BACKGROUND

Oligonucleotides have various uses in molecular biology, including, for example, 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 restore defective or missing genes using gene therapy techniques, or as nucleic acid inhibitor molecules 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 infections, and genetic disorders. Nucleoside and nucleotide analogs are also commonly used therapeutically, particularly as antiviral or anticancer agents.

Like other drugs, therapeutic oligonucleotides need, among other things, stability in biological systems and sufficient potency at the intended site of action. The in vivo environment presents challenges to the stability of therapeutic oligonucleotides because of the conditions that these molecules 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, 18:305-320. Nucleic acid inhibitor molecules having a single-stranded 3′-overhang, such as certain canonical 21-mer siRNA and other siRNA designs known in the art and described herein may, consequently, be particularly susceptible to degradation by such 3′-exonucleases. Behlke, M. A.,Oligonucleotides,2008, 18:305-320. In addition, an RNase A-like activity 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, pH-dependent 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 with in vivo administration of nucleic acid inhibitor molecules has been to introduce certain irreversible, covalent chemical modification 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′) of the 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 example, many groups have modified the 2′ position of the sugar moiety from a hydroxyl group to a 2′-fluoro (2′-F) or a 2′-O-methyl (2′-OMe), and such changes have effectively increased nuclease resistance of RNA oligonucleotides. See Behlke, M. A.,Oligonucleotides,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 inhibitor molecule include a phosphoramidate or a chemical moiety that mimics the electrostatic and steric properties of a phosphate group (“phosphate mimic”). See Prakash et al., 2015, 43(6):2993-3011. Typically, these 5′-phosphate mimics contain phosphatase-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 half-life of oligonucleotides in plasma from minutes to days. See Shen et al.Nucleic Acids Res.,2015, 43:4569-4578; Eckstein, F.Nucleic Acid Thera.,2014, 24(6):374-387.

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

While these irreversible modifications may help to improve the stability of a nucleic acid and/or protect it from enzymes 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 nucleic acid inhibitor molecule once it reaches the cytosol of the cell. See Behlke, M. A.,Oligonucleotides,2008, 18:305-320. 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 reduced potency or activity, they can limit the therapeutic efficacy of nucleic acid inhibitor molecules containing them.

While research and drug development 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 molecules have been chemically modified with cyclic disulfide moieties to mask the negative charge created by the internucleotide diphosphate linkages and improve cellular uptake and nuclease resistance. See U.S. Published Application No. 2011/0294869 originally assigned to Traversa Therapeutics, Inc. (“Traversa”), PCT Publication No. WO 2015/188197 to Solstice Biologics, Ltd. (“Solstice”), Meade et al.,Nature Biotechnology,2014, 32:1256-1263 (“Meade”), PCT Publication No. WO 2014/088920 to Merck Sharp & Dohme Corp. This reversible modification of the internucleotide diphosphate linkages is designed to be cleaved intracellularly by the reducing environment of the cytosol (e.g. glutathione). Earlier examples include neutralizing phosphotriester modifications that were reported to be cleavable inside cells (Dellinger et al.J. Am. Chem. Soc.2003, 125:940-950).

There has been less effort in the art to reversibly modify other positions in the sugar moiety of nucleotides, such as the 2′-carbon (also referred to as C2′). C2′ has been reversibly modified using modifications that are sensitive to enzymatic cleavage (Lavergne et al.,J Org. Chem.,2011, 76:5719-5731) and light-stimulated cleavage (Johnsson et al.,Bioorganic&Med. Chem. Letters,2011, 21:3721-25). 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, 26:845-848. However, the authors in Ochi found that nucleoside phosphoramidites containing 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 Biscans et al.,Org. Biomol. Chem.,2016, 14:7010-17, 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 nucleoside 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 improve 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 of nucleic acid molecules, particularly reversible modifications that are compatible with standard, phosphoramidite oligonucleotide synthesis.

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 oligonucleotides, microRNA, ribozymes, antagomirs, and aptamers. They can also be incorporated into other oligonucleotides, such as, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) 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), including other oligonucleotides that are to be administered in vivo.

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 methods. In addition, glutathione-sensitive nucleotide and nucleoside monomers without a phosphoramidite can be used therapeutically, for example as anti-viral agents.

Typically, the glutathione-sensitive moiety is used to reversibly modify the 2′-carbon 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 vivo administration (e.g., transit through the blood and/or the lysosomal/endosomal compartments of a cell) where the oligonucleotide will be exposed to nucleases and other harsh environmental conditions (e.g., pH). When the reversibly modified oligonucleotide is released into the cytosol, the intracellular conditions, including a high level of glutathione, cause the glutathione-sensitive moiety to be removed from the oligonucleotide. In certain embodiments, the removal of the glutathione-sensitive moiety yields a hydroxyl group at the 2′-carbon position, which is the natural substituent for a ribonucleotide at that position (see, e.g., Scheme 7 in Example 3).

Using reversible, glutathione-sensitive moieties 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 glutathione-sensitive moiety can be engineered to modify the kinetics of its release.

Moreover, the present reversibly modified, glutathione-sensitive oligonucleotides, 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 solid-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 glutathione-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 possible 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 oligonucleotides 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 attached 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 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), IVb(ii), IVc(i), or IVd(i); or Formula IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(v), IVe(vi), IVe(vii), IVe(viii), IVe(ix), IVe(x), or IVe(xi). In embodiments where the oligonucleotide contains more than one reversibly modified nucleotide, each reversibly modified nucleotide may comprise the same glutathione-sensitive moiety or at least one of the reversibly modified nucleotides may contain 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 moiety.

In certain 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 thereof).

In certain embodiments, the glutathione-sensitive oligonucleotide comprises at least one nucleotide represented by Formula I, as described herein, 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), IVb(ii), IVc(i), or IVd(i); or Formula IVe(i), IVe(ii), IVe(iii), IVe(iv), IVe(v), 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 O; wherein Z is NR′, wherein R′ is hydrogen or substituted or unsubstituted aliphatic; and wherein V is C and optionally wherein X2and X3are 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 glutathione-sensitive oligonucleotide, L is represented by Formula III, as described herein, wherein Y is O, S or NH; wherein Z1is N or CH; wherein V is C; and optionally, wherein M1and M2are substituted or unsubstituted C2to C6alkyl or are taken together with P1to Q1to form a 5-8 membered ring, wherein the ring is substituted or unsubstituted cycloalkyl.

In certain embodiments, L is represented by Formula IIIa, as described herein, wherein Y is O, S or NH; and Z1is 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. In one embodiment, Y is O and Z1is N (see Formula IIIa(i)).

In certain embodiments, L is represented by Formula IIIb, as described herein, wherein Y is O, S or NH; Z1is 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 Taand Tbare 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 spacer to a sulfur atom. In one embodiment, L is represented by Formula IIIb(i), as described herein.

In certain embodiments of the glutathione-sensitive oligonucleotide, L is represented by Formula IV, as described herein, wherein Y is O, S or NH; wherein Z is NH or NCH3; wherein V is C; wherein G is CH2and E is absent or G is absent and E is CH2; and optionally wherein M3and M4are independently substituted or unsubstituted C2to C6alkyl 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 R7are each independently selected from OAcyl, NHR′, NR′, CR′R″, wherein R′ and R″ are each independently selected from hydrogen, 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-C6alkyl 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, S, NH; Z is O, S or NH; V is C; M3and M4are 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 C2to C6alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or T is a ligand optionally connected 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 unsubstituted aliphatic, aryl, heteroaryl, cycloalkyl or a heterocycle or R is a targeting ligand optionally connected via a spacer.

In certain embodiments, L is represented by Formula IVc, as described herein, 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; M3and M4are 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 C2to C6alkyl; n is 0-5; T is substituted or unsubstituted C2to C6alkyl, 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 aliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl or a substituted or unsubstituted heterocycle or R is a targeting ligand optionally connected via a spacer. In one embodiment, L is represented by Formula IVc(i), as described herein, wherein R is selected from hydrogen, CH3, substituted or unsubstituted aliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl 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, wherein Y is O, S, NH; Z is selected from O, S, NH, or NCH3; T is substituted or unsubstituted C2to C6alkyl, 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, CH3or a substituted or unsubstituted C2to C6alkyl. In one embodiment, L is represented by Formula IVd(i), as described herein.

In certain embodiments of the glutathione-sensitive oligonucleotide, L is represented by Formula IVe, as described herein, 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 independently selected from hydrogen, halogen, a substituted or unsubstituted aliphatic, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocycle or R′ and R″ are taken together to form a heterocyclic ring; K is C or CH; n is 0-5; T is substituted or unsubstituted C2to C6alkyl, 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 aliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl or a substituted or unsubstituted heterocycle or R is a targeting ligand optionally connected via a spacer. In certain embodiments, Z is NH or NCH3and one or both of G and E are absent, CH2, or CR′R″, NH, NR′, wherein R′ and R″ are each independently 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, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted 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 U1is O or an internucleotide 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 U2is 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 U1or W is an internucleotide linking group attaching the at least one nucleotide represented by Formula VIIe(ix) to an oligonucleotide and provided that if U1is an internucleotide 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 certain embodiments, A is hydrogen and W is an internucleotide linking group attaching the at least one nucleotide represented by Formula VIIe(ix) to an oligonucleotide and the at least one nucleotide represented by Formula VIIe(ix) is located at nucleotide position 1 of the antisense strand.

In certain embodiments, 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 U1is a internucleotide 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 nucleotide position 14 of the antisense strand.

In certain embodiments, the glutathione-sensitive oligonucleotide comprises at least one nucleotide represented by Formula VIIe(xi), wherein A is absent, a hydrogen, a phosphate group, or a phosphate mimic; wherein U1is O or an internucleotide linking group attaching the at least one nucleotide represented by Formula VIIe(xi) to a nucleotide or an oligonucleotide; wherein B is a natural nucleobase; wherein U2is 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 U1or W is an internucleotide linking group attaching the at least one nucleotide represented by Formula VIIe(xi) to an oligonucleotide and provided that if U1is an internucleotide 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 certain embodiments, the internucleotide 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 strand. In certain embodiments, the double stranded RNAi inhibitor molecule comprises a region of complementarity 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 nucleotides.

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 embodiments, the at least one nucleotide represented by Formula I is located at nucleotide position 14 of the antisense strand. In certain embodiments, the at least one nucleotide represented by Formula I is located at one or more nucleotide positions at or adjacent to the Ago2 cleavage site of the sense strand. In certain embodiments, the at least one nucleotide represented by Formula I is located at one, two, or three nucleotides that are immediately 5′ or 3′ of the Ago2 cleavage site. In certain embodiments, the at least one nucleotide represented by Formula I is located on both sides of the Ago2 cleavage site, e.g., at one or more nucleotides that are immediately 5′ of the Ago2 cleavage site and at one or more nucleotides that are immediately 3′ of the Ago2 cleavage site.

In certain embodiments, the double stranded RNAi inhibitor molecule contains a tetraloop.

In certain embodiments, the glutathione-sensitive oligonucleotide is a single stranded oligonucleotide. 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 embodiments, 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 transport of the glutathione-sensitive oligonucleotide across an outer membrane of a cell. In certain embodiments, the delivery agent is selected from the group consisting of carbohydrates, peptides, lipids, vitamins and antibodies. In certain embodiments, the delivery agent is selected from N-Acetylgalactosamine (GalNAc), mannose-6-phosphate, galactose, oligosaccharide, polysaccharide, cholesterol, polyethylene glycol, folate, vitamin A, vitamin E, lithocholic acid and a cationic lipid.

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

In certain embodiments, the glutathione-sensitive oligonucleotide comprises at least one nucleotide having a glutathione-sensitive moiety bound to an oxygen atom that is covalently bound to a 2′-carbon of a sugar moiety of the nucleotide, wherein 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 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. In certain embodiments, the guide sequence is a chimeric guide sequence, wherein the crRNA sequence is fused to the tracrRNA 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 glutathione-sensitive oligonucleotide comprises at least one glutathione-sensitive nucleotide, wherein the at least one glutathione-sensitive nucleotide comprises a substitution of a hydroxyl group at the 2′-carbon 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 directed to a method for reducing expression of a target gene in a subject comprising administering a pharmaceutical composition comprising a glutathione-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 administering comprises systemic administration.

In certain aspects, the present disclosure is directed to a nucleoside 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 nucleoside. 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), IVb(ii), IVc(i), IVd(i), 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.

In certain aspects, the present disclosure is directed to a glutathione-sensitive nucleoside phosphoramidite, wherein the nucleoside phosphoramidite is represented by Formula VIII, as described herein. In certain embodiments, the nucleoside phosphoramidite is represented by Formula IX. In certain embodiments, the glutathione-sensitive moiety (L1) comprises a disulfide bridge or a sulfonyl group.

In certain embodiments, the nucleoside phosphoramidite is represented by Formula VIII, wherein J is O; B is a natural nucleobase; U2is O; I is CH2; W1is a phosphoramidite; A1is a protecting group, hydrogen, or solid support; and U3is O and optionally, wherein X is O and R1, R2, R3and R4are hydrogen.

In certain embodiments, the nucleoside phosphoramidite is represented by Formula VIII, wherein J is O; B is a natural nucleobase; U2is O; I is CH2; W1is a protecting group, hydrogen or solid support; A1is a phosphoramidite, and U3is O and optionally, wherein X is O and R1, R2, R3and R4are hydrogen.

In certain embodiments, the nucleoside phosphoramidite is represented by Formula X, wherein R8is H or a protecting group; R7is a phosphoramidite; B is a natural nucleobase; X is O; and wherein L1is represented by Formula IVe(ix).

In certain embodiments, the nucleoside phosphoramidite is represented by Formula X, wherein R8is H or a protecting group; R7is a phosphoramidite; B is a natural nucleobase; and X is O; and wherein L1is represented by Formula IVe(xi).

In certain embodiments, the phosphoramidite has the formula —P(ORx)—N(Ry)2, wherein Rxis selected from the group consisting of an optionally substituted methyl, 2-cyanoethyl and benzyl, wherein each of Ryis 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) attaching a nucleoside to a solid support 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 therebetween, wherein any uncoupled nucleoside on the solid support is capped with a capping reagent; (c) oxidizing said phosphorus nucleoside linkage with an oxidizing reagent; and (d) repeating steps (b) to (d) iteratively with one or more subsequent glutathione-sensitive nucleoside 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 glutathione-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 subgenera 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(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. In certain aspects, the present disclosure is directed to a glutathione-sensitive nucleoside or nucleotide that does not contain a phosphoramidite, wherein the glutathione-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 herein. In certain embodiments, J is O; X is O; L2is a glutathione-sensitive moiety represented by Formula II, III, or IV; W2is 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 heteroaryl, a heterocycle or are taken together to form a heterocyclic ring; and A2is absent, hydrogen, a phosphate group, a phosphate mimic, or a phosphoramidate; and optionally wherein R1, R2, R3, and R4are hydrogen; U2is oxygen; W2is hydrogen; I is CH2; U3is O; and A2is hydrogen or a phosphate group.

