Compositions for modulating KRAS expression and uses thereof

Abstract

Described herein are compounds, compositions, and methods for modulating KRAS expression, such as mutant KRAS expression, and / or downstream signaling pathways. Also described herein are compounds, compositions, and methods for treating diseases or conditions associated with mutant KRAS. In some embodiments, the compounds comprise at least one antisense oligonucleotide, which upon delivery into cells hybridizes to endogenous KRAS mRNA, thereby causing degradation of the KRAS mRNA. In some embodiments, the antisense oligonucleotide hybridizes to an mRNA encoding a mutant KRAS protein, such as a KRAS protein containing a G12C mutation, a G12V mutation, a G12D mutation, or a G12A mutation.

Description

[Technical Field]

[0001] Priority This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 480,467, filed January 18, 2023, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing The entire contents of the text file submitted electronically herewith are incorporated herein by reference: Copy of Sequence Listing in Computer Readable Format (Filename: "MAX-008PC_133279-5008_SequenceListing", Recording Date: January 17, 2024, File Size: 45,380 bytes). [Background technology]

[0003] Certain diseases or pathological conditions are caused by gene mutations or dysregulation of signaling pathways. One of the most sought-after therapeutic options for treating such diseases or pathological conditions involves directly editing gene mutations or regulating transcription / translation using gene silencing means or methods. Oligonucleotide-induced gene silencing can control the RNA expression of target genes in various embodiments, including transcriptional inactivation, mRNA degradation, and transcriptional attenuation. Existing RNA-induced gene silencing tools can be inefficient and / or nonspecific. Therefore, there remains a need for compositions and methods for effectively controlling gene expression, particularly for reducing the RNA levels of mutant genes such as KRAS and / or downstream signaling pathways. Summary of the Invention

[0004] In aspects and embodiments, a compound is provided comprising an antisense oligonucleotide that inhibits expression of KRAS mRNA, the antisense oligonucleotide comprising 10 to 30 linked nucleotides and having a sequence complementary to KRAS mRNA, the oligonucleotide having at least 8 consecutive nucleotides of any one of SEQ ID NOs: 1-10 and 14-21.

[0005] In embodiments, the oligonucleotide is at least 12 nucleotides in length. In embodiments, the oligonucleotide is at least 14 nucleotides in length. In embodiments, the oligonucleotide is 10-24 nucleotides in length, or 10-16 nucleotides in length, or 12-16 nucleotides in length.

[0006] In embodiments, the oligonucleotide is 12, 13, 14, 15, or 16 nucleotides in length. In embodiments, the oligonucleotide is 14 nucleotides in length. In embodiments, the oligonucleotide comprises at least 12 contiguous nucleobases of any one of SEQ ID NOs: 1-10 and 14-21. In embodiments, the oligonucleotide comprises or consists of the nucleobase sequence of any one of SEQ ID NOs: 1-10 and 14-21. In embodiments, the oligonucleotide has a nucleobase sequence selected from SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0007] In embodiments, the antisense oligonucleotide has a stretch of at least six DNA nucleotides sufficient to recruit RNase H. In embodiments, one or more DNA nucleotides independently comprise a 2' chemical modification selected from 2'-fluoro, 2'-methyl, and 2'-ethyl. In embodiments, the DNA nucleotides do not comprise a 2' chemical modification. In embodiments, the antisense oligonucleotide is a gapmer having 5' and 3' segments, each of which is 2 to 6 nucleotides or 2 to 4 nucleotides, and the 5' and 3' segments contain no DNA nucleotides. In embodiments, the lengths of the 5' and 3' segments are independently selected from 2 or 3 nucleotides, and the 5' and 3' segments are flanked by an internal sequence of eight DNA nucleotides. In embodiments, one or more nucleotides in the 5' and 3' segments comprise a 2'-O substituent, and optionally, all nucleotides in the 5' and 3' segments comprise a 2'-O substituent. In embodiments, the 2'-O substituents are independently selected from 2'-O methyl, 2'-O ethyl, 2'-O methoxyethyl (MOE), and bridged nucleotides having a 2' to 4' bridge. In embodiments, the bridged nucleotide has a methylene bridge (LNA) or a cEt (constrained ethyl) bridge. In embodiments, the antisense oligonucleotide has a modified backbone. In embodiments, the antisense oligonucleotide comprises one or more phosphorothioate or phosphorodithioate nucleotides. In embodiments, the oligonucleotide is fully phosphorothioate or phosphorodithioate-linked. In embodiments, the oligonucleotide is fully phosphorothioate-linked.

[0008] In embodiments, the cytosine nucleobase in the antisense oligonucleotide is a modified cytosine, which is optionally 5-methylcytosine or 5-hydroxymethylcytosine. In embodiments, the antisense oligonucleotide has a nucleobase sequence and structure (i.e., modification pattern) shown in one or more of Tables 1, 2, 3, 6, 7, 8, 9, 10, and 11. In embodiments, the compound further comprises a cell-targeting moiety or a cell-penetrating moiety. In embodiments, the cell-targeting moiety or the cell-penetrating moiety is directly or indirectly conjugated to the 3'-end of the oligonucleotide, optionally via a linker. In embodiments, the moiety comprises a sterol conjugate or a fatty acid conjugate, which is optionally a cholesteryl, palmitoyl, or stearyl conjugate. In embodiments, the compound further comprises a cell-targeting aptamer.

[0009] In embodiments, the compound does not include any encapsulation or transfection reagent. In embodiments, the antisense oligonucleotide is encapsulated in a particle. In embodiments, the particle is a liposome, a polymeric nanoparticle, or a lipid nanoparticle. In embodiments, the compound is formulated for parenteral administration. In embodiments, a pharmaceutical composition is provided comprising a compound of the present disclosure and a pharmaceutically acceptable carrier or vehicle.

[0010] In an embodiment, a method for treating a subject having a pathology associated with abnormal expression of KRAS or a pathology associated with mutant KRAS is provided, comprising administering an effective amount of a compound of the present disclosure or a pharmaceutical composition of the present disclosure to the subject. In an embodiment, the subject has a malignant tumor associated with an abnormality in the KRAS-mediated signaling pathway. In an embodiment, the malignant tumor is associated with KRAS or a mutant KRAS. In an embodiment, the mutant KRAS mRNA encodes a mutant KRAS protein comprising a G12C mutation. In an embodiment, the mutant KRAS mRNA encodes a mutant KRAS protein comprising a G12D mutation or a G12V mutation. In an embodiment, the malignant tumor associated with an abnormality in the KRAS-mediated signaling pathway is non-metastatic. In an embodiment, the malignant tumor associated with an abnormality in the KRAS-mediated signaling pathway is metastatic.

[0011] In an embodiment, the malignant tumor is a carcinoma. In an embodiment, the malignant tumor is breast cancer, cervical cancer, pancreatic cancer, squamous cell carcinoma, head and neck cancer, thyroid cancer, gastric cancer, colon cancer, or liver cancer. In an embodiment, the malignant tumor is pancreatic cancer. In an embodiment, the malignant tumor is cervical squamous cell carcinoma, endocervical adenocarcinoma, bile duct carcinoma, esophageal cancer, colon adenocarcinoma, oral squamous cell carcinoma, head and neck squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, liver hepatocellular carcinoma, lung squamous cell carcinoma, rectal adenocarcinoma, gastric adenocarcinoma, thyroid cancer, or pancreatic adenocarcinoma. In an embodiment, the malignant tumor is pancreatic cancer, such as pancreatic adenocarcinoma. In an embodiment, the antisense oligonucleotide is administered parenterally. [Brief explanation of the drawings]

[0012] [Figure 1] 1 illustrates knockdown of KRAS mRNA encoding a mutant KRAS protein containing a G12C mutation mediated by an antisense oligonucleotide (ASO) described herein: (a) STN-016, (b) STN-017, (c) STN-018, (d) STN-019, (e) STN-001, (f) STN-002, and (g) STN-003. [Figure 2]Figure 1 illustrates three-dimensional (3D) cell growth inhibition resulting from inhibition of mutant KRAS expression by contacting cells harboring a KRAS G12C mutation (Mia Paca-2) with the oligonucleotides described herein: (left to right) STN-022(a), STN-016(b), STN-017(c), STN-025(d), STN-018(e), STN-020(f), STN-021(g), STN-019(h), STN-001(i), STN-002(j), and STN-003(k). [Figure 3] Figure 1 shows Western blot analysis of G12V KRAS knockdown in the LCLC-97TM1 cell line using ASOs. For reference, the loaded ASOs are, from left to right, STN-100080, STN-100993, STN-100994, STN-100987, and STN-100989. DETAILED DESCRIPTION OF THE INVENTION

[0013] Described herein are compounds, compositions, and methods for modulating KRAS expression (e.g., mutant KRAS expression) and / or downstream signaling pathways. Also described herein are compounds, compositions, and methods for treating a disease or condition by modulating gene expression or signaling pathways associated with the disease or condition. In some embodiments, the compounds comprise at least one antisense oligonucleotide, which upon delivery into a cell binds (e.g., hybridizes) to endogenous KRAS mRNA, thereby resulting in degradation of the KRAS mRNA. In some embodiments, the present disclosure describes methods utilizing the compounds or oligonucleotides described herein. In some embodiments, the methods are used to treat a disease or condition by contacting a cell with the oligonucleotide to reduce KRAS gene expression (e.g., mutant KRAS) or an associated signaling pathway. In some embodiments, the antisense oligonucleotide hybridizes to mRNA encoding a mutant KRAS protein, such as a KRAS protein comprising a G12C mutation, a G12V mutation, a G12D mutation, or a G12A mutation.

[0014] In some embodiments, the oligonucleotide is an antisense oligonucleotide, and the oligonucleotide is complementary to and binds (e.g., hybridizes) to a segment of at least one endogenous nucleic acid (e.g., mRNA). In some embodiments, the binding of the oligonucleotide to the endogenous nucleic acid leads to the degradation of the endogenous nucleic acid or blocks the translation of the target protein derived from the endogenous nucleic acid. Thus, the expression of the gene encoded by the endogenous nucleic acid is reduced. For example, the binding of the oligonucleotide to the endogenous mRNA creates a double-stranded nucleic acid molecule, which can then recruit endogenous nucleases (e.g., RNase H) that degrade the mRNA.

[0015] In some embodiments, the oligonucleotide comprises a stretch of DNA nucleotides (e.g., a stretch of 5-10 DNA nucleotides) sufficient to recruit RNase H upon hybridization to a target mRNA. In embodiments, the stretch of DNA nucleotides is a gap segment. In some embodiments, the oligonucleotide comprises at least one wing segment. In some embodiments, the oligonucleotide comprises at least one gap segment flanked by two wing segments. For example, the oligonucleotide comprises a gap segment flanked by a 5'-wing segment and a 3'-wing segment. In some embodiments, the gap segment or wing segment comprises at least one chemical modification. In some embodiments, the antisense oligonucleotide is a gapmer.

[0016] In some embodiments, the oligonucleotide modulates the KRAS signaling pathway. In some embodiments, the KRAS signaling pathway includes the KRAS-RAF-MEK-ERK signaling pathway. In some embodiments, the KRAS signaling pathway includes the phosphoinositide 3-kinase (PI3K) signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, or the Ral guanine nucleotide exchange factor (Ral-GEF) signaling pathway. Thus, in some embodiments, reduced gene expression resulting from binding of the oligonucleotide to an endogenous nucleic acid may further reduce expression of a signaling pathway including the gene regulated by the oligonucleotide. In some embodiments, reduced expression of a gene or signaling pathway provides a therapeutic effect for treating a disease or condition. In some embodiments, the disease or condition is caused by increased expression of a gene or signaling pathway. In some embodiments, the disease or condition described herein is caused by a genetic mutation associated with a gene or signaling pathway (e.g., mutant KRAS).

[0017] In aspects and embodiments, compounds are provided that comprise an antisense oligonucleotide that inhibits expression of KRAS mRNA (including in some embodiments, mRNA encoding mutant KRAS), the antisense oligonucleotide comprising 10 to 30 linked nucleotides and having a sequence complementary to KRAS mRNA, the oligonucleotide having at least 8 consecutive nucleotides of any one of SEQ ID NOs: 1-10 and 14-21.

[0018] In embodiments, the oligonucleotide comprises at least 10, or at least 12, or at least 14 contiguous nucleobases of any one of SEQ ID NOS: 1-10 and 14-21. In embodiments, the oligonucleotide is 10-24 nucleotides in length (e.g., 10-16 or 12-16 nucleotides in length). In embodiments, the oligonucleotide is 12, 13, 14, 15, or 16 nucleotides in length. For example, in embodiments, the oligonucleotide is 13 or 14 nucleotides in length. In embodiments, the oligonucleotide comprises at least 12 contiguous nucleobases of any one of SEQ ID NOS: 1-10 and 14-21. In embodiments, the oligonucleotide comprises or consists of the nucleobase sequence of any one of SEQ ID NOS: 1-10 and 14-21. In embodiments, the oligonucleotide has a nucleobase sequence selected from SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0019] In some embodiments, the oligonucleotides described herein are antisense oligonucleotides that target endogenous nucleic acids and bind (e.g., hybridize) to endogenous nucleic acids. In some embodiments, binding of the oligonucleotide to the endogenous nucleic acid recruits an endogenous nuclease (e.g., RNase H) that degrades the endogenous nucleic acid. In some embodiments, degradation of the endogenous nucleic acid reduces expression of the gene encoded by the endogenous nucleic acid. In some embodiments, degradation of the endogenous nucleic acid can treat a disease or condition described herein.

[0020] In some embodiments, the oligonucleotide comprises 11 nucleobases. In some embodiments, the oligonucleotide comprises 12 nucleobases. In some embodiments, the oligonucleotide comprises 13 nucleobases. In some embodiments, the oligonucleotide comprises 14 nucleobases. In some embodiments, the oligonucleotide comprises 15 nucleobases. In some embodiments, the oligonucleotide comprises 16 nucleobases. In some embodiments, the oligonucleotide is 20 nucleotides or less in length. In some embodiments, the oligonucleotide is 19 nucleotides or less in length. In some embodiments, the oligonucleotide is 18 nucleotides or less in length. In some embodiments, the oligonucleotide is 17 nucleotides or less in length. In some embodiments, the oligonucleotide is 16 nucleotides or less in length. In some embodiments, the oligonucleotide is 15 nucleotides or less in length. In some embodiments, the oligonucleotide is 14 nucleotides or less in length. In some embodiments, the oligonucleotide is 13 nucleotides or less in length.

[0021] In some embodiments, the oligonucleotide comprises at least one gap segment. In some embodiments, the gap segment comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleobases. In some embodiments, the gap segment comprises 4 nucleobases. In some embodiments, the gap segment comprises 5 nucleobases. In some embodiments, the gap segment comprises 6 nucleobases. In some embodiments, the gap segment comprises 7 nucleobases. In some embodiments, the gap segment comprises 8 nucleobases. In some embodiments, the gap segment comprises 9 nucleobases. In some embodiments, the gap segment comprises 10 nucleobases. In some embodiments, the gap segment comprises 11 nucleobases. In some embodiments, the gap segment comprises 12 nucleobases. In some embodiments, the gap segment comprises 13 nucleobases. In some embodiments, the gap segment comprises 14 nucleobases. For example, in embodiments, the antisense oligonucleotide has a stretch of at least 6 DNA nucleotides (gap segment) sufficient to recruit RNase H. In embodiments, the gap segment is 8 nucleotides.

[0022] In some embodiments, the oligonucleotide comprises at least one wing segment (e.g., a 5' wing segment and a 3' wing segment). In some embodiments, at least one wing segment is a 5'-terminal wing segment, which is covalently attached to a gap segment at the 5'-end of the gap segment. In some embodiments, at least one wing segment is a 3'-terminal wing segment, which is covalently attached to a gap segment at the 3'-end of the gap segment. In some embodiments, the gap segment is adjacent to wing segments at both the 5'-end and the 3'-end of the gap segment. In some embodiments, the wing segments independently comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleobases. In embodiments, the wing segments are independently 2 to 6 nucleotides or 2 to 4 nucleotides in length. In some embodiments, at least one wing segment comprises one nucleobase. In some embodiments, at least one wing segment comprises two nucleobases. In some embodiments, at least one wing segment comprises three nucleobases. In some embodiments, at least one wing segment comprises four nucleobases. In some embodiments, at least one wing segment comprises five nucleobases. In some embodiments, at least one wing segment comprises six nucleobases. In some embodiments, at least one wing segment comprises seven nucleobases. In embodiments, the lengths of the wing segments are independently selected from 2 to 5 nucleobases. In embodiments, the nucleotides in the wing segments include RNA nucleotides and / or 2'-O-modified nucleotides as described in detail herein.

[0023] In some embodiments, the KRAS mRNA sequence (e.g., encoded by the target cell) comprises any one of SEQ ID NOs: 11-13, 22, and 23. In some embodiments, the wild-type and mutant KRAS mRNA sequences comprise any of those listed in Table 4.

[0024] In embodiments, the oligonucleotide comprises at least 8 contiguous nucleobases of any one of SEQ ID NOS: 1-10, 14-21, and the oligonucleotide is substantially (i.e., at least 90%) or entirely complementary to a segment of KRAS mRNA. In embodiments, the oligonucleotide comprises at least 10 or at least 12 contiguous nucleobases of any one of SEQ ID NOS: 1-10, 14-21, and the oligonucleotide is substantially (i.e., at least 90%) or entirely complementary to a segment of KRAS mRNA. In certain embodiments, the oligonucleotide comprises or consists of the nucleobase sequence of any one of SEQ ID NOS: 1-10 and 14-21.

[0025] In embodiments, the oligonucleotide comprises at least 8 consecutive nucleobases of any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, and 10, and the oligonucleotide is substantially (i.e., at least 90%) or entirely complementary to a segment of KRAS mRNA. In embodiments, the oligonucleotide comprises at least 10 or at least 12 consecutive nucleobases of any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, and 10, and the oligonucleotide is substantially (i.e., at least 90%) or entirely complementary to a segment of KRAS mRNA. In certain embodiments, the oligonucleotide comprises or consists of the nucleobase sequence of any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0026] For simplicity, nucleotide sequences may be presented herein using DNA nucleotide sequences (i.e., containing thymine nucleobases, "T" or "t") or as RNA nucleotide sequences (i.e., containing uracil nucleobases, "U" or "u"). It is understood from the context that if a nucleotide or sequence is intended to be RNA, T nucleotides may be substituted with U (or modified U, such as pseudouridine or 1-methylpseudouridine), and if a nucleotide or sequence is intended to be DNA, U nucleotides may be substituted with T or modified T. However, in embodiments, RNA nucleotides in antisense oligonucleotides may employ T (thymine) bases, and DNA nucleotides in antisense oligonucleotides may employ U (uracil) bases. In some embodiments, one or more T nucleotides in the antisense oligonucleotides disclosed herein are replaced with U or modified U.

[0027] In some embodiments, the oligonucleotide has a nucleobase sequence (or chemical structure) selected from Tables 1-3, and 6-11. In embodiments, the oligonucleotide may have a nucleobase sequence selected from SEQ ID NOs: 1-10 and 14-21. In embodiments, the oligonucleotide may have a nucleobase sequence selected from SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0028] In some embodiments, binding (e.g., hybridization) of an antisense oligonucleotide to a target mRNA results in degradation of the target mRNA or blocks translation of the target mRNA. In some embodiments, binding of an antisense oligonucleotide to a target mRNA creates a double-stranded nucleic acid molecule, which then recruits endogenous nucleases that degrade the mRNA. In some embodiments, the antisense oligonucleotide has a stretch of DNA nucleotides sufficient to induce degradation of the target mRNA by recruiting RNase H. In embodiments, the antisense oligonucleotide may have a stretch (e.g., a central stretch) of at least six or at least eight DNA nucleotides, optionally a stretch of nine or ten DNA nucleotides. In some embodiments, one or more DNA nucleotides independently comprise a 2' chemical modification selected from 2'-fluoro, 2'-methyl, and 2'-ethyl. In embodiments, the DNA nucleotides do not comprise a 2' chemical modification.

[0029] In embodiments, the antisense oligonucleotide may be a gapmer having 5' and 3' segments, each of which is 2 to 6 nucleotides or 2 to 4 nucleotides, and the 5' and 3' segments do not contain any DNA nucleotides or do not contain only DNA nucleotides. In some embodiments, the gapmer is a 3-8-3 gapmer, having a central block (DNA nucleotides) and 5' and 3' segments (three RNA nucleotides each). In other embodiments, the gapmer is a 2-10-2 gapmer, having a central block (10 DNA nucleotides) and 5' and 3' segments (two RNA nucleotides each). Other gapmer formats, such as 3-10-2 and 2-10-3, may be used. In some embodiments, one or more nucleotides in the 5' and 3' segments contain a 2'-O substituent, and optionally, all nucleotides in the 5' and 3' segments contain a 2'-O substituent. Exemplary 2'-O substituents are independently selected from 2'-O methyl, 2'-O ethyl, 2'-O methoxyethyl (MOE), and bridged nucleotides (e.g., locked nucleotides or bicyclic nucleotides) having a 2' to 4' bridge. In some embodiments, the bridged nucleotide has a methylene bridge (LNA) or a cEt (constrained ethyl) bridge.

[0030] The term "gapmer" refers to an oligonucleotide having a central block of deoxynucleotides (also referred to herein as "DNA nucleotides") with 5' and 3' segments (at least two nucleotides) comprising RNA nucleotides or 2'-O-modified nucleotides. As used herein, the term "DNA nucleotide" refers to a nucleotide that is not an RNA nucleotide. DNA nucleotides typically have a 2'H, but may alternatively have a variety of 2' chemical modifications, including 2'-halo and 2'-lower alkyl (e.g., C1-4). In some embodiments, the 2' chemical modifications of the DNA nucleotides are independently selected from 2'-fluoro, 2'-methyl, and 2'-ethyl.

[0031] Locked nucleic acids (LNA) or "locked nucleotides" are described, for example, in U.S. Patent Nos. 6,268,490, 6,316,198, 6,403,566, 6,770,748, 6,998,484, 6,670,461, and 7,034,133 (all of which are incorporated herein by reference in their entirety). LNA is a modified nucleotide that contains a bridge between the 2' and 4' carbons of the sugar moiety, resulting in a "locked" conformation and / or bicyclic structure. Other suitable locked nucleotides that can be incorporated into the oligonucleotides of the present disclosure include those described in U.S. Patent Nos. 6,403,566 and 6,833,361 (both of which are incorporated herein by reference in their entirety). In exemplary embodiments, the locked nucleotides are independently selected from 2' to 4' methylene bridges and cEt (constrained ethyl) bridges (see U.S. Pat. Nos. 7,399,845 and 7,569,686, which are incorporated by reference in their entireties).

[0032] In some embodiments, the antisense oligonucleotide has a modified backbone or modified internucleotide linkage. The term "internucleotide linkage" refers to the linkage between two adjacent nucleosides in a polynucleotide molecule. Naturally, the internucleotide linkage is a phosphodiester bond, which occurs between two oxygen atoms of the phosphate group and the oxygen atom of the sugar (either the 3' or 5' position), forming two ester bonds bridging two adjacent nucleosides. Modification of the internucleotide linkage can provide various properties, including, but not limited to, enhanced stability. For example, phosphorothioate or phosphorodithioate linkages increase the resistance of the internucleotide linkage to nucleases. Another example is a phosphoacetate linkage (PACE), which improves transfection properties and enhances nuclease resistance. The internucleotide linkage and oligonucleotide backbone modification that can be adopted in the oligonucleotide herein include but are not limited to phosphodiester, phosphorothioate, phosphorodithioate, methyl phosphonate, alkyl phosphonate, alkyl phosphonothioate, phosphotriester, phosphoramidate, phosphoramidate, phosphorodiamidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, peptide nucleic acid, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate and sulfone internucleoside linkage.In some embodiments, antisense oligonucleotide comprises one or more phosphorothioate or phosphorodithioate nucleotide.

[0033] In some embodiments, the antisense oligonucleotide contains one or more phosphorothioate or phosphorodithioate internucleotide linkages. In some embodiments, phosphorothioate or phosphorodithioate linkages may be introduced between the last 3 to 5 nucleotides at the 5'-end and / or 3'-end of the oligonucleotide to reduce exonuclease degradation. In some embodiments, the antisense oligonucleotide has a combination of phosphodiester and phosphorothioate / phosphorodithioate linkages. In some embodiments, the antisense oligonucleotide contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten phosphorothioate or phosphorodithioate internucleotide linkages. In some embodiments, the antisense oligonucleotide contains substantially alternating phosphodiester and phosphorothioate internucleotide linkages. In some embodiments, the backbone modifications described in this paragraph are used in the internucleotide linkages of at least the nucleotides in the wing segments. In some embodiments, the antisense oligonucleotide is fully phosphorothioate / phosphorodithioate linked (i.e., all linkages are either phosphorothioate or phosphorodithioate). In some embodiments, the ASO is fully phosphorothioate linked.

[0034] In some embodiments, particularly when recruitment of RNase H is undesirable, the antisense oligonucleotide has a morpholino backbone. Morpholino oligonucleotides generally do not induce degradation of their target RNA molecules and can be effective in sterically blocking the target RNA sequence. Morpholino oligonucleotides and their synthesis are generally disclosed in U.S. Patent Nos. 11,028,386, 10,947,533, and 10,927,378 (each of which is incorporated herein by reference in its entirety). In some embodiments, the antisense oligonucleotide comprises thiomorpholino nucleotides and / or other substituted or modified nucleotides, such as those described in International Patent Application Publication Nos. WO / 2019 / 060522 and WO / 2018 / 057430 (each of which is incorporated herein by reference in its entirety). For example, Langner et al. describe methods for synthesizing oligonucleotide analogs called thiophosphoramidate morpholino oligonucleotides (TMOs), which incorporate morpholino nucleosides and phosphorothioate linkages ("Synthesis and characterization of thiophosphoramidate morpholino oligonucleotides and chimeras." JACS 142.38 (2020): 16240-16253; see also Dumbovic, Gabrijela, et al. "Nuclear compartmentalization of TERT mRNA and TUG1 lncRNA is driven by intron retention." Nature Communications 12.1 (2021): 1-19, both of which are incorporated herein by reference in their entireties). Thus, the antisense oligonucleotides described herein may contain fully or partially TMO-modified nucleotides, or may contain chimeras of TMO-modified nucleotides with unmodified nucleotides and / or other nucleotides containing different modifications (e.g., LNA).

[0035] In some embodiments, antisense oligonucleotides may contain one or more modified bases. In some embodiments, cytosine is replaced with a modified cytosine, such as 5-methylcytosine or 5-hydroxymethylcytosine, which can enhance base pairing. If necessary, other modified bases (particularly cytosine or guanine) can be employed to reduce immunogenicity. Other modified bases are described in U.S. Pat. No. 10,064,959 (incorporated herein by reference). In various embodiments, the cytidine nucleobase in the antisense oligonucleotide is 5-methylcytidine. Thus, when a sequence contains a cytidine nucleobase ("C"), those skilled in the art will understand that the term includes 5-methylC. Furthermore, when a uracil base is included in the sequence, the term "U" is understood to include pseudouridine and N1-methylpseudouridine.

[0036] In some embodiments, upon binding to a target nucleic acid, the oligonucleotide forms a duplex with the target nucleic acid and recruits a nuclease that degrades the target nucleic acid. In some embodiments, the nuclease is a deoxyribonuclease. In some embodiments, the nuclease is a ribonuclease. In some embodiments, the ribonuclease is an endoribonuclease. In some embodiments, the endoribonuclease includes endoribonuclease A, P, H, I, III, T1, T2, U2, V1, PhyM, or V. In some embodiments, the ribonuclease is an exoribonuclease. In some embodiments, the exoribonuclease includes RNase PH, II, R, D, or T. In some embodiments, the nuclease includes polynucleotide phosphorylase (PNPase), oligoribonuclease, exoribonuclease I, or exoribonuclease II. In some embodiments, the ribonuclease recruited by the oligonucleotide that binds to the endogenous nucleic acid is RNase H.

[0037] In some embodiments, the oligonucleotide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, and 0, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more chemical modifications. In some embodiments, the oligonucleotide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, at least one gap segment comprising 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more chemical modifications. In some embodiments, the oligonucleotide comprises at least one wing segment comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more chemical modifications.In some embodiments, the oligonucleotide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more chemical modifications.

[0038] In some embodiments, the oligonucleotides described herein bind to a nucleic acid (e.g., mRNA) encoding KRAS, and binding of the oligonucleotide to the KRAS nucleic acid (e.g., KRAS mRNA) reduces the expression of the nucleic acid encoding KRAS in a cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to expression of KRAS not regulated by the oligonucleotide. In some embodiments, the oligonucleotides described herein bind to a nucleic acid (e.g., mRNA) encoding a mutant KRAS, and binding of the oligonucleotide to the nucleic acid encoding the mutant KRAS reduces the endogenous expression of mutant KRAS in a cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to expression of mutant KRAS not regulated by the oligonucleotide. In some embodiments, the mutant KRAS nucleic acid encodes a mutant KRAS protein that includes a G12C mutation, a G12V mutation, a G12A mutation, or a G12D mutation.

[0039] In some embodiments, the oligonucleotides described herein bind to a nucleic acid (e.g., mRNA) encoding KRAS, and binding of the oligonucleotide to KRAS reduces the activity or expression of a molecule in or associated with the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway in a cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the activity or expression of a molecule in or associated with the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway that is not modulated by the oligonucleotide.

[0040] In some embodiments, the oligonucleotides described herein bind to a nucleic acid (e.g., mRNA) encoding mutant KRAS, and binding of the oligonucleotide to the nucleic acid encoding the mutant KRAS results in a reduction in endogenous gene expression or activity in the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway in a cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to gene expression or activity in the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway that is not regulated by the oligonucleotide.

[0041] In some embodiments, the compound comprises at least two oligonucleotides, a first oligonucleotide that binds to a nucleic acid (e.g., mRNA) encoding KRAS, and a second oligonucleotide that binds to another nucleic acid encoding a mutant KRAS. In some embodiments, the nucleic acid encoding the mutant KRAS encodes a mutant KRAS protein comprising a G12C mutation, a G12V mutation, a G12A mutation, or a G12D mutation. In some embodiments, the compound comprises at least two oligonucleotides, a first oligonucleotide that binds to a nucleic acid (e.g., mRNA) encoding a first mutant KRAS, and a second oligonucleotide that binds to another nucleic acid encoding a second mutant KRAS, where the first mutant KRAS and the second mutant KRAS are different. The two different mutant KRASs may have different amino acid sequences. In some embodiments, the first and second mutant KRASs may be the same, but the nucleic acids encoding them have different sequences. In some embodiments, the compound comprises at least two oligonucleotides, a first oligonucleotide that binds to a nucleic acid encoding a different KRAS.

[0042] In some embodiments, binding of the oligonucleotide to both KRAS and mutant KRAS results in a reduction in endogenous gene expression or activity in the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway in a cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to endogenous gene expression or activity in the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway that is not regulated by the oligonucleotide.

[0043] In some embodiments, the compound is formulated for administration to a subject via an appropriate route, including, but not limited to, intravenous, intratumoral, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal. Pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate-release formulations, controlled-release formulations, fast-dissolving formulations, tablets, capsules, pills, delayed-release formulations, sustained-release formulations, pulsatile-release formulations, multiparticulate formulations, and mixed formulations of immediate-release and controlled-release formulations. In some embodiments, the compound is formulated into a dosage form. In some embodiments, the compound is formulated to include at least one excipient. In some embodiments, the excipient is a pharmaceutically acceptable excipient.

[0044] In some embodiments, the compounds comprising the oligonucleotides described herein treat a disease or condition by reducing the expression of a gene or signaling pathway associated with the disease or condition. In some embodiments, the compounds comprising the oligonucleotides described herein treat a disease or condition described herein by directly reducing the expression of a gene associated with the disease or condition. In some embodiments, the compounds comprising the oligonucleotides described herein treat a disease or condition by reducing gene expression as part of a signaling pathway described herein. In some embodiments, the compounds comprising the oligonucleotides described herein treat a disease or condition by reducing KRAS expression (e.g., mutant KRAS expression). In some embodiments, the compounds comprising the oligonucleotides described herein treat a disease or condition by reducing KRAS expression. In some embodiments, the compounds comprising the oligonucleotides described herein treat a disease or condition by reducing both KRAS expression and mutant KRAS expression. In some embodiments, the compounds comprising the oligonucleotides described herein treat a disease or condition by reducing endogenous KRAS expression. In some embodiments, the compounds comprising the oligonucleotides described herein treat a disease or condition by decreasing the expression or activity of the endogenous KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway. In some embodiments, the disease or condition described herein is cancer.

[0045] In some embodiments, the chemical modification of the oligonucleotide includes a chemical modification listed in Tables 1-3 and 6-11. In some embodiments, the oligonucleotide is an oligonucleotide listed in Tables 1-3 and 6-11.

[0046] In some embodiments, chemical modifications may occur at the 3'-OH group, 5'-OH group, backbone, sugar moiety, or nucleotide base. Chemical modifications may include non-naturally occurring linker molecules in interstrand or intrastrand crosslinks. In one aspect, chemically modified nucleic acids include modifications of one or more of the 3'-OH or 5'-OH group, backbone, sugar moiety, or nucleotide base, or the addition of non-naturally occurring linker molecules. In some embodiments, the chemically modified backbone includes a backbone other than a phosphodiester backbone. In some embodiments, the modified sugar includes a sugar other than deoxyribose (in modified DNA) or ribose (in modified RNA). In some embodiments, the modified base includes a base other than adenine, guanine, cytosine, thymine, or uracil. In some embodiments, the oligonucleotide includes at least one chemically modified base. In some examples, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more modified bases are included. In some embodiments, chemical modifications to a base moiety include natural and synthetic modifications of adenine, guanine, cytosine, thymine, or uracil, as well as natural and synthetic modifications of purine or pyrimidine bases.

[0047] In some embodiments, at least one chemical modification of the oligonucleotide includes any one of the following modifications, or any combination of the following modifications: 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-ON-methylacetamido (2′-O- 2'-modified nucleotides, including NMA, modifications of one or both of the non-linking phosphate oxygens in a phosphodiester backbone linkage, modifications of one or more of the linking phosphate oxygens in a phosphodiester backbone linkage, modifications of the components of the ribose sugar, substitution of a phosphate moiety with a "dephospho" linker, modifications or substitutions of naturally occurring nucleobases, modifications of the ribose-phosphate backbone, modifications of the 5' end of a polynucleotide, modifications of the 3' end of a polynucleotide, modifications of the deoxyribose phosphate backbone, substitution of a phosphate group, modifications of the ribophosphate backbone, modifications of the sugar of a nucleotide, modifications of the base of a nucleotide, or stereochemically pure nucleotides. Non-limiting examples of chemical modifications to oligonucleotides include modification of one or both of the unlinked or linked phosphate oxygens in the phosphodiester backbone linkage (e.g., sulfur (S), selenium (Se), BR3 (R can be, e.g., hydrogen, alkyl, or aryl), C (e.g., alkyl groups, aryl groups, and the like), H, NR2 (R can be, e.g., hydrogen, alkyl, or aryl, or R can be, e.g., alkyl or aryl), substitution of the phosphate moiety with a "dephospho" linker (e.g., methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo,substitution with methylenedimethylhydrazo, or methyleneoxymethylimino), modification or substitution of naturally occurring nucleobases with nucleic acid analogs, modification of the deoxyribose-phosphate or ribose-phosphate backbone (e.g., modifying the ribose-phosphate backbone to incorporate phosphorothioates, phosphonothioacetates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphates, phosphonocarboxylates, phosphoramidates, alkyl or aryl phosphonates, phosphonoacetates, or phosphotriesters), 5'-end modifications of nucleic acid sequences (e.g., 5'-cap or 5'-cap-OH modifications) or 3'-end modifications (3'-tail or 3'-end-OH modifications) modifications), methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, or methyleneoxymethylimino, modification of the ribophosphate backbone to incorporate morpholino (phosphorodiamidate morpholino oligomers (PMO)), thiomorpholino, cyclobutyl, pyrrolidine, or peptide nucleic acid (PNA) nucleoside surrogates, locked nucleic acid (LNA), non-locked nucleic acid (UNA), ethylene-bridged nucleic acid (ENA), cEt (constrained Modification of the sugar of a nucleotide to incorporate 2′-O-methoxyethoxy (2′-MOEr), 2′-O-methyl, 2′-O-methoxy-ethyl (2′-O-MOE), 2′-fluoro, 2′-aminoethyl, 2′-deoxy-2′-fluoroarabinonucleic acid, 2′-deoxy, 2′-O-methyl, 3′-phosphorothioate, 3′-phosphonoacetate (PACE),or 3'-phosphonothioacetate (thioPACE), modifications to the nucleotide base (A, T, C, G, or U) (e.g., 5-hydroxymethyl-modified nucleotides such as 5-hydroxymethylcytosine), and stereochemically pure nucleotides (e.g., phosphorothioate S conformation or phosphorothioate R conformation).

[0048] In some embodiments, the chemical modification of the oligonucleotide comprises at least one substitution of one or both of the non-linking phosphate oxygen atoms in the phosphodiester backbone linkages of the oligonucleotide. In some embodiments, the at least one chemical modification of the oligonucleotide comprises one or more substitutions of the linking phosphate oxygen atoms in the phosphodiester backbone linkages of the oligonucleotide. A non-limiting example of a chemical modification of a phosphate oxygen atom is a sulfur atom. In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification to the sugar of a nucleotide of the oligonucleotide. In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification to the sugar of a nucleotide of the oligonucleotide, the chemical modification comprising at least one locked nucleic acid (LNA). In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification to the sugar of a nucleotide of the oligonucleotide, the chemical modification comprising at least one non-locked nucleic acid (UNA). In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification to the sugar of a nucleotide of the oligonucleotide, the sugar comprising at least one ethylene-bridged nucleic acid (ENA). In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification to the sugar, including a modification of a sugar moiety, the sugar being a ribose sugar. In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification to the ribose sugar moiety of a nucleotide of the oligonucleotide, comprising a 2'-O-methyl group. In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification comprising a substitution of a phosphate moiety of the oligonucleotide with a dephosphoryl linker. In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification of the phosphate backbone of the oligonucleotide. In some embodiments, the oligonucleotide comprises a phosphorothioate group. In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification comprising a modification to a base of a nucleotide of the oligonucleotide.In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification comprising a non-natural base of the nucleotide. In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification comprising a morpholino group (e.g., a phosphorodiamidate morpholino oligomer, PMO), a cyclobutyl group, a pyrrolidine group, or a peptide nucleic acid (PNA) nucleoside surrogate. In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification comprising at least one stereochemically pure nucleic acid. In some embodiments, at least one chemical modification may be located near the 5' end of the oligonucleotide. In some embodiments, at least one chemical modification may be located near the 3' end of the oligonucleotide. In some embodiments, at least one chemical modification may be located near both the 5' and 3' ends of the oligonucleotide.

[0049] In some embodiments, the oligonucleotide comprises a backbone comprising multiple sugar and phosphate moieties covalently linked to one another, hi some embodiments, the backbone of the oligonucleotide comprises a phosphodiester bond between the linkage of the first hydroxyl group of the phosphate group to the 5' carbon of the deoxyribose in DNA or the ribose in RNA and the linkage of the second hydroxyl group to the 3' carbon of the deoxyribose in DNA or the ribose in RNA.

[0050] In some embodiments, the backbone of the oligonucleotide may lack a 5' reducing hydroxyl, a 3' reducing hydroxyl, or both that may be exposed to solvents. In some embodiments, the backbone of the oligonucleotide may lack a 5' reducing hydroxyl, a 3' reducing hydroxyl, or both that may be exposed to nucleases. In some embodiments, the backbone of the oligonucleotide may lack a 5' reducing hydroxyl, a 3' reducing hydroxyl, or both that may be exposed to hydrolases. In some examples, the backbone of the oligonucleotide may be represented as a polynucleotide sequence in a circular two-dimensional format, where one nucleotide follows another. In some examples, the backbone of the oligonucleotide may be represented as a polynucleotide sequence in a circular two-dimensional format, where one nucleotide follows another. In some embodiments, the 5' hydroxyl, the 3' hydroxyl, or both are connected via a phosphorus-oxygen bond. In some embodiments, the 5' hydroxyl, the 3' hydroxyl, or both are modified to a phosphoester at a phosphorus-containing moiety.

[0051] In some embodiments, the oligonucleotides described herein comprise at least one chemical modification.The chemical modification can be substitution, insertion, deletion, chemical modification, physical modification, stabilization, purification, or any combination thereof.In some embodiments, the modification is chemical modification. Suitable chemical modifications include any one of the following: 5'-adenylate, 5'-guanosine triphosphate cap, 5'-N7-methylguanosine triphosphate cap, 5'-triphosphate cap, 3'-phosphate, 3'-thiophosphate, 5'-phosphate, 5'-thiophosphate, cis-scinthymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, d spacer, PC spacer, r spacer, spacer 18, spacer 9, 3'-3' modification, 5'-5' modification, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP. TEG, DNP-X, DOTA, dT-biotin, dual biotin, PC-biotin, psoralen C2, psoralen C6, TINA, 3′-DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linker, 2′-deoxyribonucleoside analog purine, 2′-deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2′-O-methylribonucleoside analog, sugar modification analog, wobble / universal base, fluorescent dye label, 2′-fluoroRNA, 2′-O-methylRNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphorothioate DNA, phosphorothioate RNA, UNA, LNA, cEt, pseudouridine-5′-triphosphate, 5′-methylcytidine-5′-triphosphate, 2′-O-methyl-phosphorothioate, or a combination thereof.

[0052] In some embodiments, modifications to oligonucleotides can alter the physiochemical properties of the nucleotides, such as nucleotide conformation, polarity, hydrophobicity, chemical reactivity, base-pairing interactions, or any combination thereof. The chemical modification can also be a phosphorothioate substitution. In some embodiments, natural phosphodiester bonds can be susceptible to rapid degradation by cellular nucleases, and modifying internucleotide linkages with phosphorothioate (PS) bond substitutions can make them more stable against hydrolysis by cellular degradation. The modification can increase the stability of the polynucleic acid. The modification can also enhance biological activity. In some embodiments, phosphorothioate-enhanced RNA polynucleic acids can inhibit RNase A, RNase T1, bovine serum nuclease, or any combination thereof. These properties can enable the use of PS-RNA polynucleic acids of interest in applications likely to be exposed to nucleases in vivo or in vitro. For example, phosphorothioate (PS) linkages can be introduced into the last 3 to 5 nucleotides at the 5'-end or 3'-end of a polynucleic acid, thereby inhibiting exonucleolytic degradation. In some embodiments, phosphorothioate linkages can be added throughout a polynucleic acid to reduce endonuclease attack. In some embodiments, the oligonucleotides described herein contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 100, or more internucleotide linkages containing PS linkages. In some embodiments, the oligonucleotides described herein contain only PS linkages as internucleotide linkage modifications. In some embodiments, all internucleotide linkages of the oligonucleotides described herein are fully PS-modified or contain phosphorothioate internucleotide linkages.In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising one nucleobase. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising two nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising three nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising four nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising five nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising six nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising seven nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising eight nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising 9 nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising 10 nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 3'-terminal wing segment comprising 1 nucleobase. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 3'-terminal wing segment comprising 2 nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 3'-terminal wing segment comprising 3 nucleobases.In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 3'-terminal wing segment comprising four nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 3'-terminal wing segment comprising five nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 3'-terminal wing segment comprising six nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 3'-terminal wing segment comprising seven nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 3'-terminal wing segment comprising eight nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 3'-terminal wing segment comprising nine nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 3'-terminal wing segment comprising ten nucleobases.

[0053] In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising one nucleobase and a 3'-terminal wing segment comprising one nucleobase. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising two nucleobases and a 3'-terminal wing segment comprising two nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising three nucleobases and a 3'-terminal wing segment comprising three nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising four nucleobases and a 3'-terminal wing segment comprising four nucleobases. In some embodiments, an oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising five nucleobases and a 3'-terminal wing segment comprising five nucleobases.

[0054] In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising one nucleobase, a gapmer, and a 3'-terminal wing segment comprising one nucleobase, and the internucleotide linkages of the oligonucleotide connecting the 5'-terminal wing segment, gapmer, and 3'-terminal wing segment comprise only PS linkages. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising two nucleobases, a gapmer, and a 3'-terminal wing segment comprising two nucleobases, and the internucleotide linkages of the oligonucleotide connecting the 5'-terminal wing segment, gapmer, and 3'-terminal wing segment comprise only PS linkages. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising three nucleobases, a gapmer, and a 3'-terminal wing segment comprising three nucleobases, and the internucleotide linkages of the oligonucleotide connecting the 5'-terminal wing segment, gapmer, and 3'-terminal wing segment comprise only PS linkages. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising four nucleobases, a gapmer, and a 3'-terminal wing segment comprising four nucleobases, and the internucleotide linkages of the oligonucleotide connecting the 5'-terminal wing segment, gapmer, and 3'-terminal wing segment comprise only PS linkages. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising five nucleobases, a gapmer, and a 3'-terminal wing segment comprising five nucleobases, and the internucleotide linkages of the oligonucleotide connecting the 5'-terminal wing segment, gapmer, and 3'-terminal wing segment comprise only PS linkages. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising six nucleobases, a gapmer, and a 3'-terminal wing segment comprising six nucleobases, and the internucleotide linkages of the oligonucleotide connecting the 5'-terminal wing segment, gapmer, and 3'-terminal wing segment comprise only PS linkages.

[0055] In some embodiments, oligonucleotides comprising a 5'-terminal wing segment, a gapmer, a 3'-terminal wing segment, and a PS linkage as an internucleotide linkage comprise a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NOs: 1-10, and 14-21. In some embodiments, oligonucleotides comprising a 5'-terminal wing segment, a gapmer, a 3'-terminal wing segment, and a PS linkage as an internucleotide linkage comprise a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the oligonucleotide comprising a 5'-terminal wing segment, a gapmer, a 3'-terminal wing segment, and a PS linkage as an internucleotide linkage comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any of the sequences listed in Tables 1-3, and 6-11.

[0056] The oligonucleotides may be circular, substantially circular, or otherwise linked in a contiguous manner (e.g., arranged in a ring), and the oligonucleotides may also retain substantially similar secondary structures as substantially similar oligonucleotides that may not be circular or may not be ring-shaped.

[0057] In some embodiments, chemical modifications include modification of one or both of the non-linking phosphate oxygens in a phosphodiester backbone linkage or modification of one or more linking phosphate oxygens in a phosphodiester backbone linkage. As used herein, "alkyl" refers to a saturated hydrocarbon group that is straight-chain or branched. Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl or isopropyl), butyl (e.g., n-butyl, isobutyl, or t-butyl), or pentyl (e.g., n-pentyl, isopentyl, or neopentyl). Alkyl groups can contain 1 to about 20, 2 to about 20, 1 to about 12, 1 to about 8, 1 to about 6, 1 to about 4, or 1 to about 3 carbon atoms. As used herein, "aryl" refers to a monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) aromatic hydrocarbon, such as phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, or indenyl. In some embodiments, an aryl group has 6 to about 20 carbon atoms. As used herein, "alkenyl" refers to an aliphatic group containing at least one double bond. As used herein, "alkynyl" refers to a straight or branched hydrocarbon chain containing 2 to 12 carbon atoms and characterized by one or more triple bonds. Examples of alkynyl groups include ethynyl, propargyl, or 3-hexynyl. "Arylalkyl" or "aralkyl" refers to an alkyl moiety in which an alkyl hydrogen atom is replaced with an aryl group. Aralkyl also includes groups in which more than one hydrogen atom is replaced by an aryl group. Examples of "arylalkyl" or "aralkyl" include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl. "Cycloalkyl" refers to a cyclic, bicyclic, tricyclic, or polycyclic non-aromatic hydrocarbon group having 3 to 12 carbons. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl. "Heterocyclyl" refers to a monovalent radical of a heterocyclic ring system. Representative heterocyclyls include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, and morpholinyl. "Heteroaryl" refers to a monovalent radical of an aromatic heterocyclic ring system. Examples of heteroaryl moieties include imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, indolyl, thiophenyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, quinolyl, and pteridinyl.

[0058] In some embodiments, the phosphate group of a chemically modified nucleotide can be modified by replacing one or more oxygen atoms with different substituents. In some embodiments, a chemically modified nucleotide can include replacing an unmodified phosphate moiety with a modified phosphate described herein. In some embodiments, the modification of the phosphate backbone can include modifications that result in either an uncharged linker or a charged linker with an asymmetric charge distribution. Examples of modified phosphate groups include phosphorothioate, phosphonothioacetate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphate, phosphoramidate, alkyl or aryl phosphonate, and phosphotriester. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced with any of the following groups: sulfur (S), selenium (Se), BR (R can be, for example, hydrogen, alkyl, or aryl), C (e.g., alkyl, aryl, and the like), H, NR (R can be, for example, hydrogen, alkyl, or aryl), or (R can be, for example, alkyl or aryl). The phosphorus atom in an unmodified phosphate group can be achiral. However, replacing one of the non-bridging oxygens with one of the above atoms or atomic groups can make the phosphorus atom chiral. The phosphorus atom in such modified phosphate groups is an asymmetric center. The asymmetric center phosphorus atom can have either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). In some embodiments, oligonucleotides contain stereochemically pure nucleotides containing a phosphorothioate S conformation or a phosphorothioate R conformation. In some embodiments, the chiral phosphate product is present in greater than 50%, 60%, 70%, 80%, 90% or more diastereomers. In some embodiments, the chiral phosphate product is present in greater than 95% diastereomers. In some embodiments, the chiral phosphate product is present in greater than 96% diastereomers.In some embodiments, the chiral phosphate product is greater than 97% diastereomeric. In some embodiments, the chiral phosphate product is greater than 98% diastereomeric. In some embodiments, the chiral phosphate product is greater than 99% diastereomeric. In some embodiments, both non-bridging oxygens of the phosphorodithioate can be substituted with sulfur. The phosphorus center in the phosphorodithioate can be achiral, preventing oligoribonucleotide diastereomers from forming. In some embodiments, modifications to one or both non-bridging oxygens can also include replacing the non-bridging oxygen with a group independently selected from S, Se, B, C, H, N, and OR (R can be, for example, alkyl or aryl). In some embodiments, the phosphate linker can also be modified by substituting the bridging oxygen (i.e., the oxygen connecting the phosphate to the nucleoside) with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene phosphonates). Substitutions can occur at either or both of the linking oxygens.

[0059] Nucleic acids can be linked to each other using any internucleic acid linkage. There are two main classes of internucleic acid linkage groups, defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleic acid linkage groups include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Representative non-phosphorus-containing internucleic acid linkage groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-), siloxane (-O-Si(H)2-O-), and N,N*-dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In certain embodiments, internucleic acid linkage groups containing chiral atoms, such as alkylphosphonates and phosphorothioates, can be prepared as racemic mixtures or as separate enantiomers. Non-naturally occurring nucleic acids can contain a single modification. Non-naturally occurring nucleic acids can contain multiple modifications within one moiety or between different moieties.

[0060] Phosphate backbone modifications to nucleic acids include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramidates (bridged or non-bridged), phosphotriesters, phosphorodithioates, phosphodithioates, and boranophosphates, which may be used in any combination. Other non-phosphate linkages may also be used.

[0061] In some embodiments, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoramidate, and phosphorodithioate internucleotide linkages) may confer immunomodulatory activity to the modified nucleic acid and / or enhance its stability in vivo.

[0062] In some examples, the phosphorus derivative (or modified phosphate group) is attached within the sugar or sugar analog moiety and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, or the like.

[0063] In some embodiments, backbone modifications include replacing phosphodiester linkages with alternative moieties such as anionic, neutral, or cationic groups. Examples of such modifications include anionic internucleoside linkages, N3'→P5' phosphoramidate modifications, boranophosphate DNA, prooligonucleotides, neutral internucleoside linkages such as methylphosphonates, amide-linked DNA, methylene (methylimino) linkages, formacetal and thioformacetal linkages, sulfonyl-group-containing backbones, morpholino oligos, peptide nucleic acids (PNAs), and positively charged deoxyribonucleic guanidine (DNG) oligos. Modified nucleic acids can include chimeric or mixed backbones containing one or more modifications (e.g., combinations of phosphate linkages, such as combinations of phosphodiester and phosphorothioate linkages).

[0064] Substituents for the phosphate include, for example, short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamic acid backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with a mixture of N, O, S, and CH component moieties. It is also understood in nucleotide substitution that both the sugar and phosphate moieties of the nucleotide can be substituted, for example, with amide-type linkages (aminoethylglycine) (PNAs). Other types of molecules (conjugates) can also be linked to nucleotides or nucleotide analogs, for example, to enhance cellular uptake. Conjugates can be chemically linked to nucleotides or nucleotide analogs. Such conjugates include, but are not limited to, lipid moieties (cholesterol moieties, thioethers (e.g., hexyl-S-tritylthiol), thiocholesterol, aliphatic chains (e.g., dodecanediol or undecyl residues), phospholipids (e.g., di-hexadecyl-rac-glycerol or triethylammonium 1-di-O-hexadecyl-rac-glycero-SH-phosphonate, polyamines, or polyethylene glycol chains), or adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterin moieties.

[0065] In some embodiments, the chemical modifications described herein include modifications of the phosphate backbone. In some embodiments, the oligonucleotides described herein include at least one chemically modified phosphate backbone. Exemplary chemical modifications of the phosphate group or phosphate backbone can include replacing one or more oxygens with different substituents. Furthermore, modified nucleotides present in the oligonucleotide can include replacing unmodified phosphate moieties with modified phosphates described herein. In some embodiments, modifications of the phosphate backbone can include modifications that result in either uncharged linkers or charged linkers with asymmetric charge distribution. Exemplary modified phosphate groups can include phosphorothioates, phosphonothioacetates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced with any of the following groups: sulfur (S), selenium (Se), BR (R can be, for example, hydrogen, alkyl, or aryl), C (e.g., alkyl, aryl, and the like), H, NR (R can be, for example, hydrogen, alkyl, or aryl), or (R can be, for example, alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygens with one of the above atoms or groups of atoms can make the phosphorus atom chiral. That is, the phosphorus atom in such a modified phosphate group is an asymmetric center. The asymmetric center phosphorus atom can have either the "R" configuration (referred to herein as Rp) or the "S" configuration (referred to herein as Sp). In such cases, the chemically modified oligonucleotide can be stereochemically pure (e.g., S conformation or R conformation). In some embodiments, the chemically modified oligonucleotide comprises a stereochemically pure phosphate modification.For example, the chemically modified oligonucleotide comprises a phosphorothioate S conformation or a phosphorothioate R conformation.

[0066] In phosphorodithioates, both non-bridging oxygens are replaced with sulfur. The phosphorus center in phosphorodithioates is achiral, which prevents oligoribonucleotide diastereomers from forming. In some embodiments, modifications to one or both non-bridging oxygens can also include replacing the non-bridging oxygens with a group independently selected from S, Se, B, C, H, N, and OR (R can be, for example, alkyl or aryl).

[0067] The phosphate linker can also be modified by substituting the bridging oxygen (i.e., the oxygen that connects the phosphate to the nucleoside) with nitrogen (bridging phosphoramidate), sulfur (bridging phosphorothioate), and carbon (bridging methylene phosphonate). Substitution can occur at either or both of the linking oxygens.

[0068] In some embodiments, at least one phosphate group of an oligonucleotide may be chemically modified. In some embodiments, the phosphate group may be replaced with a non-phosphorus-containing linker. In some embodiments, the phosphate moiety may be replaced with a dephosphorylated linker. In some embodiments, the charged phosphate group may be replaced with a neutral group. In some embodiments, the phosphate group may be replaced with methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino. In some embodiments, the nucleotide analogs described herein may also be modified at the phosphate group. Modified phosphate groups can include modifications of the linkage between two nucleotides with phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, and other alkylphosphonates, including 3'-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidate and aminoalkylphosphoramidate), thionophosphoramidate, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. The phosphate linkage or modified phosphate linkage between two nucleotides can be via a 3'-5' or 2'-5' linkage, and the linkage can include inversions of direction, such as from 3'-5' to 5'-3' or from 2'-5' to 5'-2'.

[0069] In some embodiments, the chemical modifications described herein include modifications by substitution of the phosphate group. In some embodiments, the oligonucleotides described herein include at least one chemical modification, including substitution or replacement of the phosphate group. Exemplary phosphate group substitutions can include non-phosphorus-containing linkers. In some embodiments, substitution or replacement of the phosphate group can include replacing the charged phosphate group with a neutral moiety. Exemplary moieties that can replace the phosphate group include methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino.

[0070] In some embodiments, the chemical modifications described herein include modifying the ribophosphate backbone of an oligonucleotide. In some embodiments, the oligonucleotides described herein comprise at least one chemically modified ribophosphate backbone. Examples of chemically modified ribophosphate backbones include scaffolds that can mimic nucleic acids, in which the phosphate linker and ribose sugar are replaced with nuclease-resistant nucleoside or nucleotide surrogates. In some embodiments, the nucleobases can be linked by the surrogate backbone. Examples include morpholino, cyclobutyl, pyrrolidine, and peptide nucleic acid (PNA) nucleoside surrogates, such as phosphorodiamidate morpholino oligomers (PMOs).

[0071] In some embodiments, the chemical modifications described herein include sugar modifications. In some embodiments, the oligonucleotides described herein include at least one chemically modified sugar. Exemplary chemically modified sugars can include a 2' hydroxyl group (OH) modified or substituted with a number of different "oxy" or "deoxy" substituents. In some embodiments, modifications to the 2' hydroxyl group can enhance nucleic acid stability because the hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion. 2'-alkoxides can catalyze decomposition by intramolecular nucleophilic attack on the linker atom. Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, where "R" can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), polyethylene glycol (PEG), O(CHCHO), and hydroxyl groups. n Examples include CH2CH2OR (where R can be, for example, H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16, and 4 to 20)). In some embodiments, "oxy"-2' hydroxyl group modifications include LNA (where the 2' hydroxyl can be connected to the 4' carbon of the same ribose sugar, for example, by a Ci-6 alkylene or Cj-6 heteroalkylene bridge. Exemplary bridges can include methylene, propylene, ether, or amino bridges), O-amino (where amino can be, for example, NH2 (alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino)), and aminoalkoxy, O(CH2) n-amino (wherein amino can be, for example, NH2 (alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino)). In some embodiments, the "oxy"-2' hydroxyl group modification can include a methoxyethyl group (MOE), (OCH2CHOCH3, e.g., a PEG derivative). In some embodiments, the deoxy modification can include hydrogen (i.e., a deoxyribose sugar, e.g., an overhanging portion of a partial dsRNA), halo (e.g., bromo, chloro, fluoro, or iodo), amino (wherein amino can be, for example, NH2 (alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid)), NH(CH2CH2NH) nExamples of such sugars include CH2CH2-amino (wherein amino can be, for example, as described herein), NHC(O)R (wherein R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), cyano, mercapto, alkyl-thio-alkyl, thioalkoxy, and alkyl, cycloalkyl, aryl, alkenyl, and alkynyl, which can be optionally substituted with, for example, amino, as described herein. In some examples, the sugar group can also contain one or more carbons with the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, modified nucleic acids can include, for example, nucleotides containing arabinose as the sugar. A nucleotide "monomer" can have an alpha linkage at the Γ-position of the sugar, e.g., an alpha-nucleoside. Modified nucleic acids can also include "abasic" sugars, which lack a nucleobase at C-. Abasic sugars can also be further modified at one or more of the sugar atoms. Modified nucleic acids can also include one or more L-form sugars, e.g., L-nucleosides. In some embodiments, the oligonucleotides described herein comprise a 5-membered ribose sugar group with oxygen. Exemplary modified nucleosides and nucleotides can include oxygen substitution in ribose (e.g., with sulfur (S), selenium (Se), or alkylene (e.g., methylene or ethylene), addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl), ring contraction of ribose (e.g., to form a 4-membered cyclobutane or oxetane ring), ring expansion of ribose (e.g., to form a 6- or 7-membered ring with additional carbon or heteroatoms, such as anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino, which also have phosphoramidate backbones). In some embodiments, modified nucleotides can include polycyclic forms (e.g., tricyclic), as well as "unlocked" forms such as glycol nucleic acids (GNAs) (e.g., R-GNAs or S-GNAs, in which the ribose is replaced with a glycol unit attached to a phosphodiester bond), threose nucleic acids, etc.In some embodiments, modification of the sugar of the oligonucleotide comprises modifying the oligonucleotide to include a locked nucleic acid (LNA), a non-locked nucleic acid (UNA), an ethylene-bridged nucleic acid (ENA), a cEt (constrained ethyl) sugar, or a bridged nucleic acid (BNA).

[0072] In some embodiments, the oligonucleotides described herein comprise at least one chemical modification of the ribose sugar moiety. In some embodiments, chemical modifications of the ribose sugar moiety can include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-fluoro, 2'-aminoethyl, 2'-deoxy-2'-fluoroarabinonucleic acid, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 3'-phosphorothioate, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), 3'-phosphonoacetate (PACE), or 3'-phosphonothioacetate (thioPACE). In some embodiments, chemical modifications of the ribose sugar moiety include non-natural nucleic acids. In some instances, non-natural nucleic acids include modifications at the 5' and 2' positions of the sugar ring, such as 5'-CH2-substituted 2'-O-protected nucleosides. In some embodiments, non-natural nucleic acids include amide-linked nucleoside dimers prepared for incorporation into oligonucleotides, where the 3'-linked nucleosides (5' to 3') of the dimers include 2'-OCH3 and 5'-(S)-CH3. Non-natural nucleic acids can include 2'-substituted 5'-CH2 (or O)-modified nucleosides. Non-natural nucleic acids can include 5'-methylene phosphonate DNA and RNA monomers and dimers. Non-natural nucleic acids can include 5'-phosphonate monomers with 2'-substitutions and other modified 5'-phosphonate monomers. Non-natural nucleic acids can include 5'-modified methylene phosphonate monomers. Non-naturally occurring nucleic acids can include 5'- or 6'-phosphonate ribonucleoside analogs containing a hydroxyl group at the 5' and / or 6' position. Non-naturally occurring nucleic acids can include 5'-phosphonate deoxyribonucleoside monomers and dimers, which have a 5'-phosphate group.The non-natural nucleic acids may contain nucleosides having a 6'-phosphonate group, and the 5' and / or 6' positions are unsubstituted or substituted with a thio-tert-butyl group (SC(CH3)3) (and its analogs), a methyleneamino group (CH2NH2) (and its analogs), or a cyano group (CN) (and its analogs).

[0073] In some embodiments, non-natural nucleic acids also include modifications to the sugar moiety. In some embodiments, the nucleic acid contains one or more nucleosides in which the sugar group has been modified. Such sugar-modified nucleosides may confer enhanced nuclease stability, improved binding affinity, or some other beneficial biological property. In certain embodiments, the nucleic acid includes a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and / or 2' substituents), bridging of two ring atoms to form a bicyclic nucleic acid, S, N(R), or C(R1)(R2) (R = H, C1-C 12 and substitution of the ribosyl ring oxygen atom with an alkyl or protecting group, and combinations thereof.

[0074] In some examples, the oligonucleotides described herein contain modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a sugar "analog" cyclopentyl group. The sugar can be in pyranosyl or furanosyl form. The sugar moiety can be a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, and the sugar can be attached to the respective heterocyclic base in either the [alpha] or [beta] anomeric configuration. Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy- or amino-RNA / DNA chimeras. For example, sugar modifications can include 2'-O-methyl-uridine or 2'-O-methyl-cytidine. Sugar modifications include 2'-O-alkyl substituted deoxyribonucleosides and 2'-O-ethylene glycol-like ribonucleosides.

[0075] Modifications to the sugar moiety include natural and non-natural modifications of the ribose and deoxyribose, including, but not limited to, the following modifications at the 2' position: OH, F, O-, S-, or N-alkyl, O-, S-, or N-alkenyl, O-, S-, or N-alkynyl, or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl or alkynyl. 2' sugar modifications include, but are not limited to, -O[(CH) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n ONH2 and -O(CH2) nAlso included are ON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other chemical modifications at the 2' position include, but are not limited to, C1 to C 10 Examples of suitable sugars include lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups that improve the pharmacokinetic properties of oligonucleotides, or groups that improve the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3'-position of the sugar in 3'-terminal nucleotides or 2'-5'-linked oligonucleotides, and the 5'-position of 5'-terminal nucleotides. Chemically modified sugars also include those containing modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar. Examples of nucleic acids with modified sugar moieties include, but are not limited to, nucleic acids containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH, and 2'-O(CH)OCH substituents. Substituents at the 2' position include allyl, amino, azido, thio, O-allyl, O-(C1-C2). 10 alkyl), OCF3, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), and O-CH2-C(=O)-N(R m )(R n ) can also be selected from each R m and R n are independently H or substituted or unsubstituted C1-C 10 It is alkyl.

[0076] In certain embodiments, the nucleic acids described herein comprise one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acid comprises a bridge between the 4'-ribosyl ring atom and the 2'-ribosyl ring atom. In certain embodiments, the nucleic acids provided herein comprise one or more bicyclic nucleic acids, wherein the bridge constitutes a 4'-to-2' bicyclic nucleic acid. Examples of such 4'-to-2' bicyclic nucleic acids include, but are not limited to, one of the following formulas: 4'-(CH2)-O-2' (LNA), 4'-(CH2)-S-2', 4'-(CH2)2-O-2' (ENA), 4'-CH(CH3)-O-2', and 4'-CH(CHOCH3)-O-2', as well as analogs thereof, 4'-C(CH3)(CH3)-O-2' and analogs thereof.

[0077] In some embodiments, the chemical modifications described herein include modifications of the bases (e.g., nucleobases) of nucleotides. Exemplary nucleobases can include adenine (A), thymine (T), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or substituted in the oligonucleotides described herein. The nucleobases of the nucleotides can be independently selected from purines, pyrimidines, purine analogs, or pyrimidine analogs. In some embodiments, the nucleobases can be naturally occurring bases or synthetic base derivatives. In embodiments, nucleotide sequences may be presented herein using DNA nucleotide sequences (i.e., containing thymine nucleobases, "T" or "t") or as RNA nucleotide sequences (i.e., containing uracil nucleobases, "U" or "u"). It is understood from the context that if the nucleotide or sequence is intended to be RNA, the T nucleotide may be substituted with U (or a modified U, such as pseudouridine or 1-methylpseudouridine), and if the nucleotide or sequence is intended to be DNA, the U nucleotide may be substituted with T or a modified T. However, in embodiments, RNA nucleotides in antisense oligonucleotides may employ T (thymine) bases, and DNA nucleotides in antisense oligonucleotides may employ U (uracil) bases. In some embodiments, one or more T nucleotides in the antisense oligonucleotides disclosed herein are replaced with U or a modified U.

[0078] In some embodiments, the chemical modifications described herein include modifications of uracil. In some embodiments, the oligonucleotides described herein include at least one chemically modified uracil. Exemplary chemically modified uracils include pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine, 5 -Methoxy-uridine, Uridine 5-oxyacetic acid, Uridine 5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine, 5-carboxyhydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine, 5-methoxycarbonylmethyl-2-thio-uridine, 5-aminomethyl-2-thio-uridine, 5-methylaminomethyl-uridine, 5-methylaminomethyl-2-thio-uridine, 5-methylaminomethyl-2-seleno-uridine, 5-carbamoylmethyl-uridine, 5-carboxymethylaminomethyl-uridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl thyl-4-thio-pseudouridine, 5-methyl-uridine, 1-methyl-pseudouridine, 5-methyl-2-thio-uridine, 1-methyl-4-thio-pseudouridine, 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 5,6-Dihydrouridine, 5-methyl-dihydrouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine, 1-methyl-3-(3-amino-3-carboxypropylpseudouridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thio-uridine, α-thio-uridine, 2′-O-methyl-uridine, 5,2′-O-dimethyl-uridine, 2′-O-methyl-pseudouridine Examples of suitable uridine include uridine, 2-thio-2'-O-methyl-uridine, 5-methoxycarbonylmethyl-2'-O-methyl-uridine, 5-carbamoylmethyl-2'-O-methyl-uridine, 5-carboxymethylaminomethyl-2'-O-methyl-uridine, 3,2'-O-dimethyl-uridine, 5-(isopentenylaminomethyl)-2'-O-methyl-uridine, 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, pyrazolo[3,4-d]pyrimidine, xanthine, and hypoxanthine.

[0079] In some embodiments, the chemical modifications described herein comprise modifications of cytosines. In some embodiments, the oligonucleotides described herein comprise at least one chemically modified cytosine. Exemplary chemically modified cytosines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5-methyl-cytidine, 5-halo-cytidine, 5-hydroxymethyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, and the like. lysine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine, a-thio-cytidine, 2'-O-methyl-cytidine, 5,2'-O-dimethyl-cytidine, N4-acetyl-2'-O-methyl-cytidine, N4,2'-O-dimethyl-cytidine, 5-formyl-2'-O-methyl-cytidine, N4,N4,2'-O-trimethyl-cytidine, 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0080] In some embodiments, the chemical modifications described herein comprise modifications of adenines. In some embodiments, the oligonucleotides described herein comprise at least one chemically modified adenine.Exemplary chemically modified adenines include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diamino Purine, 1-methyl-adenosine, 2-methyl-adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6-isopentenyl-adenosine, 2-methylthio-N6-isopentenyl-adenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyl-adenosine, N6-threonylcarbamoyl-adenosine, N6-methyl α-N6-threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6-dimethyl-adenosine, N6-hydroxynorvalylcarbamoyl-adenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine, N6-2′-O-dimethyl-adenosine, N6-methyl Examples of 2'-amino-N6-methyl-adenosine include 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0081] In some embodiments, the chemical modifications described herein comprise modifications of guanines. In some embodiments, the oligonucleotides described herein comprise at least one chemically modified guanine. Exemplary chemically modified guanosines include inosine, 1-methyl-inosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine, wybutosine, peroxywybutosine, hydroxywybutosine, modified hydroxywybutosine, 7-deaza-guanosine, queuosine, epoxyqueuosine, galactosyl-queuosine, mannosyl-queuosine, 7-cyano-7-deaza-guanosine, 7-aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2 ... Chil-guanosine, N2,N2-dimethyl-guanosine, N2,7-dimethyl-guanosine, N2,N2,7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methylthio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2′-O-methyl-guanosine, N2-methyl- 2′-O-methyl-guanosine, N2,N2-dimethyl-2′-O-methyl-guanosine, 1-methyl-2′-O-methyl-guanosine, N2,7-dimethyl-2′-O-methyl-guanosine, 2′-O-methyl-inosine, 1,2′-O-dimethyl-inosine, 6-O-phenyl-2′-deoxyinosine, 2′-O-ribosylguanosine, 1-thio-guanosine, 6-O-methylguanosine, O 6 -methyl-2'-deoxyguanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.

[0082] In some embodiments, chemical modification of oligonucleotides can include the introduction or substitution of nucleic acid analogs or non-natural nucleic acids into oligonucleotides. In some embodiments, nucleic acid analogs can be any one of the chemically modified nucleic acids described herein, all of which are expressly incorporated by reference in their entirety. The chemically modified nucleotides described herein can include variants of guanosine, uridine, adenosine, thymidine, and cytosine, including any naturally occurring or non-naturally occurring guanosine, uridine, adenosine, thymidine, or cytidine that have been chemically modified, for example, by acetylation, methylation, or hydroxylation. Exemplary chemically modified nucleotides include 1-methyl-adenosine, 1-methyl-guanosine, 1-methyl-inosine, 2,2-dimethyl-guanosine, 2,6-diaminopurine, 2′-amino-2′-deoxyadenosine, 2′-amino-2′-deoxycytidine, 2′-amino-2′-deoxyguanosine, 2′-amino-2′-deoxyuridine, 2-amino-6-chloropurine ribonucleotides, and the like. 2'-Aminopurine-riboside, 2'-ara adenosine, 2'-ara cytidine, 2'-ara uridine, 2'-azido-2'-deoxyadenosine, 2'-azido-2'-deoxycytidine, 2'-azido-2'-deoxyguanosine, 2'-azido-2'-deoxyuridine, 2-chloroadenosine, 2'-fluoro-2'-deoxyadenosine, 2'-fluoro-2'-deoxy Cytidine, 2′-fluoro-2′-deoxyguanosine, 2′-fluoro-2′-deoxyuridine, 2′-fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine, 2-methyl-thio-N6-isopentenyl-adenosine, 2′-O-methyl-2-aminoadenosine, 2′-O-methyl-2′-deoxyadenosine, 2′-O-methyl-2′-deoxycytidine, 2′- O-methyl-2′-deoxyguanosine, 2′-O-methyl-2′-deoxyuridine, 2′-O-methyl-5-methyluridine, 2′-O-methylinosine, 2′-O-methylpseudouridine, 2-thiocytidine, 2-thio-cytidine, 3-methyl-cytidine, 4-acetyl-cytidine, 4-thiouridine, 5-(carboxyhydroxymethyl)-uridine, 5,6-dihydrouridine,5-aminoallylcytidine, 5-aminoallyl-deoxyuridine, 5-bromouridine, 5-carboxymethylaminomethyl-2-thio-uracil, 5-carboxymethylaminomethyl-uracil, 5-chloro-ara-cytosine, 5-fluoro-uridine, 5-iodouridine, 5-methoxycarbonylmethyl-uridine, 5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-azacytidine, 6-azauridine, 6-chloro-7-deaza-guanosine, 6-chloropurine riboside, 6-mercapto-guanosine, 6-methyl-mercaptopurine-riboside, 7-deaza-2′-deoxy-guanosine, 7-deazaadenosine, 7-methyl-guanosine, 8- Examples include azaadenosine, 8-bromo-adenosine, 8-bromo-guanosine, 8-mercapto-guanosine, 8-oxoguanosine, benzimidazole-riboside, beta-D-mannosyl-queuosine, dihydro-uridine, inosine, N1-methyladenosine, N6-([6-aminohexyl]carbamoylmethyl)-adenosine, N6-isopentenyl-adenosine, N6-methyl-adenosine, N7-methyl-xanthosine, N-uracil-5-oxyacetic acid methyl ester, puromycin, queuosine, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, wybutoxosine, xanthosine, and xylo-adenosine. In some embodiments, the chemically modified nucleic acids described herein are 2-amino-6-chloropurine riboside-5′-triphosphate, 2-aminopurine-riboside-5′-triphosphate, 2-aminoadenosine-5′-triphosphate, 2′-amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate, 2-thiouridine-5′-triphosphate, 2′-fluorouracil ... 2'-O-methyl-inosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate,5-bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate, 5-iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate, 5-iodo-2′-deoxyuridine-5′-triphosphate, 5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate, 5-propynyl-2′-deoxycytidine-5′-triphosphate, 5-propynyl-2′-deoxyuridine-5′-triphosphate, 6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate, 6-chloropurine riboside-5′-triphosphate The nucleotides include at least one chemically modified nucleotide selected from the group consisting of 7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate, 8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate, benzimidazole-riboside-5′-triphosphate, N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate, N6-methyladenosine-5′-triphosphate, 6-methylguanosine-5′-triphosphate, pseudouridine-5′-triphosphate, puromycin-5′-triphosphate, and xanthosine-5′-triphosphate. In some embodiments, the chemically modified nucleic acids described herein are selected from the group consisting of pyridin-4-one ribonucleosides, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 1-hydroxyur ... Idouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine,In some embodiments, the artificial nucleic acids described herein comprise at least one chemically modified nucleotide selected from dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In some embodiments, the artificial nucleic acids described herein comprise at least one chemically modified nucleotide selected from 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine. The compound comprises at least one chemically modified nucleotide selected from cytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine. In some embodiments, the chemically modified nucleic acids described herein are selected from the group consisting of 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyl adenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine,and 2-methoxy-adenine. In other embodiments, the chemically modified nucleic acids described herein comprise at least one chemically modified nucleotide selected from inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. In certain embodiments, the chemically modified nucleic acids described herein are selected from the group consisting of 6-aza-cytidine, 2-thio-cytidine, alpha-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, alpha-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, and the like. , alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, alpha-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.

[0083] Modified bases of non-natural nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthine-9-yl (I), 2-aminoadenin-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and and cytosine, 6-azo-uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Certain non-naturally occurring nucleic acids include 5-substituted pyrimidines, 6-azapyrimidines, and N-2 substituted purines, N-6 substituted purines, and O-6 substituted purines, 5-aminopropyl adenine, 5-propynyl uracil, 5-propynyl cytosine, 5-methyl cytosine, those that enhance the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 2-aminopropyl adenine, 5-propynyl uracil, and 5-propynyl cytosine. Methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl (-C≡C-CH3)uracil, 5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, fluorouracils, fluoroisopropyl ... Phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamp, phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), [3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), those in which the purine or pyrimidine base is replaced by other heterocycles, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine , hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-iodouracil, 2-amino-adenine, 6-thio-guanine, 2-thio-thymine, 4-thio-thymine, 5-propynyl-uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, or 2-amino-2'-deoxyadenosine.

[0084] In some embodiments, at least one chemical modification comprises a chemical modification of the 5' or 3' end. In some embodiments, the oligonucleotide comprises a chemical modification, including a 3' nucleotide, which can be stabilized against degradation, for example, by incorporating one or more of the modified nucleotides described herein. In this embodiment, uridine can be substituted with a modified uridine, such as 5-(2-amino)propyluridine and 5-bromouridine, or any of the modified uridines described herein, and adenosine and guanosine can be substituted with a modified adenosine and guanosine, for example, a modification at the 8th position (e.g., 8-bromoguanosine), or any of the modified adenosines or guanosines described herein. In some embodiments, deazanucleotides (e.g., 7-deaza-adenosine) can be incorporated into the oligonucleotide. In some embodiments, O-alkylated nucleotides and N-alkylated nucleotides (e.g., N6-methyladenosine) can be incorporated into the oligonucleotide. In some embodiments, sugar-modified ribonucleotides may be incorporated, e.g., the 2'OH group is replaced with a group selected from H, -OR, -R (where R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a sugar), halo, -SH, -SR (where R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a sugar), amino (where amino can be, e.g., NH2 (alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid)), or cyano (-CN).

[0085] Chemical means for introducing oligonucleotides into cells include colloidal dispersion systems such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, spherical nucleic acids (SNAs), liposomes, or lipid nanoparticles). An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle). Other cutting-edge methods for targeting nucleic acids are available, such as delivery of oligonucleotides using targeted nanoparticles or other suitable submicron-sized delivery systems.

[0086] An exemplary delivery vehicle is a liposome. The use of lipid formulations to introduce oligonucleotides into cells (in vitro, ex vivo, or in vivo) is contemplated. In another aspect, the oligonucleotide may be associated with a lipid. In some embodiments, lipid-associated oligonucleotides are encapsulated within the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped within the liposome, complexed with the liposome, dispersed in a lipid-containing solution, mixed with the lipid, combined with the lipid, contained in the lipid as a suspension, contained in or complexed with the micelle, or otherwise associated with the lipid. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, in some embodiments, they exist in a bilayer structure, as micelles, or with a "collapsed" structure. Alternatively, they may simply be interspersed in the solution, forming aggregates that are not uniform in size or shape. In some embodiments, lipids are fatty substances that are naturally occurring or synthetic lipids, including lipid droplets that occur naturally in the cytoplasm, and compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0087] "Liposome" is a generic term encompassing a variety of unilamellar and multilamellar lipid vesicles formed by the formation of closed lipid bilayers or aggregates. Liposomes are often characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement to form a closed structure, encapsulating water and dissolved solutes between the lipid bilayers. However, compositions with structures in solution that differ from the typical vesicular structure are also encompassed. For example, in some embodiments, lipids assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0088] In some embodiments, delivery methods include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, exosomes, polycation or lipid:cargo conjugates (or aggregates), naked polypeptides (e.g., recombinant polypeptides), naked DNA, artificial virions, and drug-enhanced uptake of polypeptides or DNA. In some embodiments, delivery methods include conjugating or encapsulating a compound or oligonucleotide described herein with at least one polymer, such as a natural polymer or a synthetic material. The polymer may be biocompatible or biodegradable. Non-limiting examples of suitable biocompatible, biodegradable synthetic polymers include aliphatic polyesters, poly(amino acids), copoly(ether-esters), polyalkylene oxalates, polyamides, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamide-esters, polyoxaesters containing amine groups, and poly(anhydrides). Such synthetic polymers can be homopolymers or copolymers (e.g., random, block, segmented, graft) of multiple different monomers (e.g., two or more of lactic acid, lactide, glycolic acid, glycolide, epsilon-caprolactone, trimethylene carbonate, p-dioxanone, etc.). In one example, the scaffold can be composed of a polymer comprising glycolic acid and lactic acid, such as a 90 / 10 or 5 / 95 ratio of glycolic acid to lactic acid. Non-limiting examples of naturally occurring biocompatible, biodegradable polymers can include glycoproteins, proteoglycans, polysaccharides, glycosaminoglycans (GAGs) and fragment(s) derived from these components, elastin, laminin, decorin, fibrinogen / fibrin, fibronectin, osteopontin, tenascin, hyaluronic acid, collagen, chondroitin sulfate, heparin, heparan sulfate, ORC, carboxymethylcellulose, and chitin.

[0089] In some embodiments, the oligonucleotides described herein can be packaged and delivered to cells via extracellular vesicles. The extracellular vesicles can be any membrane-bound particle. In some embodiments, the extracellular vesicles can be any membrane-bound particle secreted by at least one cell. In some examples, the extracellular vesicles can be any membrane-bound particle synthesized in vitro. In some examples, the extracellular vesicles can be any membrane-bound particle synthesized without the use of cells. In some embodiments, the extracellular vesicles can be exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apoptosomes, oncosomes, exosomes, enveloped viruses, exomers, or other ultra-large extracellular vesicles.

[0090] In some embodiments, the oligonucleotides described herein are conjugated. In some embodiments, the oligonucleotides are conjugated to an aptamer, peptide, antibody, lipid, carbohydrate, or polymer. In some embodiments, the oligonucleotides are conjugated to an aptamer, peptide, antibody, lipid, carbohydrate, or polymer at the 5'-end of the oligonucleotide. In some embodiments, the oligonucleotides are conjugated to an aptamer, peptide, antibody, lipid, carbohydrate, or polymer at the 3'-end of the oligonucleotide. In some embodiments, the oligonucleotides are conjugated to an aptamer, peptide, antibody, lipid, carbohydrate, or polymer at any nucleic acid residue of the oligonucleotide. In some embodiments, the aptamer, peptide, antibody, lipid, carbohydrate, or polymer conjugated to the oligonucleotide confers a therapeutic effect. For example, the peptide, antibody, lipid, carbohydrate, or polymer conjugated to the oligonucleotide can be a cytotoxic agent or an agent for treating cancer. In some embodiments, an aptamer, peptide, antibody, lipid, carbohydrate, or polymer conjugated to an oligonucleotide increases the binding efficiency of the oligonucleotide to endogenous nucleic acids. In some embodiments, an aptamer, peptide, antibody, lipid, carbohydrate, or polymer conjugated to an oligonucleotide confers targeting specificity to a particular type of cell (e.g., cancer cell) to the oligonucleotide. In some embodiments, an aptamer, peptide, antibody, lipid, carbohydrate, or polymer conjugated to an oligonucleotide confers in vitro, ex vivo, or in vivo stability to the oligonucleotide. For example, an oligonucleotide can be conjugated to polyethylene glycol (PEG) or an endosomolytic agent to reduce immunogenicity or degradation.In some embodiments, aptamers, peptides, antibodies, lipids, carbohydrates, or polymers are conjugated to oligonucleotides to facilitate their entry into cells. In some embodiments, aptamers, peptides, antibodies, lipids, carbohydrates, or polymers are conjugated to oligonucleotides to facilitate their release within cells. In some embodiments, the aptamers, peptides, antibodies, lipids, carbohydrates, or polymers conjugated to the oligonucleotides comprise at least one targeting moiety for targeting cells. Non-limiting examples of targeting moieties include signaling peptides, chemokines, chemokine receptors, adhesion molecules, antigens, or antibodies.

[0091] In embodiments, the antisense oligonucleotides described herein are conjugated to aptamers that target pancreatic cells, for example, as described in PCT / US2023 / 026980 (incorporated herein by reference in its entirety).

[0092] The linker for conjugating an oligonucleotide to an aptamer, peptide, antibody, lipid, or polymer can be any linker that connects biomolecules. In some embodiments, the linker described herein is a cleavable linker or a non-cleavable linker. In some examples, the linker is a cleavable linker. In other examples, the linker is a non-cleavable linker. In some embodiments, the linker is a non-polymeric linker. A non-polymeric linker refers to a linker that does not contain repeating units of a monomer produced by a polymerization process. In some embodiments, the linker includes a peptide moiety. In some examples, the peptide moiety includes at least 2, 3, 4, 5, or 6 or more amino acid residues. In some embodiments, the linker includes a benzoic acid group or a derivative thereof. In some embodiments, the linker can include a nucleic acid linker, such as a DNA linker. In such cases, the aptamer, peptide, antibody, lipid, or polymer can be conjugated to one end of the nucleic acid linker or inserted between the nucleic acid base pairs of the nucleic acid linker. In some embodiments, the linker can be a peptide linker. Peptide linkers can be flexible (e.g., polyglycine linkers) or rigid (e.g., EAAAK repeat linkers). In some embodiments, the peptide linker can be cleaved (e.g., disulfide bonds). In some embodiments, the linker comprises a polymer such as PEG, polylactic acid (PLA), or polyacrylic acid (PAA).

[0093] In embodiments, the melting temperature of the antisense oligonucleotide hybridized to the target sequence is at least about 35° C. m T is the temperature at which 50% of the oligonucleotide forms a duplex with its perfect complement and 50% is free in solution. T can be determined experimentally by measuring the change in absorbance of the oligonucleotide and its complement as a function of temperature. m Also, there are publicly available known T mIt can also be estimated using a computer. In some embodiments, the T of an oligonucleotide hybridized to a target sequence m is at least about 40° C., or at least about 45° C., or at least about 50° C. In some embodiments, the T of an oligonucleotide hybridized to a target sequence m In some embodiments, the T of the oligonucleotide hybridized to the target sequence is about 35° C. to about 60° C. m The temperature is about 40°C to about 60°C, or about 50°C to about 60°C.

[0094] In some embodiments, the antisense oligonucleotide further comprises a cell-penetrating moiety, which in some embodiments is directly or indirectly conjugated to the 3'-end or to its 3'-end of the oligonucleotide, optionally via a linker (e.g., polyethylene glycol linker or alkyl linker).In some embodiments, the compound further comprises a sterol conjugate (e.g., cholesterol conjugate) or a fatty acid conjugate, such as palmitoyl or stearyl lipid conjugate, which is optionally conjugated to the 3'-end of the antisense oligonucleotide.These moieties can enhance cell permeability.See US 9,012,225 (the entirety of which is incorporated herein by reference).

[0095] In some embodiments, the compound does not include any encapsulation or transfection reagents.

[0096] In some embodiments, the antisense oligonucleotide is encapsulated in a particle. In various embodiments, the particle is a liposome, a polymeric nanoparticle, or a lipid nanoparticle. Exemplary polymeric nanoparticles can be formed from PLA, PLGA, or their PEG copolymers. In some embodiments, the particle comprises a poly(beta amino ester) polymer. In various embodiments, the LNP comprises a cationic or ionizable lipid, a neutral lipid, cholesterol or a cholesterol moiety, and a PEGylated lipid.

[0097] In some embodiments, the lipid nanoparticle (or LNP) comprises a structured lipid. Exemplary structured lipids may be selected from one or more of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and tocopherols (e.g., alpha-tocopherol). In some embodiments, the structured lipid is cholesterol.

[0098] In some embodiments, the LNP comprises one or more phospholipids. Exemplary phospholipids are selected from cardiolipin, sterol-modified lipids (modified with a cholesterol moiety attached to the sn-2 carbon of the glycerol backbone), mixed acylglycerophospholipids, and symmetric acylglycerophospholipids. Head groups of acylglycerophospholipids include, for example, phosphatidic acid, lysophosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphoinositides, and phosphatidylserine.Exemplary phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

[0099] In some embodiments, the lipid nanoparticle composition further comprises one or more PEG-lipids. The PEG-lipids are lipids modified with polyethylene glycol. Exemplary PEG-lipids are selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. The PEG-lipid may be selected from PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-cholesterol, PEG-tocopherol, or PEG-DSPE lipids.

[0100] Lipid particle formulations that find use in embodiments of the present disclosure include those described in US 9,738,593, US 10,221,127, and US 10,166,298, which are incorporated herein by reference in their entireties. In some embodiments, the liposome or nanoparticle further comprises a targeting moiety as described.

[0101] In other embodiments, the compound is formulated for parenteral administration. In some embodiments, the compound for parenteral administration is configured encapsulated in particles as described.

[0102] In some embodiments, disclosed herein is a method for reducing the expression of KRAS or mutant KRAS protein or KRAS or mutant KRAS mRNA in cancer cells by treating cancer cells with a composition comprising the antisense oligonucleotide, composition, or pharmaceutical composition described herein, or contacting cancer cells with the composition, thereby regulating the KRAS-mediated signal transduction pathway in cancer cells.In some embodiments, the mutant KRAS protein comprises G12C mutation, G12V mutation, G12A mutation, or G12D mutation.

[0103] Also disclosed herein, in some embodiments, are methods of treating a subject in need thereof by administering to the subject a therapeutically effective amount of an oligonucleotide, composition, or pharmaceutical composition described herein. In some embodiments, the method is a method of treating a subject by modulating gene expression or the activity of signaling pathway expression associated with a signaling pathway in the subject. In some embodiments, the method comprises reducing gene expression by contacting a nucleic acid (e.g., endogenous mRNA) or a cell (e.g., a cancer cell) containing the nucleic acid with an oligonucleotide described herein. In some embodiments, the method comprises reducing KRAS, mutant KRAS, or a combination of KRAS and mutant KRAS in a subject or cancer cell by contacting KRAS mRNA or mutant KRAS mRNA with an oligonucleotide described herein, where binding of the oligonucleotide to the mRNA recruits endogenous nucleases that degrade the mRNA. In some embodiments, the method comprises reducing expression of a signaling pathway, such as a KRAS-mediated signaling pathway. In some embodiments, the method comprises decreasing expression of a gene in or activity of the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway.

[0104] In some embodiments, the oligonucleotide, composition, or pharmaceutical composition may be administered alone to a subject (e.g., as a single treatment). In some embodiments, the oligonucleotide, composition, or pharmaceutical composition is administered in combination with an additional agent. In some embodiments, the additional agent used herein is administered alone. The oligonucleotide, composition, or pharmaceutical composition and the additional agent may be administered together or sequentially. Non-limiting examples of additional agents include N-(2-(4-(4-bis(2-chloroethyl)aminophenyl)butyryl)aminoethyl)-5-(4-amidinophenyl)-2-furancarboxamide hydrochloride, allyl isothiocyanate, benzyl isothiocyanate, phenethyl isothiocyanate, belinostat, berberine, casticin, chrysin, bufalin, fisetin, fucoidan, gallic acid, gemcitabine, keishibukuryogan, JOTO1007, quercetin, lasfonin, 2,3,7,8-tetrachlorodibenzodioxin, triptolide, 4-hydroxybutenolide, or combinations thereof. Combination therapy can occur on the same day or can be separated by one or more days, one or more weeks, one or more months, or one or more years.

[0105] In some embodiments, the oligonucleotide, composition, or pharmaceutical composition is a first-line treatment for a disease or condition. In some embodiments, the oligonucleotide, composition, or pharmaceutical composition is a second-line, third-line, or fourth-line treatment. Generally, the methods disclosed herein involve administering the oligonucleotide, composition, or pharmaceutical composition via oral administration. However, in some examples, the methods involve administering the oligonucleotide, composition, or pharmaceutical composition via intraperitoneal injection. In some examples, the methods involve administering the pharmaceutical composition in the form of a rectal suppository. In some examples, the methods involve administering the oligonucleotide, composition, or pharmaceutical composition via intravenous ("iv") administration. It is contemplated that in some cases, the oligonucleotide, composition, or pharmaceutical composition disclosed herein may also be administered via other routes (such as subcutaneous injection, intramuscular injection, intradermal injection, transdermal administration, intranasal administration, intralymphatic injection, rectal administration, intragastric administration, or any other suitable parenteral administration). In some embodiments, a local delivery route close to the site of injury or inflammation is preferred over a systemic route. The route, dosage, time point, and duration of administration of the therapeutic agent can be adjusted. In some embodiments, the therapeutic agent is administered before or after the onset of acute and / or chronic symptoms of a disease or condition.

[0106] The appropriate dose and dosage to be administered to a subject will be determined by factors including, but not limited to, the particular oligonucleotide, composition, or pharmaceutical composition, the disease state and its severity, the identity of the subject requiring treatment (e.g., weight, sex, age), and can be determined depending on the particular circumstances surrounding the situation (including, for example, the specific agent being administered, the route of administration, the condition being treated, and the subject being treated).

[0107] The range of effective dosages can be adjusted based on the subject's response to treatment. Some routes of administration may require higher concentrations of the therapeutic agent to be effective than others.

[0108] In some embodiments, administration of an oligonucleotide, composition, or pharmaceutical composition described herein inhibits tumor growth by at least 10%, 15%, 20%, 30%, 40%, 50%, or more. In some embodiments, an oligonucleotide, composition, or pharmaceutical composition described herein is administered at a dose that inhibits tumor growth by at least 10%, 15%, 20%, 30%, 40%, 50%, or more. In some embodiments, an oligonucleotide, composition, or pharmaceutical composition described herein is administered on a schedule that inhibits tumor growth by at least 10%, 15%, 20%, 30%, 40%, 50%, or more. In some embodiments, an oligonucleotide, composition, or pharmaceutical composition described herein is administered at a dose and schedule that inhibits tumor growth by at least 10%, 15%, 20%, 30%, 40%, 50%, or more.

[0109] In some embodiments, the oligonucleotides, compositions, or pharmaceutical compositions described herein are administered to a subject at a dose sufficient to inhibit tumor growth. In some embodiments, the oligonucleotides, compositions, or pharmaceutical compositions described herein are administered to a subject on a schedule sufficient to inhibit tumor growth. In some embodiments, the oligonucleotides, compositions, or pharmaceutical compositions described herein are administered to a subject at a dose and on a schedule sufficient to inhibit tumor growth.

[0110] In some embodiments, the disease or condition described herein is cancer. In some embodiments, the cancer is associated with KRAS. In some embodiments, the cancer is associated with mutant KRAS. In some embodiments, the cancer is associated with KRAS. In some embodiments, the cancer is associated with abnormalities in the KRAS-mediated signaling pathway. In some embodiments, the cancer is lung cancer, pancreatic cancer, or colon cancer. Other non-limiting examples of cancer include acute lymphocytic leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenal carcinoma, adrenocortical carcinoma, adult leukemia, AIDS-related lymphoma, amyloidosis, anal cancer, astrocytoma, ataxia-telangiectasia, atypical nevus syndrome, atypical teratoma / atypical rhabdomyosarcoma, basal cell tumor, cholangiocarcinoma, Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor (gastrointestinal), cancer of unknown primary origin, cardiac tumor (heart tumor), cervical cancer, bile duct carcinoma, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia, and chronic myeloid leukemia.Leukemia, chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, nasal neuroblastoma, Ewing's sarcoma, extracranial germ cell tumors, extragonadal germ cell tumors, eye tumors, fallopian tube cancer, fibrous histiocytoma of bone, malignant osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, HER2-positive breast cancer, histiocytosis, Langerhans cells, Hodgkin's lymphoma tumor, hypopharyngeal carcinoma, intraocular melanoma, islet cell tumor, juvenile polyposis syndrome, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lobular carcinoma, lung cancer (non-small cell and small cell), lymphoma, malignant fibrous histiocytoma and osteosarcoma of bone, malignant glioma, melanoma, intraocular melanoma, meningioma, Merkel cell carcinoma, mesothelioma, malignant metastatic carcinoma, primary and occult metastatic squamous cell carcinoma of the neck, midline carcinoma, multiple endocrine neoplasia syndrome, multiple myeloma, plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome (MDS), bone Myeloproliferative neoplasms, chronic nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumors, non-Hodgkin's lymphoma, oral cavity cancer, lip and oral cavity cancer and oropharyngeal cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian germ cell tumors, pancreatic cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, peritoneal cancer, Peutz-Jeghers syndrome, pharyngeal cancer, pheochromocytoma, pituitary tumors, plasma cell neoplasms / multiple myeloma, pleuropulmonary blastoma, polycythemia vera, breast cancer during pregnancy, primary central nervous system (CNS) lymphoma, primary abdominal The cancer may include membranous cancer, prostate cancer, rectal cancer, recurrent carcinoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer, small intestine cancer, soft tissue sarcoma, solid tumors, squamous cell carcinoma of the skin, primary occult squamous cell carcinoma of the cervix, metastatic gastric cancer, T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, rare cancers of childhood, ureter and renal pelvis, transitional cell carcinoma, urethral cancer, uterine cancer (endometrial cancer), uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer, Wilms' tumor, or a combination thereof.

[0111] In some embodiments, the cancer is pancreatic cancer.

[0112] In some embodiments, the present disclosure provides pharmaceutical compositions comprising the oligonucleotides or compounds described herein. As used herein, a pharmaceutical composition refers to a mixture of a pharmaceutical composition with other chemical components (i.e., pharmaceutically acceptable inactive ingredients), such as carriers, excipients, binders, fillers, suspending agents, flavoring agents, sweeteners, disintegrants, dispersants, surfactants, lubricants, colorants, diluents, solubilizers, moistening agents, plasticizers, stabilizers, penetration enhancers, wetting agents, antifoaming agents, antioxidants, preservatives, or one or more combinations thereof. Optionally, the compound comprises two or more pharmaceutical compositions described herein. In practicing the methods of treatment or use provided herein, a therapeutically effective amount of a pharmaceutical composition described herein is administered as a pharmaceutical composition to a mammal having a disease, disorder, or condition (e.g., an inflammatory disease, a fibrostenosing disease, and / or a fibrotic disease) to be treated. In some embodiments, the mammal is a human. The therapeutically effective amount can vary widely depending on the severity of disease, the age and relative health condition of the subject, the efficacy of the pharmaceutical composition used, and other factors.The pharmaceutical composition can be used alone or can be used in combination with one or more pharmaceutical compositions as a component of a mixture.The pharmaceutical composition described herein comprises oligonucleotide, compound, cell contacted with oligonucleotide or cell contacted with compound containing oligonucleotide, or combinations thereof.

[0113] The pharmaceutical formulations described herein are administered to a subject by a suitable route of administration, including but not limited to, intravenous, intratumoral, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal. The pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast-dissolving formulations, tablets, capsules, pills, delayed release formulations, sustained release formulations, pulsatile release formulations, multiparticulate formulations, and mixed formulations of immediate release and controlled release formulations.

[0114] The pharmaceutical compositions, including the drug composition, are manufactured in a conventional manner, such as, by way of example only, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compressing processes.

[0115] The pharmaceutical composition may contain at least one pharmaceutical compound as an active ingredient in free acid or free base form, or in pharmaceutically acceptable salt form. In addition, the methods and pharmaceutical compositions described herein include the use of N-oxides (if appropriate), crystalline forms, amorphous phases, and active metabolites of these compounds having the same type of activity. In some embodiments, the pharmaceutical composition exists in a nonsolvated form or in a solvated form with a pharmaceutically acceptable solvent, such as water, ethanol, and the like. Solvated forms of the pharmaceutical composition are also considered to be disclosed herein.

[0116] In some embodiments, pharmaceutical compositions exist as enantiomers, diastereomers, or other stereoisomeric forms. The agents disclosed herein include all enantiomeric, diastereomeric, and epimeric forms, and mixtures thereof.

[0117] The use of absolute or chronological terms, such as "shall," "shall not," "shall not," "must," "must not," "first," "initial," "next," "subsequently," "before," "after," "finally," and "finally," is not meant to be limiting on the scope of the embodiments disclosed herein, but is exemplary.

[0118] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, to the extent that the terms "including," "include," "having," "has," "with," or variations thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0119] As used herein, the phrases "at least one," "one or more," and "and / or" are open-ended expressions that are conjunctive and disjunctive in operation. For example, each of the phrases "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" means A only, B only, C only, both A and B, both A and C, both B and C, or both A, B, and C.

[0120] As used herein, "or" can refer to "and," "or," or "and / or," and can be used both exclusively and inclusively. For example, the term "A or B" can refer to "A or B," "A but B," "A but B," and "A and B." In some embodiments, context may dictate a particular meaning.

[0121] When referring to a number or range of numbers, the term "about" means that the stated number or range of numbers is approximate within experimental variation (or within statistical experimental error), and that the number or range of numbers may vary, for example, by 1% to 15% of the stated number or range of numbers. For example, the term "about" refers to ±10% of the stated number or value.

[0122] As used herein, the terms "increased," "increase," or "increase" generally refer to an increase by a statistically significant amount. In some embodiments, the term "increased" or "increase" refers to an increase of at least 10% compared to a reference level, e.g., an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase of up to 100% (including 100%), or any increase between 10 and 100%, compared to a reference level, standard, or control. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more, compared to a reference level.

[0123] As used herein, the terms "decreased," "reduced," or "reduction" generally refer to a statistically significant decrease. In some embodiments, "decreased" or "reduction" refers to a decrease of at least 10% compared to a reference level, e.g., a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference level, or a decrease of up to 100% (e.g., absent or undetectable levels compared to a reference level), or any decrease between 10% and 100%. In the context of a marker or symptom, these terms refer to a statistically significant decrease in such level. For example, the decrease can be at least 10%, at least 20%, at least 30%, at least 40%, or more, preferably to a level that is accepted as being within the normal range for individuals without a given disease. [Example]

[0124] The following illustrative examples are representative of embodiments of the present disclosure. Example 1. Exemplary KRAS Antisense Oligonucleotides and KRAS mRNA Knockdown [Table 1] [Table 2]

[0125] KRAS mRNA knockdown Cell culture conditions and in vitro transfection Figure 1 shows mutant KRAS mRNA knockdown by KRAS G12C-specific antisense oligonucleotides (ASOs). The y-axis shows the percentage of KRAS mRNA knockdown compared to KRAS mRNA in cells treated with the non-KRAS-targeting ASO control, STN-030. The x-axis indicates the ASOs used, from left to right: a-STN-016, b-STN-017, c-STN-018, d-STN-019, e-STN-001, f-STN-002, and g-STN-003. STN-001 has a base shift relative to STN-018 to avoid a TCC nucleotide at the 3′-terminus. STN-002 is a 13-mer in a 3-8-2 wing-gap-wing configuration. STN-003 is a 13-mer in a 2-8-3 wing-gap-wing configuration. Mia PaCa-2 (ATCC) cells carrying the KRAS G12C mutation were seeded at a density of 20,000 cells per well in RPMI 1640 containing 10% FBS without antibiotics in clear, flat-bottom 96-well plates. Cells were transfected with 50 nM, 25 nM, 12.5 nM, or 6.25 nM antisense oligonucleotides complexed with RNAiMAX (Thermo Fisher). After overnight incubation at 37°C, the transfection mixture was removed and replaced with RPMI 1640 supplemented with 10% FBS and penicillin / streptomycin for an additional 48 hours. mRNA quantification was performed using QuantiGene (Thermo Fisher) according to the manufacturer's instructions. To normalize for inter- and intra-experimental variability, STN-018 was selected as a benchmark ASO and included in all plates to normalize mRNA knockdown.

[0126] Example 2. Antisense oligonucleotide-mediated inhibition of cancer cell growth Figure 2 and Table 3 below show the 3D growth inhibition of G12C-specific ASOs in Mia PaCa-2 cells. The y-axis shows the percentage of growth inhibition compared to the non-KRAS-targeting ASO control, STN-030. The x-axis shows the ASOs used, from left to right: a-STN-022, b-STN-016, c-STN-017, d-STN-025, e-STN-018, f-STN-020, g-STN-021, h-STN-019, i-STN-001, j-STN-002, and k-STN-003.

[0127] Cells were seeded overnight at 800 cells per well in RPMI 1640 containing 10% FBS in clear 384-well plates (S-Bio, #MS-9384UZ) and treated with ASOs at 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.313 μM, 0.156 μM, or 0.078 μM. After 5 days, cell proliferation was determined by measuring total ATP content using Cell Titer Glo reagent (Promega, G7570) according to the manufacturer's instructions. In Figure 2, for each group of cells treated with a specific ASO, individual bars represent the highest to lowest concentrations of ASO used (2-fold dilutions from left to right). The ASOs used in this experiment demonstrate significant growth inhibitory activity against cancer cell lines. [Table 3]

[0128] Example 3. Mutant KRAS mRNA knockdown and KRAS pathway modulation cell line [Table 4]

[0129] mRNA knockdown The ability of ASOs to knockdown desired mRNAs was assessed as follows. Cells were seeded at 20,000 cells per well in growth medium containing 10-20% FBS with 100 IU / mL Pen / Strep in a clear, flat-bottom 96-well plate. ASOs were added to the cells naked at various concentrations. Treated cells were incubated at 37°C for 96 hours. A pan-KRAS ASO (STN-100019) was used as a positive control. mRNA quantification was performed using Thermo Fisher's QuantiGene according to the manufacturer's instructions. The results are shown in Tables 6-11 below. [Table 5-1] [Table 5-2] [Table 5-3] [Table 6-1] [Table 6-2] [Table 7-1] [Table 7-2]

[0130] Inhibition of cell proliferation: The ability of ASOs to inhibit cell proliferation was assessed as follows: Cell lines were seeded overnight at 800 cells per well in growth medium containing 10-20% FBS and 100 IU / mL Pen / Strep in clear 384-well plates (S-Bio, #MS-9384UZ). Cells were treated with various concentrations of ASOs. After 7-10 days, cell viability was determined by measuring total ATP content using Cell Titer Glo reagent (Promega, G7570) according to the manufacturer's instructions. Growth inhibition results are shown in the table and graph below. [Table 8-1] [Table 8-2] [Table 8-3] [Table 9-1] [Table 9-2] [Table 10-1] [Table 10-2]

[0131] Figure 3 shows Western blot analysis following ASO-mediated G12V KRAS knockdown in the LCLC-97TM1 cell line (day 4), demonstrating KRAS protein knockdown and pathway modulation. [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5] [Table 11-6] [Table 11-7] [Table 11-8] Table 11-9 Table 11-10 Table 11-11 Table 11-12 Table 11-13 Table 11-14 Table 11-15 Table 11-16 Table 11-17 Table 11-18 Table 11-19 Table 11-20 Table 11-21 Table 11-22 Table 11-23 Table 11-24 [Table 11-25]

[0132] Although the foregoing disclosure has been described in some detail for purposes of clarity and understanding, it will be apparent to those skilled in the art from a reading of this disclosure that various changes in form and detail can be made therein without departing from the true scope of the present disclosure. For example, all of the techniques and devices described above may be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document was individually and separately indicated to be incorporated by reference for all purposes.

Claims

1. A compound comprising an antisense oligonucleotide that inhibits expression of KRAS mRNA, wherein the antisense oligonucleotide comprises 10 to 30 linked nucleotides and has a sequence complementary to KRAS mRNA, and the oligonucleotide has at least 8 consecutive nucleotides of any one of SEQ ID NOs: 1-10 and 14-21.

2. The compound of claim 2 , wherein the oligonucleotide is at least 12 nucleotides in length.

3. The compound of claim 3 , wherein the oligonucleotide is at least 14 nucleotides in length.

4. 3. The compound of claim 2, wherein the oligonucleotide is 10 to 24 nucleotides in length, or 10 to 16 nucleotides in length, or 12 to 16 nucleotides in length.

5. 6. The compound of claim 5, wherein the oligonucleotide is 12, 13, 14, 15, or 16 nucleotides in length.

6. The compound of claim 6 , wherein the oligonucleotide is 14 nucleotides in length.

7. The compound of any one of claims 1 to 6, wherein the oligonucleotide comprises at least 12 consecutive nucleobases of any one of SEQ ID NOs: 1-10 and 14-21.

8. 8. The compound of claim 7, wherein the oligonucleotide comprises or consists of the nucleobase sequence of any one of SEQ ID NOs: 1-10 and 14-21.

9. 9. The compound of claim 8, wherein the oligonucleotide has a nucleobase sequence selected from SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, and 10.

10. The compound of any one of claims 1 to 9, wherein the antisense oligonucleotide has a stretch of at least six DNA nucleotides sufficient to recruit RNase H.

11. 11. The compound of claim 10, wherein one or more DNA nucleotides comprise a 2' chemical modification independently selected from 2'-fluoro, 2'-methyl, and 2'-ethyl.

12. The compound of claim 10, wherein the DNA nucleotide does not contain a 2' chemical modification.

13. 13. The compound of any one of claims 10 to 12, wherein the antisense oligonucleotide is a gapmer having 5' and 3' segments, each of the 5' and 3' segments being 2 to 6 nucleotides or 2 to 4 nucleotides, and the 5' and 3' segments containing no DNA nucleotides.

14. 14. The compound of claim 13, wherein the lengths of the 5' and 3' segments are independently selected from 2 or 3 nucleotides, and the 5' and 3' segments are flanked by an internal sequence of 8 DNA nucleotides.

15. 15. The compound of claim 14, wherein one or more nucleotides of the 5' segment and the 3' segment comprise a 2'-O substituent, and optionally, all of the nucleotides of the 5' segment and the 3' segment comprise a 2'-O substituent.

16. 16. The compound of claim 15, wherein the 2'-O substituents are independently selected from 2'-O methyl, 2'-O ethyl, 2'-O methoxyethyl (MOE), and bridged nucleotides having a 2' to 4' bridge.

17. 17. The compound of claim 16, wherein the bridged nucleotide has a methylene bridge (LNA) or a cEt (constrained ethyl) bridge.

18. The compound of any one of claims 1 to 17, wherein the antisense oligonucleotide has a modified backbone.

19. 19. The compound of claim 18, wherein the antisense oligonucleotide comprises one or more phosphorothioate or phosphorodithioate nucleotides.

20. 20. The compound of claim 19, wherein the oligonucleotide is fully phosphorothioate or phosphorodithioate linked.

21. 21. The compound of claim 20, wherein the oligonucleotide is fully phosphorothioate linked.

22. 22. The compound of any one of claims 1 to 21, wherein the cytosine nucleobase in said antisense oligonucleotide is a modified cytosine, which is optionally 5-methylcytosine or 5-hydroxymethylcytosine.

23. 2. The compound of claim 1, wherein the antisense oligonucleotide has a structure shown in one or more of Tables 1, 2, 3, 6, 7, 8, 9, 10, and 11.

24. The compound of any one of claims 1 to 23, further comprising a cell-targeting or cell-penetrating moiety.

25. 25. The compound of claim 24, wherein the cell-targeting or cell-penetrating moiety is conjugated directly or indirectly, optionally via a linker, at the 3' end of the oligonucleotide.

26. 26. The compound of claim 24 or claim 25, wherein the moiety comprises a sterol conjugate or a fatty acid conjugate, which is optionally a cholesteryl, palmitoyl, or stearyl conjugate.

27. 25. The compound of claim 24, wherein the compound further comprises a cell-targeting aptamer.

28. The compound of any one of claims 1 to 27, wherein the compound does not include any encapsulation or transfection reagents.

29. The compound of any one of claims 1 to 27, wherein the antisense oligonucleotide is encapsulated in a particle.

30. 30. The compound of claim 29, wherein the particle is a liposome, a polymeric nanoparticle, or a lipid nanoparticle.

31. The compound of any one of claims 1 to 30, wherein the compound is formulated for parenteral administration.

32. A pharmaceutical composition comprising a compound according to any one of claims 1 to 31 and a pharmaceutically acceptable carrier or vehicle.

33. A method for treating a subject having a pathology associated with the aberrant expression of KRAS or a pathology associated with mutated KRAS, comprising administering to the subject an effective amount of a compound according to any one of claims 1 to 32, or a pharmaceutical composition according to claim 32.

34. 34. The method of claim 33, wherein the subject has a malignancy associated with an abnormality in the KRAS-mediated signaling pathway.

35. 35. The method of claim 34, wherein the malignancy is associated with KRAS or mutated KRAS.

36. 36. The method of claim 35, wherein the mutant KRAS mRNA encodes a mutant KRAS protein comprising a G12C mutation.

37. 36. The method of claim 35, wherein the mutant KRAS mRNA encodes a mutant KRAS protein comprising a G12D mutation or a G12V mutation.

38. The method of any one of claims 34 to 37, wherein the malignant tumor associated with an abnormality in the KRAS-mediated signaling pathway is non-metastatic.

39. The method of any one of claims 34 to 37, wherein the malignant tumor associated with an abnormality in the KRAS-mediated signaling pathway is metastatic.

40. The method of any one of claims 34 to 39, wherein the malignant tumor is a carcinoma.

41. 41. The method of claim 40, wherein the malignant tumor is breast cancer, cervical cancer, pancreatic cancer, squamous cell carcinoma, head and neck cancer, thyroid cancer, gastric cancer, colon cancer, or liver cancer.

42. 42. The method of claim 41, wherein the malignant tumor is pancreatic cancer.

43. 41. The method of claim 40, wherein the malignant tumor is cervical squamous cell carcinoma, endocervical adenocarcinoma, bile duct carcinoma, esophageal carcinoma, colon adenocarcinoma, oral squamous cell carcinoma, head and neck squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung carcinoma, liver hepatocellular carcinoma, lung squamous cell carcinoma, rectal adenocarcinoma, gastric adenocarcinoma, thyroid carcinoma, or pancreatic adenocarcinoma.

44. The method of any one of claims 33 to 43, wherein the compound or composition is administered parenterally.

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