e.g., for cleavage mediated by an RNAi molecule that contains a sequence within its guide/antisense region that is lly, substantially, or fully or sufficiently complementary to that target sequence.

Tetraloop: As used herein, the term “tetraloop” refers to a loop (a single stranded region) that forms a stable secondary structure that contributes to the stability of an adjacent -Crick hybridized nucleotides. Without being limited to theory, a tetraloop may stabilize an adjacent Watson-Crick base pair by stacking interactions. In on, interactions among the nucleotides in a tetraloop include but are not limited to non-Watson-Crick base pairing, stacking interactions, en bonding, and contact interactions (Cheong et al., Nature, 1990,346(6285):680-2, Heus and Pardi, Science, 1991,253(5016):191-4). A oop confers an se in the melting temperature (Tm) of an adjacent duplex that is higher than ed from a simple model loop sequence consisting of random bases. For example, a oop can confer a melting temperature of at least 50° C, at least 55° C., at least 56° C, at least 58° C, at least 60° C, at least 65° C or at least 75° C in 10 mM NaHPO4 to a hairpin comprising a duplex of at least 2 base pairs in . A tetraloop may contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. In certain embodiments, a tetraloop consists of four nucleotides. In certain embodiments, a tetraloop consists of five nucleotides.

Examples ofRNA tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop. (Woese et al., PNAS, 1990,87(21):8467-71, Antao et al., Nucleic Acids Res., 1991,19(21):5901-5). Examples of DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA), the d(GNRA)) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family oftetraloops, and the d(TNCG) family of tetraloops (e.g., d(TTCG)). (Nakano et al. mistry, 2002,41(48):14281-14292. Shinji et al., Nippon ai Koen Yokoshu, 8(2):73l).

I. Introduction This application provides various new glutathione-sensitive nucleotides and nucleosides that can be incorporated into any oligonucleotide of interest, including, but not limited to, nucleic acid inhibitor molecules, such as dsRNAi, antisense, miRNA, and ssRNAi , as well as methods of using the hione-sensitive nucleic acid inhibitor molecules to modulate the expression of target genes and to treat patients in need thereof Other oligonucleotides that can be reversibly ed with one or more glutathione-sensitive moieties in accordance with the disclosure of this application include, but are not limited to, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) c acids, nucleic acids for gene therapy, nucleic acids for DNA editing, and probes.

The reversibly modified oligonucleotide comprises one or more nucleotides having a glutathione-sensitive moiety, typically at the 2’-carbon of a sugar moiety. The one or more glutathione-sensitive nucleotides in the oligonucleotide help to ize the oligonucleotide during transit through the blood and the lysosomal/endosomal compartments of a cell and protect the oligonucleotide from nucleases and other environmental conditions (e.g., pH) encountered during in viva administration. Unlike irreversible approaches used to protect therapeutic oligonucleotides, the reversible, glutathione-sensitive modifications disclosed herein are removed from the oligonucleotide when it reaches the reducing environment of the cytosol of the cell. In n embodiments, ng the glutathione-sensitive moiety at the 2’-carbon leaves a hydroxyl group at the 2’-carbon, which is the natural substituent for a ribonucleotide at that position. As a result, when they reach the cytosol of the cell, the reversibly modified, glutathione-sensitive ucleotides can carry out their intended biological activity without any interference from the reversible, glutathione-sensitive moiety, which is removed in the cytosol. The reversible, glutathione-sensitive ations disclosed in this ation represent a powerful new tool for tic oligonucleotides that can used in place of or in combination with irreversible ations to generate stable oligonucleotides having enhanced biological activity within the cytosol of a cell.

Moreover, the glutathione-sensitive nucleoside oramidites disclosed herein are compatible with conventional solid-phase synthesis. Thus, the present reversibly modified, glutathione-sensitive oligonucleotides can be synthesized using conventional phosphoramidite based synthetic methods. Using this synthetic approach, one can select the nucleotide position at which the glutathione-sensitive nucleotides is incorporated into the oligonucleotide. As it is may be ble to modify specific nucleotide ons in an oligonucleotide, this discovery facilitates the rational design of oligonucleotides having reversible modifications at c nucleotide positions of interest.

This application also provides glutathione-sensitive nucleotides and sides that can be used as therapeutic agents (e.g., antiviral or anticancer agents). 11. Glutathione—Sensitive Oligonucleotides One aspect of the present disclosure relates to an oligonucleotide comprising at least one glutathione-sensitive moiety. Typically, the glutathione-sensitive moiety is attached to the sugar moiety of the nucleotide, e.g. a deoxyribose or ribose (or analogs thereof). Typically, the glutathione-sensitive moiety is located at the 2’-carbon of a deoxyribose or ribose (or analogs thereof). In some embodiments, the glutathione-sensitive moiety is located at the 5’- carbon of a ribose or deoxyribose (or analogs thereof), particularly when the modified nucleotide is the 5’-terminal nucleotide of the oligonucleotide. In other embodiments, the glutathione-sensitive moiety is located at the 3’-carbon of a ribose or deoxyribose (or s thereof), particularly when the modified nucleotide is the 3’-terminal nucleotide of the oligonucleotide.

In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group. In other embodiments, the glutathione-sensitive moiety comprises a disulfide bridge.

In certain ments, the oligonucleotide ses at least one nucleotide having a glutathione-sensitive moiety ntly bound to an oxygen atom that is covalently bound to the 2’-carbon of the sugar moiety (e.g., ribose) of the nucleotide. In n embodiments, the glutathione-sensitive moiety is represented by Formula II, III, or IV. In certain embodiments, the glutathione-sensitive moiety is represented by Formula IIa, IIIa, IIIb, ), ), IVa, IVb, IVc, IVd, IVe, IVa(i), IVb(i), IVb(ii), IVc(i), IVd(i), IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(v), IVe(vi), i), IVe(viii), IVe(iX), IVe(X), or ). 1. Formulal In one embodiment, the glutathione-sensitive oligonucleotide comprises at least one nucleotide ented by Formula I: A—U1—1 B R4 R1 R3 R2 /U2 X\L wherein X is O, S, Se or NR’, wherein R’ is selected from hydrogen, halogen, a substituted or unsubstituted aliphatic, an aryl, a substituted or unsubstituted heteroaryl or a substituted or unsubstituted heterocycle, wherein R1, R2, R3 and R4 are each independently selected from en, halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl or wherein two of R1, R2, R3 and R4 are taken together to form a -8 membered ring, wherein the ring optionally contains a heteroatom, 2017/048239 wherein J is O, S, NR’, CR’R”, wherein each of R’ and R” is independently ed from hydrogen, halogen, a tuted or tituted aliphatic, aryl or heteroaryl, wherein B is selected from hydrogen, a natural nucleobase, a modified nucleobase or a universal nucleobase, wherein U2 is absent or selected from O, S, NR’, or CR’R”, wherein R’ and R” are each independently hydrogen, a substituted or unsubstituted aliphatic, a substituted or unsubstituted aryl, a substituted or unsubstituted aryl, a tuted or unsubstituted heterocycle or a substituted or unsubstituted cycloalkyl, wherein W is hydrogen, a phosphate group, an internucleotide linking group attaching the at least one nucleotide represented by Formula I to a tide or an oligonucleotide, a halogen, OR’, SR’, NR’R”, a substituted or unsubstituted aliphatic, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycle, wherein R’ and R” are each independently selected from hydrogen, n, a substituted or unsubstituted aliphatic, an aryl, a heteroaryl, a heterocycle or are taken together to form a heterocyclic ring, wherein I is absent or is selected from O, S, NR’, CR’R”, wherein R’ and R” are each independently hydrogen, a substituted or unsubstituted aliphatic, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or tituted heterocycle and a substituted or unsubstituted cycloalkyl, wherein U1 is absent, hydrogen, an internucleotide linking group attaching the at least one nucleotide represented by FormulaI to a nucleotide or an oligonucleotide, or selected from O, S, NR’, or CR’R”, wherein R’ and R” are each independently hydrogen, a substituted or unsubstituted aliphatic, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycle and a substituted or tituted cycloalkyl and wherein at least one of U1 or W is an internucleotide linking group attaching the at least one nucleotide represented by aI to a nucleotide or an oligonucleotide and provided that if U1 is an ucleotide linking group, A is absent, wherein I and U1 can be combined to form CR’-CR” alkyl, CR’-CR” alkenyl, CR’-CR” l, a tuted or unsubstituted aliphatic, an aryl, a heteroaryl a heterocycle or taken together to form cycloalkyl or heterocyclic ring, wherein A is absent, a hydrogen, a phosphate group, a phosphate mimic or a phosphoramidate, and 2017/048239 wherein L is a glutathione-sensitive moiety selected from a II, III, or IV, as described below.

In certain embodiments, X is O. [0155l In certain embodiments, R1, R2, R3 and R4 are en. [0156l In certain embodiments, J is O.

In certain embodiments, B is a natural nucleobase. [0158l In certain embodiments, U2 is absent or O.

In certain embodiments, W is hydrogen, a phosphate group, an intemucleotide linking group attaching the at least one nucleotide represented by Formula I to a nucleotide or an oligonucleotide. Typically, W is an cleotide linking group attaching the at least one tide represented by Formula I to a nucleotide or an oligonucleotide.

In certain embodiments, I is CH2.

In certain embodiments, U1 is hydrogen or an intemucleotide linking group attaching the at least one nucleotide represented by Formula I to a nucleotide or an oligonucleotide.

Typically, U1 is an intemucleotide linking group ing the at least one nucleotide represented by Formula I to a nucleotide or an oligonucleotide and A is absent.

In certain embodiments, A is , a phosphate group or a phosphate mimic. In some embodiments, the phosphate group is a monophosphate, a diphosphate or a triphosphate. In some embodiments, the phosphate mimic is Vinylphosphonate, hylenephosphonate, or a 4’-oxymethylphosphonate.

In certain embodiments, A is hydrogen and U1 is O.

In certain embodiments, X is 0, R1, R2, R3 and R4 are hydrogen, and J is O. In certain embodiments, X is 0, R1, R2, R3 and R4 are hydrogen, J is O, B is a natural nucleobase, U2 is absent or O, A is absent, I is CH2, W is hydrogen, a phosphate group, or an intemucleotide linking group attaching the at least one nucleotide represented by Formula I to a nucleotide or an oligonucleotide, and U1 is hydrogen or an cleotide g group attaching the at least one nucleotide represented by Formula I to a nucleotide or an oligonucleotide, wherein at least one of U1 or W is an intemucleotide linking group attaching the at least one nucleotide represented by Formula I to an oligonucleotide.

In certain embodiments, the oligonucleotide containing at least one nucleotide of Formula I has 2-2500 nucleotides. In certain ments, the oligonucleotide of Formula I has 500-1500 tides. In certain embodiments, the ucleotide containing at least one nucleotide of Formula I has 7-100 nucleotides. In another ment, the oligonucleotide containing at least one nucleotide of Formula I has 15-50 nucleotides. In yet another embodiment, the oligonucleotide containing at least one tide of Formula I has 19-25 nucleotides.

In certain ments, the oligonucleotide containing at least one nucleotide of Formula I is a c acid inhibitor le, as discussed in further detail throughout the ation. In other embodiments, the oligonucleotide containing at least one nucleotide of Formula I is a CRISPR nucleic acid, a nucleic acid for gene therapy, a nucleic acid for DNA editing, a probe, or any other oligonucleotide that is susceptible to degradation by nucleases and/or harsh environmental conditions (e.g., pH), ing other oligonucleotides that are to be administered in viva.

In certain embodiments, the ring structure of the sugar moiety of at least one nucleotide in the nucleic acid inhibitor molecule is modified and encompasses, for example, Locked Nucleic Acid ) structures, Bridged Nucleic Acid (“BNA”) structures, and Unlocked Nucleic Acid (“UNA”) structures, as discussed previously. a. Glutathione—sensitive moiety - Formula II As discussed above, the glutathione-sensitive oligonucleotides comprise at least one tide represented by Formula I, where the hione-sensitive moiety is selected from Formula II, III, or IV. In some embodiments, the glutathione-sensitive moiety is represented by Formula II as follows: wherein Y is O, S, Se, or NR’, wherein R’ is ed from en, halogen, a substituted or unsubstituted aliphatic, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocycle, wherein Z is selected from O, S, NR’, or CR’R”, wherein R’ and R” are each independently selected from hydrogen, halogen, CH3, substituted or unsubstituted aliphatic, substituted or tituted aryl, substituted or unsubstituted aryl, tuted or unsubstituted heterocycle, or R’ and R” are taken together to form a heterocyclic ring, n V is C or SO, wherein X2 and X3 are independently selected from hydrogen, halogen, nitro, amino, acyl, substituted or unsubstituted aliphatic, ORIO, CORio, CO2R10, NQ1Q2, wherein R10 is independently hydrogen, substituted or unsubstituted aliphatic, hydroxyl or alkoxy substituted aliphatic, arylaliphatic, hydroxyl or an alkoxy substituted aryl or an alkoxy substituted heterocyclic, wherein P and Q are taken together to form a disulfide bridge or a sulfonyl group, and wherein T is a substituted or unsubstituted aliphatic, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl or T is a ligand optionally connected via a spacer to P or Q.

In certain embodiments, said disulfide bridge or said sulfonyl group is ble in the cytosol by glutathione at a pH of at least about 7.5.

In some embodiments, Y is O, S or NH. Typically, Y is O.

In some embodiments, Z is O, S or NR’. Typically, Z is NH. [017 8] In some embodiments, V is SO, Typically, V is C.

In some embodiments, X2 and X3 are independently selected from hydrogen, halogen, nitro, amino or acyl or C3 to C6 branched or unbranched alkyl. Typically, X2 and X3 are independently selected from hydrogen, halogen, nitro or amino.

Typically, P and Q are taken together to form a disulfide bond.

In some ments, T is a C3 to C6 branched or unbranched alkyl or T is a ligand optionally connected via a spacer to P or Q. Typically, T is a C4 branched alkyl.

In certain embodiments, the oligonucleotide having a glutathione-sensitive moiety of a II has 2-2500 nucleotides. In certain embodiments, the oligonucleotide having a glutathione-sensitive moiety of Formula II has 500-1500 nucleotides. In certain embodiments, the ucleotide having a hione-sensitive moiety of Formula II has 7-100 nucleotides.

In another ment, the oligonucleotide having a glutathione-sensitive moiety of Formula II has 15-50 nucleotides. In yet r embodiment, the oligonucleotide having a glutathione- sensitive moiety of Formula II has 19-25 nucleotides.

In certain embodiments, the oligonucleotide having a glutathione-sensitive moiety of Formula II is a nucleic acid inhibitor molecule, as discussed in r detail throughout the application. In other embodiments, the oligonucleotide having a glutathione-sensitive moiety ofFormula II is a CRISPR c acid, a nucleic acid for gene therapy, a nucleic acid for DNA editing, a probe, or any other ucleotide that is susceptible to degradation by nucleases and/or harsh environmental conditions (e.g., pH), including other oligonucleotides that are to be administered in vivo. i. Formula Ho In some embodiments, the glutathione-sensitive moiety is represented by the following Formula: S/S7< 1121 b. Glutathione—sensitive moiety - Formula III In other embodiments of the glutathione-sensitive oligonucleotides comprising at least one nucleotide represented by Formula I, the glutathione-sensitive moiety is represented by Formula 111 as follows: wherein Y is O, S, Se, or NR’, wherein R’ is selected from hydrogen, n, a substituted or unsubstituted tic, a tuted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted heterocycle, wherein Zl is N or CR’, wherein R’ is selected from hydrogen, halogen, substituted or unsubstituted aliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, wherein V is C or SO, wherein M1 and M2 are each independently ed from tuted or unsubstituted aliphatic, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, wherein Pi and Qi are taken together to form a disulfide bridge or a sulfonyl group or wherein Pi and Qi are each independently a disulfide bridge or a yl group; wherein when Pi and Qi form a disulfide bridge, M1, M2, Pi, and Qi can form a 4-9 membered ring, n the ring can be tuted or unsubstituted aromatic, substituted or unsubstituted cycloalkyl, wherein the aromatic or cycloalkyl ring can optionally contain a heteroatom, and wherein Ta and Th are each independently absent or selected from CH3, substituted or substituted aliphatic, substituted or unsubstituted aryl, tuted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle or a ligand optionally connected Via any spacer to P1 or Q1.

In some embodiments, M1, M2, Pi, and Qi are taken together to form a 5-8 membered ring containing alkoxy substituted arylaliphatic, alkoxy substituted heteroaryl or alkoxy substituted heterocyclic.

In some ments, Y is O, S or NH. Typically, Y is O.

In some embodiments, 21 is N or CH. lly, 21 is N.

In some ments, V is SO. lly, V is C.

In some embodiments, M1 and M2 are each independently selected from substituted or unsubstituted aliphatic, or M1, M2, Pi, and Qi are taken together to form a 4-9 membered ring, wherein the ring is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycle. Typically, M1 and M2 are substituted or unsubstituted C2 to C6 alkyl or are taken together with P1 and Qi to form a 5-8 membered ring, wherein the ring is substituted or unsubstituted cycloalkyl.

In some ments, Pi and Qi are taken together to form a disulfide bridge or a sulfonyl group. Typically, Pi and Qi are taken together to form a disulfide bridge. In some embodiments, the de bridge or the sulfonyl group is cleavable in the cytosol by glutathione at a pH of at least about 7.5.

In some embodiments, Ta and Th are absent or are each independently absent or selected from CH3, a branched or unbranched C3 to C6 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle or a ligand optionally connected Via any spacer to P1 or Q1. Typically, Ta and Th are absent, a branched C3 to C6 alkyl, or a ligand connected Via any spacer to P1 or Q1.

In some embodiments, the glutathione-sensitive moiety is represented by Formula 111, wherein Y is O, S or NH, Z1 is N, V is C, M1 and M2 are each independently selected from substituted or unsubstituted tic, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaryl, or M1, M2, P1, and Q1 are taken together to form a 4-9 membered ring, n the ring is substituted or unsubstituted aromatic, substituted or unsubstituted cycloalkyl, substituted or tituted heterocycle or substituted or unsubstituted heteroaryl, P1 and Q1 are taken together to form a disulfide bridge, and Ta and Th are absent or a ligand optionally connected Via any spacer to P1 or Q1.

Typically, the glutathione-sensitive moiety is represented by Formula 111, wherein Y is O, S or NH, Z1 is N, V is C, M1, M2, P1, and Q1 are taken together to form a 5-8 membered ring, wherein the ring is a substituted or unsubstituted aromatic ring or a substituted or unsubstituted cycloalkyl, wherein the ic ring or cycloalkyl can optionally contain any heteroatom, P1 and Q1 are taken together to form a disulfide bridge, and Ta and Tb are absent or a ligand ally connected Via any spacer to P1 or Q1.

More typically, the glutathione-sensitive moiety is represented by Formula 111, wherein Y is O, S or NH, Z1 is N, V is C, M1, M2, P1, and Q1 are taken together to form a 7 membered ring, wherein the ring is a substituted or unsubstituted cycloalkyl, P1 and Q1 are taken together to form a disulfide bridge, and Ta and Tb are .

In some embodiments, the glutathione-sensitive moiety is represented by Formula 111, wherein Y is O, S or NH, Z1 is N, V is C, M1 and M2 are each independently selected from substituted or unsubstituted aliphatic, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaryl, P1 and Q1 are each independently a disulfide , and Ta and Th are selected from CH3, substituted or substituted aliphatic, substituted or unsubstituted aryl, substituted or tituted aryl, substituted or unsubstituted heterocycle or a ligand optionally ted Via any spacer to P1 or Q1.

More typically, the glutathione-sensitive moiety is represented by Formula 111, wherein Y is O, S or NH, Z1 is N, V is C, M1 and M2 are substituted or unsubstituted tic, P1 and Q1 are each independently a disulfide bridge, and Ta and Th are each independently ed from CH3, substituted or tuted aliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle or a ligand optionally connected Via any spacer to P1 or Q1.

Yet even more typically, the glutathione-sensitive moiety is represented by Formula 111, wherein Y is O; S or NH; Z1 is N; V is C; M1 and M2 are substituted or unsubstituted tic; Pi and Q1 are each independently a de bridge; and Ta and Th are each independently absent or selected from CH3; a branched or unbranched C3 to C6 alkyl; or a ligand optionally connected via any spacer to P1 or Q1.

In certain embodiments; the oligonucleotide having a glutathione-sensitive moiety of Formula III has 2-2500 nucleotides. In certain ments; the oligonucleotide having a glutathione-sensitive moiety ofFormula III has 500-1500 nucleotides. In certain ments; the oligonucleotide having a glutathione-sensitive moiety ofFormula III has 7-100 tides.

In another embodiment; the oligonucleotide having a hione-sensitive moiety of Formula III has 15-50 nucleotides. In yet another embodiment; the oligonucleotide having a glutathione-sensitive moiety of Formula III has 19-25 nucleotides.

In certain embodiments; the oligonucleotide having a glutathione-sensitive moiety of Formula III is a nucleic acid inhibitor molecule; as sed in further detail throughout the application. In other embodiments; the oligonucleotide having a glutathione-sensitive moiety of a III is a CRISPR nucleic acid; a nucleic acid for gene therapy; a nucleic acid for DNA editing; a probe; or any other oligonucleotide that is susceptible to degradation by nucleases and/or harsh environmental conditions (e.g.; pH); including other oligonucleotides that are to be administered in viva. i. Formula 111a In some embodiments; the glutathione-sensitive moiety is represented by the following Formula: Fig/v (S)S 11121 wherein Y is O, S or NH, Z1 is N or CR’, wherein R’ is selected from hydrogen, n, substituted or unsubstituted aliphatic, substituted or unsubstituted aryl, substituted or tituted heteroaryl, tuted or unsubstituted heterocycle.

More typically, the glutathione-sensitive moiety is represented by the ing ii. Formula 111b In some embodiments, the glutathione-sensitive moiety is represented by the following Formula: /S S T/ S\ IIIb wherein Y is O, S or NH, Z1 is N or CR’, wherein R’ is selected from hydrogen, halogen, substituted or unsubstituted aliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, and Ta and Tb are each independently absent or selected from CH3, substituted or substituted aliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle or a ligand optionally connected Via any spacer to a sulfur atom.

More typically, the glutathione-sensitive moiety is represented by the ing Formula: ,y" o / S\ 7 ‘ S c. Glutathione—sensitive moiety - Formula IV In yet other ments of the glutathione-sensitive oligonucleotides comprising at least one tide represented by Formula I, the glutathione-sensitive moiety is represented by Formula IV as follows: givéY Q P\/ \T 2' E \ / ; E in \M4 wherein Y is O, S, Se, or NR’, n R’ is selected from hydrogen, halogen, a substituted or unsubstituted aliphatic, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocycle wherein Z is selected from O, S, NR’, or CR’R”, wherein R’ and R” are each ndently selected from hydrogen, halogen, CH3, substituted or unsubstituted aliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, or R’ and R” are taken together to form a cyclic ring, wherein V is C or SO, wherein G and E can be each independently absent, or selected from CH2, CHR’, CR’R”, NH, or NR’, wherein R’ and R” are each independently selected from hydrogen, halogen, a substituted or unsubstituted aliphatic, a substituted or tituted aryl, a substituted or unsubstituted aryl, a substituted or unsubstituted heterocycle or R’ and R” are taken together to form a heterocyclic ring, wherein M3 and M4 can be taken to form a 4-9 membered ring, wherein the ring can be tuted or unsubstituted ic, substituted or unsubstituted cycloalkyl, wherein the aromatic or cycloalkyl ring can optionally contain a heteroatom, or M3 and M4 are each independently selected from hydrogen, substituted or unsubstituted aliphatic, substituted or unsubstituted aromatic, substituted or tituted heteroaryl, substituted or tituted cycloalkyl or COOR, wherein R is selected from hydrogen, CH3, or substituted or unsubstituted aliphatic, wherein K is C, CH, or a tuted or unsubstituted aliphatic, wherein n is 0 — 5, wherein P and Q are taken together to form a disulfide bridge or a sulfonyl group, and wherein T is substituted or unsubstituted aliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or T can be a ligand optionally connected Via any spacer to P or Q.

In some embodiments, Y is O, S or NH. Typically, Y is O.

In some embodiments, Z is O, S, NH, NR’, or CR’R”, wherein R’ and R” are each independently selected from en, CH3, or tuted or unsubstituted aliphatic. In certain embodiments, Z is NH or N-CH3 In some embodiments, V is SO. Typically, V is C.

In some embodiments, M3 and M4 are each independently selected from hydrogen or substituted or unsubstituted aliphatic, such as a C2 to C6 alkyl, or M3 and M4 are taken together to form a 4-9 membered ring, wherein the ring is substituted or unsubstituted cycloalkyl or tuted or unsubstituted heterocycle. Typically, M3 and M4 are independently substituted or tituted C2 to C6 alkyl or taken er to form a 5-8 membered ring, wherein the ring is substituted or unsubstituted cycloalkyl.

In some embodiments, G is , CH2, or CHR’, wherein R’ is substituted or unsubstituted aliphatic. Typically, G is absent or CH2.

In some embodiments, E is absent, NH, NR’, CH2, or CHR’, wherein R’ is substituted or unsubstituted aliphatic. Typically, E is absent, NH, or CH2.

In some embodiments, G and E are absent.

In some embodiments, K is C, or CH. Typically, K is CH.

WO 39364 In some embodiments, n is 0.

In some embodiments, P and Q are taken together to form a disulfide bridge or a sulfonyl group. Typically, P and Q are taken together to form a disulfide bridge. In some embodiments, the disulfide bridge or the sulfonyl group is cleavable in the cytosol by glutathione at a pH of at least about 7.5.

In some embodiments, T is a substituted or unsubstituted C2-C6 alkyl, tuted or unsubstituted aryl, substituted or unsubstituted heteroaryl or T is a ligand optionally connected to P or Q via any spacer. Typically, T is a substituted or unsubstituted C2-C6 alkyl or a ligand optionally connected to P or Q via any .

In some embodiments, the glutathione-sensitive moiety is represented by Formula IV, wherein Y is O, S, NH, wherein Z is O, S NH, or NCH3, V is C, G is CH2 and E is absent or G is absent and E is CH2, M3 and M4 are taken together to form a 5-8 membered ring, wherein the ring is a cycloalkyl substituted with a heteroatom or an tituted cycloalkyl or M3 and M4 are each ndently a substituted or unsubstituted C2 to C6 alkyl, K is CH, 11 is 0, P and Q are taken together to form a disulfide bridge, T is CH3, substituted or unsubstituted C2 to C6 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or T is a ligand ally connected via any spacer to P or Q.

In certain embodiments, the oligonucleotide having a hione-sensitive moiety of Formula IV has 2-2500 nucleotides. In certain embodiments, the oligonucleotide having a glutathione-sensitive moiety ofFormula IV has 500-1500 nucleotides. In certain embodiments, the oligonucleotide having a glutathione-sensitive moiety ula IV has 7-100 nucleotides.

In another embodiment, the oligonucleotide having a glutathione-sensitive moiety of Formula IV has 15-50 nucleotides. In yet another embodiment, the oligonucleotide having a glutathione-sensitive moiety of Formula IV has 19-25 nucleotides.

In certain embodiments, the oligonucleotide having a glutathione-sensitive moiety of Formula IV is a nucleic acid inhibitor molecule, as sed in further detail throughout the application. In other embodiments, the oligonucleotide having a glutathione-sensitive moiety of Formula IV is a CRISPR nucleic acid, a c acid for gene therapy, a nucleic acid for DNA editing, a probe, or any other oligonucleotide that is tible to degradation by nucleases and/or harsh environmental conditions (e.g., pH), including other oligonucleotides that are to be administered in vivo. i. Formula IVa In some embodiments, the glutathione-sensitive moiety may be represented by the ing Formula: T_ _S S R5 R7 R6 wherein Y is O, S, NH, wherein Z is O, S or NH, wherein R5, R6, and R7 are each independently ed from OAcyl, NHR’, NR’, or CR’R”, n R’ and R” are each independently selected from en, halogen, substituted aliphatic or unsubstituted aliphatic, aryl, heteroaryl, heterocyclic, or can be taken together to form a heterocyclic ring, and, wherein T is a branched or unbranched C2-C6 alkyl or a ligand optionally connected Via any spacer to a sulfur atom.

For example, the glutathione-sensitive moiety may be represented by the following Formula: H3CH2C— s— s 0 IVa(i) ii. Formula IVb In some embodiments, the glutathione-sensitive moiety may be represented by the following Formula: gm 0 V || \\ K—E—S—T Y || M4 0 wherein Y is O; 8, NH; Z is O; S or NH; V is C; M3 and M4 are hydrogen; K is CH or a substituted or unsubstituted aliphatic; E is NH or NR’; wherein R’ is tuted or unsubstituted tic; n is 0-5; T is substituted or unsubstituted C2 to C6 alkyl; substituted or tituted aryl; substituted or unsubstituted heteroaryl or T is a ligand optionally connected Via any spacer.

In some ments; the glutathione-sensitive moiety is represented by Formula IVb; n Y is 0; Z is NH; V is C; M3 and M4 are hydrogen; K is CH; E is NH; 11 is l; P and Q are taken together to form a sulfonyl group; and T is substituted aryl.

For example; the glutathione-sensitive moiety may be ented by the following Formula: H H NWN\S//O R O 0/ IVb(i) wherein R is selected from hydrogen; CH3; N02; substituted or unsubstituted aliphatic; aryl; heteroaryl; cycloalkyl or a heterocycle or R is a targeting ligand optionally connected Via any spacer.

In some embodiments; the glutathione-sensitive moiety is represented by Formula IVb; wherein Y is 0; Z is NH; V is C; M3 and M4 are hydrogen; K is CH; E is NH; 11 is 0; P and Q are taken together to form a sulfonyl group; and T is substituted aryl.

For example; the glutathione-sensitive moiety may be represented by the following Formula: 2017/048239 \/\NH R \ // IVb(ii) n R is selected from hydrogen, CH3, N02, substituted or tituted aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected Via any spacer. In certain embodiments, R is hydrogen. iii. a IVc In some embodiments, the hione-sensitive moiety is represented by Formula IV, wherein Y is O, S, NH, Z is ed from O, S, or NR’, wherein R’ is selected from hydrogen, halogen, CH3, substituted or unsubstituted aliphatic, substituted or tituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, G and E are absent, V is C, M3 and M4 are taken together to form a 5-8 membered ring, wherein the ring is a substituted or unsubstituted cycloalkyl, optionally substituted with a heteroatom, K is CH, 11 is 0-5, P and Q are taken together to form a disulfide bridge, T is substituted or unsubstituted C2 to C6 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or T is a ligand optionally connected Via any spacer. Typically, Y is O and Z is NH or NCH3.

In some embodiments, the glutathione-sensitive moiety is represented by the following Formula: wherein Y is O, S, NH, Z is selected from O, S, or NR’, wherein R’ is selected from hydrogen, halogen, CH3, substituted or unsubstituted aliphatic, substituted or unsubstituted WO 39364 aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, V is C, M3 and M4 are taken er to form a 5-8 ed ring, wherein the ring is a substituted or unsubstituted cycloalkyl, optionally substituted with a heteroatom, K is a branched or unbranched tuted or unsubstituted C2 to C6 alkyl, 11 is 0 or 1, T is substituted or unsubstituted C2 to C6 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or T is a ligand optionally connected Via any spacer; wherein R is selected from hydrogen, CH3, substituted or unsubstituted aliphatic, substituted or unsubstituted aryl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl or a substituted or unsubstituted heterocycle or R is a targeting ligand optionally connected Via any spacer.

Typically, Y is O and Z is NH or NCH3.

For example, the glutathione-sensitive moiety may be represented by the following Formula: IVc(i) wherein R is ed from hydrogen, CH3, substituted or unsubstituted aliphatic, substituted or unsubstituted aryl, substituted or tituted heteroaryl, substituted or unsubstituted cycloalkyl or a substituted or unsubstituted heterocycle or R is a ing ligand optionally connected Via any spacer. iv. Formula IVd In some embodiments, the glutathione-sensitive moiety is represented by Formula IV, n Y is O, S, NH, wherein Z is selected from O, S, or NR’, wherein R’ is selected from hydrogen, halogen, CH3, substituted or unsubstituted aliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, tuted or unsubstituted heterocycle, V is C, G and E are absent, M3 is COOR, wherein R is selected from hydrogen, CH3 or a substituted or unsubstituted C2 to C6 alkyl, M4 is hydrogen, K is CH, 11 is 0, P and Q are taken together to form a de bridge, T is substituted or unsubstituted C2 to C6 alkyl, substituted or tituted aryl, substituted or unsubstituted heteroaryl or T is a ligand ally ted Via any spacer to P or Q. Typically, Y is O and Z is NH or NCH3.

In one embodiment, the glutathione-sensitive moiety is represented by the following Formula: ZY\S/ \TS COOR wherein Y is O, S, NH, Z is O, S, NH, or NCH3, T is substituted or unsubstituted C2 to C6 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or T is a ligand optionally connected Via any spacer, and R is selected from hydrogen, CH3 or a substituted or tituted C2 to C6 alkyl.

For example, the glutathione-sensitive moiety may be represented by the following Formula: HNW/\S/ S COZCHZCH3 IVd(i) v. a IVe In some embodiments, the glutathione-sensitive moiety is represented by Formula IV, wherein Y is O, S, NH, Z is selected from O, S, or NR’, wherein R’ is selected from hydrogen, halogen, CH3, substituted or unsubstituted aliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, V is C or SO, 11 is 0, M3 and M4 are taken er to form a 4-9 membered ring, n the ring is a substituted or unsubstituted aryl, K is C, CH, N, NH, or a branched or unbranched substituted or unsubstituted C2 to C6 alkyl, T is substituted or unsubstituted C2 to C6 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or T is a ligand optionally connected Via any spacer. Typically, Y is O and Z is NH or NCH3.

In some embodiments, the glutathione-sensitive moiety is represented by the following Formula: | E Z\ K/ G )n wherein Y is O, S, NH, Z is selected from O, S, or NR’, wherein R’ is selected from hydrogen, halogen, CH3, substituted or unsubstituted aliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, V is C or SO, G and E can be each independently absent, or selected from CH2, CHR’, CR’R”, NH, NR’, wherein R’ and R” are each ndently selected from hydrogen, halogen, a tuted or unsubstituted aliphatic, a substituted or unsubstituted aryl, a substituted or tituted heteroaryl, a substituted or unsubstituted heterocycle or R’ and R” are taken together to form a heterocyclic ring, K is C or CH, 11 is 0-5, T is substituted or unsubstituted C2 to C6 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aryl or T is a ligand optionally connected Via any spacer, and n R is selected from hydrogen, CH3, substituted or unsubstituted aliphatic, substituted or unsubstituted aryl, substituted or tituted heteroaryl, substituted or tituted cycloalkyl or a substituted or unsubstituted heterocycle or R is a targeting ligand optionally connected Via any spacer.

In certain embodiments, Z is NR’, wherein R’ is hydrogen, CH3, or substituted or unsubstituted aliphatic. Typically, Z is NH or NCH3. In n embodiments, Z is S.

In certain embodiments, Y is O, S, or NH and V is C. In one embodiment, V is SO and Y is O.

In certain embodiments, one or both of G and E are absent, CH2, or CR’R”, wherein R’ and R” are ndently selected from hydrogen or substituted or unsubstituted aliphatic. In n embodiments, G and E are both absent or G is CH2 and E is absent or G is absent and E is CH2 or branched alkyl.

For example, the glutathione-sensitive moiety may be represented by one of the following Formulas: 2017/048239 IVe(i) IVe(ii) :5 5—3 IVe(iii) 2017/048239 IVe(vi) WO 39364 IVe(vii) IVe(ix) fYO J3 R ; or IVe(x) Y0 8—3 IVe(xi) wherein R is selected from hydrogen, CH3, substituted or unsubstituted aliphatic, substituted or tituted aryl, substituted or unsubstituted heteroaryl, substituted or tituted cycloalkyl or a substituted or unsubstituted heterocycle or R is a targeting ligand ally connected Via any spacer. 2. Formula V (Oligonucleotide with Formula II Glutathione—Sensitive Moietjy) In other embodiments, the glutathione-sensitive oligonucleotide comprises at least one nucleotide represented by Formula V as follows: / / W Y 2 x3 X2 P/ \T wherein A, U1, 1, B, W, and U2 are as described in Formula I, and wherein Y, V, Z, X2, X3, P, Q and T are as described in Formula II.

In certain embodiments, U1 is absent, an oxygen, or an intemucleotide linking group attaching the at least one nucleotide represented by Formula V to a nucleotide or an oligonucleotide, or hydrogen, U2 is absent or O, and W is hydrogen, a phosphate group, or an intemucleotide g group attaching the at least one nucleotide represented by a V to a tide or an oligonucleotide, provided that at least one of U1 or W is an intemucleotide linking group attaching the at least one nucleotide ented by Formula V to a nucleotide or an oligonucleotide and provided that if U1 is an internucleotide linking group, A is absent.

In certain embodiments, I is CH2. In certain embodiments, B is a l nucleobase. In certain embodiments, I is CH2 and B is a natural base.

In certain embodiments, A is hydrogen, a phosphate group or a phosphate mimic. In certain embodiments, A is hydrogen and U1 is oxygen. In certain embodiments, A is hydrogen, U1 is oxygen, and I is CH2. :1, Formula Va In some ments, the glutathione-sensitive oligonucleotide ses at least one nucleotide represented by the ing a: A—Ul—I B \ 0 wherein A, U1, U2, I, W, and B are as described above for Formula V.

In certain embodiments, the oligonucleotide containing at least one nucleotide of Formula V has 2-2500 nucleotides. In certain embodiments, the oligonucleotide containing at least one nucleotide of a V has 500-1500 nucleotides. In certain embodiments, the oligonucleotide containing at least one nucleotide of Formula V has 7-100 nucleotides. In another ment, the oligonucleotide containing at least one nucleotide of Formula V has -50 nucleotides. In yet another embodiment, the oligonucleotide containing at least one nucleotide of Formula V has 19-25 nucleotides.

In certain embodiments, the oligonucleotide containing at least one nucleotide of Formula V is a nucleic acid inhibitor molecule, as discussed in further detail throughout the application. In other embodiments, the oligonucleotide containing at least one nucleotide of Formula V is a CRISPR nucleic acid, a nucleic acid for gene therapy, a nucleic acid for DNA editing, a probe, or any other oligonucleotide that is susceptible to degradation by nucleases 2017/048239 and/or harsh environmental conditions (e.g., pH), including other oligonucleotides that are to be administered in vivo. 3. Formula VI (Oligonucleotide with Formula 111 Glutathione—Sensitive Moietjy) In some embodiments, the glutathione-sensitive oligonucleotide comprises at least one nucleotide represented by Formula VI as follows: A—Ul—I B \ O /U2 \V// W | / Zl~ TI] M2 {’1 ('21 Ta Tb [027 8] wherein A, U1, I, B, W, and U2 are as described in Formula I, and wherein Y, V, Z1, M1, M2, P1, Q1, Ta and Tb are as bed in Formula III.

In certain embodiments, U1 is absent, an , or an intemucleotide linking group ing the at least one nucleotide ented by Formula VI to a nucleotide or an oligonucleotide, or hydrogen, U2 is absent or O, and W is hydrogen, a phosphate group, or an intemucleotide linking group attaching the at least one nucleotide ented by Formula VI to a nucleotide or an ucleotide, provided that at least one of U1 or W is an intemucleotide linking group attaching the at least one nucleotide represented by Formula VI to a tide or an oligonucleotide and provided that if U1 is an intemucleotide linking group, A is absent.

In certain embodiments, I is CH2. In certain embodiments, B is a natural nucleobase.

In certain embodiments, I is CH2 and B is a natural nucleobase.

In certain embodiments, A is hydrogen, a phosphate group or a phosphate mimic. In certain embodiments, A is hydrogen and U1 is oxygen. In certain embodiments, A is hydrogen, U1 is oxygen, and I is CH2.

In certain embodiments, the ucleotide containing at least one nucleotide of Formula VI has 2-2500 nucleotides. In certain embodiments, the oligonucleotide containing WO 39364 at least one nucleotide of Formula VI has 500-1500 tides. In certain embodiments, the oligonucleotide containing at least one nucleotide of Formula VI has 7-100 nucleotides. In another embodiment, the oligonucleotide containing at least one tide of Formula VI has -50 nucleotides. In yet another embodiment, the oligonucleotide ning at least one nucleotide of Formula VI has 19-25 nucleotides.

In certain embodiments, the oligonucleotide containing at least one nucleotide of Formula VI is a nucleic acid inhibitor molecule, as discussed in further detail throughout the application. In other embodiments, the oligonucleotide containing at least one nucleotide of Formula VI is a CRISPR nucleic acid, a nucleic acid for gene therapy, a nucleic acid for DNA editing, a probe, or any other oligonucleotide that is susceptible to degradation by nucleases and/or harsh environmental conditions (e.g., pH), including other oligonucleotides that are to be administered in viva. a. Formula VIa In some embodiments, the hione-sensitive oligonucleotide comprises at least one nucleotide represented by the following Formula: A—Ul—I B \ o / YY W C} wherein A, U1, U2, W, I and B are as described above for Formula VI and Y and Z1 are as bed above for Formula 111a.

More typically, the present glutathione-sensitive oligonucleotide comprises at least one nucleotide ented by the following Formula: VIa(i) wherein A, U1, U2, W, and B are as described above for Formula VI. b. Formula VIb In some ments, the t hione-sensitive oligonucleotide comprises at least one nucleotide represented by the following Formula: A_U]—I B \ O Ta/ s\ wherein A, U1, U2, W, I, and B are as described above for Formula VI, and wherein Y, Z1 and Ta and Th are as described above for Formula IIIb.

More typically, the present glutathione-sensitive oligonucleotide comprises at least one nucleotide represented by the following Formula: 2017/048239 A—U1—\ B O O W (N3 /S S\ S s VIb(i) wherein A, U1, U2, W, and B are as described above for Formula VI. 4. Formula VII (Oligonucleotide with Formula IV Glutathione—Sensitive Moietjy) In some embodiments, the present glutathione-sensitive oligonucleotide comprises at least one nucleotide represented by Formula VII as follows: A—Ul—I B \ o Y Q /U2 \V4 P/ \T W l ‘ wherein A, U1, 1, B, W, and U2 are as described in Formula I, and wherein Y, Z, V, K, G, E, 11, M3, M4, P, Q, and T are as described above in Formula IV.

In certain embodiments, U1 is , an oxygen, or an intemucleotide linking group attaching the at least one tide represented by Formula VII to a nucleotide or an oligonucleotide, or hydrogen, U2 is absent or O, and W is en, a phosphate group, or an internucleotide linking group attaching the at least one nucleotide represented by Formula VII to a nucleotide or an oligonucleotide, provided that at least one of U1 or W is an internucleotide linking group attaching the at least one nucleotide represented by a VII to a nucleotide or an oligonucleotide and provided that if U1 is an internucleotide linking group, A is absent.

In certain embodiments, I is CH2. In certain embodiments, B is a natural nucleobase.

In certain embodiments, I is CH2 and B is a natural nucleobase.

In certain embodiments, A is hydrogen, a phosphate group or a phosphate mimic. In certain embodiments, A is hydrogen and U1 is oxygen. In certain embodiments, A is en, U1 is oxygen, and I is CH2.

In certain embodiments, the oligonucleotide containing at least one nucleotide of Formula VII has 2-2500 nucleotides. In certain embodiments, the oligonucleotide containing at least one nucleotide of Formula VII has 00 nucleotides. In n embodiments, the oligonucleotide containing at least one nucleotide of Formula VII has 7-100 tides. In r embodiment, the oligonucleotide containing at least one nucleotide of Formula VII has -50 nucleotides. In yet another embodiment, the oligonucleotide containing at least one nucleotide of Formula VII has 19-25 nucleotides.

In certain embodiments, the oligonucleotide ning at least one nucleotide of Formula VII is a nucleic acid inhibitor molecule, as discussed in further detail throughout the application. In other embodiments, the oligonucleotide containing at least one nucleotide of Formula VII is a CRISPR nucleic acid, a c acid for gene therapy, a nucleic acid for DNA editing, a probe, or any other ucleotide that is susceptible to degradation by nucleases and/or harsh nmental conditions (e.g., pH), including other oligonucleotides that are to be administered in viva. a. a VIIa In some embodiments, the glutathione-sensitive modified oligonucleotide comprises at least one tide represented by the following Formula: 2017/048239 A_U1_I B \ O O Y ,Uz Y W z T—S—S 0 R5 R7 R6 VIIa wherein A, U1, U2, W, I and B are as described above for Formula VII, and n Y, Z, R5, R6, and R7, and T are as described above in Formula IVa. In certain embodiments, B is a natural nucleobase.

More typically, the present glutathione-sensitive oligonucleotide comprises at least one nucleotide represented by the following Formula: A—U1—\ B 0 0 /U2 Y H3CH2C—S—S 0 ACO OAC VIIa(i) wherein A, U1, U2, W, and B are as described above for Formula VII. b. Formula VIIb In some embodiments, the present glutathione-sensitive oligonucleotide comprises at least one nucleotide represented by the ing Formula: A_U1_I B \ 0 gm 0 O~V H II 02 \\ / Y IK—E—ISI—T W M4 0 VIIb wherein A, U1, U2, W, I, and B are as described above for Formula VII, and wherein Y, V, Z, K, E, M3, M4, 11 and T are as bed above for Formula IVb. In certain embodiments, B is a l nucleobase.

In some ments, the present glutathione-sensitive oligonucleotide comprises at least one nucleotide represented by the following Formula: A—U1—\ B o E H N R 0 O// VIIb(i) wherein A, U1, U2, W, and B are as described above for Formula VII, and wherein R is as bed in Formula IVb(i) .

In some embodiments, the present glutathione-sensitive oligonucleotide comprises at least one nucleotide represented by the following Formula: A—Ul—\ B o N \/\ R /U2 Y HN\ //o o //s VIIb(ii) wherein A, U1, U2, W, and B are as bed above for Formula VII, and wherein R is as described in Formula IVb(ii) . c. Formula VIIc In some embodiments, the present glutathione-sensitive oligonucleotide comprises at least one nucleotide represented by the following Formula: A_U1—I B \ O O\ // /U2 V S—S—T VIIc wherein A, U1, U2, W, I and B are as described above for Formula VII, and wherein Y, Z, V, K, n, T, and R are as described above for Formula IVc. In certain embodiments, B is a l base.

More typically, the present hione-sensitive oligonucleotide comprises at least one nucleotide represented by the following Formula: A—U1—\ B VIIc(i) wherein A, U1, U2, W and B are as described above for Formula VII, and wherein R is as described in Formula IVc(i). d. Formula VIId In some embodiments, the present glutathione-sensitive oligonucleotide comprises at least one nucleotide represented by the following Formula: VIId wherein A, U1, U2, W, I, and B are as described above for Formula VII, and wherein Y, Z, R and T are as described above for Formula IVd. In certain embodiments, B is a natural nucleobase.

More lly, the present glutathione-sensitive oligonucleotide comprises at least one tide represented by the following Formula: U2 Y W HNY\S S / COZCHZCH3 VIId(i) wherein A, U1, U2, W, and B are as bed above for Formula VII. e. Formula VIIe In some embodiments, the present glutathione-sensitive oligonucleotide comprises at least one nucleotide represented by the following Formula: A_U1—I B \ 0 0\ // s—s—r ,Uz Y W \E z\ / G l“ VIIe wherein A, U1, U2, W, I, and B are as described above for Formula VII and wherein V, Y, Z, G, E, T, K, n, R, and T are as described above for Formula IVe. In certain embodiments, B is a natural base.

More typically, the t glutathione-sensitive oligonucleotide comprises at least one nucleotide selected from one of the following Formula: A—U1 B VIIe(i) VIIe(ii) 2017/048239 VIIe(iii) VIIe(iv) A_U1 B 0 s /SJ< /U2 Y S VIIe(v) 2017/048239 VIIe(vii) VIIe(viii) VIIe(ix) VIIe(x) VIIe(xi) n A, U1, U2, W, and B are as described above for Formula VII, and wherein R is selected from hydrogen, CH3, tuted or substituted aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected Via any spacer.

A. Glutathione—Sensitive Nucleic Acid tor Molecules In certain embodiments, the hione-sensitive moiety is incorporated into a nucleic acid inhibitor molecule. Various oligonucleotide structures have been used as nucleic acid inhibitor molecules, including single stranded and double stranded oligonucleotides, and any of these various ucleotides can be modified to include one or more hione-sensitive nucleotides as described herein.

In certain embodiments, the nucleic acid inhibitor molecule is a double-stranded RNAi inhibitor molecule comprising a sense (or passenger) strand and an antisense (or guide strand).

A variety of double stranded RNAi inhibitor molecule structures are known in the art. For example, early work on RNAi inhibitor molecules focused on double-stranded nucleic acid molecules with each strand having sizes of 19-25 nucleotides with at least one 3’-overhang of l to 5 nucleotides (see, e.g., US Patent No. 8,372,968). Subsequently, longer double-stranded RNAi inhibitor molecules that get processed in vivo by the Dicer enzyme to active RNAi inhibitor molecules were ped (see, e.g., US. Patent No. 8,883,996). Later work developed extended double-stranded nucleic acid inhibitor molecules where at least one end of at least one strand is extended beyond the double-stranded targeting region of the molecule, including structures where one of the strands includes a thermodynamically-stabilizing tetraloop ure (see, e.g., US. Patent No. 8,513,207, US. Patent No. 8,927,705, WO 2010/033225, and W0 2016/100401, which are incorporated by reference for their sure of these double-stranded nucleic acid inhibitor molecules). Those structures include single- ed extensions (on one or both sides of the molecule) and -stranded extensions.

In some ments ofthe dsRNAi inhibitor molecule, the sense and antisense strands range from 15-66, 25-40, or 19-25 nucleotides. In some embodiments, the sense strand is between 18 and 66 nucleotides in length. In certain ments, the sense strand is between 18 and 25 nucleotides in length. In certain embodiments, the sense strand is 18, 19, 20, 21, 22, 23, or 24 nucleotides in length. In certain of those embodiments, the sense strand is between and 45 nucleotides in length. In certain embodiments, the sense strand is between 30 and 40 nucleotides in length. In certain embodiments, the sense strand is 36, 37, 38, 39, or 40 nucleotides in length. In certain embodiments, the sense strand is between 25 and 30 nucleotides in length. In certain of those embodiments, the sense strand is 25, 26, or 27 tides in .

In some embodiments ofthe dsRNAi inhibitor molecule, the antisense strand is between 18 and 66 nucleotides in length. Typically, the antisense strand comprises a sequence that is sufficiently complementary to a sequence in the target gene mRNA to direct the effect of the nucleic acid inhibitor molecule to the target gene. In certain embodiments, the antisense strand comprises a sequence that is fully complementary with a sequence contained in the target gene mRNA where the fully complementary sequence is between 18 and 40 nucleotides long. In certain of those ments, the antisense strand is between 20 and 50 nucleotides in length.

In certain embodiments, the antisense strand is n 20 and 30 nucleotides in length. In certain embodiments, the antisense strand is 21, 22, 23, 24, 25, 26, 27, or 28 tides in length. In certain embodiments, the antisense strand is between 35 and 40 tides in length.

In certain of those embodiments, the antisense strand is 36, 37, 38, or 39 nucleotides in length.

In some embodiments ofthe dsRNAi inhibitor le, the sense and antisense strands form a duplex structure of n 15 and 50 base pairs. In certain embodiments, the duplex region is between 15 and 30 base pairs in length, such as between 19 and 30, more typically between 18 and 26, such as between 19 and 23, and in certain instances between 19 and 21 base pairs in length. In certain ments, the double-stranded region is 19, 20, 21, 22, 23, 24, 25, or 26 base pairs in length.

In some embodiments, the dsRNAi inhibitor molecule may further comprise one or more -stranded nucleotide overhang(s). Typically, the dsRNAi inhibitor molecule has a -stranded overhang of 1-10, 1-4, or 1-2 nucleotides. The single stranded overhang is typically located at the 3’-end of the sense strand and/or the 3’-end of the nse strand. In certain ments, a single-stranded overhang of 1-10, 1-4, or 1-2 nucleotides is located at the 5’-end of the antisense strand. In certain embodiments, a single-stranded overhang of 1-10, 1-4, or 1-2 nucleotides is located at the 5’-end of the sense strand. In certain ments, the single-stranded overhang of 1-2 nucleotides is located at the 3’-end of the antisense strand. In certain embodiments, the single-stranded overhang of 10 nucleotides is located at the 5’-end of the antisense . In certain embodiments, the dsRNA inhibitor molecule has a blunt end, typically at the 5’-end of the antisense strand.

In certain embodiments, the dsRNAi inhibitor molecule comprises a sense and an antisense strand and a duplex region of between 19-21 nucleotides, wherein the sense strand is 19-21 nucleotides in length and the nse strand is 21-23 nucleotides in length and comprises a single-stranded overhang of 1-2 nucleotides at its minus.

In certain embodiments, the dsRNAi inhibitor molecule has an antisense strand of 21 nucleotides in length and a sense strand of 21 nucleotides in length, where there is a two nucleotide 3’-passenger strand overhang on the right side of the molecule (3’-end of sense /5’-end of antisense strand) and a two nucleotide 3’-guide strand overhang on the left side of the molecule (5’-end of the sense strand/3’-end of the antisense strand). In such molecules, there is a 19 base pair duplex .

In certain embodiments, the dsRNAi inhibitor molecule has an antisense strand of 23 tides in length and a sense strand of 21 tides in length, where there is a blunt end on the right side of the molecule (3’-end of sense strand/5’-end of antisense strand) and a two nucleotide 3’-guide strand overhang on the left side of the molecule (5’-end of the sense strand/3’-end of the antisense strand). In such molecules, there is a 21 base pair duplex region.

In certain embodiments, the dsRNAi inhibitor molecule comprises a sense and an antisense strand and a duplex region of between 18-34 nucleotides, where the sense strand is -34 nucleotides in length and the antisense strand is 26-38 nucleotides in length and comprises 1-5 -stranded nucleotides at its 3’ terminus. In certain embodiments, the sense strand is 26 nucleotides, the antisense strand is 38 tides and has a single-stranded overhang of 2 nucleotides at its 3’ terminus and a single-stranded overhang of 10 tides at its 5’ terminus, and the sense strand and antisense strand form a duplex region of 26 nucleotides. In certain embodiments, the sense strand is 25 nucleotides, the antisense strand is 27 nucleotides and has a single-stranded overhang of 2 nucleotides at its 3’ terminus, and the sense strand and antisense strand form a duplex region of 25 nucleotides.

In some embodiments, the dsRNAi inhibitor molecules include a stem and loop.

Typically, a 3’-terminal region or 5’-terminal region of a passenger strand of a dsRNAi inhibitor molecule form a single ed stem and loop structure.

In some embodiments, the dsRNAi inhibitor molecule contains a stem and tetraloop.

In embodiments where the dsRNAi inhibitor molecule contains a stem and oop, the passenger strand contains the stem and oop and ranges from 20-66 nucleotides in length.

Typically, the guide and passenger strands are separate strands, each having a 5’ and 3’ end that do not form a contiguous oligonucleotide (sometimes referred to as a “nicked” structure).

In certain of those embodiments, the guide strand is n 15 and 40 tides in length. In certain embodiments, the extended part of the passenger strand that contains the stem and tetraloop is on 3’-end of the strand. In certain other ments, the extended part of the passenger strand that contains the stem and tetraloop is on 5’-end of the strand.

In certain embodiments, the passenger strand of a dsRNAi inhibitor molecule containing a stem and tetraloop is between 34 and 40 nucleotides in length and the guide strand ofthe dsRNAi inhibitor molecule contains between 20 and 24 nucleotides, where the passenger strand and guide strand form a duplex region of 18-24 nucleotides.

In certain embodiments, the dsRNAi inhibitor molecule comprises (a) a passenger strand that contains a stem and oop and is 36 nucleotides in length, wherein the first 20 nucleotides from the 5’-end are complementary to the guide strand and the following 16 nucleotides form the stem and tetraloop and (b) a guide strand that is 22 nucleotides in length and has a single-stranded ng of two nucleotides at its 3’ end, wherein the guide and passenger strands are te s that do not form a contiguous oligonucleotide (see e.g., Figure 1A and 1B).

In certain embodiments, the c acid inhibitor molecule includes one or more ibonucleotides. Typically, the nucleic acid inhibitor le contains fewer than 5 deoxyribonucleotides. In certain embodiments, the nucleic acid inhibitor molecule includes one or more ribonucleotides. In n embodiments, all of the nucleotides of the nucleic acid inhibitor molecule are ribonucleotides.

In some embodiments, the at least one glutathione-sensitive nucleotide of a double stranded nucleic acid inhibitor molecule, e.g., a dsRNAi inhibitor molecule, is located on the passenger strand. In another embodiment, the at least one glutathione-sensitive nucleotide is located on the guide strand. In some embodiments, the at least one glutathione-sensitive nucleotide is located in a duplex . In some embodiments, the at least one glutathione- sensitive nucleotide is located in an overhang region.

In certain embodiments, the nucleic acid tor molecule is a -stranded nucleic acid inhibitor molecule comprising at least one nucleotide having a glutathione-sensitive moiety, as described herein. Single stranded nucleic acid inhibitor molecules are known in the art. For example, recent efforts have demonstrated activity of ssRNAi inhibitor molecules (see, e.g., Matsui etal.,M01ecular Therapy, 2016,24(5):946-55. And, antisense molecules have been used for decades to reduce expression of ic target genes. Pelechano and Steinmetz, Nature Review Genetics, 2013,14z880-93. A number of variations on the common themes of these structures have been developed for a range of targets. Single stranded nucleic acid inhibitor molecules include, for example, conventional nse oligonucleotides, microRNA, ribozymes, aptamers, antagomirs, and ssRNAi inhibitor molecules, all of which are known in the art.

In certain embodiments, the nucleic acid inhibitor molecule is a ssRNAi inhibitor molecule having 14-50, 16-30, or 15-25 nucleotides. In other embodiments, the ssRNAi tor molecule has 18-22 or 20-22 nucleotides. In certain embodiments, the ssRNAi inhibitor molecule has 20 tides. In other embodiments, the ssRNAi inhibitor molecule has 22 nucleotides. In certain embodiments, the nucleic acid inhibitor molecule is a single- stranded oligonucleotide that inhibits ous RNAi inhibitor molecules or natural miRNAs.

In certain embodiments, the nucleic acid tor le is a single-stranded antisense oligonucleotide having 8-80, 14-50, 16-30, 12-25, 12-22, 14-20, 18-22, or 20-22 nucleotides. In certain embodiments, the single-stranded antisense oligonucleotide has 18-22, such as 18-20 nucleotides.

In certain embodiments, the antisense oligonucleotide or a portion thereof is fully complementary to a target nucleic acid or a specific portion thereof. In certain embodiments, the antisense oligonucleotide or a portion thereof is complementary to at least l2, l3, 14, 15, l6, l7, l8, 19, 20, or more contiguous nucleotides of the target nucleic acid. In certain embodiments, the nse oligonucleotide contains no more than 5, 4, 3, 2, or 1 non- complementary nucleotides relative to the target nucleic acid or portion thereof It is possible to decrease the length ofthe antisense oligonucleotide and/or introduce mismatch bases without eliminating activity.

As described herein, the sugar moiety of one or more nucleotides can be modified with a glutathione-sensitive moiety, typically at the 2’-carbon of the sugar moiety. Typically one or two nucleotides of a nucleic acid inhibitor molecule are ibly modified with a glutathione- sensitive . In certain embodiments, more than two nucleotides of a nucleic acid inhibitor molecule, such as three, four, five nucleotides, or more, are reversibly modified with a glutathione-sensitive moiety. In n embodiments, most of the nucleotides are ibly modified with a hione-sensitive moiety. In certain embodiments, all or substantially all of the nucleotides of the oligonucleotide contain a glutathione-sensitive moiety.

In certain embodiments, the passenger strand of a dsRNAi inhibitor molecule contains one or more nucleotides that are ibly modified with a glutathione-sensitive . In certain embodiments, the guide strand of a dsRNAi inhibitor molecule contains one or more nucleotides that are reversibly modified with a glutathione-sensitive moiety. In certain embodiments, the guide and passenger strands of a dsRNAi inhibitor le each contain one or more nucleotides that are reversibly modified with a glutathione-sensitive moiety.

In some embodiments, the presence of at least one glutathione-sensitive moiety in a nucleic acid inhibitor molecule reduces ation of the oligonucleotide resulting from nucleases in serum, for example, and/or nucleases within cells, e.g., within vesicles such as endosomal vesicles, lysosomal vesicle and/or fused mal/lysosomal vesicles. For example, placing a glutathione-sensitive moiety at either the 5’- or 3’-terminal nucleotide ofthe nucleic acid inhibitor molecule can protect against degradation from nucleases. In addition, certain double stranded nucleic acid tor les contain a single stranded overhang region on either the passenger or guide strand or both, which is more susceptible to nuclease degradation. ing this single stranded overhang region can t such double stranded nucleic acid inhibitor molecules against degradation from ses.

In some embodiments, the at least one hione-sensitive moiety is located at the 5’- terminal nucleotide of a single stranded c acid inhibitor molecule or the 5’-terminal nucleotide of the passenger strand or the guide strand of a double-stranded nucleic acid inhibitor molecule. In certain embodiments, the glutathione-sensitive moiety is located at the ’-carbon of the 5’-terminal nucleotide. In other embodiments, the glutathione-sensitive moiety is located at the 2’-carbon of the 5’-terminal nucleotide. In certain embodiments of the double stranded nucleic acid inhibitor molecule, the glutathione-sensitive moiety located at the 5’- terminal tide of the passenger or guide strand is in an overhang region.

In some embodiments, the at least one glutathione-sensitive moiety is located at the 3’- terminal nucleotide of a single stranded nucleic acid inhibitor le or the 3’-terminal nucleotide ofthe passenger strand or the guide strand of a double stranded nucleic acid inhibitor molecule. In certain embodiments, the glutathione-sensitive moiety is located at the 3’-carbon of the minal nucleotide. In other embodiments, the glutathione-sensitive moiety is located at the 2’-carbon of the 3’-terminal nucleotide. In certain embodiments of the double stranded c acid inhibitor molecule, the glutathione-sensitive moiety located at the minal nucleotide of the passenger or guide strand is in an overhang region.

Irreversible chemical modifications at nucleotide position 2 and position 14 of an RNAi inhibitor molecule, such as modifications at the 2’-carbon of the sugar, are generally not well tolerated. Without intending to be bound by any theory, it is possible that these nucleotide positions are sensitive to steric bulk. In some embodiments, the at least one glutathione- sensitive moiety is d at nucleotide position 2 of a single stranded nucleic acid tor molecule or position 2 of the guide strand of a double stranded nucleic acid inhibitor molecule.

In some embodiments, the at least one glutathione-sensitive moiety is located at nucleotide position 14 of a single stranded nucleic acid inhibitor molecule or position 14 of the guide strand of a double stranded nucleic acid inhibitor molecule.

In some embodiments, the at least one glutathione-sensitive moiety is on one or more nucleotides located at or adjacent to the Ago2 cleavage site ofthe passenger strand of a dsRNAi inhibitor le. Typically, Ago2 cleaves the passenger strand at a phosphodiester bond between the two nucleotides opposing nucleotide positions 10 and 11 of the guide strand, as measured from the 5’-end of the guide strand. Thus, for example, if the guide strand has 22 nucleotides and a two-base pair overhang (or 20 nucleotides and no overhang), Ago2 should cleave between nucleotide positions 10 and 11 of the passenger strand. If the guide strand has 21 nucleotides and a two-base pair overhang (or 19 nucleotides and no overhang), Ago2 should cleave between nucleotide positions 9 and 10 of the passenger strand. In certain ments, the dsRNAi inhibitor molecule contains a hione-sensitive moiety on one, two, or three nucleotides that are immediately 5’ of the Ago2 cleavage site. In certain ments, the dsRNAi inhibitor molecule contains a hione-sensitive moiety on one, two, or three nucleotides that are immediately 3’ of the Ago2 cleavage site. In certain embodiments, the dsRNAi tor molecule contains a glutathione-sensitive moiety on both sides of the Ago2 cleavage site, including, for example, on one, two, or three nucleotides that are immediately 5’ of the Ago2 cleavage site and on one, two, or three tides that are immediately 3’ of the Ago2 cleavage site.

B. Other Modifications ofthe Glutathione—sensitive Oligonucleotides An oligonucleotide that is modified with a reversible glutathione-sensitive moiety as described , can be further modified on one or more nucleotides using, for example, other nucleotide modifications known in the art, including the irreversible modifications described herein. Typically, multiple nucleotide subunits of the oligonucleotide of st are modified to improve various characteristics of the molecule such as ance to nucleases or lowered immunogenicity. See, e.g., Bramsen et al. (2009), Nucleic Acids Res, 37, 2867-2881. Many nucleotide modifications have been used in the oligonucleotide field, particularly for nucleic acid inhibitor molecules. Such irreversible modifications can be made on any part of the nucleotide, including the sugar moiety, the phosphodiester linkage, and the base.

Typical examples of nucleotide modification e, but are not limited to, 2’-F, 2’-O-methyl (“2’-OMe” or “2’-OCH3”), ethoxyethyl OE” or “2’-OCH2CH20CH3”), and 5’- cytosine. Irreversible modifications can occur at other parts of the nucleotide, such as the 5’-carbon, as described herein.

In certain embodiments, the ring structure of the sugar moiety is d, including, but not d to, Locked Nucleic Acid (“LNA”) structures, d Nucleic Acid (“BNA”) structures, and ed Nucleic Acid (“UNA”) ures, as discussed previously. d nucleobases include nucleobases other than adenine, guanine, cytosine, thymine and uracil at the l’-position, as known in the art and as described herein. A typical example of a modified base is 5’-methylcytosine.

The natural occurring intemucleotide linkage of RNA and DNA is a 3’ to 5’ phosphodiester linkage. Modified phosphodiester linkages include non-naturally occurring intemucleotide linking groups, ing intemucleotide linkages that contain a phosphorous atom and intemucleotide es that do not contain a phosphorous atom, as known in the art and as described . Typically, the oligonucleotide contains one or more phosphorous- containing intemucleotide linking groups, as described herein. In other embodiments, one or more of the intemucleotide linking groups of the nucleic acid inhibitor molecule is a non- phosphorus containing linkage, as bed herein. In certain embodiments, the oligonucleotide contains one or more phosphorous-containing intemucleotide linking groups and one or more non-phosphorous containing intemucleotide linking groups.

The 5’-end of the glutathione-sensitive oligonucleotide can include a natural tuent, such as a hydroxyl or a phosphate group. In certain embodiments, a hydroxyl group is attached to the 5’-terminal end of the glutathione-sensitive oligonucleotide. In certain embodiments, a phosphate group is attached to the 5’-terminal end of the glutathione-sensitive oligonucleotide. Typically, the phosphate is added to a monomer prior to oligonucleotide synthesis. In other embodiments, 5’-phosphorylation is accomplished naturally after an oligonucleotide of the disclosure is uced into the cytosol, for example, by a cytosolic Clpl kinase. In some embodiments, the 5’-terminal phosphate is a phosphate group, such as ’-monophosphate [(HO)2(O)P-O-5’], 5’-diphosphate [(HO)2(O)P-O-P(HO)(O)-O-5’] or a 5’- triphosphate[(HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5’].

The 5’-end of the glutathione-sensitive ucleotide can also be modified. For example, in some embodiments, the 5’-end of the glutathione-sensitive ucleotide is attached to a phosphoramidate [(HO)2(O)P-NH-5’, (HO)(NH2)(O)P-O-5’]. In certain embodiments, the 5’-terminal end of the glutathione-sensitive oligonucleotide is attached to a phosphate mimic. le phosphate mimics include 5’-phosphonates, such as 5’- methylenephosphonate (5’-MP) and 5’-(E)-Vinylphosphonate (5’-VP). Lima et al., Cell, 2012, 15094, WO2014/130607. Other suitable phosphate mimics e 4’-phosphate analogs that are bound to the 4’-carbon of the sugar moiety (e.g., a ribose or deoxyribose or analog thereof) of the 5’-terminal nucleotide of an ucleotide, as described in US. Provisional Application No. 62/393,401, which is hereby incorporated by reference in its ty. For example, in some embodiments, the 5’-end of the nucleic acid inhibitor molecule is attached to an oxymethylphosphonate, where the oxygen atom of the oxymethyl group is bound to the 4’- carbon of the sugar moiety or analog thereof. In certain embodiments, the 4’- oxymethylphosphonate is ented by the formula —O-CH2-PO(OH)2 or —O-CH2-PO(OR)2, where R is independently selected from H, CH3, an alkyl group, or a protecting group. In certain embodiments, the alkyl group is CH2CH3. More lly, R is independently selected from H, CH3, or CH2CH3. In other embodiments, the phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate, where the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is bound to the 4’-carbon of the sugar moiety or analog f.

In certain embodiments, an oligonucleotide is fully modified, wherein every nucleotide of the fully modified oligonucleotide is modified with either an irreversible modification or a reversible, glutathione-sensitive moiety, as described herein. In certain embodiments, every nucleotide of an oligonucleotide is modified, wherein every nucleotide that is not modified with a glutathione-sensitive moiety is modified with an irreversible modification. In certain embodiments, the oligonucleotide contains ribonucleotides and deoxyribonucleotides and every ribonucleotide in the oligonucleotide is modified with either an rsible modification or a reversible, glutathione-sensitive moiety, as described herein. In certain embodiments, substantially all of the tides of an oligonucleotide are modified. In certain embodiments, more than half of the nucleotides of an oligonucleotide are modified. In certain ments, more than half of the nucleotides of an oligonucleotide contain an irreversible modification. In certain embodiments, less than half of the nucleotides of an oligonucleotide are modified. In certain embodiments, less than half of the tides of an oligonucleotide contain an irreversible modification. In certain embodiments, the oligonucleotide does not contain any modifications other than the one or more glutathione-sensitive nucleotides. Modifications can occur in groups on the oligonucleotide chain or different modified tides can be interspersed.

In some ments, the irreversible chemical modification is located at the same nucleotide as that containing the glutathione-sensitive . In other embodiments, the WO 39364 irreversible chemical modification is located at one or more tides that do not contain the glutathione-sensitive .

In some embodiments, all of the nucleotides in a single stranded nucleic acid inhibitor molecule or in the guide strand or passenger strand of a double stranded nucleic acid inhibitor molecule are modified with an irreversible chemical modification, except for one nucleotide, which is reversibly modified with a glutathione-sensitive moiety as described herein. In other embodiments, at least one, such as at least two, three, four, five, siX, seven, eight, nine, or 10 nucleotides of a single stranded nucleic acid inhibitor molecule or the guide strand or passenger strand of a double stranded c acid inhibitor molecule are reversibly modified with a glutathione-sensitive moiety and at least one, such as at least two, three, four, five, siX, seven, eight, nine, or 10 tides of a single stranded nucleic acid inhibitor molecule or the guide strand or passenger of a double stranded nucleic acid inhibitor molecule are chemically modified with an irreversible chemical ation. In some embodiments, all of the nucleotides of a single stranded nucleic acid inhibitor molecule or the guide strand or passenger strand of a double stranded nucleic acid inhibitor molecule contain at least one glutathione- sensitive moiety as described herein or at least one irreversible modification.

In certain embodiments of the nucleic acid inhibitor molecule, every nucleotide is modified at the 2’-carbon. In certain embodiments of the nucleic acid inhibitor molecule (or the sense strand and/or antisense strand thereof) every tide that is not modified with a glutathione-sensitive moiety is d is modified with 2’-F, 2’-O-Me, and/or 2’-MOE. In certain embodiments of the nucleic acid tor le, from one to every phosphorous atom is modified and from one to every cleotide is modified at the 2’-carbon.

III. Glutathione—sensitive Monomers (Nucleosides and Nucleotides) One aspect of the present disclosure relates to reversibly modified nucleosides or nucleotides comprising a glutathione-sensitive moiety, including hione-sensitive nucleoside phosphoramidites that can be used in standard oligonucleotide sis methods and glutathione-sensitive nucleosides or tides without a phosphoramidite group that have therapeutic utility, for e, as antiviral agents. Typically, the reversible modification comprises a glutathione-sensitive moiety at the sugar moiety of the nucleoside or nucleotide, e. g. a deoxyribose or ribose (or analogs thereof). Typically, the glutathione-sensitive moiety in the nucleoside or nucleotide is located at the 2’-carbon of a deoxyribose or ribose (or analogs thereof). In other ments, the glutathione-sensitive moiety in the nucleoside or nucleotide is located at the 5’-carbon of a ribose or deoxyribose (or analogs thereof). In yet other ments, the glutathione-sensitive moiety in the nucleoside or nucleotide is located at the 3’-carbon of a ribose or deoxyribose (or analogs thereof).

In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group. In other embodiments, the glutathione-sensitive moiety comprises a disulfide bridge.

A. hione—Sensitive Nucleoside Phosphoramidites This application discloses nucleosides that are reversibly d with a glutathione- sensitive moiety and that are ible with phosphoramidite ucleotide synthesis methods. Thus, in another aspect, the present sure relates to reversibly modified nucleoside phosphoramidites comprising a glutathione-sensitive moiety and methods of sizing oligonucleotides using these glutathione-sensitive nucleoside phosphoramidites.

In certain embodiments, the nucleoside comprises a phosphoramidite and a glutathione- sensitive moiety, wherein the nucleoside is compatible with phosphoramidite ucleotide synthesis methods. Typically, the phosphoramidite is bound to the 5’- or 3’-carbon of the sugar moiety of the nucleoside and the glutathione-sensitive moiety is bound to an oxygen atom that is covalently bound to the 2’-carbon of the sugar moiety (e.g., ribose) of the nucleoside. In some embodiments, the glutathione-sensitive moiety is represented by Formula II, as described previously. In certain embodiments, Formula II is Formula IIa, as described herein. In other embodiments, the glutathione-sensitive moiety is represented by Formula III, as described previously. In some embodiments, Formula III is ed from Formula IIIa or IIIb, as described previously. In some embodiments, Formula III is selected from a IIIa(i) or IIIb(i), as described usly. In yet other embodiments, the glutathione-sensitive moiety is represented by Formula IV, as bed usly. In some ments, Formula IV is selected from a IVa, IVb, IVc, IVd, or IVe, as described previously. In some embodiments, Formula IV is selected from Formula IVa(i), IVb(i), IVb(ii), IVc(i), or IVd(i), as described previously. In some embodiments, Formula IVe is selected from Formula IVe(i), ), IVe(iii), IVe(iv), IVe(v), IVe(vi), IVe(vii), IVe(viii), IVe(iX), IVe(X), or IVe(Xi), as described previously. 1. Formula VIII In some embodiments, the nucleoside phosphoramidite is represented by the following Formula: Al—U3_I B R4 R1 R3 R2 /U2 X W1 L1 VIII wherein L1 is a hione-sensitive moiety; n A1 is , hydrogen, a phosphate group, a phosphate mimic, a phosphoramidate, a phosphoramidite, a protecting group, or a solid t, wherein W1 is a phosphoramidite, a protecting group, a solid support, hydrogen, halogen, OR’, SR’, NR’R”, a substituted or unsubstituted aliphatic, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycle, wherein R’ and R” are each independently selected from hydrogen, halogen, a substituted or unsubstituted aliphatic, an aryl, a aryl, a cycle or are taken together to form a heterocyclic ring, wherein U3 is hydrogen or selected from O, S, NR’ or CR’R”, wherein R’ and R” are each independently hydrogen, a substituted or unsubstituted aliphatic, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocycle and a substituted or unsubstituted cycloalkyl, wherein at least A1 is a phosphoramidite and U3 is O or at least W1 is a phosphoramidite and U2 is O, wherein X is O, S, Se or NR’, n R’ is selected from en, halogen, a tuted or unsubstituted aliphatic, an aryl, a substituted or unsubstituted heteroaryl or a substituted or unsubstituted heterocycle, wherein R1, R2, R3 and R4 are each independently selected from hydrogen, halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl or wherein two of R1, R2, R3 and R4 are taken together to form a -8 membered ring, n the ring optionally contains a heteroatom, wherein J is O, S, NR’, CR’R”, wherein each of R’ and R” is independently selected from hydrogen, halogen, a substituted or unsubstituted aliphatic, aryl or heteroaryl, wherein B is selected from hydrogen, a tuted or unsubstituted aliphatic, a natural nucleobase, a modified nucleobase or a universal nucleobase, wherein U2 is absent or selected from O, S, NR’, or CR’R”, wherein R’ and R” are each independently hydrogen, a substituted or unsubstituted aliphatic, a substituted or tituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocycle or a substituted or unsubstituted cycloalkyl, wherein I is absent or is selected from O, S, NR’, CR’R”, wherein R’ and R” are each independently en, a substituted or unsubstituted aliphatic, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocycle and a substituted or unsubstituted cycloalkyl, and wherein I and U3 can be combined to form CR’-CR” alkyl, CR’-CR” alkenyl, CR’-CR” l, a substituted or unsubstituted aliphatic, an aryl, a heteroaryl a heterocycle or taken together to form lkyl or heterocyclic ring.

Typically, the glutathione-sensitive moiety (Li) comprises a disulfide bridge or a sulfonyl group. In certain embodiments, the glutathione-sensitive moiety comprises a disulfide bridge. In other embodiments, the glutathione-sensitive moiety comprises a sulfonyl group.

In some embodiments, the glutathione-sensitive moiety (L1) is represented by Formula II, Formula III, or a IV, as described previously.

In certain embodiments, the glutathione-sensitive moiety (L1) is represented by Formula IIa, as bed previously.

In some embodiments, the glutathione-sensitive moiety (L1) is represented by Formula IIIa or IIIb, as described previously.

In some embodiments, the glutathione-sensitive moiety (L1) is represented by Formula IIIa(i) or ), as described previously.

In some embodiments, the glutathione-sensitive moiety (L1) is represented by Formula IVa, IVb, IVc, IVd, or We, as described previously.

In some embodiments, the glutathione-sensitive moiety (L1) is represented by a IVa(i), IVb(i), IVb(ii), IVc(i), or IVd(i), as bed previously.

In some ments, the glutathione-sensitive moiety (L1) is represented by Formula IVe(i), IVe(ii), i), IVe(iv), IVe(v), IVe(vi), IVe(vii), IVe(viii), ), IVe(X), or IVe(Xi), as described previously.

In n ments, X is O.

In certain embodiments, R1, R2, R3 and R4 are hydrogen.

In certain embodiments, J is O. [0389l In certain embodiments, B is a natural nucleobase.

In certain embodiments, U2 is O.

In certain embodiments, W1 is a phosphoramidite, a protecting group, or a hydrogen.

In certain embodiments, A1 is a phosphoramidite, a protecting group, or a en.

In certain embodiments, W1 is a phosphoramidite and A1 is a ting group.

In n ments, W1 is a protecting group and A1 is a phosphoramidite.

In certain embodiments, I is CH2.

In certain embodiments, U3 is O.

In certain embodiments, X is 0, R1, R2, R3 and R4 are hydrogen, and J is O.

In n embodiments, X is 0, R1, R2, R3 and R4 are hydrogen, J is O, B is a natural base, U2 is O, I is CH2, W1 is a phosphoramidite, A1 is a protecting group, and U3 is O.

In certain embodiments, X is 0, R1, R2, R3 and R4 are hydrogen, J is O, B is a natural nucleobase, U2 is O, I is CH2, W1 is a protecting group, A1 is a phosphoramidite, and U3 is O.

In certain embodiments, the phosphoramidite has the formula —P(ORX)—N(Ry)2, wherein RX is ed from the group consisting of an optionally substituted methyl, 2- cyanoethyl and benzyl, n each of Ry is selected from the group consisting of an optionally substituted ethyl and isopropyl. In certain embodiments, RXis 2-cyanoethyl and Ry is isopropyl. 2. Formula IX In certain embodiments, the nucleoside phosphoramidite is represented by the ing Formula: A —U —13 3 B R4 R1 R3 R2 R90 X\ wherein L1 is a glutathione-sensitive moiety, wherein R9 is a phosphoramidite, wherein X is O, S, Se or NR’, wherein R’ is selected from hydrogen, halogen, a substituted or unsubstituted aliphatic, an aryl, a substituted or unsubstituted heteroaryl or a substituted or unsubstituted heterocycle, wherein R1, R2, R3 and R4 are each independently selected from hydrogen, halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl or wherein two of R1, R2, R3 and R4 are taken together to form a -8 membered ring, n the ring optionally contains a heteroatom, wherein J is O, S, NR’, CR’R”, wherein each of R’ and R” is independently ed from hydrogen, halogen, a substituted or unsubstituted tic, aryl or heteroaryl, wherein B is hydrogen, a natural nucleobase, a modified nucleobase or a universal nucleobase, wherein I is absent or is selected from O, S, NR’, CR’R”, wherein R’ and R” are each independently en, a substituted or unsubstituted aliphatic, a substituted or unsubstituted aryl, a substituted or unsubstituted aryl, a substituted or unsubstituted heterocycle and a substituted or unsubstituted cycloalkyl, wherein U3 is a hydrogen or selected from O, S, NR’ or CR’R”, wherein R’ and R” are each independently hydrogen, a tuted or unsubstituted tic, a substituted or unsubstituted aryl, a substituted or tituted aryl, a substituted or unsubstituted heterocycle and a substituted or unsubstituted cycloalkyl, n I and U3 can be combined to form CR’-CR” alkyl, CR’-CR” alkenyl, CR’-CR” alkynyl, a substituted or tituted aliphatic, an aryl, a heteroaryl a heterocycle or taken together to form lkyl or cyclic ring, and wherein A3 is absent, hydrogen, a phosphate group, a phosphate mimic, a phosphoramidate, a protecting group, or a solid support.

Typically, L1 comprises a disulfide bridge or a sulfonyl group. In certain embodiments, the glutathione-sensitive moiety comprises a disulfide bridge. In other embodiments, the glutathione-sensitive moiety comprises a sulfonyl group.

In some embodiments, L1 is represented by Formula II, as described previously. In some embodiments, L1 is represented by Formula IIa, as described previously.

In other embodiments, L1 is represented by Formula III, as described previously. In some embodiments, a III is selected from Formula IIIa, IIIa(i), IIIb, or IIIb(i),, as described previously.

In yet other embodiments, L1 is represented by Formula IV, as described previously.

In some embodiments, Formula IV is selected from Formula IVa, IVb, IVc, IVd, or IVe, as described previously. In some embodiments, Formula IV is selected from Formula IVa(i), IVb(i), IVb(ii), IVc(i), or IVd(i), as described previously. In some embodiments, Formula IVe is selected from Formula IVe(i), IVe(ii), IVe(iii), IVe(iV), IVe(V), IVe(Vi), IVe(Vii), IVe(Viii), IVe(iX), IVe(X), or ), as bed previously.

In certain embodiments, X is O. [0417l In certain embodiments, R1, R2, R3 and R4 are hydrogen. [0418l In certain embodiments, J is O. [0419l In certain embodiments, B is a natural nucleobase. [0420l In certain embodiments, A3 is a ting group or a hydrogen. [0421 l In certain embodiments, I is CH2. [0422l In certain embodiments, U3 is O.

In certain embodiments, X is 0, R1, R2, R3 and R4 are hydrogen, and J is O.

In certain embodiments, X is 0, R1, R2, R3 and R4 are hydrogen, J is O, B is a natural nucleobase, I is CH2, A3 is a protecting group, and U3 is O.

In certain embodiments, the phosphoramidite has the formula —P(ORX)—N(Ry)2, wherein RX is selected from the group consisting of an optionally substituted methyl, 2- cyanoethyl and , wherein each of Ry is selected from the group consisting of an optionally substituted ethyl and isopropyl. In certain embodiments, RXis 2-cyanoethyl and Ry is isopropyl. 3. FormulaX In certain embodiments, the side phosphoramidite is ented by the following Formula: L1 is a hione-sensitive moiety, R8 is H, a protecting group, a solid support, or a phosphoramidite, R7 is H, a protecting group, a solid support, or a oramidite, wherein if Rs is a phosphoramidite, R7 is H, a solid support, or a protecting group or if R8 is H, a solid support, or a protecting group, R7 is a phosphoramidite, wherein B is a natural nucleobase, a modified nucleobase or a universal nucleobase, wherein X is O, S, Se, NR’, where R’ can be selected from hydrogen, n, aliphatic or tuted aliphatic, aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic. lly, Li comprises a disulfide bridge or a sulfonyl group. In certain ments, Li comprises a disulfide bridge. In other embodiments, Li comprises a sulfonyl group.

In some embodiments, L1 is represented by Formula II, as bed previously. In some embodiments, L1 is represented by Formula IIa, as described usly.

In other embodiments, L1 is represented by Formula III, as described previously. In some embodiments, Formula III is selected from Formula IIIa, IIIa(i), IIIb, or IIIb(i), as described previously.

In yet other embodiments, L1 is represented by Formula IV, as described previously.

In some embodiments, Formula IV is selected from Formula IVa, IVb, IVc, IVd, or IVe, as described usly. In some embodiments, Formula IV is selected from Formula IVa(i), IVb(i), IVb(ii), IVc(i), or IVd(i), as described previously. In some embodiments, a IVe is selected from Formula IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(v), ), IVe(vii), IVe(viii), IVe(iX), IVe(X), or IVe(Xi), as described usly.

In certain embodiments, X is O.

In n embodiments, B is a natural base.

In certain embodiments, the phosphoramidite has the formula —P(ORX)—N(Ry)2, n RX is selected from the group consisting of an optionally substituted methyl, 2- cyanoethyl and , wherein each of Ry is selected from the group consisting of an optionally substituted ethyl and isopropyl. In certain embodiments, RXis 2-cyanoethyl and Ry is isopropyl.

In certain embodiments, R8 is a phosphoramidite having the formula —P(ORX)— N(Ry)2 and R9 is H or a protecting group.

In certain embodiments, R9 is a phosphoramidite having the formula —P(ORX)— N(Ry)2 and R8 is H or a protecting group.

In certain embodiments, X is O, B is a natural nucleobase, R8 is a protecting group, and R9 is a phosphoramidite having the formula —P(ORX)—N(Ry)2.

In certain embodiments, X is O, B is a natural nucleobase, R8 is a phosphoramidite having the formula —P(ORX)—N(Ry)2 and R9 is a protecting group. In certain embodiments, ins 2-cyanoethyl and Ry is isopropyl.

B. Glutathione—Sensitive Nucleosides and Nucleotides Without a oramidite In some embodiments, the reversibly d, glutathione-sensitive monomers (nucleoside or nucleotide or s thereof) do not contain a phosphoramidite group at the 3’- carbon or 5’-carbon of the sugar moiety. Such glutathione-sensitive monomers can be used as therapeutics, for example as nucleoside or nucleotide analogs with antiviral activity. Typically, the reversible modification comprises a glutathione-sensitive moiety at the sugar moiety of the nucleotide or nucleoside (or analogs thereof), e. g. a deoxyribose or ribose (or analogs thereof). lly, the glutathione-sensitive moiety is located at the bon of a deoxyribose or ribose (or analogs thereof). In some embodiments, the glutathione-sensitive moiety is located at the ’-carbon of a ribose or deoxyribose (or analogs thereof). In other embodiments, the glutathione-sensitive moiety is located at the 3’-carbon of a ribose or deoxyribose (or analogs thereof).

In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group. In other embodiments, the glutathione-sensitive moiety comprises a disulfide .

In n embodiments, the glutathione-sensitive monomer comprises a glutathione- sensitive moiety bound to an oxygen atom that is covalently bound to the 2’-carbon of the sugar moiety (e.g., ribose) of the monomer. In some embodiments, the glutathione-sensitive moiety is represented by Formula II, as described previously. In n embodiments, Formula II is Formula IIa, as described herein. In other embodiments, the hione-sensitive moiety is represented by Formula III, as bed previously. In some ments, Formula III is selected from Formula IIIa, IIIa(i), IIIb, or IIIb(i), as described previously. In yet other embodiments, the glutathione-sensitive moiety is represented by Formula IV, as described usly. In some embodiments, Formula IV is selected from a IVa, IVb, IVc, IVd, or IVe, as described previously. In some embodiments, Formula IV is selected from Formula IVa(i), IVb(i), IVb(ii), IVc(i), or IVd(i), as described previously. In some embodiments, Formula IVe is selected from Formula IVe(i), ), IVe(iii), IVe(iv), IVe(v), IVe(vi), IVe(vii), IVe(viii), IVe(iX), IVe(X), or IVe(Xi), as described usly.

In certain embodiments, the glutathione-sensitive monomer oside or nucleotide or analog thereof) is formulated in a pharmaceutical ition comprising a therapeutically effective amount of the glutathione-sensitive nucleoside or nucleotide (or analog thereof) and a pharmaceutical carrier, as described in further detail below. 1. Formula XI In some embodiments, the glutathione-sensitive nucleoside or nucleotide is represented by the following formula: A2—U3_1 B R4 R1 R3 R2 /U2 X\ w2 L2 wherein L2 is a glutathione-sensitive moiety represented by Formula II, III or IV, or is absent if one of A2 or W2 is the glutathione-sensitive moiety; wherein if L2 is a glutathione-sensitive moiety, X is O, S, Se, or NR’, wherein R’ is selected from hydrogen, halogen, a substituted or unsubstituted aliphatic, an aryl, a tuted or unsubstituted heteroaryl or a substituted or unsubstituted cycle or if L2 is absent, X is H, OH, SH, NH2, halogen, optionally substituted alkoxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, ally substituted alkylthio, ally substituted 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), S02, N(R’), C(O), N(R’)C(O)O, OC(O)N(R’) optionally substituted aryl, optionally substituted heteroaryl, optionally tuted cyclic or optionally substituted cycloalkyl, O, S, Se or NHR’, wherein R’ is ed from hydrogen, halogen, a substituted or tituted tic, an aryl, a tuted or unsubstituted heteroaryl or a substituted or unsubstituted heterocycle, wherein R1, R2, R3 and R4 are each independently selected from hydrogen, halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl or n two of R1, R2, R3 and R4 are taken together to form a -8 membered ring, wherein the ring optionally contains a heteroatom, wherein J is O, S, NR’, CR’R”, n each of R’ and R” is independently selected from hydrogen, halogen, a substituted or unsubstituted aliphatic, aryl or heteroaryl, wherein B is selected from hydrogen, a natural nucleobase, a modified nucleobase or a universal nucleobase, wherein U2 is absent or selected from O, S, NR’, or CR’R”, wherein R’ and R” are each independently hydrogen, a substituted or unsubstituted aliphatic, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a tuted or unsubstituted heterocycle or a substituted or unsubstituted cycloalkyl, n W2 is a glutathione-sensitive moiety represented by Formula II, III or IV, hydrogen, halogen, OR’, SR’, NR’R”, a substituted or unsubstituted aliphatic, a substituted or unsubstituted aryl, a substituted or tituted heteroaryl, a substituted or unsubstituted cycloalkyl, a substituted or tituted heterocycle, wherein R’ and R” are each ndently selected from hydrogen, n, a substituted or tituted aliphatic, an aryl, a heteroaryl, a heterocycle or are taken together to form a heterocyclic ring, wherein I is absent or is selected from O, S, NR’, CR’R”, wherein R’ and R” are each independently hydrogen, a substituted or unsubstituted aliphatic, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocycle and a substituted or unsubstituted cycloalkyl, wherein U3 is hydrogen or selected from O, S, NR’ or CR’R”, n R’ and R” are each independently hydrogen, a substituted or unsubstituted aliphatic, a substituted or tituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocycle and a substituted or unsubstituted cycloalkyl, wherein I and U3 can be combined to form CR’-CR” alkyl, CR’-CR” alkenyl, CR’-CR” alkynyl, a substituted or unsubstituted aliphatic, an aryl, a heteroaryl, a heterocycle or taken together to form cycloalkyl or heterocyclic ring, and wherein A2 is absent, hydrogen, a phosphate group, a ate mimic, a phosphoramidate, or a glutathione-sensitive moiety represented by Formula II, III or IV.

In some embodiments, A2 is a glutathione-sensitive moiety represented by Formula II, III or IV. In some ments, W2 is a glutathione-sensitive moiety represented by Formula II, III or IV. In some embodiments, L2 is a glutathione-sensitive moiety ented by Formula II, III or IV and neither A2 nor W2 is a glutathione-sensitive moiety represented by a II, III or IV.

In some embodiments, the glutathione-sensitive moiety is represented by Formula II, as bed previously. In some embodiments, the glutathione-sensitive moiety is ented by Formula IIa, as described previously.

In other embodiments, the glutathione-sensitive moiety is represented by Formula III, as described previously. In some embodiments, Formula III is selected from Formula IIIa, IIIa(i), IIIb, or IIIb(i), as described previously. In yet other embodiments, the glutathione- sensitive moiety is represented by Formula IV, as described previously. In some embodiments, Formula IV is selected from Formula IVa, IVb, IVc, IVd, or IVe, as described previously. In some ments, Formula IV is selected from a , IVb(i), IVb(ii), , or IVd(i), as described previously. In some embodiments, Formula We is selected from a IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(v), IVe(vi), IVe(vii), IVe(viii), IVe(ix), IVe(X), or IV(xi), as described previously.

In certain embodiments, X is O. [0464l In certain embodiments, R1, R2, R3 and R4 are hydrogen. [0465l In certain embodiments, J is O. [0466l In certain embodiments, B is a natural nucleobase. [0467l In certain embodiments, U2 is O.

In certain embodiments, W2 is a en.

In certain embodiments, U3 is O.

In certain embodiments, I is CH2.

In certain ments, A2 is en or a phosphate group. [0472l In certain embodiments, X is 0, R1, R2, R3 and R4 are hydrogen, and J is O.

In certain embodiments, X is 0, R1, R2, R3 and R4 are hydrogen, J is O, B is a natural nucleobase, U2 is O, I is CH2, W2 is a hydrogen, U3 is O, and A2 is hydrogen or a ate group. 2. Formula XII In some embodiments, the glutathione-sensitive nucleoside or nucleotide monomer is represented by the following formula: wherein R10 is a hydroxyl, a phosphate mimic, or a phosphate group, n L is selected from Formulas II, III or IV, as described above, and wherein B is hydrogen, a natural nucleobase, a modified nucleobase or a universal nucleobase.

In some embodiments, L is represented by Formula II, as described previously. In some embodiments, L is represented by Formula IIa, as bed previously.

In other embodiments, L is represented by Formula III, as described previously. In some embodiments, Formula III is selected from Formula IIIa, IIIa(i), IIIb, or IIIb(i), as described previously.

In yet other embodiments, L is ented by Formula IV, as described previously. In some embodiments, Formula IV is selected from Formula IVa, IVb, IVc, IVd, or IVe, as described previously. In some embodiments, Formula IV is selected from Formula IVa(i), IVb(i), ), IVc(i), or IVd(i), as described previously. In some embodiments, Formula IVe is selected from Formula IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(v), IVe(vi), i), IVe(viii), IVe(iX), IVe(X), or IVe(Xi), as described previously.

C. Protecting Groups In some embodiments of the glutathione-sensitive nucleotides or nucleosides, a protecting group is attached to B, l'.e., the natural, modified or sal nucleobase. Suitable protecting groups for B include acetyl, oacetyl, trifluoroacetyl, isobutyryl, benzoyl, 9- ylmethoxycarbonyl, phenoxyacetyl, dimethylformamidine, dibutylforamidine and N, N diphenyl carbamate.

In some ments, a protecting group is attached to a hydroxyl group in the nucleosides described above, particularly for the nucleoside oramidites. Suitable ting groups for the hydroxyl groups of the above-described nucleosides e any protecting group that is compatible with solid phase oligonucleotide synthesis, including, but not limited to, dimethoxytrityl, monomethoxytrityl, and/or trityl groups. A typical example is 4, 4’-dimethoxytriphenylmethyl (DMTr) group, which may be readily cleaved under acidic ions (e.g. in the presence of dichlroacetic acid (DCA), trichloroacetic acid (TCA), trifluoracetic acid (TFA) or acetic acid).

Other typical hydroxyl protecting groups include trialkyl silyl groups, such as tertbutyldimethylsilyl (TBDMS). The TBDMS group is stable under the acidic conditions used to remove the DMT group during the synthesis cycle, but can be removed by a variety of methods after cleavage and deprotection of the RNA oligomer, e.g., with a solution of tetrabutylammonium fluoride (TBAF) in tetrahydrofurane (THF) or with triethylamine hydrofluoride. Other typical hydroxyl protecting groups include tert—butyldiphenylsilyl ether (TBDPS), which may be d with ammonium fluoride, for example.

IV. Nucleobases In the glutathione-sensitive oligonucleotides, nucleotides, and nucleosides described above, B represents a natural nucleobase, a modified nucleobase or a universal nucleobase.

Suitable natural nucleobases include purine and dine bases, e.g. adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U).

Suitable modified bases include diaminopurine and its derivatives, alkylated purines or pyrimidines, acylated purines or pyrimidines thiolated purines or pyrimidines, and the like.

Other suitable d bases include analogs of purines and pyrimidines.

Suitable analogs e, but are not limited to, l-methyladenine, 2-methyladenine, N6- methyladenine, N6-isopentyladenine, ylthio-N6-isopentyladenine, N,N— dimethyladenine, 8-bromoadenine, 2-thiocytosine, ylcytosine, ylcytosine, 5- ethylcytosine, 4-acetylcytosine, l-methylguanine, 2-methylguanine, 7-methylguanine, 2,2- dimethylguanine, 8-bromoguanine, roguanine, 8-aminoguanine, 8-methylguanine, 8- thioguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, uracil, 5-ethyluracil, 5- propyluracil, 5-methoxyuracil, oxymethyluracil, 5-(carboxyhydroxymethyl)uracil, 5- (methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 2-thiouracil, 5-methyl thiouracil, 5-(2-bromovinyl)uracil, oxyacetic acid, uraciloxyacetic acid methyl ester, pseudouracil, l-methylpseudouracil, queosine, hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine, nitropyrrolyl, nitroindolyl and difluorotolyl, 6-thiopurine and 2,6- diaminopurine nitropyrrolyl, ndolyl and difluorotolyl.

Typically a nucleobase contains a nitrogenous base. In certain embodiments, the nucleobase does not contain a nitrogen atom. See eg, US. Published Patent Application No. 20080274462.

A universal nucleobase refers to a base that can pair with more than one of the bases typically found in naturally occurring nucleic acids and can thus substitute for such naturally occurring bases in a duplex. The base need not be capable of pairing with each of the naturally occurring bases. For example, certain bases pair only or selectively With purines, or only or selectively with pyrimidines. The universal base may base pair by forming hydrogen bonds via -Crick or non-Watson-Crick interactions (e.g., Hoogsteen interactions).

Representative universal nucleobases include inosine and its derivatives.

V. Other Substituents in Formulas I-XII In Formulas I-XII, as appropriate, suitable aliphatic groups typically contain between about 2 and about 10 carbon atoms, more typically between about 2 and about 6 carbon atoms, such as between about 2 and about 5 carbon atoms.

In Formulas I-XII, as riate, suitable alkyl groups typically contain between about 1 and about 10 carbon atoms, more typically n about 2 and about 6 carbon atoms, such as between about 2 and about 5 carbon atoms.

In Formulas I-XII, as riate, le alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, xy, utoxy, neopentoxy and n-hexoxy and the like.

In Formulas I-XII, as appropriate, suitable cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like.

In Formulas I-XII, as appropriate, suitable heteroatoms include oxygen, sulfur, and nitrogen. Representative heterocycles include pyrrolidinyl, pyrazolinyl, pyrazolidinyl, olinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. Representative heteroaryls include furanyl, thienyl, pyridyl, pyrrolyl, N—lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl.

In as I-XII, as appropriate, suitable l groups include vinyl, allyl, and 2- methylheptene and suitable alkynyl groups include propyne, and 3-hexyne.

In Formulas I-XII, as appropriate, suitable aryl groups include phenyl, naphthyl and the like, while le aryl groups include pyridyl, furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, lyl, and the like.

In Formulas I-XII, as appropriate, suitable alkylaminos include -CH2CH2CH2NH- or CH2CH2NH-.

VI. Methods ofSynthesizing Oligonucleotides As sed above, this application discloses nucleosides comprising a glutathione- sensitive moiety that are compatible with standard, phosphoramidite-based oligonucleotide synthesis methods.

The glutathione-sensitive oligonucleotides described in this application can be made using a variety of synthetic methods known in the art, including standard phosphoramidite s. Any phosphoramidite synthesis method can be used to synthesize the glutathione- sensitive oligonucleotides of this invention. In certain embodiments, phosphoramidites are used in a solid phase synthesis method to yield reactive intermediate phosphite compounds, which are subsequently oxidized using known methods to produce glutathione-sensitive oligonucleotides, typically with a phosphodiester or phosphorothioate intemucleotide linkages.

The oligonucleotide synthesis of the present disclosure can be performed in either direction: from 5’ to 3’ or from 3’ to 5’ using art known methods.

Thus, in another aspect, the present disclosure relates to s of synthesizing oligonucleotides using a glutathione-sensitive nucleoside phosphoramidite, such as those discussed above and represented, for example by Formulas VIII, IX, or X. lly, the glutathione-sensitive moiety is located at the 2’-carbon of a ribose or deoxyribose (or analog thereof) and ses a yl group or a disulfide bridge, including, for example, the hione-sensitive moieties ented by Formulas II, III, and IV. In certain embodiments, the method for sizing an oligonucleotide comprises (a) attaching a nucleoside to a solid t via a covalent linkage, (b) coupling a nucleoside oramidite to a reactive hydroxyl group on the nucleoside of step (a) to form an intemucleotide bond therebetween, wherein any uncoupled nucleoside on the solid support is capped with a g reagent, (c) oxidizing said intemucleotide bond with an ing agent, and (d) repeating steps (b) to (c) iteratively with subsequent nucleoside phosphoramidites to form an oligonucleotide, wherein at least the nucleoside of step (a), the nucleoside phosphoramidite of step (b) or at least one of the subsequent nucleoside phosphoramidites of step (d) comprises a glutathione-sensitive moiety as described herein. Typically, the coupling, capping/oxidizing steps and optionally, deprotecting steps, are repeated until the oligonucleotide reaches the desired length and/or sequence, after which it is cleaved from the solid support.

In certain aspects, the oligonucleotide ses at least one nucleotide having a glutathione-sensitive moiety and is prepared by a phosphoramidite-based oligonucleotide synthesis method using a nucleoside phosphoramidite that comprises at least one glutathione- sensitive moiety. In certain embodiments, the ucleotide is prepared by a method comprising (a) attaching a nucleoside to a solid support via a covalent linkage, (b) coupling a nucleoside phosphoramidite to a reactive hydroxyl group on the nucleoside of step (a) to form an intemucleotide bond therebetween, wherein any led nucleoside on the solid support is capped with a capping reagent, (c) oxidizing said intemucleotide bond with an oxidizing agent, (d) ing steps (b) to (c) iteratively with subsequent nucleoside phosphoramidites to form an ucleotide, and (e) optionally cleaving the oligonucleotide from the solid support, wherein at least the nucleoside of step (a), the nucleoside phosphoramidite of step (b) or at least one of the subsequent nucleoside phosphoramadites of step (d) comprises a glutathione- sensitive moiety.

VII. Pharmaceutical Compositions The present sure provides pharmaceutical compositions sing a glutathione-sensitive ucleotide or a glutathione-sensitive nucleoside or nucleotide and a pharmaceutically acceptable ent.

In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient and a therapeutically effective amount of a nucleic acid inhibitor molecule, wherein the nucleic acid inhibitor molecule comprises at least one nucleotide comprising a hione-sensitive moiety, as described herein. As described elsewhere, the glutathione-sensitive moiety is lly d at the bon of the sugar moiety of the nucleotide and typically comprises a sulfonyl group or a disulfide bridge, including, such as the glutathione-sensitive moieties represented by Formulas II, III, or IV, as described previously.

In some embodiments, the ceutical composition comprises a pharmaceutically acceptable excipient and a therapeutically effective amount of a nucleic acid inhibitor le, wherein the nucleic acid inhibitor molecule comprises at least one glutathione- sensitive nucleotide represented by Formula I, V, VI, or VII, as described previously.

In other embodiments, the pharmaceutical composition ses a pharmaceutically acceptable excipient and a therapeutically effective amount of a glutathione-sensitive nucleoside or nucleotide, as represented, for example, by Formulas XI and XII, as described previously.

A. Pharmaceatically—Acceptable Excipients The pharmaceutically-acceptable excipients useful in this disclosure are conventional.

Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th n (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions. Some es of materials which can serve as pharmaceutically-acceptable ents include: , such as lactose, glucose and sucrose, starches, such as corn starch and potato starch, cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate, malt, gelatin, excipients, such as cocoa butter and suppository waxes, oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, buffering agents, such as magnesium hydroxide and um ide; (isotonic saline; Ringer's solution); ethyl alcohol; pH buffered ons; polyols; such as glycerol; propylene glycol; polyethylene glycol; and the like; and other non-toxic compatible substances employed in pharmaceutical formulations.

B. Dosage Forms The pharmaceutical compositions may be formulated with conventional excipients for any intended route of administration; which may be selected according to ordinary ce.

In one embodiment; the pharmaceutical composition contains a glutathione-sensitive ucleotide or glutathione-sensitive nucleotide or nucleoside; as described herein; and is suitable for parenteral administration. Typically; the pharmaceutical compositions of the present disclosure that contain oligonucleotides are formulated in liquid form for parenteral administration; for e; by subcutaneous; intramuscular; intravenous or al inj ection.

Dosage forms suitable for parenteral administration typically include one or more suitable vehicles for parenteral administration ing; by way of example; sterile aqueous solutions; saline; low molecular weight alcohols such as propylene glycol; polyethylene glycol; vegetable oils; gelatin; fatty acid esters such as ethyl oleate; and the like. The parenteral formulations may contain sugars; alcohols; antioxidants; buffers; bacteriostats; solutes which render the formulation ic with the blood of the intended recipient or suspending or thickening agents. Proper fluidity can be maintained; for example; by the use of surfactants. Liquid formulations can be lyophilized and stored for later use upon reconstitution with a sterile inj ectable solution.

In another embodiment; the pharmaceutical composition ns a glutathione- sensitive oligonucleotide or glutathione-sensitive nucleotide or nucleoside; as described herein; and is suitable for oral administration. Typically; the pharmaceutical compositions of the present disclosure that n nucleotides or nucleosides are formulated for oral administration. Suitable pharmaceutical itions for oral stration may be in the form of es; tablets; pills; lozenges; cachets; dragees; powders; granules and the like.

The pharmaceutical compositions may also be formulated for other routes of administration including topical or transdermal administration; rectal or vaginal stration; ocular administration; nasal administration; buccal administration; or sublingual administration using well known ques.

C. Delivery Agents The glutathione-sensitive nucleic acid inhibitor molecule; nucleotide; or side may be admixed; encapsulated; conjugated or otherwise ated with other molecules; WO 39364 molecule structures or mixtures of compounds, ing, for example, liposomes and lipids such as those disclosed in US. Patent Nos. 6,815,432, 6,586,410, 6,858,225, 7,811,602, 7,244,448 and 8,158,601, polymeric materials such as those disclosed in US. Patent Nos. 6,835,393, 7,374,778, 7,737,108, 7,718,193, 8,137,695 and US. Published Patent Application Nos. 2011/0143434, 2011/0129921, 2011/0123636, 2011/0143435, 2011/0142951, 2012/0021514, 2011/0281934, 2011/0286957 and 2008/0152661, capsids, capsoids, 0r receptor targeted molecules for ing in uptake, bution or absorption.

In certain embodiments, the glutathione-sensitive nucleic acid inhibitor molecule, nucleotide, or nucleoside is formulated in a lipid nanoparticle (LNP). nucleic acid nanoparticles typically form spontaneously upon mixing lipids with nucleic acid to form a complex. Depending on the desired particle size bution, the resultant rticle mixture can be optionally extruded through a polycarbonate membrane (e. g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as LIPEX® Extruder (Northern Lipids, Inc). To prepare a lipid nanoparticle for therapeutic use, it may desirable to remove solvent (e.g., ethanol) used to form the nanoparticle and/or exchange buffer, which can be accomplished by, for example, dialysis or tangential flow tion. s of making lipid nanoparticles containing nucleic acid interference les are known in the art, as disclosed, for example in US. Published Patent Application Nos. 374842 and 2014/0107178.

In certain embodiments, the LNP comprises a lipid core comprising a ic lipid and a pegylated lipid. The LNP can further comprise one or more envelope lipids, such as a cationic lipid, a structural or neutral lipid, a sterol, a pegylated lipid, or mixtures thereof.

In certain embodiments, an oligonucleotide of the invention is covalently conjugated to a ligand that directs delivery of the ucleotide to a tissue of interest. Many such ligands have been explored. See, e.g., r, Ther. Deliv., 4(7): 9 (2013). For example, an oligonucleotide of the invention can be conjugated to multiple sugar ligand moieties (e.g., N- acetylgalactosamine (GalNAc)) to direct uptake of the oligonucleotide into the liver. See, 6. g. , W0 2016/100401. Other ligands that can be used include, but are not limited to, mannose phosphate, cholesterol, folate, transferrin, and galactose (for other specific exemplary ligands see, e.g., ). Typically, when an oligonucleotide is conjugated to a ligand, the oligonucleotide is administered as a naked oligonucleotide, wherein the oligonucleotide is not also formulated in an LNP or other protective coating. In certain embodiments, the naked oligonucleotide contains at least one nucleotide having a glutathione—sensitive moiety, with the 2’-position of the sugar moiety of the remaining nucleotides of the naked oligonueleotide modified, typically with 2’—F or 250N163.

These pharmaceutical compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The ing aqueous solutions may be packaged for use as is, or lized, the lyophilized preparation being combined with a e aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The pharmaceutical compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above mentioned agent or agents, such as in a sealed package of tablets or capsules. The pharmaceutical compositions in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.

The pharmaceutical compositions of the t disclosure are applied for therapeutic use. Thus, one aspect of the disclosure es a pharmaceutical composition, which may be used to treat a subject including, but not limited to, a human suffering from a disease or a condition by administering to said subject an ive amount of a pharmaceutical ition of the t disclosure.

In certain embodiments, the present disclosure features the use of a eutically effective amount of a pharmaceutical composition as bed herein for the manufacture of a ment for treatment of a patient in need thereof VIII. Methods ofAdministration/Treatment The pharmaceutical compositions described herein are typically administered orally or parenterally. Pharmaceutical compositions containing the glutathione-sensitive nucleic acid inhibitor molecules of the invention are typically administered intravenously or subcutaneously. Pharmaceutical compositions containing the glutathione-sensitive nucleotides or sides ofthe invention are typically administered orally. However, the pharmaceutical itions disclosed herein may also be administered by any method known in the art, including, for example, buccal, sublingual, rectal, vaginal, intraurethral, topical, intraocular, intranasal, and/or intraauricular, which administration may include tablets, es, granules, aqueous suspensions, gels, sprays, suppositories, salves, ointments, or the like.

In certain embodiments, the pharmaceutical compositions disclosed herein may be useful for the treatment or prevention of ms related to a viral infection in a patient in need thereof. One embodiment is ed to a method of treating a viral infection, comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of a glutathione-sensitive nucleic acid inhibitor molecule, nucleotide, or nucleoside, as described herein. In certain ments, the pharmaceutical composition comprises a glutathione-sensitive nucleoside or nucleotide, as represented, for example, by Formulas XI and XII, as described previously. Non-limiting examples of such viral infections include HCV, HBV, HPV, HSV or HIV ion.

In certain embodiments, the pharmaceutical compositions disclosed herein may be useful for the treatment or prevention of symptoms related to cancer in a patient in need thereof.

One embodiment is directed to a method of treating cancer, comprising administering to a t a pharmaceutical composition sing a therapeutically ive amount of a glutathione-sensitive nucleic acid inhibitor molecule, as described . Non- limiting examples of such cancers include bilary tract cancer, bladder cancer, transitional cell carcinoma, urothelial carcinoma, brain cancer, gliomas, astrocytomas, breast carcinoma, metaplastic carcinoma, cervical cancer, cervical squamous cell carcinoma, rectal cancer, ctal carcinoma, colon , hereditary nonpolyposis colorectal cancer, colorectal arcinomas, gastrointestinal stromal tumors (GISTs), endometrial carcinoma, endometrial stromal sarcomas, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, ocular melanoma, uveal melanoma, gallbladder carcinomas, gallbladder adenocarcinoma, renal cell oma, clear cell renal cell carcinoma, transitional cell carcinoma, urothelial carcinomas, wilms tumor, leukemia, acute lymocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic (CLL), chronic myeloid (CML), c myelomonocytic (CMML), liver cancer, liver carcinoma, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, Lung , non-small cell lung cancer (NSCLC), mesothelioma, B-cell lymphomas, dgkin lymphoma, diffuse large B-cell lymphoma, Mantle cell ma, T-cell lymphomas, non-Hodgkin lymphoma, precursor T- blastic lymphoma/leukemia, peripheral T-cell lymphomas, multiple myeloma, aryngeal carcinoma (NPC), neuroblastoma, oropharyngeal , oral cavity squamous cell carcinomas, osteosarcoma, ovarian carcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, pseudopapillary neoplasms, acinar cell carcinomas. Prostate cancer, prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine carcinomas, h cancer, c carcinoma, gastrointestinal stromal tumor (GIST), uterine cancer, or uterine sarcoma. lly, the present disclosure features methods WO 39364 of treating liver cancer, liver carcinoma, hepatoma, hepatocellular carcinoma, cholangiocarcinoma and hepatoblastoma by administering a therapeutically effective amount of a pharmaceutical composition as described herein.

In certain embodiments the pharmaceutical compositions disclosed herein may be useful for treatment or prevention of symptoms related to proliferative, inflammatory, autoimmune, neurologic, , respiratory, lic, dermatological, auditory, liver, kidney, or infectious diseases. One embodiment is directed to a method of treating a proliferative, inflammatory, autoimmune, neurologic, ocular, atory, metabolic, dermatological, auditory, liver, kidney, or infectious disease, comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of a hione-sensitive nucleic acid inhibitor molecule, as described herein. Typically, the disease or condition is e of the liver.

In some embodiments, the present disclosure provides a method for reducing expression of a target gene in a subject comprising stering a pharmaceutical composition to a subject in need thereof in an amount sufficient to reduce expression of the target gene, wherein the pharmaceutical composition comprises a glutathione-sensitive nucleic acid inhibitor molecule as described herein and a pharmaceutically acceptable ent as also described herein.

In some embodiments, the glutathione-sensitive c acid inhibitor le is an RNAi inhibitor molecule as described herein, including a ssRNAi inhibitor molecule or a dsRNAi inhibitor molecule.

The target gene may be a target gene from any mammal, such as a human target gene.

Any gene may be ed according to the instant method. Exemplary target genes include, but are not limited to, Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, HBV, HCV, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, INK gene, RAF gene, Erkl/2 gene, PCNA(p2l) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-l gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, in gene, Her2/Neu gene, topoisomerase I gene, topoisomerase II alpha gene, p73 gene, p2l(WAFl/CIP1) gene, p27(KIPl) gene, PPMlD gene, RAS gene, caveolin I gene, MIB I gene, MTAI gene, M68 gene, ons in tumor suppressor genes, p53 tumor suppressor gene, LDHA, and combinations thereof.

In some embodiments the glutathione-sensitive nucleic acid inhibitor le silences a target gene and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted expression of the target gene. For example, in some embodiments, the present hione-sensitive nucleic acid tor molecule silences the beta-catenin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted beta- catenin expression, e.g., adenocarcinoma or hepatocellular carcinoma.

In certain embodiments, the pharmaceutical composition is delivered via systemic administration (such as via intravenous or subcutaneous administration) to relevant tissues or cells in a subject or organism, such as the liver. In other embodiments, the pharmaceutical composition is red via local administration or systemic administration. In certain embodiments, the pharmaceutical composition is delivered via local administration to relevant tissues or cells, such as lung cells and tissues, such as via pulmonary delivery.

The therapeutically effective amount of the compounds disclosed herein may depend on the route of stration and the physical characteristics of the patient, such as the size and weight of the subject, the extent of the disease progression or penetration, the age, health, and sex of the t. As used herein, a therapeutically effective amount means an amount of nd or compounds effective to prevent, alleviate or ameliorate disease or condition symptoms of the subject being treated.

In certain embodiments, the glutathione-sensitive oligonucleotide, nucleotide or nucleoside is administered at a dosage of 20 micrograms to 10 milligrams per kilogram body weight of the recipient per day, 100 micrograms to 5 milligrams per kilogram, 0.25 milligrams to 2.0 rams per kilogram, or 0.5 to 2.0 rams per kilogram.

A pharmaceutical composition of the instant disclosure may be administered every day, or intermittently. For example, intermittent administration of a compound of the instant disclosure may be administration one to six days per week, one to six days per month, once weekly, once every other week, once monthly, once every other month, or once or twice per year or divided into le yearly, monthly, , or daily doses. In some embodiments, ittent dosing may mean administration in cycles (e. g. daily administration for one day, one week or two to eight consecutive weeks, then a rest period with no administration for up to one week, up to one month, up to two , up to three months or up to six months or more) or it may mean administration on alternate days, weeks, months or years.

WO 39364 In any of the s of treatment of the invention, the compounds may be administered to the subject alone as a erapy or in combination with additional therapies known in the art.

EXAMPLES Example 1. Synthesis ofglutathione—sensitive compounds All drolytic reactions, unless ted otherwise, were carried out in dry solvents purchased from Sigma-Aldrich Corporation (St. Louis, MO). High Performance Liquid Chromatography (HPLC), except for the amidites, was performed at 60 °C using an Agilent ZORBAX® Eclipse Plus (Agilent Technologies Company, Santa Clara, CA) C18, 21 X 50 eter (mm), 1.8 micron column, 100 X 4.6 mm, 2.7 micron column with ammonium formate (3 millmolar) as a modifier under otherwise identical conditions. UV traces were recorded at 220 ter (nm) and mass a were obtained using an Agilent Technologies 6140 Quadrapole LC/MS mass spectrometer in both positive and negative ion mode.

Preparative purifications were performed by gradient chromatography on a Teledyne Isco COMBIFLASH® Rf using pre-packed columns (Teledyne Isco, Inc., n, NE). NMR spectra were recorded on a Varian UNITY® 600, 500 or 400 spectrometers, Varian, Inc. (Palo Alto, CA). nd 8b The below scheme 1 depicts the synthesis of a glutathione-sensitive compound comprising a disulfide bridge: (2R,3R,4R,5R)((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)(((2 cyanoethyl)(diisopropylamino)phosphino)oxy)(2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)tetrahydrofuranyl (2-(tert-butyldisulfanyl)phenyl)carbamate (Compound 8b). The glutathione-sensitive moiety of Compound 8b is encompassed by Formula IVe and more specifically is represented by Formula IVe(ix). To demonstrate the feasibility of preparing this compound, a simple model oligomer with eight nucleotides was synthesized. The key ediate, phosphoramidite 8b, was synthesized according to the procedure shown in the scheme below. In brief, commercially available tert butyl thiol was converted to activated thiosulfonate 2b, which was subsequently reacted with 2-aminothiophenol to obtain disulfide compound 4b. The 4b compound was next treated with triphosgene generated isocyanate intermediate 5b. Without prior isolation, the isocyante 5b compound was “in-situ reacted with ethoxytrityl (DMtr)-protected uridine to afford a mixture of 2’,3’-protected carbamates.

We ed migration of carbamate from the 2’- to the 3’- position during chromatography WO 39364 ation. To avoid this undesired migration, a 1% pyridine solution was used during silica gel purification. After separation of the undesired isomers, compound 7b was ted to phosphitylation conditions, as typically used with synthesized oramidite. Compound 8b was then purified by silica gel column chromatography as commonly used during the purification of standard cyanoethyl group-containing phosphoramidites. Compound 8b exhibited similar physiochemical behavior, including stability, to standard phosphoramidite compounds.

H2N i \k \k S S 3/ triphosgene 5/ x MsCl, pyridine 0%,,0 3 CHzclzv ElaN H2N O=C=N —> —> S” S MeOH step 1 step 2 step 3 1b 2b 4b 5b + . . : 3, ~ 0 ,S é c , 0 OH 0 O 8 ~ 0 + HO OH E) / HO 3 3+ 0=[\ 0=[\ 6 HN HN NH NH —, 693+ 695+ 7b' 7b" NC/\/O\,NP I \(Ny'll— 8/.

YN N (1Heq.) step 5 (1-1 eq-) pyridine (1 eq.) , CHZCIZ [lN” DMTrOVo 0 )Nfifi 0 55+ d H ‘33 (5 Scheme 1 sis ofS-tert-butyl methanesulfonothioate (2b) To a solution of tert—butylthiol (1b) (20 grams (g)); 0.22 mole (mol); 1 equivalent (equiv.) aldehyde (Ald.)) in dry pyridine (100 mL; Ald. anhydrous) was dropwise added methanesulfonyl de (17.1 milliliter (mL); 0.22 mol; Ald.). The reaction was stirred at room temperature and monitored by Thin Layer Chromatography (TLC): hexane: EtOAc = 6 :1; visualized with phosphomolybdic acid (PMA); Retardation factor (Rf) = 0.44. After overnight, the reaction was te and the reaction was diluted with Et20; then acidified with 4N HCl. The aqueous phase was extracted with Et20; separated and dried over anhydrous NazSO4. After concentrating with a Rotary Evaporator (rotavap); the crude product was purified by ISCO chromatography (ISCO REDISEP® (Teledyne Isco; Inc); 330 g) and eluted with 0% to 100% of EtOAc in hexane (monitored by UV: 254 nm; 280 nm). The desired fractions were ed and evaporated to give a colorless oil of 2b (20 g; 53%); Proton nuclear magnetic resonance (1H NMR) (300 megahertz (MHz); chloroform-d (CDCl3) spectrum is as follows: 3.33 (s; 3H); 1.58 (s; 9H)).

Synthesis 0f2-(tert-bu02ldisulfanyl)aniline (4b) To a solution of ortho-aminobenzenethiol (3)(12.8 mL; 0.12 mol; 1 equiv. Acros) in MeOH (200 mL; Ald. anhydrous) was added S—z‘ert—butyl methanesulfonothioate 2b (20 g; 0.12 mol; 1 ) and the reaction was stirred at room temperature under N2. The reaction was monitored by TLC & mass spectrometry (MS): hexane : EtOAc = 6 :1; visualized with PMA; Rf: 0.68; MS heric pressure chemical ionization (APCI) [M+1]: 214.0 (100%). After 2h; the reaction was complete and concentrated by rotavap. The crude product was purified by ISCO chromatography (ISCO REDISEP®; 330 g) and eluted with 0% to 100% of EtOAc in hexane (monitored by UV: 254 nm; 280 nm). The desired fractions were combined and evaporated to give a ess oil of 4b (25 g; 98%). 1H NMR (300 MHz; CDCl3) spectrum is as s: 7.50 (dd; J = 7.68; 1.38 Hz; 1H); 7.09 (td; J = 7.41; 1.38 Hz; 1H); 6.67 (m; 2H); 1.34 (s; 9H). MS: (APCI+) M+1 = 214.0.

Synthesis 0f1-(tert-butyl)(2-is0cyanat0phenyl)disulfane (5b) Under 0°C ice-water bath, to a solution of 2-(tert—butyldisulfanyl)aniline (4b) (10 g, 46.8 mol, 1 ) in CH2C12 (500 mL, Ald. anhydrous) was added sgene (13.9 g, 46.8 mol, 1 equiv., Acros), followed by the addition of Et3N (65.3 mL, 0.46 mol, 10 equiv., Ald. anhydrous) and the reaction was stirred at 0°C for 1h. The reaction was concentrated by p (water bath: room temp.) and the obtained crude solid was used in the next step directly.

Synthesis of (2R,3R,4R,5R)((bis(4-meth0x;yphenyl)@henmeethoxy)methyl)—2—(2,4- di0x0-3,4-dihydr0pyrimidin-1(2H)-yl)hydr0xytetrahydr0furanyl (2—(tert- butyldisulfanybphenyDcarbamate (7b) Under 0°C ter bath, to a solution of crude 1-(tert—butyl)—2-(2- isocyanatophenyl)disulfane (5b) (crude, 2 equiv.) in CH2C12 (500 mL, Ald. anhydrous ) was added 1-((2R,3R,4S,5R)((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4- dihydroxytetrahydrofuranyl)pyrimidine-2,4(1H,3H)-dione 6 (13 g, 23.78 mmol,1 equiv, carbosynth) and the reaction was d for 1.5 h, allowing 0°C to room temperature. The reaction was monitored by TLC, hexane : EtOAc = 1 :2, visualize with PMA. TLC showed the t 7b (Rf = 0.38) as well as 7b' (regioisomer, Rf = 0.19) and 7b” (dicarbamate, Rf = 0.61). After 1.5 h, the reaction was concentrated and mixed with EtOAc (100 mL) and the insoluble salt was filtered. The filtrate was diluted with EtOAc (500 mL), washed with saturated NaHCO3, H20, Brine and dried over anhydrous Na2804. After concentration by rotavap, the crude product was loaded onto a pre-equilibrated silica-gel column and purified by ISCO chromatography (ISCO REDISEP®, 120 g, pre-equilibrated with 0.5% pyridine/Hexane)1, and eluted with 0% to 100% of EtOAc in hexane (monitored by UV: 254 nm, 280 nm). The desired fractions were combined and evaporated to give a colorless foam 2.6 g of 7b (14%) with the purity of 94% (HPLC). 1H NMR (300 MHz, DMSO-d6) um is as follows: 11.42 (s, 1H), 9.38 (s, 1H), 7.72 (m, 2H), 7.24 — 7.38 (m, 13H), 6.88 — 6.91 (m, 4H), 6.01 (d, J = 4.95 Hz, 1H), 5.70 (d, J = 5.79 Hz, 1H), 5.37 (dd, J = 7.95, 2.19 Hz, 1H), .30 (t, J = 3.09 Hz, 1H), 4.41 (dd, J = 11.01, 5.49 Hz, 1H), 3.73 (s, 6H), 3.21 — 3.30 (m, 2H), 1.21 (s, 9H). MS: (APCI-) M-1 = 784.2.

(Step 5): Synthesis of (2R,3R,4R,5R)((bis(4-meth0xyphenyl)(phenmeethoxgy)methyl) (((2-cyan0ethyl)(diisopropylamin0)ph0sphin0)0xy)-2—(2,4-di0x0-3,4-dihydr0pyrimidin- 1(2H)-yl)tetrahydrofuran-3—yl (2-(tert-butyldisulfanyl)phenyl)carbamate (8b) To a solution of (2R,3R,4R,5R)((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)- 2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)hydroxytetrahydrofuranyl (2-(z‘ert- isulfanyl)phenyl)carbamate (7b) (1.6 g, 2.03 mol, 1 equiv.) in CH2C12 (40 mL, Ald. anhydrous) was added pyridine (0.16 mL, 2.03 mol, 1 equiv, Ald. ous) and 5- (ethylthio)-1H-tetrazole (265 mg, 2.03 mmol, 1 equiv, Ald.) at room temperature under N2.

Then O-cyanoethyl-N,N,N’,N’-tetraisopropyl phospharodiamidite (674.9mg, 2.23 mmol, 1.1 equiv, nes Corporation, Wilmington, MA) was added. The reaction was stirred at room temp and monitored by TLC: hexane : EtOAc = 1 :2, visualize with PMA, Rf = 0.51.

After 2 h, the reaction was te and the reaction was diluted with CH2C12 (400 mL) and washed with saturated (sat.) NaHCO3, H20, Brine and dried over anhydrous NazSO4. After concentration by rotavap, the crude product was loaded onto a pre-equilibrated silica-gel column and purified by ISCO chromatography (ISCO REDISEP®, 40 g, pre-equilibrated with 1.0% Et3N in hexane), and eluted with 0% to 100% of EtOAc in hexane (1% Et3N) (monitored by UV: 254 nm, 280 nm). The desired fractions were combined and evaporated to give a colorless foam 1.6 g of8b (77%) with the purity of 97% . 1H NMR (300 MHz, DMSO- d6) spectrum is as follows: 11.47 (s, 1H), 9.45 (m, 1H), 7.74 (m, 2H), 7.25 — 7.38 (m, 13H), 6.87 — 6.90 (m, 4H), 6.02 (m, 1H), 5.40 — 5.49 (m, 2H), 4.62 (m, 1H), 4.21 (m, 1H), 3.73 (s, 6H), 3.48 — 3.65 (m, 4H), 3.32 (m, 1H), 2.73 (m, 1H), 2.62 (t, J = 6.3 Hz, 1H), 1.19 (s, 9H), 0.94 — 1.12 (m, 12H). 31P NMR (161 MHz, DMSO-d6) 150.44, . MS: (APCI-)M-1= 984.4. nd 8d The below scheme 3 depicts the synthesis of a glutathione-sensitive compound comprising a disulfide bridge: (2R,3R,4R,5R)((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)(((2- thoxy)(diisopropylamino)phosphino)oxy)(2,4-dioxo-3 ,4-dihydropyrimidin-1(2H)- yl)tetrahydrofuranyl (2-((tert-butyldisulfanyl)methyl)phenyl)carbamate (Compound 8d).

The glutathione—sensitive moiety of Compound 8d is encompassed by Formula IVe and more specifically is represented by Formula IVe(ii). The nucleoside phosphoramidite 8d was synthesized by following the analogous procedure described for the synthesis of 8b. Briefly, commercially available 2-amino benzyl alcohol was transiently protected with a Boc group to afford 1d-2. A Mitsunobu reaction of 1d-2 in the presence of thioacetic acid afforded the thioester intermediate 1d-2. Selective hydrolysis ofthe thioester with NaOMe/MeOH followed by treatment with S-z‘ert—butyl methanesulfonothioate ed the nd 3d. After Boc deprotection with trifluoracetic acid (TFA), 4d was converted to isocyanate ediate 5d, and “in-situ” reacted with 5’-dimethoxytriphenylmethyl (DMTr)-protected uridine to afford mixture of 2’- and 3’-protected carbamates 7d and 7d’. After column chromatography separation, itylation of 7d afforded the required phosphoramidite 8d as ess foam in 50% yield.

NHz NHBoc 5 Me SH PPh3, DIAD T OH B0020,THF OH AcSH,THF 0 BOCHN _, BocHN NaOMe MeOH 1d 1d-2 1d-2 1d-3 /\OS//OS ’etriphosgene J< BocHN F—. CHZCI2 Et3N MeOH 0=C=N step 2 steps [Km 0 NH I DMTrO o N’ko I (lNH N’h /§ DMTrO N o o DMTrO’W o k V H6 bH + _ _ :- 6 o i H6 in O OH —. 0:4 step4 NC/\/o‘ ’N\( ,N_N '1’ NZ ksfi (‘LNH DMTrO Y 7/N N H AV: $1k (1 6(1) : 5 .) )\_N 0P, "< \ HN pyridine (1 eq.) , CHZCIZ OW Scheme 3 (Step 1): Synthesis aftert-buljyl (2-(hydroxymethybphenyl)carbamate (Id-1) To a solution of (2-aminophenyl)methanol (1d) (10 g, 81.2 mol, 1 equiv. Ald.) in tetrahydrofuran (THF) (200 mL, Ald. anhydrous ) was added BOC2O (18.6 g, 85.2 mmol, 1.1 W0 2018/039364 equiv. AK scientific). The reaction was d at room temperature and monitored by TLC: hexane : EtOAc = 6 :1, visualize with PMA, Rf = 0.2. After overnight, the on was complete and the reaction was diluted with EtOAc (500 mL), washed with saturated NaHCO3, H20, brine and dried over anhydrous NazSO4. After concentration by rotavap, the crude product was purified by ISCO chromatography (ISCO REDISEP®, 220 g), and eluted with 0% to 100% of EtOAc in hexane (monitored by UV: 254 nm, 280 nm). The desired fractions were combined and evaporated to give a colorless foam 15 g of 1d-1 (82%). 1H NMR (300 MHz, CDCl3) spectrum is as follows: 7.90 (d, J = 7.98 Hz, 1H), 7.61 (s, 1H), 7.30 (t, J = 7.68 Hz, 1H), 7.16 (d, J = 7.41 Hz, 1H), 7.00 (t, J = 7.41 Hz, 1H), 4.68 (s, 2H), 1.51 (s, 9H).

(Step 2): Synthesis ofS-Z-((tert-butoxycarbonyl)amino)benzyl ethanethioate (1d—Z) To a solution of Ph3P (23.6 g, 90.3 mmol, 2.1 equiv. Ald.) in THF (300 mL) was added diisopropyl azodicarboxylate (DIAD) (17.7 mL, 90.3 mmol, 2.1 equiv. Ald.) at 0°C and the mixture was stirred for 30 min. A mixture of utyl (2-(hydroxymethyl)phenyl)carbamate (1d-1) (9.6 g, 43 mol, 1 equiv.) and thioacetic acid (6.3 mL, 90.3 mmol, 2.1 equiv. Ald.) in THF (100 mL) was added dropwise to the above reaction mixture. The reaction was stirred, allowing to warm to room temperature and was monitored by TLC: hexane : EtOAc = 6 :1, visualized with PMA, Rf = 0.47. After overnight, the mixture was diluted with EtOAc (500 mL), washed with sat. , H20, brine and dried over anhydrous Na2804. After concentration by p, the crude product was purified by ISCO chromatography (ISCO REDISEP®, 220 g), and eluted with 0% to 100% of EtOAc in hexane (monitored by UV: 254 nm, 280 nm). The desired fractions were combined and evaporated to give a colorless foam g of 1d-2 (88%). 1H NMR (300 MHz, DMSO-d6) spectrum is as follows: 8.64 (s, 1H), 7.19 — 7.30 (m, 3H), 7.05 (t, J = 7.41 Hz, 1H), 4.10 (s, 2H), 2.28 (s, 3H), 1.41 (s, 9H).

(Step 3): Synthesis oftert-butyl (2-(mercaptomethprhenyl)carbamate (1d-3) To a solution of tert—butoxycarbonyl)amino)benzyl thioate (1d-2) (11.8 g, 41.9 mmol, 1 equiv.) in MeOH (200 mL) was added NaOMe (2.2 g, 41.9 mol, 1 equiv. Ald.) and the reaction was stirred at room temperature and was monitored by TLC: hexane : EtOAc = 6 :1, ized with PMA, Rf: 0.5. After 2h, the reaction was complete and was acidified with 1N HCl to pH ~6, then concentrated by rotavap. The crude product was dissolved in EtOAc (500 mL), washed with H20, brine and dried over anhydrous NazSO4. After concentration, the crude product 1d-3 was used directly in the next step.

(Step 4): Synthesis 0f2-((tert-bntyldisnlfanyl)methyl)aniline (3d) To a solution of tert—butyl (2-(mercaptomethyl)phenyl)carbamate (1d-3)(10 g, 41.9 mol, 1 equiv.) in MeOH (200 mL, Ald. anhydrous) was added S—tert—butyl methanesulfonothioate 2b (9.2 g, 54.5 mmol, 1.3 equiv.), followed by the addition of Et3N (17.5 mL, 125.8 mol, 3 equiv. Ald, anhydrous). The reaction was stirred at room temperature under N2 and was monitored by TLC: hexane : EtOAc = 6 :1, visualize with PMA, Rf = 0.6.

After 2h, the reaction was complete and concentrated by rotavap. The crude product was d by ISCO chromatography (ISCO REDISEP®, 80 g) and eluted with 0% to 100% of EtOAc in hexane (monitored by UV: 254 nm, 280 nm). The desired ons were combined and evaporated to give a white solid of 3d (5.8 g, 42%). 1H NMR (300 MHz, 6) spectrum is as follows: 8.62 (s, 1H), 7.19 — 7.30 (m, 3H), 7.05 (t, J = 7.41 Hz, 1H), 4.02 (s, 2H), 1.41 (s, 9H), 1.21 (s, 9H).

(Step 5): Synthesis 0f2-((tert-bntyldisnlfanyl)methyl)aniline (4d) 2-((tert—butyldisulfanyl)methyl)aniline (3d) (3 g, 9.16 mol, 1 equiv.) was added into a e solution of TFA/CH2C12 (15 mL/45mL) and the reaction was stirred at room temperature for 2 hours. The reaction was concentrated by rotavap (water bath: room temperature) and the obtained crude product 4d was used directly in the next step.

(Step 6): Synthesis 0f1-(tert-bntyl)(Z-isocyanatobenzyl)disnlfane (5d) Under 0°C ice-water bath, to a solution of rt—butyldisulfanyl)methyl)aniline (4d) (crude, 2 g, 8.8 mmol, 1 equiv.) in CH2C12 (100 mL, Ald. anhydrous) was added triphosgene (2.6 g, 8.8 mol, 1 equiv., Acros), followed by the addition of Et3N (12.3 mL, 0.09 mol, 10 equiv., Ald. anhydrous) and the reaction was stirred at 0°C for 1hour. The on was concentrated by rotavap (water bath: room temperature) and the obtained crude solid 5d was used in the next step directly.

(Step 7): Synthesis of ,4R,5R)((bis(4-meth0xyphenyl)(phenyl)meth0xy)methyl) i0x0-3,4-dihydr0pyrimidin-1(2H)-yl)hydr0xytetrahydr0fnranyl (2-((tert- bntyldisnlfanyl)methyl)phenyl)carbamate (7d) Under 0°C ice-water bath, to a solution of crude 1-(tert—butyl)—2-(2- isocyanatobenzyl)disulfane (5d) (crude, 2 equiv.) in CH2C12 (100 mL, Ald. anhydrous ) was W0 2018/039364 added 1-((2R,3R,4S,5R)((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4- dihydroxytetrahydrofuranyl)pyrimidine-2,4(1H,3H)-dione 6 (2.4 g, 4.39 mmol,1 equiv, carbosynth) and the reaction was stirred, allowing 0°C to room temperature. The reaction was monitored by TLC, hexane : EtOAc = 1 :4, visualize with PMA. TLC showed the product 7d (Rf: 0.35) as well as 7d' (regioisomer, Rf: 0.18) and 7d” (dicarbamate, Rf: 0.71). After overnight, the reaction was concentrated and mixed with EtOAc (100 mL) and the insoluble salt was filtered. The filtrate was diluted with EtOAc (500 mL), washed with sat. NaHCO3, H20, brine and dried over anhydrous NazSO4. After concentration by rotavap, the crude t was loaded onto a pre-equilibrated silica-gel column and purified by ISCO chromatography (ISCO REDISEP®, 80 g, pre-equilibrated with 0.5% pyridine/Hexane)1, and eluted with 0% to 100% c in hexane (monitored by UV: 254 nm, 280 nm). The desired fractions were combined and evaporated to give a colorless foam 339 mg of 7d (10%) with the purity of 79.3% (HPLC)2’3. 1H NMR (300 MHz, DMSO-d6) spectrum is as follows: 11.45 (s, 1H), 9.27(s, 1H), 7.73 (d, J = 8.25 Hz, 1H), 7.23 — 7.40 (m, 13H), 6.88 — 6.96 (m, 4H), 6.04 (d, J = 4.65 Hz, 1H), 5.72 (d, J = 5.49 Hz, 1H), 5.38 (d, J = 7.98 Hz, 1H), 5.26 (t, J = 5.22 Hz, 1H), 4.45 (dd, J = 10.44, 5.22 Hz, 1H), 4.05 (m, 1H), 3.72 (s, 6H), 3.32 (m, 1H), 3.26 (m, 1H), 1.21 (s, 9H).

(Step 8): Synthesis of ,4R,5R)((bis(4-meth0xyphenyl)(phenmeethoxy)methyl)—4— (((2-cyan0eth0xy)(diisopropylamin0)ph0sphin0)0x;y)—2—(2,4-di0x0-3,4-dihydr0pyrimidin- yl)tetrahydrofuran-3—yl (2-((tert-butyldisulfanyl)methyl)phenyl)carbamate (8d) To a solution of (2R,3R,4R,5R)((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)- 2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)hydroxytetrahydrofuranyl (2-((tert- isulfanyl)methyl)phenyl)carbamate (7d) (239 mg, 0.30 mol, 1 equiv) in CH2C12 (10 mL, Ald. anhydrous) was added pyridine (0.02 mL, 0.30 mol, 1 equiv, Ald. Anhydrous) and -(ethylthio)-1H—tetrazole (38.9 mg, 0.30 mol, 1 equiv, Combi-Blocks) at room temperature under N21. Then O-cyanoethyl-N,N,N’,N’-tetraisopropyl phospharodiamidite (99.1 mg, 0.33 mmol, 1.1 equiv, nes Corporation) was added. The on was stirred at room temperature and monitored by TLC: hexane : EtOAc = 1 :4, visualize with PMA, Rf = 0.69.

After 6 h, the reaction showed the expect product as the major spot and the reaction was diluted with CH2C12 (200 mL) and washed with sat. NaHCO3, H20, brine and dried over anhydrous . After concentration by rotavap, the crude product was loaded onto a pre-equilibrated silica-gel column and purified by ISCO chromatography (ISCO REDISEP®, 24 g, pre- WO 39364 2017/048239 equilibrated with 1.0% Et3N in hexane), and eluted with 0% to 100% of EtOAc in hexane (1% Et3N) (monitored by UV: 254 nm, 280 nm). The desired fractions were combined and evaporated to give a colorless foam 151 mg of 8d (50%) with a purity of 94.4% (HPLC)2. 1H NMR (300 MHz, DMSO-d6) spectrum is as follows: 11.49 (s, 1H), 9.41 (m, 1H), 7.71 (m, 1H), 7.13 — 7.40 (m, 14H), 6.87 — 6.90 (m, 4H), 6.05 (m, 1H), 5.41 — 5.45 (m, 2H), 4.68 (m, 1H), 4.25 (m, 1H), 4.08 (m, 1H), 3.98 (m, 1H), 3.72 (s, 6H), 3.52 — 3.65 (m, 4H), 3.34 (m, 1H), 2.63 (m, 1H), 2.53 (m, 1H), 1.21 (s, 9H), 0.96 — 1.12 (m, 12H). 31P NMR (161 MHz, DMSO- d6) 150.45, 150.34. MS: (APCI-)M-1 = 999.4.

Compound 8h The below scheme 4 depicts the synthesis of a glutathione-sensitive compound comprising a disulfide und 8h): ethyl N-((((2R,3R,4R,5R)—5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)(((2- cyanoethoxy)(diisopropylamino)phosphaneyl)oxy)(2,4-dioxo-3,4-dihydropyrimidin- 1(2H)-yl)tetrahydrofuranyl)oxy)carbonyl)—S -(tert-butylthio)-L-cysteinate. The hione-sensitive moiety of Compound 8h is encompassed by Formula IVd and more specifically is represented by Formula IVd(i).

HN SJ< | HN | I A A I S i'PKIO O N . o O N I-Pr\,- o O N -NO PhCOCI "Pr“. 2 ,Si p SI 0 I-Pr_ I ,Si O —>i—Pr’l H2N COzEt Ox.

I-Pr. I 0‘, . ,LPr’Sk O‘S' "Pr’sl’"o lP? O O o O - i-Pr’ I'~ H i—Pr DIPEA (P9 0 f ”N s’S\|< Q COZEt O N02 0 0 . 0 O N “b H”) ° N 0 1811115211: Ho 0 N i—Pr’él y' ' q 0 O ,-_pr«S|»i\O o o Dioxane/THF(2/1) ,-_pr Y 200 OH 0Y0 0H 0Y0 Yxs, KS HNWAS,S\|< Mrs/SK COzEt COzEt (:02E Y HN 0‘ / DMTO O N NON P \< r I o \ /\ \b 7/N s NOW ,3o o o o (1.1 eq) (1.0eq) \f \r \l/ HNf3 K,s ne(1.0eq),DCM c023 Scheme4 Compound 8i The below scheme 5 depicts the synthesis of a glutathione-sensitive compound comprising a sulfone (Compound 8i): ,4R,5R)((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)(((2- cyanoethoxy)(diisopropylamino)phosphaneyl)oxy)(2,4-dioxo-3,4-dihydropyrimidin- 1(2H)-yl)tetrahydrofuranyl (2-(phenylsulfonamido)ethyl)carbamate. The glutathione- sensitive moiety of Compound Si is assed by Formula IVb and more specifically is ented by Formula IVb(ii), wherein R is hydrogen. 0 o HN A\ N 19 A I | o o N o N A ’ P \s o p-NOzPhCOCI r\,_ u IPK: o H NN 2 0 0 - o O N ,SI ,SI I—Pr\ I Pr 6 I , —> I Pr Si O \ IPr’I 1-Pr’3.' o o DIPEA IPr’SI' H O Q 0:3 o o o \ IPr Y I r N\/\ )8 IPr ,' N OH O \b iPr D O H TBAF (3.0 equiv) 0 DMTrCl/Py DMTrO O Py.HC| (3.0 Eqiv) —, o H 0 J3 OH 0 N \‘ \ll/ \/\N S\\ OH O H Q‘s/Q o H 0 731/ \AN/“O Y O N HN 0‘ / NC/\/ P \< N-N A I [l] [\l, \ /\ DMTrO O N N s o (1.1eq) (1.0 eq) H O Q 0 O N “ O‘P/ \n/ \/\ ,3 .. \ N Pyrldlne(1.Oeq)xDCM NCN “ N\( H O \( 8i Schemes Example 2. Synthesis of glutathione—sensitive oligonucleotides Oligonucleotides were synthesized on a commercial oligo synthesizer. Test Compounds 1 and 2 (Figure 1B) were synthesized using 2’-modified nucleoside oramidites, i.e., 2’-F and 2’-OMe d nucleoside phosphoramidites, and 2’- glutathione-sensitive nucleoside phosphoramidites. Test Compounds 1 and 2 contained one tide having a reversible, glutathione-sensitive modification at the 2’-carbon, while the remaining nucleotides contained irreversible 2’-F or 2’-OMe modifications. 2017/048239 Oligonucleotide synthesis was carried out on a solid support in the 3’ to 5’ direction.

The standard oligonucleotide synthesis protocol was employed. The ng time was 300 seconds with lthio-1H-tetrazole (ETT) as an activator. Iodine solution was used for phosphite triester oxidation. Synthesized oligonucleotides were treated with concentrated aqueous ammonium at 55°C for 10 hours. After removal of ammonia in the suspensions, CPG’s were removed by filtration. After the addition of triethylammonium acetate (TEAA), the crude oligonucleotides were analyzed and d by strong anion ge high performance liquid chromatography (SAX-HPLC). The obtained oligonucleotide ons were pooled and concentrated and were desalted with water. Finally, oligonucleotides were lyophilized to a powder.

An oligonucleotide guide strand was synthesized for each of the two test compounds.

One guide strand had a single 2’-glutathione-sensitive nucleoside located at nucleotide position 1 (i.e., the 5’-terminal nucleotide). The other guide strand had a single 2’-glutathione-sensitive nucleoside located at nucleotide on 14. The two guide strands contained the same nucleotide sequence that was complementary to a target mRNA sequence. ingly, the two oligonucleotide guide strands for Test Compound 1 and 2 were identical except for the nucleotide position of the 2’-glutathione-sensitive nucleoside moiety.

The above-described s was then repeated to prepare mentary oligonucleotide passenger strands, which did not n a glutathione-sensitive moiety. The passenger strands were further modified by ating a phosphoramidite to a polyethylene glycol-GalNAc ligand via a spacer. The GalNAc terminated polyethylene glycol was conjugated via click chemistry to the 2’-carbon of four nucleotides of the tetraloop ure in the passenger strand using methods known in the art (see, e.g., W0 2016/100401).

Duplexes were formed by mixing each of the two complementary strands (guide and passenger) in a 1:1 molar ratio to obtain two dsRNAi inhibitor molecules: Test Compound 1 and Test Compound 2. See Figure 1B. Test Compound 1 contains a 22-base pair guide strand having a 2’-glutathione-sensitive moiety at nucleotide positon 1 and a 36-base pair ger strand without any glutathione-sensitive moiety, where the passenger strand contains four nucleotides in the tetraloop that are each conjugated to a polyethylene glycol-GalNAc ligand (see Figure 1B). Test Compound 1 also had a free hydroxyl group (5’-OH) at the 5’-carbon at the 5’-end of the guide strand. Except for the glutathione-sensitive nucleotide at nucleotide position 1 ofthe guide , the remaining nucleotides in Test Compound 1 were irreversibly modified with either 2’-F or 2’-OMe.

Test Compound 2 contains a 22-base pair guide strand having a 2’-glutathione-sensitive moiety at tide positon 14 and a 36-base pair passenger strand without any glutathione- sensitive moiety, where the passenger strand contains four nucleotides in the tetraloop that are each conjugated to a polyethylene -GalNAc ligand (see Figure 1B). Except for the hione-sensitive nucleotide at nucleotide position 14 of the guide strand, the remaining nucleotides in Test Compound 2 were irreversibly modified with either 2’-F or 2’-OMe.

It has been reported that bulky if1ed nucleosides are generally not well tolerated at nucleotide position 14 of double stranded RNAi inhibitor molecules (Zheng et al., FASEB Journal, 2013, 27(2):1-10), and that small functional moieties, such as 2’-F or 2’-OMe, are preferably used to modify nucleosides at position 14. In Test Compound 2, the bulky 2’- glutathione-sensitive moiety at on 14 is cleaved by glutathione in the cytosol to yield a much smaller hydroxyl group at the 2’-carbon, which also happens to be the natural substituent for a ribonucleotide at that carbon position. Thus, it was expected that Test Compound 2 would have little to no RNA inhibition activity unless the glutathione-sensitive moiety was ed from Test Compound 2. As such, Test Compound 2 es a test for in viva removal of the glutathione-sensitive moiety.

Two control double stranded RNAi inhibitor molecules (Control Compound A and Control Compound B) were also prepared as described above except that none of the nucleotides in the control compounds included a glutathione-sensitive moiety. See Figure 1A.

All of the nucleotides in the control compounds were irreversibly modified with 2’-F or 2’- OMe in the same pattern as the Test Compounds (other than the position modified with the glutathione-sensitive moiety). Control Compound A was synthesized with natural phosphate (5’-PO42') at the 5’-carbon of the 5’-terminal nucleotide of the guide strand, whereas l Compound B contained a free hydroxyl group ) at the 5’-carbon of the 5’-terminal nucleotide of the guide strand. The guide strands of Control Compounds A and B ned the same nucleotide sequence and, therefore, recognized the same target mRNA sequence as Test Compounds 1 and 2.

Example 3. Release kinetics of 2'-reversibly modified side and oligonucleotide A reversibly-modified nucleoside (uridine) ning a glutathione-sensitive moiety at the 2’-carbon, as shown below in Scheme 7, was ed. e studies of the 2’- glutathione-sensitive uridine were conducted by dissolving the modified nucleoside in PBS buffer containing a 500-fold excess of glutathione (5 mM glutathione) at pH 7.5. The progress ofthe disulfide release s was monitored by RP-HPLC. RP-HPLC showed two new peaks corresponding to the intermediate species “int. A” and “int. B”, which are depicted in Scheme 7 below. The intermediate species were slowly converted to the desired e and the benzothiazolone release product, as shown in Scheme 7 below.

As shown in Scheme 7 below, the release mechanism for the 2’-glutathione-sensitive uridine proceeds through a two-step reaction. The first step is a disulfide exchange reaction after re to glutathione, which is rapid, and results in full conversion to the glutathione adducts within 30-60 minutes. The initial disulfide cleavage produces two intermediates “int.

A” and “int. B.” The second step is rapid intramolecular ation via O->S acyl transfer reaction to release benzothiazolone from the nucleoside, leaving a hydroxyl group at the 2’- position of the nucleoside. The reaction c data for benzothiazolone formation supported the biphasic profile of uridine formation. The half-life (t 1/2) for cyclization and release of benzothiazolone (resulting in free uridine) was approximately 4 hours. See Figure 2.

O O HN HN 0:< O=< HN / Glutathione HO / HO N N O=< / I: D,O O HO ; ; Glutathione g 3 ' ' OH 0Y0 s—sk (~5mM) OH O O ECi 3 OH 0 U \KJSH ——_ CH3 HN pH~7.4 HN LEN '“l-A Int.B _ 2'-PD _ Ho =<N / 6H 6H Uridine Scheme7 The release rate was also determined for a glutathione-sensitive oligonucleotide (i.e., Test Compound 2), as shown in Figure 3, which shows a time course of the percentage of Test nd 2 ing. 500 equivalents of glutathione (21 mg) was added to the Test Compound 2 (1 mg) at pH 7.5 in 10 mM phosphate buffer (volume of 10 mL). The rate of Test Compound 2 disappearance was monitored by RP-HPLC. As is evident in Figure 3, the reaction is bi-phasic. The half-life (ti/2) for cyclization and release of the glutathione-sensitive 2017/048239 moiety from Test Compound 2 was approximately 6.5 hours (about 400 minutes). See Figure Example 4. In vitro potency of Test Compound 1 Murine hepatocytes The ability of Test Compound 1 to own expression of a target mRNA was tested in vitro. As noted above, the test oligonucleotides and l oligonucleotides recognize the same target sequence. Test Compound 1 and Control Compounds A and B were reverse transfected into murine hepatocytes using LIPOFECTAMINE® RNAiMax (Thermo Fisher Scientific Inc., Rockville, MD) in a 96 well plate as per manufacturer’s protocol. The final concentration of the test and control oligonucleotides ranged from 1000pM to 0.06pM. 12000 cells/well were added to the plate. The plate was incubated at 37 0C for 48 hours. At the end of 48 hours, the cells were lysed by adding 30ul of ISCRIPTTM lysis buffer per well. 22 ul of the lysate was transferred to a fresh plate and used to make cDNA as per the manufacturer’s protocol. Quantitative PCR was performed with the target sequence normalized to human SFR69 gene (hSFRS9-F569 (HEX)) at 55 0C. Graphs were plotted using GraphPad Prism (GraphPad Software Inc., La Jolla, CA), and the ICso values were calculated.

Figure 4 s the potency of different concentrations of Test Compound 1 in the lipid ection assay after 48 hours. Control Compound A (having a 5’-natural phosphate at the ’-terminal nucleotide of the guide strand) had an ICso of about 8.7 pM, s Control Compound B (having a 5’-hydroxyl at the 5’-terminal nucleotide of the guide strand) was less effective at reducing expression of the target mRNA, with an ICso of about 24.5 pM. For Test nd 1, the ICso was about 13.5 pM. This IC50 value of Test Compound 1 was more comparable to Control Compound A, suggesting that the 5’-hydroxyl at the 5’-terminal nucleotide of the guide strand of Test Compound 1 was phosphorylated by a kinase in the cytosol. It is expected that release of the glutathione-sensitive moiety at the 2’-carbon at nucleoside position 1 of the guide strand of Test Compound 1 in the l makes the 5’- hydroxyl more amenable to kinase phosphorylation, which in turn should tate Ago2- mediated RISC loading of the guide strand for target mRNA knockdown.

Monkey hepatocytes Primary monkey hepatocytes were obtained from Life Technologies Corporation bad, CA) and thawed and plated as per manufacturer’s protocol in CORNING® BIOCOATTM 96 well plates. After 4-6 hours of plating, the media was ed with 90ul of Williams E incubation media per well. Test Compound 1 was serially diluted starting with a 2017/048239 concentration of luM to 12.8pM (5-fold reduction). 10ul of Test nd 1 was added to the tive wells in the absence of LIPOFECTAMINE® o Fisher Scientfic, Inc).

The plate was incubated at 37°C and knockdown of an RNA target was tested at 24 hours. At the end of 24 hours, target RNA was extracted and purified using SV96 Total RNA Isolation System (Promega, Madison, WI) as per the manufacturer’s protocol. cDNA was prepared using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems Corporation).

Quantitative PCR was carried out at 60 0C with the target sequence normalized to Homo s peptidyl prolyl isomerase B PPIB. Graphs were plotted using the GraphPad Prism software (GraphPad Software Inc.) and the IC50 values were calculated. Figure 5 shows the potency at 24 hours of different concentrations of Test Compound I delivered to primary monkey hepatocytes in the absence of any lipid transfection agent. Test Compound 1 had an ICso of 1.6 nM at 24 hours.

Example 5. In vivo potency and duration of effect for Test Compounds 1 and 2 Test Compound 1 and Test Compound 2 were diluted in PBS to a 1 mg/kg working solution. On the same day as the PBS dilution, CD-l female mice were injected subcutaneously with a single 1 mg/kg dose of Test nd 1, Test Compound 2, or a control PBS solution.

Post-dosing (3, 10, 21 and 28 days) s were exsanguinated by cardiac puncture after euthanasia in C02. The left medial lobe of the liver was removed and a 1-4 mm punch was removed and placed into a 96 well plate on dry ice. After all samples were collected, RNA and cDNA were prepared for quantitative PCR (qPCR). All samples were ed in triplicate and qPCR was performed using the CFX384 TOUCHTM Real-Time PCR Detection System (BioRad Laboratories, Inc., Hercules, CA). All samples were then normalized to the PBS treated control animals and blotted using GraphPad Prism software.

Figure 6 depicts an in viva duration study of Test Compound 1. Subcutaneous injection of Test Compound 1 at 1 mg/kg resulted in more than 50% own of target RNA at day 3. Increased levels of target RNA knockdown were observed at day 10, suggesting slow release of the glutathione-sensitive moiety to generate an oligonucleotide substrate that is more amenable to kinase phosphorylation and subsequent Ago-2 mediated RISC loading for target gene knockdown. These results te that conjugating a glutathione-sensitive moiety to the 2’-carbon of the nucleoside at position 1 of the guide strand of a dsRNAi tor le can stabilize the dsRNAi tor molecule during transit to the l ofthe cell and facilitate effective knockdown of target RNA in the cytosol, where the glutathione-sensitive moiety of the oligonucleotide is removed in the presence of glutathione.

WO 39364 Figure 7 depicts an in viva duration study of Test nd 2. As noted above, Test Compound 2 is modified with a glutathione-sensitive moiety at the 2’-carbon at nucleotide position 14 of the guide strand (see Figure 1B), a nucleotide position that generally does not tolerate bulky modifications at the 2’-carbon. Thus, it was expected that Test Compound 2 would have little to no RNA knockdown effect unless the bulky, hione-sensitive moiety was released from Test Compound 2. As shown in Figure 7, subcutaneous injection of Test Compound 2 at 1 mg/kg resulted in about 50% knockdown oftarget RNA by day 10, suggesting slow in viva release of the glutathione-sensitive moiety in the cytosol to generate a l 2’- OH in place of the reversible, glutathione-sensitive moiety at nucleotide position 14 of the guide strand.

We

Claims (55)

Claim:

1. A glutathione-sensitive oligonucleotide, wherein the glutathione-sensitive oligonucleotide comprises at least one nucleotide represented by Formula I: A U1 I B R4 R1 R3 R2 U2 X wherein X is O, S, Se or NR′, wherein R′ is selected from hydrogen, halogen, an aliphatic, an aryl, a heteroaryl or a heterocycle; wherein R1, R2, R3 and R4 are each independently selected from hydrogen, halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl or wherein two of R1, R2, R3 and R4 are taken together to form a 5-8 membered ring, wherein the ring optionally contains a heteroatom; n J is O, S, NR′, CR′R″, wherein each of R′ and R″ is independently ed from hydrogen, n, an aliphatic, aryl or aryl; wherein B is selected from hydrogen, a natural nucleobase, a modified nucleobase or a universal nucleobase; wherein U2 is absent or selected from O, S, NR′, or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a aryl, a heterocycle or a lkyl; wherein W is hydrogen, a phosphate group, an ucleotide 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, a cycloalkyl, a cycle, wherein R′ and R″ are each independently selected from hydrogen, halogen, an aliphatic, an aryl, a heteroaryl, a heterocycle or are taken together to form a heterocyclic ring; wherein I is absent or is selected from O, S, NR′, CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl, a heterocycle and a cycloalkyl; wherein U1 is absent, hydrogen, an internucleotide linking group attaching the at least one nucleotide represented by a I to a nucleotide or an oligonucleotide, or selected from O, S, NR′ or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl, a cycle and a cycloalkyl and wherein at least one of U1 or W is an ucleotide linking group attaching the at least one nucleotide represented by Formula I to a nucleotide or an oligonucleotide and provided that if U1 is an ucleotide linking group, A is absent; wherein I and U1 can be combined to form ″ alkyl, CR′-CR″ alkenyl, CR′- CR″ alkynyl, an aliphatic, an aryl, a heteroaryl a heterocycle or taken together to form cycloalkyl or heterocyclic ring; n A is absent, a hydrogen, a phosphate group, a phosphate mimic or a phosphoramidate; and wherein L is a glutathione-sensitive moiety represented by: Formula IIa: IIa; Formula IIIa(i): IIIa(i); Formula IIIb(i): IIIb(i); Formula IVa(i): IVa(i); Formula IVb(i): IVb(i); Formula IVb(ii): IVb(ii); wherein, in Formula IVb(i) and Formula IVb(ii), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand ally connected via a ; Formula IVc(i): IVc(i); wherein, in Formula , R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected via a Formula IVd(i): IVd(i); Formula IVe(i): IVe(i); Formula IVe(ii): IVe(ii); Formula IVe(iii): IVe(iii); Formula ): IVe(iv); Formula IVe(ix): IVe(ix); Formula IVe(x): IVe(x); Formula IVe(xi): IVe(xi); wherein, in Formulae IVe(iii), IVe(iv), and IVe(x), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected via a spacer.

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

3. The glutathione-sensitive oligonucleotide ing to claim 1 or 2, wherein the oligonucleotide is a double-stranded oligonucleotide comprising a first strand and a second strand.

4. The glutathione-sensitive oligonucleotide according to claim 3, wherein the double ed oligonucleotide is a double-stranded RNAi inhibitor molecule and the first strand comprises a sense strand and the second strand comprises an antisense strand.

5. The glutathione-sensitive oligonucleotide ing to claim 4, wherein the double stranded RNAi tor molecule comprises a region of complementarity between the sense strand and the antisense strand of about 15 to 45 nucleotides.

6. The glutathione-sensitive oligonucleotide according to claim 5, wherein the region of complementarity n the sense strand and the antisense strand is 20 to 30, 21 to 26, 19 to 24, or 19 to 21 nucleotides.

7. The glutathione-sensitive oligonucleotide according to any one of claims 4-6, wherein the at least one nucleotide represented by Formula I is located on the antisense .

8. The glutathione-sensitive oligonucleotide according to any one of claims 4-6, wherein the at least one nucleotide represented by a I is located on the sense strand.

9. The glutathione-sensitive oligonucleotide according to claim 7, wherein the at least one nucleotide represented by Formula I is located at nucleotide position 1 of the antisense strand.

10. The glutathione-sensitive ucleotide according to claim 7, wherein the at least one nucleotide ented by Formula I is located at nucleotide on 14 of the antisense strand.

11. The hione-sensitive oligonucleotide according to any one of claims 4-6 or 8, wherein the at least one tide represented by Formula I is located at a nucleotide position at or adjacent to the Ago2 cleavage site of the sense strand.

12. The glutathione-sensitive oligonucleotide according to any one of claims 4-11, wherein the double stranded RNAi inhibitor molecule ns a tetraloop.

13. The glutathione-sensitive oligonucleotide according to claim 1 or 2, wherein the ucleotide is a single stranded oligonucleotide.

14. The glutathione-sensitive oligonucleotide according to claim 13, wherein the single stranded oligonucleotide is a single stranded RNAi inhibitor molecule.

15. The glutathione-sensitive oligonucleotide according to claim 13, wherein the single-stranded oligonucleotide is a conventional antisense oligonucleotide, a ribozyme, a microRNA, an antagomir, or an aptamer.

16. The glutathione-sensitive oligonucleotide according to claim 14 or 15, n the single stranded RNAi inhibitor molecule is about 14-50, 16-30, 18-22, or 20-22 tides in length.

17. The glutathione-sensitive oligonucleotide according to any one of the preceding claims, wherein the glutathione-sensitive ucleotide contains 1-5 nucleotides represented by Formula I.

18. The glutathione-sensitive oligonucleotide according to any one of the preceding claims, 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 modification.

19. The glutathione-sensitive oligonucleotide according to any one of the preceding claims, further comprising a delivery agent, wherein the delivery agent facilitates transport of the hione-sensitive oligonucleotide across an outer membrane of a cell.

20. The glutathione-sensitive oligonucleotide according to claim 19, n the delivery agent is selected from the group consisting of ydrates, peptides, , vitamins and antibodies.

21. The glutathione-sensitive oligonucleotide ing to claim 19 or 20, wherein the delivery agent is selected from N-Acetylgalactosamine (GalNAc), mannosephosphate, galactose, oligosaccharide, polysaccharide, cholesterol, polyethylene glycol, , vitamin A, vitamin E, lithocholic acid and a cationic lipid.

22. The glutathione-sensitive oligonucleotide according to any one of the preceding claims, wherein the glutathione-sensitive oligonucleotide is a naked, glutathione-sensitive oligonucleotide.

23. The glutathione-sensitive oligonucleotide according to claim 1 or 2, wherein the hione-sensitive oligonucleotide is a red Regularly Interspaced Short Palindromic Repeats “CRISPR” nucleic acid sequence having a crRNA sequence having a first portion capable of hybridizing to a target sequence in a cell and/or a tracrRNA sequence that hybridizes with a second portion of the crRNA sequence to form a guide sequence.

24. The glutathione-sensitive oligonucleotide according to claim 23, wherein the guide sequence is a chimeric guide sequence and wherein the crRNA sequence is fused to the tracrRNA sequence.

25. A pharmaceutical composition comprising the glutathione-sensitive ucleotide according to any one of the preceding claims and a pharmaceutically acceptable carrier.

26. A ceutical composition comprising the hione-sensitive oligonucleotide according to any one of claims 3-22 and a ceutically acceptable carrier.

27. Use of the glutathione-sensitive oligonucleotide according to any one of claims 3- 22 and a ceutically acceptable carrier in the cture of a medicament for reducing expression of a target gene.

28. The use of claim 27, wherein the medicament is formulated for systemic administration.

29. A nucleoside phosphoramidite, wherein the nucleoside phosphoramidite is represented by a VIII: VIII wherein L1 is a glutathione-sensitive moiety represented by Formula IIa, IIIa(i), IIIb(i), IVa(i), IVb(i), IVb(ii), IVc(i), IVd(i), IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(ix), IVe(x) or IVe(xi); IIa; IIIb(i); IVa(i); IVb(i); IVb(ii); wherein, in Formula IVb(i) and Formula ), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a cycle or R is a targeting ligand optionally connected via a spacer; IVc(i); wherein, in Formula IVc(i), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally ted via a spacer; IVd(i); IVe(i); IVe(ii); IVe(iv); IVe(x); IVe(xi); wherein, in Formulae IVe(iii), ), and IVe(x), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected via a ; wherein A1 is absent, hydrogen, a phosphate group, a phosphate mimic, a phosphoramidate, a protecting group, or a solid support; wherein W1 is a phosphoramidite, a protecting group, a solid support, hydrogen, halogen, OR′, SR′, NR′R″, an aliphatic, an aryl, a heteroaryl, a cycloalkyl, a heterocycle, wherein R′ and R″ are each independently selected from hydrogen, halogen, an aliphatic, an aryl, a heteroaryl, a heterocycle or are taken together to form a heterocyclic ring; wherein U3 is a hydrogen or selected from O, S, NR′ or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl, a cycle and a cycloalkyl; wherein at least A1 is a phosphoramidite and U3 is O or at least W1 is a phosphoramidite and U2 is O; wherein X is O, S, Se or NR′, wherein R′ is selected from hydrogen, halogen, an aliphatic, an aryl, a heteroaryl or a heterocycle; wherein R1, R2, R3 and R4 are each independently selected from hydrogen, halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl or wherein two of R1, R2, R3 and R4 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″, n each of R′ and R″ is independently selected from hydrogen, halogen, an aliphatic, aryl or heteroaryl; wherein B is selected from hydrogen, an aliphatic, a natural nucleobase, a modified nucleobase or a universal nucleobase; wherein U2 is absent or selected from O, S, NR′, or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl, a heterocycle or a cycloalkyl; wherein I is absent or is selected from O, S, NR′, CR′R″, n R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl, a heterocycle and a cycloalkyl; and wherein I and U3 can be combined to form CR′-CR″ alkyl, CR′-CR″ alkenyl, CR′-CR″ alkynyl, an aliphatic, an aryl, a heteroaryl a heterocycle or taken together to form lkyl or cyclic ring.

30. The nucleoside phosphoramidite of claim 29, wherein the nucleoside phosphoramidite is represented by Formula IX: wherein L1 is a glutathione-sensitive moiety represented by Formula IIa, IIIa(i), IIIb(i), IVa(i), IVb(i), IVb(ii), , IVd(i), IVe(i), IVe(ii), i), IVe(iv), IVe(ix), IVe(x) or IVe(xi); wherein R9 is a phosphoramidite; wherein X is O, S, Se or NR′, wherein R′ is ed from hydrogen, halogen, an tic, an aryl, a heteroaryl or a heterocycle; wherein R1, R2, R3 and R4 are each independently selected from hydrogen, halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl or wherein two of R1, R2, R3 and R4 are taken together to form a 5-8 membered ring, wherein the ring optionally contains a atom; n J is O, S, NR′, CR′R″, wherein each of R′ and R″ is independently selected from hydrogen, halogen, an aliphatic, aryl or aryl; wherein B is hydrogen, a natural nucleobase, a modified nucleobase or a universal nucleobase; wherein I is absent or is selected from O, S, NR′, CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl, a cycle and a cycloalkyl; wherein U3 is a en or selected from O, S, NR′ or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl, a cycle and a cycloalkyl; wherein I and U3 can be combined to form CR′-CR ″ alkyl, CR′-CR ″ alkenyl, CR′-CR ″ alkynyl, an aliphatic, an aryl, a heteroaryl a cycle or taken together to form cycloalkyl or heterocyclic ring; and wherein A3 is absent, hydrogen, a phosphate group, a phosphate mimic, a phosphoramidate, a protecting group, or a solid support.

31. The nucleoside phosphoramidite of claim 29 or 30, wherein the hionesensitive moiety (L1) is represented by Formula IVb(ii), IVd(i), IVe(ii) or IVe(ix).

32. The nucleoside phosphoramidite of claim 29, wherein J is O; B is a natural base; U2 is O; I is CH2; W1 is a phosphoramidite; A1 is a protecting group, hydrogen, or solid support; and U3 is O.

33. The nucleoside phosphoramidite of claim 32, wherein X is O and R1, R2, R3 and R4 are hydrogen.

34. The nucleoside phosphoramidite of claim 29, n J is O; B is a natural nucleobase; U2 is O; I is CH2; W 1 is a protecting group, hydrogen or solid support; A1 is a phosphoramidite, and U3 is O.

35. The nucleoside phosphoramidite of claim 34, wherein X is O and R1, R2, R3 and R4 are hydrogen.

36. The nucleoside oramidite of any one of claims 29-35, wherein the phosphoramidite has the a —P(ORx)—N(R y) 2, wherein Rx is selected from the group consisting of a methyl, 2-cyanoethyl and benzyl, wherein each of Ry is selected from the group consisting of an ethyl and isopropyl.

37. A method for preparing a glutathione-sensitive oligonucleotide comprising: (a) attaching a nucleoside to a solid t via a nt linkage; (b) coupling the nucleoside phosphoramidite according to any one of claims 29-36 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) ing said phosphorus nucleoside e with an oxidizing reagent; and (d) repeating steps (b) to (d) iteratively with one or more subsequent nucleoside phosphoramidites according to any one of claims 29-36 or one or more subsequent side phosphoramidites that do not contain a glutathione-sensitive moiety, to form the glutathionesensitive oligonucleotide; and (f) optionally removing said glutathione-sensitive oligonucleotide from said solid support.

38. The method according to claim 37, wherein the glutathione-sensitive moiety comprises a disulfide bridge or sulfonyl group.

39. A glutathione-sensitive nucleoside or nucleotide, wherein the glutathione-sensitive nucleoside or nucleotide comprises a glutathionesensitive moiety; wherein the glutathione-sensitive moiety is bound to an oxygen atom that is covalently bound to the 2′-carbon of the sugar moiety of the nucleotide or side; and wherein the glutathione-sensitive moiety is represented by Formula IIa, IIIa(i), IIIb(i), IVa(i), IVb(i), IVb(ii), IVc(i), IVd(i), IVe(i), IVe(ii), IVe(iii), ), IVe(ix), IVe(x) or IVe(xi): IIa; IIIa(i); IVa(i); IVb(i); IVb(ii); wherein, in Formula IVb(i) and Formula IVb(ii), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected via a spacer; IVc(i); wherein, in Formula IVc(i), R is ed from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally ted via a IVd(i); IVe(i); IVe(ii); IVe(iv); IVe(ix); IVe(x); IVe(xi); wherein, in Formulae IVe(iii), IVe(iv), and IVe(x), R is ed from en, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected via a spacer.

40. The glutathione-sensitive nucleoside or nucleotide of claim 39, wherein the glutathione-sensitive moiety is ented by Formula IVb(ii), IVd(i), IVe(ii) or IVe(ix).

41. A glutathione-sensitive nucleoside or nucleotide, wherein the glutathionesensitive nucleoside or nucleotide is represented by Formula XI: wherein L2 is a glutathione-sensitive moiety represented by Formula IIa, IIIa(i), IIIb(i), IVa(i), IVb(i), IVb(ii), , IVd(i), IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(ix), IVe(x) or IVe(xi): IIa; IIIa(i); IIIb(i); IVa(i); IVb(i); IVb(ii); wherein, in Formula IVb(i) and Formula IVb(ii), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand ally connected via a spacer; IVc(i); wherein, in Formula , R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected via a spacer; IVd(i); IVe(i); IVe(ii); IVe(iii); IVe(iv); IVe(ix); IVe(xi); wherein, in Formulae IVe(iii), IVe(iv), and IVe(x), R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected via a spacer; or n L2 is absent if one of A2 or W2 is the glutathione-sensitive moiety represented by a IIa, IIIa(i), IIIb(i), IVa(i), IVb(i), IVb(ii), IVc(i), IVd(i), IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(ix), IVe(x) or IVe(xi); wherein if L2 is a glutathione-sensitive moiety, X is O, S, Se, or NR′, wherein R′ is selected from hydrogen, halogen, an aliphatic, an aryl, a heteroaryl or a heterocycle or if L2 is absent, X is H, OH, SH, NH2, halogen, alkoxy, alkyl, alkenyl, alkynyl, alkylthio, mino or lamino wherein one or more methylenes in the alkyl, alkenyl, and alkynyl may be interrupted with one or more of O, S, S(O), SO2, 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 selected from en, halogen, an aliphatic, an aryl, a aryl or a heterocycle; wherein R1, R2, R3 and R4 are each independently selected from en, halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl or wherein two of R1, R2, R3 and R4 are taken together to form a 5-8 membered ring, wherein the ring optionally contains a atom; wherein J is O, S, NR′, CR′R″, wherein each of R′ and R″ is independently selected from hydrogen, halogen, an aliphatic, aryl or heteroaryl; n B is selected from hydrogen, a natural nucleobase, a modified nucleobase or a universal nucleobase; wherein U2 is absent or selected from O, S, NR′, or CR′R″, wherein R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl, a heterocycle or a cycloalkyl; wherein W2 is a glutathione-sensitive moiety represented by Formula IIa, IIIa(i), IIIb(i), IVa(i), IVb(i), IVb(ii), IVc(i), , IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(ix), IVe(x) or IVe(xi); hydrogen, halogen, OR′, SR′, NR′R″, an aliphatic, an aryl, a heteroaryl, a cycloalkyl, a heterocycle, wherein R′ and R″ are each independently selected from hydrogen, halogen, an aliphatic, an aryl, a heteroaryl, a heterocycle or are taken together to form a heterocyclic ring; wherein I is absent or is selected from O, S, NR′, CR′R″, wherein R′ and R″ are each ndently hydrogen, an aliphatic, an aryl, a heteroaryl, a cycle and a cycloalkyl; wherein U3 is hydrogen, or selected from O, S, NR′ or CR′R″, n R′ and R″ are each independently hydrogen, an aliphatic, an aryl, a heteroaryl, a heterocycle and a cycloalkyl; wherein I and U3 can be combined to form CR′-CR ″ alkyl, CR′-CR ″ alkenyl, CR′-CR ″ alkynyl, an aliphatic, an aryl, a heteroaryl, a heterocycle or taken er to form cycloalkyl or heterocyclic ring; and n A2 is absent, hydrogen, a phosphate group, a phosphate mimic, a phosphoramidate, or a glutathione-sensitive moiety represented by Formula IIa, ), IIIb(i), IVa(i), IVb(i), ), IVc(i), IVd(i), IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(ix), IVe(x) or IVe(xi).

42. The glutathione-sensitive nucleoside or nucleotide of claim 41, wherein J is O; X is O; L2 is a glutathione-sensitive moiety represented by Formula IIa, IIIa(i), IIIb(i), IVa(i), IVb(i), IVb(ii), IVc(i), IVd(i), IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(ix), IVe(x) or IVe(xi); W2 is hydrogen, halogen, OR′, SR′, NR′R″, an aliphatic, an aryl, a heteroaryl, a cycloalkyl, a heterocycle, n R′ and R″ are each independently selected from hydrogen, halogen, an aliphatic, an aryl, a heteroaryl, a heterocycle or are taken together to form a heterocyclic ring; and A2 is absent, hydrogen, a phosphate group, a phosphate mimic, or a oramidate.

43. The glutathione-sensitive nucleoside or nucleotide of claim 42, n R1, R2, R3, and R4 are hydrogen; U2 is oxygen; W2 is hydrogen; I is CH2; U3 is O; and A2 is hydrogen or a ate group.

44. The glutathione-sensitive nucleoside or nucleotide of any one of claims 41-43, wherein the glutathione-sensitive moiety is represented by Formula IVb(ii), IVd(i), IVe(ii) or

45. The glutathione-sensitive oligonucleotide of claim 1, wherein the glutathionesensitive ucleotide comprises at least one nucleotide represented by a VIIe(ix): A U1 B O O S U2 S VIIe(ix) wherein A is absent, a hydrogen, a phosphate group, or a phosphate mimic; wherein U1 is O or an internucleotide linking group attaching the at least one nucleotide represented by Formula x) to a nucleotide or an oligonucleotide; wherein B is a natural base; n U2 is O; wherein W is hydrogen or an internucleotide linking group attaching the at least one nucleotide represented by Formula VIIe(ix) to a nucleotide or an oligonucleotide, wherein at least one of U1 or W is an internucleotide linking group attaching the at least one nucleotide represented by a VIIe(ix) to an oligonucleotide and provided that if U 1 is an internucleotide g group, A is absent; and wherein the glutathione-sensitive oligonucleotide is a double-stranded RNAi inhibitor molecule comprising a sense strand and an antisense strand.

46. The glutathione-sensitive oligonucleotide of claim 45, wherein A is hydrogen and W is an ucleotide linking group attaching the at least one nucleotide represented by Formula VIIe(ix) to an oligonucleotide and n the at least one nucleotide represented by a VIIe(ix) is d at tide position 1 of the antisense strand.

47. The glutathione-sensitive oligonucleotide of claim 45, wherein A is absent; W is a internucleotide linking group attaching the at least one nucleotide represented by Formula VIIe(ix) to a first oligonucleotide; and U1 is a internucleotide linking group attaching the at least one nucleotide represented by a VIIe(ix) to a second oligonucleotide; and wherein the at least one nucleotide represented by Formula x) is located at nucleotide position 14 of the antisense strand.

48. The glutathione-sensitive oligonucleotide of claim 1, wherein the glutathionesensitive oligonucleotide comprises at least one nucleotide represented by Formula VIIe(xi): VIIe(xi) wherein A is absent, a hydrogen, a phosphate group, or a phosphate mimic; wherein U1 is O or an internucleotide linking group ing the at least one nucleotide represented by Formula VIIe(xi) to a nucleotide or an oligonucleotide; wherein B is a natural nucleobase; wherein U2 is O; wherein W is hydrogen or an internucleotide linking group attaching the at least one nucleotide represented by Formula VIIe(xi) to a nucleotide or an oligonucleotide, wherein at least one of U1 or W is an internucleotide linking group attaching the at least one nucleotide represented by Formula VIIe(xi) to an oligonucleotide and provided that if U 1 is an internucleotide linking group, A is absent; and wherein the hione-sensitive oligonucleotide is a double-stranded RNAi inhibitor molecule comprising a sense strand and an antisense strand.

49. The nucleoside phosphoramidite of claim 29, n the nucleoside phosphoramidite is represented by Formula X: n R8 is H or a protecting group; R7 is a oramidite; B is a natural nucleobase; and X is O; and wherein L1 is represented by Formula IVe(ix): IVe(ix).

50. The nucleoside phosphoramidite of claim 29, n the nucleoside phosphoramidite is represented by Formula X: wherein R8 is H or a protecting group; R7 is a phosphoramidite; B is a natural nucleobase; and X is O; and wherein L1 is represented by a IVe(xi): IVe(xi).

51. The nucleoside phosphoramidite of claim 49 or 50, wherein the oramidite has the formula —P(ORx)—N(Ry)2, wherein Rx is selected from the group consisting of a methyl, 2-cyanoethyl and , wherein each of Ry is selected from the group consisting of an ethyl and isopropyl.

52. An oligonucleotide, wherein the oligonucleotide comprises at least one nucleotide having a glutathione-sensitive moiety covalently bound to an oxygen atom that is covalently bound to the 2′-carbon of the sugar moiety of the at least one nucleotide having the glutathione-sensitive moiety, wherein the hione-sensitive moiety is represented by Formula IVe(ii): O S S IVe(ii).

53. An oligonucleotide, wherein the oligonucleotide comprises at least one nucleotide having a glutathione-sensitive moiety covalently bound to an oxygen atom that is covalently bound to the bon of the sugar moiety of the at least one nucleotide having the glutathione-sensitive moiety, n the glutathione-sensitive moiety is represented by Formula IVe(ix) or IVe(xi): IVe(ix), or IVe(xi).

54. An oligonucleotide, wherein the oligonucleotide comprises at least one nucleotide having a hione-sensitive moiety covalently bound to an oxygen atom that is covalently bound to the 2′-carbon of the sugar moiety of the at least one nucleotide having the glutathione-sensitive moiety, wherein the glutathione-sensitive moiety is represented by Formula IVb(i) or IVb(ii): IVb(i), or IVb(ii), n R is selected from hydrogen, CH3, aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected via a spacer.

55. An oligonucleotide, wherein the ucleotide ses at least one nucleotide having a glutathione-sensitive moiety covalently bound to an oxygen atom that is covalently bound to the 2′-carbon of the sugar moiety of the at least one nucleotide having the glutathione-sensitive moiety, wherein the glutathione-sensitive moiety is represented by Formula IVd(i): IVd(i). WO 39364 258800 33:00 .mmcwmmmm <—. 3:25 22.5 .825 .0."— WO 39364 »258800 _o=:oo 3208 .mmcwmmmm <_. 3:25 22.5 .825 .0."— WO 39364 owl2: 012Lo//_\2 .m.\ I ,o» v 22:80. 0: 22.5 25258 .mmcwmmmm mr 3:25 22:6 .825 .0."—