Compositions for modulating KRAS expression and uses thereof

Abstract

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

Description

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

[0002] Sequence Listing The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: Computer-readable format copy of the Sequence Listing (file name: "MAX-008PC_133279-5008_SequenceListing"; record date: January 17, 2024; file size: 45,380 bytes). Background Art

[0003] Certain diseases or disorders are caused by gene mutations or dysregulation of signal transduction pathways. To treat such diseases or disorders, one of the most popular treatment options involves directly editing gene mutations or using gene silencing tools or methods for transcriptional / translational regulation. In various embodiments, oligonucleotide-induced gene silencing can control the RNA expression of target genes, including transcriptional inactivation, mRNA degradation, and transcriptional attenuation. Existing RNA-induced gene silencing tools may be inefficient and / or non-specific. Therefore, there is still a need for compositions and methods for effectively controlling gene expression, and in particular reducing the RNA levels of mutant genes such as KRAS and / or downstream signal transduction pathways. Summary of the Invention

[0004] In aspects and embodiments, a compound is provided 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 wherein the oligonucleotide has at least 8 consecutive nucleotides of any one of SEQ ID NOs: 1-10 and 14-21.

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

[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 consecutive nucleobases of any one of SEQ ID NOs: 1-10 and 14-21. In embodiments, the oligonucleotide comprises or consists of a 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 an embodiment, the antisense oligonucleotide has an extension of at least 6 DNA nucleotides sufficient to recruit RNase H. In an embodiment, one or more DNA nucleotides include 2' chemical modifications independently selected from 2'-fluoro, 2'-methyl and 2'-ethyl. In an embodiment, DNA nucleotides do not include 2' chemical modifications. In an embodiment, the antisense oligonucleotide is a gap polymer with a 5' segment and a 3' segment, each of the 5' segment and the 3' segment being 2 to 6 nucleotides or 2 to 4 nucleotides, and wherein the 5' segment and the 3' segment do not contain DNA nucleotides. In an embodiment, the length of the 5' segment and the 3' segment is each independently selected from 2 or 3 nucleotides, and the 5' segment and the 3' segment are flanked by an internal sequence of 8 DNA nucleotides. In an embodiment, one or more nucleotides of the 5' segment and the 3' segment include 2'-O substituents, optionally wherein all nucleotides of the 5' segment and the 3' segment include 2'-O substituents. In an embodiment, the 2'-O substituent is independently selected from 2'-O methyl, 2'-O ethyl, 2'-O methoxyethyl (MOE) and a bridged nucleotide with a 2' to 4' bridge. In an embodiment, the bridged nucleotide has a methylene bridge (LNA) or a constrained ethyl bridge (cEt). In an embodiment, the antisense oligonucleotide has a modified backbone. In an embodiment, the antisense oligonucleotide comprises one or more phosphorothioate or phosphorodithioate nucleotides. In an embodiment, the oligonucleotide is completely phosphorothioate or phosphorodithioate connected. In an embodiment, the oligonucleotide is completely phosphorothioate connected.

[0008] In an embodiment, the cytosine nucleobase in the antisense oligonucleotide is a modified cytosine, and the modified cytosine is optionally 5-methylcytosine or 5-hydroxymethylcytosine. In an embodiment, the antisense oligonucleotide has a core base 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 an embodiment, the compound further comprises a cell targeting or penetrating portion. In an embodiment, the cell targeting or penetrating portion is directly or optionally indirectly conjugated through a joint at the 3' end of the oligonucleotide. In an embodiment, the portion comprises a sterol conjugate or a fatty acid conjugate, and the conjugate is optionally a cholesterol base, a palmitoyl group or a stearoyl group conjugate. In an embodiment, the compound further comprises a cell targeting aptamer.

[0009] In an embodiment, the compound does not comprise any encapsulation or transfection reagent. In an embodiment, the antisense oligonucleotide is encapsulated in a particle. In an embodiment, the particle is a liposome, a polymer nanoparticle, or a lipid nanoparticle. In an embodiment, the compound is formulated for parenteral administration. In an embodiment, 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 suffering from a disease associated with abnormal expression of KRAS or associated with mutated KRAS is provided, the method comprising administering to the subject an effective amount of a compound of the present disclosure or a pharmaceutical composition of the present disclosure. In an embodiment, the subject suffers from a malignant tumor associated with an abnormality in a KRAS-mediated signaling pathway. In an embodiment, the malignant tumor is associated with KRAS or a mutated KRAS. In an embodiment, the mutated KRAS mRNA encodes a mutant KRAS protein comprising a G12C mutation. In an embodiment, the mutated 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 a KRAS-mediated signaling pathway is non-metastatic. In an embodiment, the malignant tumor associated with an abnormality in a KRAS-mediated signaling pathway is metastatic.

[0011] In embodiments, the malignant tumor is a cancer. In embodiments, 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 embodiments, the malignant tumor is pancreatic cancer. In embodiments, the malignant tumor is cervical squamous cell carcinoma, endocervical adenocarcinoma, bile duct cancer, 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, hepatocellular carcinoma, lung squamous cell carcinoma, rectal adenocarcinoma, gastric adenocarcinoma, thyroid cancer or pancreatic adenocarcinoma. In embodiments, the malignant tumor is pancreatic cancer, such as pancreatic adenocarcinoma. In embodiments, the antisense oligonucleotide is administered parenterally. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Illustration of knockdown of KRAS mRNA encoding a mutant KRAS protein comprising a G12C mutation mediated by antisense oligonucleotides (ASOs) described herein, from left to right: STN-016 (a), STN-017 (b), STN-018 (c), STN-019 (d), STN-001 (e), STN-002 (f), and STN-003 (g).

[0013] Figure 2 Illustration of three-dimensional (3D) inhibition of cell proliferation due to inhibition of mutant KRAS expression by contacting cells carrying the KRAS G12C mutation (Mia Paca-2) with oligonucleotides described herein, from 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).

[0014] Figure 3 Figure 1 illustrates Western blot analysis of G12V KRAS knockdown in LCLC-97TM1 cell line using ASOs. For reference, the ASOs loaded from left to right are STN-100080, STN-100993, STN-100994, STN-100987, and STN-100989. DETAILED DESCRIPTION

[0015] Described herein are compounds, compositions, and methods for regulating KRAS expression (e.g., mutated KRAS expression) and / or downstream signaling pathways. Also described herein are compounds, compositions, and methods for treating the disease or disorder by regulating gene expression or signaling pathways associated with the disease or disorder. In some embodiments, the compound comprises at least one antisense oligonucleotide that binds (e.g., hybridizes) to endogenous KRAS mRNA after being delivered to the cell, thereby causing degradation of KRAS mRNA. In some embodiments, described herein is a method of utilizing the compounds or oligonucleotides described herein. In some embodiments, the method is used to treat a disease or disorder by contacting the cell with an oligonucleotide to reduce KRAS gene expression (e.g., mutated KRAS) or related signaling pathways. In some embodiments, the antisense oligonucleotide hybridizes with an mRNA encoding a mutated KRAS protein, such as a KRAS protein comprising a G12C mutation, a G12V mutation, a G12D mutation, or a G12A mutation.

[0016] In some embodiments, the oligonucleotide is an antisense oligonucleotide, wherein the oligonucleotide is complementary to and binds to (e.g., hybridizes to) a segment of at least one endogenous nucleic acid (e.g., mRNA). In some embodiments, the combination of the oligonucleotide and the endogenous nucleic acid results in degradation of the endogenous nucleic acid or blocks translation of the target protein from the endogenous nucleic acid, thereby reducing the expression of the gene encoded by the endogenous nucleic acid. For example, the combination of the oligonucleotide and the endogenous mRNA produces a duplex nucleic acid molecule, which can then recruit endogenous nucleases (e.g., RNase H) to degrade the mRNA.

[0017] In some embodiments, the oligonucleotide comprises an extension of DNA nucleotides (such as an extension of 5 to 10 DNA nucleotides) sufficient to recruit RNase H when hybridized with the target mRNA. In embodiments, the extension 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 gap polymer.

[0018] In some embodiments, the oligonucleotides regulate 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. Therefore, in some embodiments, the reduction in gene expression due to the binding of the oligonucleotide to the endogenous nucleic acid can further reduce the expression of the signaling pathway comprising the gene regulated by the oligonucleotide. In some embodiments, reducing the expression of a gene or signaling pathway results in a therapeutic effect for treating a disease or disorder. In some embodiments, the disease or disorder is caused by an increase in expression of a gene or signaling pathway. In some embodiments, the disease or disorder described herein is caused by a genetic mutation (such as a mutated KRAS) associated with a gene or signaling pathway.

[0019] In aspects and embodiments, compounds are provided comprising an antisense oligonucleotide that inhibits expression of KRAS mRNA, including, in some embodiments, mRNA encoding a mutant KRAS, wherein the antisense oligonucleotide comprises 10 to 30 linked nucleotides and has a sequence complementary to KRAS mRNA, and wherein the oligonucleotide has at least 8 contiguous nucleotides of any one of SEQ ID NOs: 1-10 and 14-21.

[0020] In embodiments, the oligonucleotide comprises at least 10, or at least 12, or at least 14 continuous core bases of any one of SEQ ID NO: 1-10 and 14-21. In embodiments, the length of the oligonucleotide is 10-24 nucleotides, such as 10 to 16 or 12 to 16 nucleotides. In embodiments, the length of the oligonucleotide is 12, 13, 14, 15 or 16 nucleotides. For example, in embodiments, the length of the oligonucleotide is 13 or 14 nucleotides. In embodiments, the oligonucleotide comprises at least 12 continuous core bases of any one of SEQ ID NO: 1-10 and 14-21. In embodiments, the oligonucleotide comprises or consists of the core base sequence of any one of SEQ ID NO: 1-10 and 14-21. In embodiments, the oligonucleotide has a core base sequence selected from SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0021] In some embodiments, oligonucleotides as described herein are antisense oligonucleotides for targeting and binding (e.g., hybridization) to endogenous nucleic acids. In some embodiments, the combination of oligonucleotides and endogenous nucleic acids raises endogenous nucleases (e.g., RNase H) to degrade endogenous nucleic acids. In some embodiments, the degradation of endogenous nucleic acids reduces the expression of genes encoded by endogenous nucleic acids. In some embodiments, the degradation of endogenous nucleic acids can treat diseases or illnesses as described herein.

[0022] In some embodiments, the oligonucleotide comprises 11 nucleic acid bases. In some embodiments, the oligonucleotide comprises 12 nucleic acid bases. In some embodiments, the oligonucleotide comprises 13 nucleic acid bases. In some embodiments, the oligonucleotide comprises 14 nucleic acid bases. In some embodiments, the oligonucleotide comprises 15 nucleic acid bases. In some embodiments, the oligonucleotide comprises 16 nucleic acid bases. In some embodiments, the oligonucleotide has a length of no more than 20 nucleotides. In some embodiments, the oligonucleotide has a length of no more than 19 nucleotides. In some embodiments, the oligonucleotide has a length of no more than 18 nucleotides. In some embodiments, the oligonucleotide has a length of no more than 17 nucleotides. In some embodiments, the oligonucleotide has a length of no more than 16 nucleotides. In some embodiments, the oligonucleotide has a length of no more than 15 nucleotides. In some embodiments, the oligonucleotide has a length of no more than 14 nucleotides. In some embodiments, the oligonucleotide has a length of no more than 13 nucleotides.

[0023] In some embodiments, the oligonucleotide comprises at least one gap segment. In some embodiments, the gap segment comprises four, five, six, seven, eight, nine, 10, 11, 12, 13, or 14 nucleic acid bases. In some embodiments, the gap segment comprises four nucleic acid bases. In some embodiments, the gap segment comprises five nucleic acid bases. In some embodiments, the gap segment comprises six nucleic acid bases. In some embodiments, the gap segment comprises seven nucleic acid bases. In some embodiments, the gap segment comprises eight nucleic acid bases. In some embodiments, the gap segment comprises nine nucleic acid bases. In some embodiments, the gap segment comprises 10 nucleic acid bases. In some embodiments, the gap segment comprises 11 nucleic acid bases. In some embodiments, the gap segment comprises 12 nucleic acid bases. In some embodiments, the gap segment comprises 13 nucleic acid bases. In some embodiments, the gap segment comprises 14 nucleic acid bases. For example, in some embodiments, the antisense oligonucleotide has an extension (gap segment) of at least 6 DNA nucleotides sufficient to recruit RNase H. In some embodiments, the gap segment is 8 nucleotides.

[0024] 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' end wing segment, which is covalently linked to the gap segment at the 5' end of the gap segment. In some embodiments, at least one wing segment is a 3' end wing segment, which is covalently linked to the gap segment at the 3' end of the gap segment. In some embodiments, the gap segment is flanked by wing segments at the 5' and 3' ends of the gap segment. In some embodiments, the wing segment independently comprises at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12 or more nucleic acid bases. In embodiments, the length of the wing segment is independently 2 to 6 nucleotides or 2 to 4 nucleotides. In some embodiments, at least one wing segment comprises one nucleic acid base. In some embodiments, at least one wing segment comprises two nucleic acid bases. In some embodiments, at least one wing segment comprises three nucleic acid bases. In some embodiments, at least one wing segment comprises four nucleic acid bases. In some embodiments, at least one wing segment comprises five nucleic acid bases. In some embodiments, at least one wing segment comprises six nucleic acid bases. In some embodiments, at least one wing segment comprises seven nucleic acid bases. In embodiments, the length of the wing segment is independently selected from 2 to 5 nucleic acid bases. In embodiments, the nucleotides in the wing segment comprise RNA nucleotides and / or 2'-O-modified nucleotides as described in detail herein.

[0025] 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 one of those described in Table 4.

[0026] In embodiments, the oligonucleotide comprises at least 8 consecutive nucleobases of any one of SEQ ID NOs: 1-10 to 14-21, and the oligonucleotide is substantially (i.e., at least 90%) or fully complementary to a segment of the KRAS mRNA. In embodiments, the oligonucleotide comprises at least 10 or at least 12 consecutive nucleobases of any one of SEQ ID NOs: 1-10 to 14-21, and the oligonucleotide is substantially (i.e., at least 90%) or fully complementary to a segment of the 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.

[0027] 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 fully complementary to a segment of the 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 fully complementary to a segment of the 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.

[0028] For simplicity, nucleotide sequences can be shown herein using DNA nucleotide sequences (i.e., including thymine nucleobases, "T" or "t") or as RNA nucleotide sequences (i.e., including uracil nucleobases, "U" or "u"). It will be understood from the context that when a nucleotide or sequence is intended to be RNA, the T nucleotide can be replaced by U (or a modified U, such as pseudouridine or 1-methylpseudouridine); and when a nucleotide or sequence is intended to be DNA, the U nucleotide can be replaced by T or a modified T. However, in embodiments, the RNA nucleotides in the antisense oligonucleotide can adopt T (thymine) bases, while the DNA nucleotides in the antisense oligonucleotide can adopt U (uracil) bases. In some embodiments, one or more T nucleotides in the antisense oligonucleotides disclosed herein are exchanged with U or modified U.

[0029] In some embodiments, the oligonucleotide has a nucleobase sequence (or chemical structure) selected from Tables 1-3 and Tables 6-11. In some 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.

[0030] In some embodiments, the combination of antisense oligonucleotide and target mRNA (for example, hybridization) causes the degradation of target mRNA or blocks the translation of target mRNA. In some embodiments, the combination of antisense oligonucleotide and target mRNA produces duplex nucleic acid molecules, which then recruit endogenous nucleases to degrade mRNA. In some embodiments, antisense oligonucleotides have extensions of DNA nucleotides sufficient to recruit RNase H, thereby triggering the degradation of target mRNA. In embodiments, antisense oligonucleotides can have extensions (for example, central extensions) of at least 6 or at least 8 DNA nucleotides, and they are optionally extensions of 9 or 10 DNA nucleotides. In some embodiments, one or more DNA nucleotides include 2' chemical modifications independently selected from 2'-fluoro, 2'-methyl and 2'-ethyl. In embodiments, DNA nucleotides do not include 2' chemical modifications.

[0031] In an embodiment, the antisense oligonucleotide can be a gap polymer with a 5' segment and a 3' segment, each of the 5' segment and the 3' segment being 2 to 6 nucleotides or 2 to 4 nucleotides, and wherein the 5' segment and the 3' segment are free of DNA nucleotides or not only contain DNA nucleotides. In some embodiments, the gap polymer is a 3-8-3 gap polymer with a central block of DNA nucleotides and a 5' segment and a 3' segment of 3 RNA nucleotides respectively. In some embodiments, the gap polymer is a 2-10-2 gap polymer with a central block of 10 DNA nucleotides and a 5' segment and a 3' segment of 2 RNA nucleotides respectively. Other gap polymer formats can be used, such as 3-10-2 and 2-10-3. In some embodiments, one or more nucleotides of a 5' segment and a 3' segment include a 2'-O substituent, and optionally all nucleotides of a 5' segment and a 3' segment include a 2'-O substituent. Exemplary 2'-O substituents are independently selected from 2'-O methyl, 2'-O ethyl, 2'-O methoxyethyl (MOE) and a bridged nucleotide having a 2' to 4' bridge (e.g., a locked nucleotide or a bicyclic nucleotide). In some embodiments, the bridged nucleotide has a methylene bridge (LNA) or a constrained ethyl bridge (cEt).

[0032] The term "gap polymer" refers to an oligonucleotide having a central block of deoxynucleotides (also referred to herein as "DNA nucleotides") and a 5' segment and a 3' segment (of at least 2 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 2'H, but may also have various 2' chemical modifications, including 2'-halo and 2'-low alkyl (e.g., C1-4). In some embodiments, the 2' chemical modifications of DNA nucleotides are independently selected from 2'-fluoro, 2'-methyl, and 2'-ethyl.

[0033] Locked nucleic acids (LNAs) 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 hereby incorporated by reference in their entireties. LNAs are modified nucleotides that contain a bridge between the 2' and 4' carbons of the sugar moiety, resulting in a "locked" conformation and / or a 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, each of which is hereby incorporated by reference in its entirety. In exemplary embodiments, the locked nucleotide is independently selected from a 2' to 4' methylene bridge and a constrained ethyl (cEt) bridge (see US Pat. Nos. 7,399,845 and 7,569,686, which are hereby incorporated by reference in their entireties).

[0034] 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 is formed between two oxygen atoms of a phosphate group and an oxygen atom of a sugar (at the 3' position or the 5' position) to form two ester bonds bridged between two adjacent nucleosides. The modification of the internucleotide linkage can provide different features, 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 phosphoacetate linkage (PACE), which can improve transfection characteristics and enhance nuclease resistance. The internucleotide linkage and oligonucleotide backbone modification that can be used in the oligonucleotide of this specification include but are not limited to phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, alkylphosphonate, alkylthiophosphonate, phosphotriester, phosphoramidate, phosphoramidite, phosphorodiamidate, siloxane, carbonate, alkoxycarbonyl, acetamido ester, carbamate, morpholino, peptide nucleic acid, borane, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphorothioate and sulfone nucleoside interlinkage.In some embodiments, antisense oligonucleotide comprises one or more phosphorothioate or phosphorodithioate nucleotides.

[0035] In some embodiments, antisense oligonucleotide comprises one or more phosphorothioate or phosphorodithioate nucleotides and is connected.In some embodiments, phosphorothioate bond or phosphorodithioate bond can be introduced between the last three to five nucleotides at the 5' end and / or 3' end of oligonucleotide to reduce the degradation of exonuclease.In some embodiments, antisense oligonucleotide has the combination of phosphodiester bond and phosphorothioate bond / phosphorodithioate bond.In some embodiments, 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 nucleotides and is connected.In some embodiments, antisense oligonucleotide comprises substantially alternating phosphodiester and phosphorothioate nucleotides and is connected.In some embodiments, the backbone described in this section is modified at least for the nucleotides of nucleotides in the wing section and is connected.In some embodiments, antisense oligonucleotide is that completely phosphorothioate / phosphorodithioate connects (that is, all bonds are phosphorothioate or phosphorodithioate). In some embodiments, the ASO is fully phosphorothioate-linked.

[0036] In some embodiments, especially when RNase H is not required to be recruited, the antisense oligonucleotide has a morpholino backbone.Morpholino oligonucleotides generally do not trigger the degradation of their target RNA molecules, and can effectively block the target RNA sequence spatially.Morpholino oligonucleotides and their synthesis are generally disclosed in U.S. Patent No. 11,028,386, U.S. Patent No. 10,947,533 and U.S. Patent No. 10,927,378, each of which is hereby incorporated by reference in its entirety. In some embodiments, antisense oligonucleotides include thiomorpholino oligonucleotides and / or other substituted or modified nucleotides, such as those described in International Patent Application Publication No. WO / 2019 / 060522 and International Patent Application Publication No. WO / 2018 / 057430, each of which is hereby incorporated by reference in its entirety. For example, Langner et al. describe methods for synthesizing oligonucleotide analogs termed thiophosphoramidate morpholino oligonucleotides (TMOs) comprising a morpholino nucleoside and a phosphorothioate linkage (“Synthesis and characterization of thiophosphoramidate morpholino oligonucleotides and chimeras.” JACS 142.38 (2020): 16240-16253; see also Dumbović, 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 hereby incorporated by reference in their entireties). Thus, the antisense oligonucleotides described herein may comprise all or part of TMO-modified nucleotides, or may comprise chimeras of TMO-modified nucleotides with unmodified nucleotides and / or other nucleotides comprising different modifications (e.g., LNA).

[0037] In some embodiments, antisense oligonucleotides may contain one or more modified bases. In some embodiments, cytosine is replaced by a modified cytosine, such as 5-methylcytosine or 5-hydroxymethylcytosine, which can enhance base pairing. If necessary, other modified bases (especially cytosine or guanine) can be used to reduce immunogenicity. Other modified bases are described in U.S. Patent No. 10,064,959, which is hereby incorporated by reference. In various embodiments, the cytidine nucleobase in the antisense oligonucleotide is 5-methylcytidine. Therefore, it will be understood by those skilled in the art that when the sequence includes cytidine nucleobase ("C"), the term includes 5-methyl C. In addition, it will be understood that when the sequence includes uracil bases, the term "U" includes pseudouridine and N1-methyl pseudouridine.

[0038] In some embodiments, after being bound to the target nucleic acid, the oligonucleotide forms a duplex with the target nucleic acid and recruits a nuclease for degrading 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 comprises an endoribonuclease or RNase 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 comprises RNase PH, II, R, D or T. In some embodiments, the nuclease comprises polynucleotide phosphorylase (PNPase), oligoribonuclease, exoribonuclease I or exoribonuclease II. In some embodiments, the ribonuclease recruited by the oligonucleotide bound to the endogenous nucleic acid is RNase H.

[0039] In some embodiments, the oligonucleotide comprises at least one, two, three, four, five, six, seven, eight, nine, 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 or more chemical modifications. In some embodiments, the oligonucleotide comprises at least one gap segment comprising at least one, two, three, four, five, six, seven, eight, nine, 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 or more chemical modifications. In some embodiments, the oligonucleotide comprises at least one wing segment comprising at least one, two, three, four, five, six, seven, eight, nine, 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 one gap segment and at least one wing segment comprising at least one, two, three, four, five, six, seven, eight, nine, 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 94, 95, 96, 97, 98, 99, 100 or more chemical modifications.

[0040] In some embodiments, the oligonucleotides described herein bind to nucleic acids encoding KRAS (e.g., mRNA), wherein binding of the oligonucleotide to the KRAS nucleic acid encoding KRAS (e.g., KRAS mRNA) reduces expression of the KRAS nucleic acid in the cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to expression of KRAS not modulated by the oligonucleotide. In some embodiments, the oligonucleotides described herein bind to nucleic acids encoding mutant KRAS (e.g., mRNA), wherein binding of the oligonucleotide to the nucleic acid encoding mutant KRAS reduces endogenous expression of the mutant KRAS in the cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to expression of the mutant KRAS not modulated by the oligonucleotide. In some embodiments, the mutant KRAS nucleic acid encodes a mutant KRAS protein comprising a G12C mutation, a G12V mutation, a G12A mutation, or a G12D mutation.

[0041] In some embodiments, the oligonucleotides described herein bind to a nucleic acid (e.g., mRNA) encoding KRAS, wherein 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 the 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.

[0042] In some embodiments, an oligonucleotide described herein binds to a nucleic acid (e.g., mRNA) encoding a mutant KRAS, wherein binding of the oligonucleotide to the nucleic acid encoding the mutant KRAS reduces endogenous expression of genes in the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway, or the activity of these signaling pathways in the cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, compared to expression of genes in the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway, or the activity of these signaling pathways not modulated by the oligonucleotide.

[0043] In some embodiments, the compound comprises at least two oligonucleotides, wherein the first oligonucleotide is bound to a nucleic acid encoding KRAS (e.g., mRNA), and the second oligonucleotide is bound to another nucleic acid encoding a mutated KRAS. In some embodiments, the nucleic acid encoding the mutated KRAS encodes a mutated KRAS protein, and the mutated KRAS protein comprises a G12C mutation, a G12V mutation, a G12A mutation, or a G12D mutation. In some embodiments, the compound comprises at least two oligonucleotides, wherein the first oligonucleotide is bound to a nucleic acid encoding a first mutated KRAS (e.g., mRNA), and the second oligonucleotide is bound to another nucleic acid encoding a second mutated KRAS, wherein the first and second mutated KRAS are different. Two different mutated KRAS may have different amino acid sequences. In some embodiments, the first and second mutated KRAS may be the same, but the nucleic acids encoding them have different sequences. In some embodiments, the compound comprises at least two oligonucleotides, wherein the first oligonucleotide and the second oligonucleotide are bound to a nucleic acid encoding another KRAS.

[0044] In some embodiments, binding of the oligonucleotide to both KRAS and mutant KRAS reduces endogenous expression of genes in the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway, or the activity of these pathways in the cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, compared to endogenous expression of genes 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 modulated by the oligonucleotide.

[0045] In some embodiments, the compound is formulated for administration to a subject by an appropriate route of administration, including but not limited to intravenous, intratumoral, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal administration. Pharmaceutical formulations as 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, extended release formulations, pulse release formulations, multi-particulate formulations, and mixed immediate release and controlled release formulations. In some embodiments, the compound is formulated into a dosage form. In some embodiments, the compound is formulated into and comprises at least one excipient. In some embodiments, the excipient is a pharmaceutically acceptable excipient.

[0046] In some embodiments, the compound comprising the oligonucleotides described herein treats the disease or disorder by reducing the expression of a gene or signaling pathway associated with the disease or disorder. In some embodiments, the compound comprising the oligonucleotides described herein treats the disease or disorder described herein by directly reducing the expression of a gene associated with the disease or disorder described herein. In some embodiments, the compound comprising the oligonucleotides treats the disease or disorder by reducing the expression of a gene that is part of a signaling pathway described herein. In some embodiments, the compound comprising the oligonucleotides described herein treats the disease or disorder by reducing KRAS expression (e.g., mutated KRAS expression). In some embodiments, the compound comprising the oligonucleotides described herein treats the disease or disorder by reducing KRAS expression. In some embodiments, the compound comprising the oligonucleotides described herein treats the disease or disorder by reducing both KRAS and mutated KRAS expression. In some embodiments, the compound comprising the oligonucleotides described herein treats the disease or disorder by reducing endogenous KRAS expression. In some embodiments, the compounds comprising the oligonucleotides described herein treat a disease or condition by reducing the expression or activity of an endogenous KRAS-RAF-MEK-ERK signaling pathway, a PI3K signaling pathway, a MAPK signaling pathway, or a Ral-GEF signaling pathway. In some embodiments, the disease or condition described herein is cancer.

[0047] In some embodiments, the chemical modifications of the oligonucleotide include the chemical modifications listed in Tables 1-3 and Tables 6-11. In some embodiments, the oligonucleotide is an oligonucleotide listed in Tables 1-3 and Tables 6-11.

[0048] In some embodiments, chemical modification may occur at 3' OH groups, 5' OH groups, main chains, sugar components or nucleotide bases. Chemical modification may include non-naturally occurring linker molecules for interchain or intrachain crosslinking. In one aspect, chemically modified nucleic acids include modifications of one or more of 3' OH or 5' OH groups, main chains, sugar components or nucleotide bases, or add non-naturally occurring linker molecules. In some embodiments, the chemically modified main chain includes a main chain other than a phosphodiester main chain. In some embodiments, the modified sugar includes a sugar other than deoxyribose (in modified DNA) or ribose (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 cases, at least one, two, three, four, five, six, seven, eight, nine, 10, 15, 20 or more modified bases are included. In some embodiments, chemical modifications to base moieties include adenine, guanine, cytosine, thymine, or uracil, as well as natural and synthetic modifications of purine or pyrimidine bases.

[0049] In some embodiments, at least one chemical modification of the oligonucleotide comprises any one or any combination of the following modifications: a 2'-modified nucleotide comprising 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA); modification of one or both non-linked phosphate oxygens in a phosphodiester backbone linkage; modification of one or more linked phosphate oxygens in a phosphodiester backbone linkage; modification of the ribose moiety; replacement of the phosphate moiety with a "dephospho" linker; modification or replacement of a naturally occurring nucleobase; modification of the ribose-phosphate backbone; modification of the 5' end of a polynucleotide; modification of the 3' end of a polynucleotide; modification of the deoxyribose-phosphate backbone; substitution of a phosphate group; modification of the ribose-phosphate backbone; modification of a nucleotide sugar; modification of a nucleotide base; or a stereopure nucleotide.Non-limiting examples of chemical modifications to oligonucleotides can include: modification of one or both of the non-linked or linked phosphate oxygens in the phosphodiester backbone linkage (e.g., sulfur (S), selenium (Se), BR3 (wherein R can be, for example, hydrogen, alkyl, or aryl), C (e.g., alkyl, aryl, etc.), H, NR2, where R can be, for example, hydrogen, alkyl, or aryl, or where R can be, for example, alkyl or aryl); replacement of the phosphate moiety with a "dephospho" linker (e.g., with a methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformal, methylal, oxime, methyleneimino, etc.); , methylenemethylimino, methylenehydrazine, methylenedimethylhydrazine or methyleneoxymethylimino replacement); modification or replacement of naturally occurring nucleobases with nucleic acid analogs; modification of the deoxyribose-phosphate or ribose-phosphate backbone (e.g., modification of the ribose-phosphate backbone to incorporate phosphorothioates, phosphonothioacetates, selenophosphates, boranophosphates, boranophosphates, hydrogen phosphonates, phosphonocarboxylates, phosphoramidates, alkyl or aryl phosphonates, phosphonoacetates or phosphotriesters); modification of the 5' end (e.g., modification of the 5' cap or 5' cap-OH) or 3' end (modification of the 3' tail or 3' end-OH) of the nucleic acid sequence; modification of the 5' end (e.g., modification of the 5' cap or 5' cap-OH) or 3' end (modification of the 3' tail or 3' end-OH) of the nucleic acid sequence with methylphosphonates, hydroxylamino groups, siloxanes, The phosphate group can be substituted with a carbonate, carboxymethyl, carbamate, amide, thioether, oxirane linker, sulfonate, sulfonamide, thioformal, methylal, oxime, methyleneimino, methylenemethylimino, methylenehydrazinyl, methylenedimethylimino, or methyleneoxymethylimino; the ribose phosphate backbone can be modified to incorporate morpholino (phosphodiamidate morpholino oligomer PMO), thiomorpholino, cyclobutyl, pyrrolidine, or peptide nucleic acid (PNA) nucleoside surrogates; the sugar of the nucleotide can be modified to incorporate locked nucleic acid (LNA), unlocked nucleic acid (UNA), ethylene nucleic acid (ENA), constrained ethyl (cEt) sugar, or bridged nucleic acid (BNA); modification of the ribose moiety Modifications of the nucleotide base (e.g., 2'-O-methoxyethoxy (2'-MOEr), 2'-O-methyl, 2'-O-methoxy-ethyl (2'-O-MOE), 2'-fluoro, 2'-aminoethyl, 2'-deoxy-2'-fluoroarabinoic acid, 2'-deoxy, 2'-O-methyl, 3'-phosphorothioate, 3'-phosphonoacetate (PACE), or 3'-phosphonothioacetate (thioPACE)); modifications of the nucleotide base (A, T, C, G, or U; e.g., 5-hydroxymethyl modified nucleotides such as 5-hydroxymethylcytosine); and stereopure nucleotides (e.g., the S conformation of the phosphorothioate or the R conformation of the phosphorothioate).

[0050] In some embodiments, the chemical modification of the oligonucleotide includes at least one substitution of one or two non-connected phosphate oxygen atoms in the phosphodiester backbone linkage of the oligonucleotide. In some embodiments, the at least one chemical modification of the oligonucleotide includes the substitution of one or more connected phosphate oxygen atoms in the phosphodiester backbone linkage of the oligonucleotide. A non-limiting example of the chemical modification of the phosphate oxygen atom is a sulfur atom. In some embodiments, the chemical modification of the oligonucleotide includes at least one chemical modification of the sugar of the nucleotides of the oligonucleotide. In some embodiments, the chemical modification of the oligonucleotide includes at least one chemical modification of the sugar of the nucleotides, wherein the chemical modification includes at least one locked nucleic acid (LNA). In some embodiments, the chemical modification of the oligonucleotide includes at least one chemical modification of the sugar of the nucleotides of the oligonucleotide including at least one unlocked nucleic acid (UNA). In some embodiments, the chemical modification of the oligonucleotide includes at least one chemical modification of the sugar of the nucleotides of the oligonucleotide including at least one ethylene nucleic acid (ENA). In some embodiments, the chemical modification of the oligonucleotide includes at least one chemical modification of the sugar, including the modification of the component of the sugar, wherein the sugar is ribose sugar. In some embodiments, the chemical modification of the oligonucleotide includes at least one chemical modification of the ribose sugar component of the nucleotide of the oligonucleotide comprising 2'-O-methyl. In some embodiments, the chemical modification of the oligonucleotide includes at least one chemical modification, including replacing the phosphate moiety of the oligonucleotide with a dephosphorylation linker. In some embodiments, the chemical modification of the oligonucleotide includes at least one chemical modification of the phosphate backbone of the oligonucleotide. In some embodiments, the oligonucleotide includes a thiophosphate group. In some embodiments, the chemical modification of the oligonucleotide includes at least one chemical modification, which includes the modification of the base of the nucleotide of the oligonucleotide. In some embodiments, the chemical modification of the oligonucleotide includes at least one chemical modification of the non-natural base comprising nucleotide. In some embodiments, the chemical modification of the oligonucleotide includes at least one chemical modification, which includes a morpholino group (such as phosphorodiamidate morpholino oligomer, PMO), cyclobutyl, pyrrolidinyl or peptide nucleic acid (PNA) nucleoside substitute. In some embodiments, the chemical modification of the oligonucleotide includes at least one chemical modification, which includes at least one stereo pure nucleic acid. In some embodiments, at least one chemical modification can 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.

[0051] In some embodiments, the oligonucleotide comprises a backbone comprising a plurality of sugar and phosphate moieties covalently linked together. In some embodiments, the backbone of the oligonucleotide comprises a phosphodiester linkage between a first hydroxyl group in the phosphate group on the 5' carbon of the deoxyribose in DNA or the ribose in RNA and a second hydroxyl group on the 3' carbon of the deoxyribose in DNA or the ribose in RNA.

[0052] In some embodiments, the main chain of oligonucleotide can lack 5' reduced hydroxyl group, 3' reduced hydroxyl group or both that can be exposed to solvent.In some embodiments, the main chain of oligonucleotide can lack 5' reduced hydroxyl group, 3' reduced hydroxyl group or both that can be exposed to nuclease.In some embodiments, the main chain of oligonucleotide can lack 5' reduced hydroxyl group, 3' reduced hydroxyl group or both that can be exposed to hydrolase.In some cases, the main chain of oligonucleotide can be expressed as the polynucleotide sequence of circular two-dimensional format, in which one of Nucleotide is followed by another Nucleotide.In some cases, the main chain of oligonucleotide can be expressed as the polynucleotide sequence of ring-type two-dimensional format, in which one of Nucleotide is followed by another Nucleotide.In some embodiments, 5' hydroxyl group, 3' hydroxyl group or both are connected by phosphorus-oxygen bond.In some embodiments, 5' hydroxyl group, 3' hydroxyl group or both are modified to the phosphate with phosphorus-containing moiety.

[0053] In some embodiments, the oligonucleotides described herein comprise at least one chemical modification. The chemical modification can be a substitution, insertion, deletion, chemical modification, physical modification, stabilization, purification, or any combination thereof. In some embodiments, the modification is a chemical modification. Suitable chemical modifications include any of the following: 5' adenylate, 5' guanosine-triphosphate cap, 5' N7-methylguanosine-triphosphate cap, 5' triphosphate cap, 3' phosphate, 3' phosphorothioate, 5' phosphate, 5' phosphorothioate, cis-Syn thymidine 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, cholesterol TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, bis-biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3'DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDyeQC-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-methyl ribonucleoside analog, sugar modification analog, wobble / universal base, fluorescent dye label, 2' fluoro RNA, 2'O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphorothioate DNA, phosphorothioate RNA, UNA, LNA, cEt, pseudouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 2'-O-methyl-phosphorothioate, or any combination thereof.

[0054] In some embodiments, oligonucleotide modifications can alter the physiochemical properties of the nucleotides, such as their conformation, polarity, hydrophobicity, chemical reactivity, base pairing interactions, or any combination thereof. Chemical modifications can also be phosphorothioate substitutes. In some embodiments, natural phosphodiester bonds can be readily degraded rapidly by cellular nucleases; modifying internucleotide linkages with phosphorothioate (PS) bond substitutes can be more stable to hydrolysis by cellular degradation. Modifications can increase the stability of polynucleic acids. Modifications can also enhance biological activity. In some embodiments, phosphorothioate-enhanced RNA polynucleic acids can inhibit RNase A, RNase T1, calf serum nuclease, or any combination thereof. These properties make PS-RNA polynucleic acids useful in applications where exposure to nucleases is highly likely in vivo or in vitro. For example, phosphorothioate (PS) bonds can be introduced between the last 3-5 nucleotides at the 5' or 3' end of the polynucleic acid, which can inhibit exonuclease degradation. In some embodiments, phosphorothioate linkages can be added throughout the polynucleic acid to reduce attack by endonucleases. In some embodiments, the oligonucleotides described herein comprise at least one, two, three, four, five, six, seven, eight, nine, 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 comprising PS bonds. In some embodiments, the oligonucleotides described herein comprise only PS bonds as internucleotide linkage modifications. In some embodiments, all internucleotide linkages of the oligonucleotides described herein are fully PS modified or comprise phosphorothioate internucleotide linkages. In some embodiments, oligonucleotides comprising PS bonds as internucleotide linkage modifications comprise a 5' wing segment comprising one nucleic acid base. In some embodiments, oligonucleotides comprising PS bonds as internucleotide linkage modifications comprise a 5' wing segment comprising two nucleic acid bases. In some embodiments, the oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' wing segment comprising three nucleic acid bases. In some embodiments, the oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' wing segment comprising four nucleic acid bases. In some embodiments, the oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' wing segment comprising five nucleic acid bases. In some embodiments, the oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' wing segment comprising six nucleic acid bases. In some embodiments, the oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' wing segment comprising seven nucleic acid bases. In some embodiments, the oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' wing segment comprising eight nucleic acid bases.In some embodiments, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' wing segment comprising nine nucleic acid bases. In some embodiments, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' wing segment comprising 10 nucleic acid bases. In some embodiments, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' wing segment comprising one nucleic acid base. In some embodiments, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' wing segment comprising two nucleic acid bases. In some embodiments, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' wing segment comprising three nucleic acid bases. In some embodiments, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' wing segment comprising four nucleic acid bases. In some embodiments, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' wing segment comprising five nucleic acid bases. In some embodiments, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' wing segment comprising six nucleic acid bases. In some embodiments, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' wing segment comprising seven nucleic acid bases. In some embodiments, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' wing segment comprising eight nucleic acid bases. In some embodiments, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' wing segment comprising nine nucleic acid bases. In some embodiments, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' wing segment comprising 10 nucleic acid bases.

[0055] In some embodiments, the oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' wing segment containing one nucleic acid base and a 3' wing segment containing one nucleic acid base. In some embodiments, the oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' wing segment containing two nucleic acid bases and a 3' wing segment containing two nucleic acid bases. In some embodiments, the oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' wing segment containing three nucleic acid bases and a 3' wing segment containing three nucleic acid bases. In some embodiments, the oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' wing segment containing four nucleic acid bases and a 3' wing segment containing four nucleic acid bases. In some embodiments, the oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' wing segment containing five nucleic acid bases and a 3' wing segment containing five nucleic acid bases.

[0056] In some embodiments, the oligonucleotide comprises a 5' wing segment comprising one nucleic acid base, a gapmer, and a 3' wing segment comprising one nucleic acid base, wherein the internucleotide linkages connecting the 5' wing segment, the gapmer, and the 3' wing segment of the oligonucleotide comprise only PS bonds. In some embodiments, the oligonucleotide comprises a 5' wing segment comprising two nucleic acid bases, a gapmer, and a 3' wing segment comprising two nucleic acid bases, wherein the internucleotide linkages connecting the 5' wing segment, the gapmer, and the 3' wing segment of the oligonucleotide comprise only PS bonds. In some embodiments, the oligonucleotide comprises a 5' wing segment comprising three nucleic acid bases, a gapmer, and a 3' wing segment comprising three nucleic acid bases, wherein the internucleotide linkages connecting the 5' wing segment, the gapmer, and the 3' wing segment of the oligonucleotide comprise only PS bonds. In some embodiments, the oligonucleotide comprises a 5' wing segment comprising four nucleic acid bases, a gapmer, and a 3' wing segment comprising four nucleic acid bases, wherein the internucleotide linkages connecting the 5' wing segment, the gapmer, and the 3' wing segment of the oligonucleotide comprise only PS bonds. In some embodiments, the oligonucleotide comprises a 5' wing segment comprising five nucleic acid bases, a gapmer, and a 3' wing segment comprising five nucleic acid bases, wherein the internucleotide linkages connecting the 5' wing segment, the gapmer, and the 3' wing segment of the oligonucleotide comprise only PS bonds. In some embodiments, the oligonucleotide comprises a 5' wing segment comprising six nucleic acid bases, a gapmer, and a 3' wing segment comprising six nucleic acid bases, wherein the internucleotide linkages connecting the 5' wing segment, the gapmer, and the 3' wing segment of the oligonucleotide comprise only PS bonds.

[0057] In some embodiments, the oligonucleotide comprising a 5' wing segment, a gapmer, a 3' wing segment, and a PS bond as an internucleotide linkage comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NOs: 1-10 and 14-21. In some embodiments, the oligonucleotide comprising a 5' wing segment, a gapmer, a 3' wing segment, and a PS bond as an internucleotide linkage comprises a nucleic acid sequence that is 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' wing segment, a gapmer, a 3' wing segment, and a PS bond 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 Tables 6-11.

[0058] Oligonucleotides may be circular, substantially circular, or otherwise linked in a continuous manner (eg, may be arranged as a loop), and may also retain a substantially similar secondary structure as a substantially similar oligonucleotide that may not be circular or may not be a loop.

[0059] In some embodiments, chemical modification includes modification of one or two non-connected phosphate oxygens in the phosphodiester backbone bond or modification of one or more connected phosphate oxygens in the phosphodiester backbone bond. As used herein, "alkyl" means a straight or branched saturated hydrocarbon group. Exemplary alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl or isopropyl), butyl (e.g., n-butyl, isobutyl or tert-butyl) or pentyl (e.g., n-pentyl, isopentyl or neopentyl). Alkyl 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 monocyclic or polycyclic (e.g., with 2, 3 or 4 fused rings) aromatic hydrocarbons, such as phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl or indenyl. In some embodiments, aryl 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 2-12 carbon atoms, characterized by having one or more triple bonds. Examples of alkynyl groups may include ethynyl, propargyl, or 3-hexynyl. "Arylalkyl" or "aralkyl" refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl groups include 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, diphenylmethyl, and triphenylmethyl. "Cycloalkyl" refers to a cyclic, bicyclic, tricyclic, or polycyclic non-aromatic hydrocarbon group having 3 to 12 carbon atoms. 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 heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl, dioxolanyl, diazepine, oxazepanyl, thiazepanyl, and morpholinyl. "Heteroaryl" refers to a monovalent group of a heteroaromatic ring system. Examples of heteroaryl moieties can include imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furyl, indolyl, thiophenylpyrazolyl, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, quinolinyl, and pteridinyl.

[0060] In some embodiments, the phosphate group of the chemically modified nucleotide can be modified by replacing one or more oxygens with different substituents. In some embodiments, the chemically modified nucleotide may include replacing an unmodified phosphate moiety with a modified phosphate as described herein. In some embodiments, the modification of the phosphate backbone may include causing a change in an uncharged joint or a charged joint with an asymmetric charge distribution. The example of the modified phosphate group may include phosphorothioate, phosphonothioacetate, selenophosphate, borane phosphate, borane phosphate, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate and phosphotriester. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone portion may be replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (wherein R can be, for example, hydrogen, alkyl or aryl), C (such as alkyl, aryl, etc.), H, NR2 (wherein R can be, for example, hydrogen, alkyl or aryl) or (wherein R can be, for example, alkyl or aryl). The phosphorus atom in the unmodified phosphate group can be achiral. However, replacing one of the non-bridging oxygens with one of the above-mentioned atoms or atomic groups can make the phosphorus atom have chirality. The phosphorus atom in the phosphate group modified in this way is a stereocenter. The stereogenic phosphorus atom can have an "R" configuration (herein Rp) or an "S" configuration (herein Sp). In some embodiments, the oligonucleotide comprises a stereopure nucleotide comprising an S conformation of a thiophosphate or an R conformation of a thiophosphate. In some embodiments, the chiral phosphoric acid product is present in an excess of 50%, 60%, 70%, 80%, 90% or more. In some embodiments, the chiral phosphoric acid product is present in an excess of 95%. In some embodiments, the chiral phosphoric acid product is present in an excess of 96%. In some embodiments, the chiral phosphoric acid product is present in an excess of 97%. In some embodiments, the chiral phosphoric acid product is present in an excess of 98%. In some embodiments, the chiral phosphoric acid product is present in an excess of 99%. In some embodiments, two non-bridging oxygens of dithiophosphate can be replaced by sulfur. In some embodiments, the phosphorus center in the dithiophosphate can be achiral, which has stopped the formation of oligoribonucleotide diastereomers.In some embodiments, the modification of one or two non-bridging oxygens can also include replacing non-bridging oxygens with a group independently selected from S, Se, B, C, H, N and OR (R can be, for example, an alkyl or aryl group).In some embodiments, the phosphate joint can also be modified by replacing bridging oxygen (that is, phosphoric acid is connected to the oxygen of nucleosides) with nitrogen (phosphoramidate of bridging), sulphur (phosphorothioate of bridging) and carbon (methylene phosphonate of bridging).Replacement can occur on one or two connection oxygens.

[0061] Nucleic acids can be linked together using any internucleic acid bond. Two major classes of internucleic acid linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleic acid bonds include, but are not limited to, phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing internucleic acid linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-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 bonds with chiral atoms can be prepared as racemic mixtures as individual enantiomers, such as alkylphosphonates and phosphorothioates. Non-natural nucleic acids can contain a single modification. Non-natural nucleic acids can contain multiple modifications within a portion or between different portions.

[0062] The backbone phosphate modification of nucleic acids includes but is not limited to methylphosphonate, phosphorothioate, phosphoramidate (bridged or non-bridged), phosphotriester, phosphorodithioate, phosphorodithioate and boranephosphate, and can be used in any combination. Other non-phosphate linkages can also be used.

[0063] In some embodiments, backbone modifications (eg, methylphosphonate, phosphorothioate, phosphoramidate, and phosphorodithioate internucleotide linkages) can confer immunomodulatory activity to the modified nucleic acids and / or enhance their in vivo stability.

[0064] In some cases, the phosphorus derivative (or modified phosphate group) is attached to the sugar or sugar analog moiety and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, or the like.

[0065] In some embodiments, the backbone modification includes replacing the phosphodiester bond with an alternative moiety such as an anion, neutral or cationic group. Examples of such modifications include: anionic internucleoside bonds; N3' to P5' phosphoramidate modifications; borane phosphate DNA; oligonucleotide precursors; neutral internucleoside bonds, such as methylphosphonate; amide-connected DNA; methylene (methylimino) bonds; methylal and thioformal bonds; backbones containing sulfonyl groups; morpholino oligonucleotides; peptide nucleic acids (PNAs); and positively charged deoxyribonucleic acid guanidine (DNG) oligonucleotides. Modified nucleic acids may include chimeric or mixed backbones comprising one or more modifications, such as combinations of phosphate bonds, such as combinations of phosphodiester and thiophosphate bonds.

[0066] Phosphoric acid substitutes include, for example, short chain alkyl or cycloalkyl nucleoside bond, mixed heteroatoms and alkyl or cycloalkyl nucleoside bond or one or more short chain heteroatoms or heterocyclic nucleoside bond.These include those with the following: morpholino bond (partially formed from the sugar moiety of nucleoside); siloxane backbone; sulfide, sulfoxide and sulfone backbone; formyl acetyl and thioformyl acetyl backbone; methylene formyl acetyl and thioformyl acetyl backbone; olefin-containing backbone; sulfamic acid ester backbone; methylene imine and methylene hydrazine backbone; sulfonic acid ester and sulfonamide backbone; amide backbone; and with mixed N, O, S and CH2 other backbones of component parts. It should also be understood that in nucleotide substitutes, the sugar and phosphate moieties of nucleotide can be replaced by, for example, amide type bond (aminoethylglycine) (PNA). Other types of molecules (conjugates) can also be connected to nucleotides or nucleotide analogs to enhance, for example, cellular uptake. Conjugates can be chemically linked to nucleotides or nucleotide analogs. Such conjugates include, but are not limited to, lipid moieties such as cholesterol moieties, thioethers such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chains such as dodecandiol or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1-di-O-hexadecyl-rac-glycero-SH-phosphonate, polyamines or polyethylene glycol chains, or adamantaneacetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

[0067] In some embodiments, chemical modification as described herein includes modification of phosphate backbone. In some embodiments, oligonucleotides as described herein include at least one chemically modified phosphate backbone. Exemplary chemical modification of phosphate group or backbone may include replacing one or more oxygen with different substituents. In addition, the modified nucleotides present in the oligonucleotide may include replacing unmodified phosphate moieties with modified phosphate as described herein. In some embodiments, modification of the phosphate backbone may include causing uncharged joints or changes in charged joints with asymmetric charge distribution. Exemplary modified phosphate groups may include phosphorothioate, phosphonothioacetate, selenophosphate, borane phosphate, borane phosphate, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate and phosphotriester. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety may be replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (wherein R may be, for example, hydrogen, alkyl or aryl), C (such as alkyl, aryl, etc.), H, NR2 (wherein R may be, for example, hydrogen, alkyl or aryl) or (wherein R may be, for example, alkyl or aryl). The phosphorus atom in the unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygens with one of the above-mentioned atoms or atomic groups can make the phosphorus atom chiral; that is, the phosphorus atom in the phosphate group modified in this way is a stereocenter. The stereophosphorus atom can have an "R" configuration (herein Rp) or an "S" configuration (herein Sp). In this case, the chemically modified oligonucleotide can be stereopure (e.g., S or R conformation). In some embodiments, the chemically modified oligonucleotide comprises a stereopure phosphate modification. For example, the chemically modified oligonucleotide includes the S conformation of phosphorothioate or the R conformation of phosphorothioate.

[0068] Phosphorodithioate has two non-bridging oxygens replaced by sulfur.The phosphorus center in phosphorodithioate is achiral, and this has stopped the formation of oligoribonucleotide diastereomers.In some embodiments, the modification of one or two non-bridging oxygens can also include replacing 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).

[0069] The phosphate linker can also be modified by replacing the bridging oxygen (i.e., the oxygen that connects the phosphate to the nucleoside) with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate), and carbon (bridged methylenephosphonate). The replacement can occur on either or both connecting oxygens.

[0070] In some embodiments, at least one phosphate group of the oligonucleotide can be chemically modified. In some embodiments, the phosphate group can be replaced by a phosphorus-free connector. In some embodiments, the phosphate moiety can be replaced by a dephosphorylation joint. In some embodiments, the charged phosphate group can be replaced by a neutral group. In some embodiments, the phosphate group can be replaced by methyl phosphonate, hydroxyamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide joint, sulfonate, sulfonamide, thioformal, methylal, oxime, methyleneimino, methylenemethylimino, methylenehydrazine, methylenedimethylhydrazine and methyleneoxymethylimino. In some embodiments, nucleotide analogs as herein described can also be modified at the phosphate group. The phosphate group of modification can be included in the linkage place between two Nucleotide and use phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, phosphonic acid methyl ester and other phosphonic acid alkyl ester (comprising phosphonic acid 3 ' alkylene ester and chiral phosphonate), phosphinate, phosphoramidate (comprising 3 '-amino phosphoramidate and aminoalkyl phosphoramidate), thionyl phosphoramidate, thionyl phosphate, thionyl phosphotriester and borane phosphate to modify.The phosphoric acid between two Nucleotide or the phosphate linkage of modification can be through 3 '-5 ' linkage or 2 '-5 ' linkage, and this linkage comprises reverse polarity, for example 3 '-5 ' to 5 '-3 ' or 2 '-5 ' to 5 '-2 '.

[0071] In some embodiments, chemical modification as herein described includes modification by replacing phosphate group. In some embodiments, oligonucleotide as herein described includes at least one chemical modification, which includes phosphate group replacement or replacement. Exemplary phosphate group replacement may include a phosphorus-free linker. In some embodiments, phosphate group replacement or replacement may include replacing a charged phosphate group with a neutral moiety. Exemplary portions of replaceable phosphate groups may include methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide joint, sulfonate, sulfonamide, thiomethyl acetal, methylal, oxime, methylene imino, methylene methyl imino, methylene hydrazine, methylene dimethyl hydrazine and methyleneoxy methyl imino.

[0072] In some embodiments, chemical modification as herein described includes the ribose phosphate backbone of modified oligonucleotide.In some embodiments, oligonucleotide as herein described includes at least one chemically modified ribose phosphate backbone.The ribose phosphate backbone of exemplary chemical modification can include the support that can simulate nucleic acid, and can also construct the support in which the phosphate linker and ribose sugar are replaced by nuclease-resistant nucleoside or nucleotide substitute.In some embodiments, core base can be bound by alternative backbone.Example can include morpholino, such as phosphorodiamidate morpholino oligomer (PMO), cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside substitute.

[0073] In some embodiments, the chemical modifications described herein include modifications of sugars. In some embodiments, the oligonucleotides described herein comprise at least one chemically modified sugar. Exemplary chemically modified sugars may include a 2' hydroxyl (OH) modified or replaced with many different "oxy" or "deoxy" substituents. In some embodiments, modification of the 2' hydroxyl can enhance the stability of the nucleic acid because the hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion. The 2'-alkoxide can catalyze degradation by intramolecular nucleophilic attack on the phosphorus atom of the linker. Examples of "oxy"-2' hydroxyl modifications may include alkoxy or aryloxy (OR, where "R" can be, for example, an alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH2CH2O); n CH2CH2OR, where R can be, for example, H or an optionally substituted alkyl group, 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, the "oxy"-2'hydroxy modification can include (LNA, where the 2'hydroxyl can be connected to the 4' carbon of the same ribose sugar, for example, via a C1-6 alkylene or C1-6 heteroalkylene bridge, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein the amino group 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'hydroxy modification can include methoxyethyl (MOE) (OCH2CH2OCH3, such as a PEG derivative). In some embodiments, the deoxy modification can include hydrogen (i.e., deoxyribose sugar, such as in the overhang of a portion of a dsRNA); halogen (e.g., bromine, chloride, fluorine, or iodine); amino (wherein amino can be, for example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or an amino acid); NH(CH2CH2NH) nCH2CH2-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; sulfhydryl; 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 cases, the sugar group can also contain one or more carbons having a stereochemical configuration opposite to the corresponding carbon in ribose. Thus, the modified nucleic acid can include nucleotides containing, for example, arabinose as a sugar. The nucleotide "monomer" can have an α linkage at the Γ position on the sugar, such as an α-nucleoside. The modified nucleic acid can also include a "abasic" sugar, which lacks a nucleobase at the C-position. The abasic sugar can also be further modified at one or more constituent sugar atoms. The modified nucleic acid can also include one or more L-forms of sugars, such as L-nucleosides. In some embodiments, the oligonucleotides described herein include a glycosyl ribose, which is a 5-membered ring with oxygen. Exemplary modified nucleosides and modified nucleotides can include replacement of the oxygen in the ribose sugar (e.g., with sulfur (S), selenium (Se), or an alkylene group, such as a methylene or ethylene group); addition of a double bond (e.g., replacement of the ribose sugar with a cyclopentenyl or cyclohexenyl group); ring contraction of the ribose sugar (e.g., to form a 4-membered ring of a cyclobutane or oxetane); ring expansion of the ribose sugar (e.g., to form a 6- or 7-membered ring having additional carbon or heteroatoms, such as anhydrohexitol, arabitol, mannitol, cyclohexyl, cyclohexenyl, and morpholino, which also has a phosphoramidate backbone). In some embodiments, modified nucleotides can include polycyclic forms (e.g., tricyclic; and "unlocked" forms, such as glycol nucleic acids (GNAs) (e.g., R-GNA or S-GNA, in which the ribose sugar is replaced by a glycol unit linked to a phosphodiester bond), threose nucleic acids. In some embodiments, modifications to the sugar of the oligonucleotide include modifying the oligonucleotide to include locked nucleic acid (LNA), unlocked nucleic acid (UNA), ethylene nucleic acid (ENA), constrained ethyl (cEt) sugar, or bridged nucleic acid (BNA).

[0074] In some embodiments, the oligonucleotides described herein include chemical modifications of at least one ribose sugar component. In some embodiments, the chemical modifications of the ribose sugar component may include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-fluoro, 2'-aminoethyl, 2'-deoxy-2'-fluoroarabinoic 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-N-methylacetamido (2'-O-NMA), 3'-phosphonoacetate (PACE), or 3'-phosphonothioacetate (thioPACE). In some embodiments, the chemical modification of the ribose sugar component comprises non-natural nucleic acids. In some cases, non-natural nucleic acids include modifications at the 5'-position and 2'-position 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, wherein the 3' connection nucleosides (5' to 3') in the dimer include 2'-OCH3 and 5'-(S)-CH3. Non-natural nucleic acids may include 2'-substituted 5'-CH2 (or O) modified nucleosides. Non-natural nucleic acids may include 5'-methylene phosphonate DNA and RNA monomers and dimers. Non-natural nucleic acids may include 5'-phosphonate monomers with 2'-substituted and other modified 5'-phosphonate monomers. Non-natural nucleic acids may include 5'-substituted methylene phosphonate monomers. Non-natural nucleic acids may include analogs of 5' or 6'-phosphonic acid ribonucleosides comprising hydroxyl groups at 5' and / or 6'-positions. Non-natural nucleic acids may include 5'-phosphonate deoxyribonucleoside monomers and dimers having a 5'-phosphate group. Non-natural nucleic acids may include nucleosides having a 6'-phosphonate group, wherein the 5' or / and 6'-positions are unsubstituted or substituted with t-butylthio (SC(CH3)3) (and its analogs); methyleneamino (CH2NH2) (and its analogs) or cyano (CN) (and its analogs).

[0075] In some embodiments, non-natural nucleic acids further include modifications of sugar moieties. In some embodiments, the nucleic acid contains one or more nucleosides in which the sugar group has been modified. Such sugar-modified nucleosides can impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological properties. In certain embodiments, the nucleic acid comprises a chemically modified ribofuranose ring portion. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and / or 2' substituents; bridging two ring atoms to form a bicyclic nucleic acid; using S, N(R), or C(R1)(R2) (R=H, C1-C12 alkyl or protecting group) to replace the ribosyl ring oxygen atom; and combinations thereof.

[0076] In some cases, oligonucleotides as described herein comprise modified sugars or sugar analogs. Therefore, in addition to ribose and deoxyribose, the sugar moiety can be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose or sugar "analog" cyclopentyl. Sugar can be a pyranosyl or furanosyl form. The sugar moiety can be a furanoside of ribose, deoxyribose, arabinose or 2'-O-alkyl ribose, and the sugar can be connected to each heterocyclic base with [α] or [β] 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.

[0077] Modifications to the sugar moiety include natural modifications of ribose and deoxyribose as well as non-natural modifications. Sugar modifications include, but are 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 alkyl, alkenyl, and alkynyl groups can be substituted or unsubstituted C1 to C 10 Alkyl or C2 to C 10 Alkenyl and alkynyl. 2' sugar modifications also include but are not limited to -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n ONH2 and -O(CH2) n ON[(CH2)n CH3)]2, wherein n and m are 1 to about 10. Other chemical modifications at the 2' position include, but are not limited to: C1 to C 10Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of oligonucleotides or group for improving the pharmacodynamic properties of oligonucleotides and other substituents with similar properties. Similar modifications can also be carried out at other positions of the sugar, particularly at the 3 ' terminal nucleotide or at the 3 ' position of the sugar in the 2 '-5 ' connected oligonucleotides and at the 5 ' position of the 5 ' terminal nucleotide. Chemically modified sugars also include sugars containing modifications on the bridging ring oxygen, such as CH 2 and S. Nucleotide sugar analogs can also have sugar mimetics, such as the cyclobutyl moiety replacing 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'-OCH3, and 2'-O(CH2)2OCH3 substituents. The substituent at the 2' position can also be selected from allyl, amino, azido, thio, O-allyl, O-(C1-C 1O alkyl), OCF3, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), and O-CH2-C(=O)-N(R m )(R n ), where each R m and R n are independently H or substituted or unsubstituted C1-C 10 alkyl.

[0078] In certain embodiments, nucleic acid as described herein includes one or more bicyclic nucleic acids. In some of the embodiments, bicyclic nucleic acids include bridging between 4' and 2' ribosyl ring atoms. In certain embodiments, nucleic acid provided herein includes one or more bicyclic nucleic acids, wherein bridging includes 4' to 2' bicyclic nucleic acids. Examples of such 4' to 2' bicyclic nucleic acids include, but are not limited to, one of the following formulae: 4'-(CH2)-O-2'(LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2'(ENA); 4'-CH(CH3)-O-2' and 4'-CH(CH2OCH3)-O-2' and analogs thereof; 4'-C(CH3)(CH3)-O-2' and analogs thereof.

[0079] In some embodiments, chemical modification as described herein includes the modification of nucleotide bases (e.g., core bases). Exemplary core bases may include adenine (A), thymine (T), guanine (G), cytosine (C) and uracil (U). These core bases may be modified or replaced with oligonucleotides as described herein. The core base of nucleotides may be independently selected from purine, pyrimidine, purine or pyrimidine analogs. In some embodiments, core bases may be naturally occurring bases or synthetic derivatives of bases. In embodiments, nucleotide sequences may be shown using DNA nucleotide sequences (i.e., including thymine core bases, "T" or "t") or as RNA nucleotide sequences (i.e., including uracil core bases, "U" or "u"). From the context, it can be understood that when nucleotides or sequences are intended to be RNA, T nucleotides may be replaced by U (or modified U, such as pseudouridine or 1-methyl pseudouridine); and when nucleotides or sequences are intended to be DNA, U nucleotides may be replaced by T or modified T. In some embodiments, the RNA nucleotides in the antisense oligonucleotides may be based on T (thymine) bases, while the DNA nucleotides in the antisense oligonucleotides may be based on U (uracil) bases. In some embodiments, one or more T nucleotides in the antisense oligonucleotides disclosed herein may be exchanged with U or modified U.

[0080] In some embodiments, the chemical modifications described herein include modified uracil. In some embodiments, the oligonucleotides described herein comprise at least one chemically modified uracil.Exemplary chemically modified uracils can 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 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-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 Uridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrobaicalin, dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine, 2-thiodihydrouridine, 2-thiodihydropseudouridine, 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-carboxypropyl)pseudouridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thiouridine, a-thiouridine, 2'-O-methyl uridine, 5-(2-carboxymethyloxyvinyl)uridine, 5-[3-(1E-propyleneamino)uridine, pyrazolo[3,4-d]pyrimidine, xanthine and hypoxanthine.

[0081] In some embodiments, the chemical modifications described herein include modified cytosines. In some embodiments, the oligonucleotides described herein comprise at least one chemically modified cytosine. Exemplary chemically modified cytosines may include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5-methyl-cytidine, 5-halocytidine, 5-hydroxymethyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrole-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebulin, 5-aza-zebulin, 5-methyl N4,N4,2'-O-trimethylcytidine, 1-thiocytidine, 2'-F-cytosine, 2'-F-cytidine, and 2'-OH-cytosine.

[0082] In some embodiments, chemical modification as described herein includes modification of adenine. In some embodiments, oligonucleotides as described herein include at least one chemically modified adenine. Exemplary chemically modified adenines can 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-chloropurine), 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-diaminopurine, 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 glycosides, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylcarbamoyl-adenosine, N6-threonylcarbamoyl-adenosine, N6-methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6-dimethyl-adenosine, N6-hydroxynorvalylcarbamoyl-adenosine, 2-methylthio-N6-hydroxynorvalyl Carbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine, N6,2'-O-dimethyladenosine, N6-methyl-2'-deoxyadenosine, N6,N6,2'-O-trimethyladenosine, 1,2'-O-dimethyladenosine, 2'-O-riboadenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-arabinoadenosine, 2'-F-adenosine, 2'-OH-arabinoadenosine and N6-(19-amino-pentaoxacyclododecyl)-adenosine.

[0083] In some embodiments, the chemical modifications described herein include modified guanines. In some embodiments, the oligonucleotides described herein comprise at least one chemically modified guanine. Exemplary chemically modified guanines can include inosine, 1-methyl-inosine, wyosine, methyl wyosine, 4-demethyl-wyosine, isowyosine, wyobutosine, peroxidized wyobutosine, hydroxy wyobutosine, suboptimally modified hydroxy wyobutosine, 7-deaza-guanosine, quiubutosine, epoxyquiubutosine, galactosyl-quiubutosine, mannosyl-quiubutosine, 7-cyano-7-deazaguanosine, 7-aminomethyl-7-deazaguanosine, archaeosine, 7-deaza-8-azaguanosine, 6-thio-guanosine, 6-thio-7-deazaguanosine, 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-methyl-guanosine, N2,N2-dimethylguanosine, Methyl-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-arabinoguanosine and 2'-F-guanosine.

[0084] In some embodiments, the chemical modification of the oligonucleotide can include introducing or substituting a nucleic acid analog or a non-natural nucleic acid into the oligonucleotide. In some embodiments, the nucleic acid analog can be any chemically modified nucleic acid described herein. All of these are explicitly 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 has been chemically altered (e.g., by acetylation, methylation, hydroxylation).Exemplary chemically modified nucleotides can 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 nucleoside, 2-aminopurine nucleoside, 2'-arabinoadenosine, 2'-arabinocytidine, 2'-arabinouridine, 2'-azido-2'-deoxyadenosine, 2'-azido-2'-deoxycytidine, 2'-azido-2'-deoxyguanosine, 2'-azido-2'-deoxyuridine, Oxyuridine, 2-chloroadenosine, 2'-fluoro-2'-deoxyadenosine, 2'-fluoro-2'-deoxycytidine, 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-thiocytidine, 3-methylcytidine, 4-acetylcytidine, 4-thiouridine, 5-(carboxyhydroxymethyl)-uridine, 5,6-dihydrouridine, 5-aminoallylcytidine, 5-aminoallyldeoxyuridine, 5-bromouridine, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 5-chloro-arabinoside, 5-fluorouridine, 5-iodouridine, 5-methoxycarbonylmethyl-uridine, 5-methoxy-uridine, 5-methyl-2-thiouridine, 6-azacytidine, 6-azauridine, 6-chloro-7-deaza-guanosine, 6-chloropurine nucleoside, 6-mercapto-guanosine, 6-methyl -mercaptopurine-nucleoside, 7-deaza-2'-deoxyguanosine, 7-deazaadenosine, 7-methylguanosine, 8-azaadenosine, 8-bromoadenosine, 8-bromoguanosine, 8-mercaptoguanosine, 8-oxoguanosine, benzimidazole nucleoside, β-D-mannosyl-quercetin, dihydro-uridine, inosine, N1-methyladenosine, N6-([6-aminohexyl]carbamoylmethyl)-adenosine, N6-isopentenyl-adenosine, N6-methyl-adenosine, N7-methyl-xanthosine, N-uracil-5-hydroxyacetic acid methyl ester, puromycin, quercetin, uracil-5-hydroxyacetic acid, uracil-5-hydroxyacetic acid methyl ester, wibutosine, xanthosine and xyloadenosine.In some embodiments, the chemically modified nucleic acids described herein comprise at least one chemically modified nucleotide selected from the group consisting of 2-amino-6-chloropurine nucleoside-5'-triphosphate, 2-aminopurine-nucleoside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 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-iodine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate Acid, 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 nucleoside-5'-triphosphate, 7-deazaadenosine- 5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole nucleoside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, 6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, puromycin-5'-triphosphate or xanthosine-5'-triphosphate. In some embodiments, the chemically modified nucleic acids described herein comprise at least one chemically modified nucleotide selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethylpseudouridine, 5-propynyluridine, 1-propynylpseudouridine, 5-taurinemethyluridine, 1-taurinemethyl-pseudouridine, 5-taurinemethyl-2-thio uridine, 1-methoxy-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, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine and 4-methoxy-2-thio-pseudouridine.In some embodiments, the artificial nucleic acid described herein comprises at least one chemically modified nucleotide selected from the group consisting of 5-azacytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methylpseudoisocytidine, pyrrolocytidine, pyrrolopseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio 1-Methyl-1-deaza-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-Methyl-1-deaza-pseudoisocytidine, zebulline, 5-aza-zebulline, 5-methyl-zebulline, 5-aza-2-thio-zebulline, 2-thio-zebulline, 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 comprise at least one chemically modified nucleotide 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-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine and 2-methoxyadenine. In other embodiments, the chemically modified nucleic acids described herein comprise at least one chemically modified nucleotide selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wyobutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazaguanosine, 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 comprise at least one chemically modified nucleotide selected from the group consisting of 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, desaturase, urea-1-dextrin, urea-2-dextrin, urea-3-dextrin, urea-4-dextrin, urea-5-dextrin, urea-6-dextrin, urea-7-dextrin, urea-8-dextrin, urea-9-dextrin, urea-10-dextrin, urea-11-dextrin, urea-12-dextrin, urea-13-dextrin, urea-14-dextrin, urea-15-dextrin, urea-16-dextrin, urea-17-dextrin, urea-18-dextrin, urea-19-dextrin, urea-20-dextrin, urea-21-dextrin, urea-22-dextrin, urea-23-dextrin, urea-24-dextrin, urea-25-dextrin, urea-26-dextrin, urea-27-dextrin, urea-28-dextrin, urea-29-dextrin, urea-30-dextrin, urea-31-dextrin, urea-32-dextrin, urea-33-dextrin, urea-34-dextrin, urea Oxy-thymidine, 5-methyl-uridine, pyrrolocytidine, inosine, α-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, pseudoisocytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.

[0085] Modified bases of non-natural nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthine-9-yl (I), 2-aminoadenine-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-propynyl uracil and cytosine uracil, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Certain non-natural nucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine, those nucleic acids that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 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-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halogen, in particular 5-bromo, 5-trifluoromethyl, 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, phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimidine [5,4-b] [l,4]benzothiazin-2(3H)-one), G-clamp, phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimidinyl[5,4-b][l,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimidinyl[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), compounds in which purine or pyrimidine bases are substituted with other heterocycles, 7-deazaadenine, 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, 5-iodouracil, 2-aminoadenine, 6-thioguanine, 2-thiothymine, 4-thiothymine, 5-propynyluracil, 4-thiouracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, or 2-amino-2'-deoxyadenosine.

[0086] 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 containing a 3' nucleotide, which can be stabilized to prevent degradation, for example, by incorporating one or more modified nucleotides described herein. In this embodiment, uridine can be replaced by modified uridines such as 5-(2-amino)propyluridine and 5-bromouridine or any modified uridine described herein; adenosine and guanosine can be replaced by modified adenosine and guanosine, for example, with a modification at position 8, such as 8-bromoguanosine, or with any modified adenosine or guanosine described herein. In some embodiments, deazanucleotides, such as 7-deaza-adenosine, can be incorporated into the oligonucleotide. In some embodiments, O- and N-alkylated nucleotides, such as N6-methyladenosine, can be incorporated into the oligonucleotide. In some embodiments, sugar-modified ribonucleotides can be incorporated, for example, wherein the 2'OH- group is replaced by a group selected from H, -OR, -R (wherein R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a sugar), halo, -SH, -SR (wherein R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a sugar), amino (wherein the amino group can be, for example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or an amino acid); or cyano (-CN).

[0087] Chemical means for introducing oligonucleotides into cells can include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, spherical nucleic acids (SNA), liposomes, or lipid nanoparticles. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other state-of-the-art nucleic acid targeted delivery methods are available, such as delivering oligonucleotides with targeted nanoparticles or other suitable submicron-sized delivery systems.

[0088] In one embodiment, oligonucleotides are delivered to cells in a manner that allows them to be delivered to the cell ... For example, lipids include the fat droplets naturally present in the cytoplasm as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0089] "Liposome" is a general term encompassing a variety of unilamellar and multilamellar lipid carriers formed by forming closed lipid bilayers or aggregates. Liposomes are generally characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components rearrange themselves before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers. However, compositions that have structures that differ from normal vesicular structures in solution are also included. For example, in some embodiments, lipids exhibit micellar structures or exist only as heterogeneous aggregates of lipid molecules. Lipid amine-nucleic acid complexes are also contemplated.

[0090] In some embodiments, the delivery method includes lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, exosomes, polycations or lipid: cargo conjugates (or aggregates), naked polypeptides (e.g., recombinant polypeptides), naked DNA, artificial virions, and drug-enhanced polypeptide or DNA uptake. In some embodiments, the delivery method includes conjugating or encapsulating the compounds or oligonucleotides described herein with at least one polymer such as a natural polymer or a synthetic material. The polymer can be biocompatible or biodegradable. Non-limiting examples of suitable biocompatible, biodegradable synthetic polymers can include aliphatic polyesters, poly(amino acids), copoly(ether-esters), polyalkylene oxalates, polyamides, poly(iminocarbonates), polyorthoesters, polyoxyesters, polyamide esters, polyoxyesters containing amine groups, and poly(anhydrides). Such synthetic polymers can be homopolymers or copolymers (e.g., random, block, segmented, grafted) of a variety of different monomers, such as two or more of lactic acid, lactide, glycolic acid, glycolide, ε-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 those having a ratio of 90 / 10 or 5 / 95 glycolic acid to lactic acid. Non-limiting examples of naturally occurring biocompatible, biodegradable polymers can include glycoproteins, proteoglycans, polysaccharides, glycosaminoglycans (GAGs), and fragments derived from these components, elastin, laminin, decrorin, fibrinogen / fibrin, fibronectin, osteopontin, tendin, hyaluronic acid, collagen, chondroitin sulfate, heparin, heparan sulfate, ORC, carboxymethyl cellulose, and chitin.

[0091] 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 particles. In some embodiments, the extracellular vesicles can be any membrane-bound particles secreted by at least one cell. In some cases, the extracellular vesicles can be any membrane-bound particles synthesized in vitro. In some cases, the extracellular vesicles can be any membrane-bound particles synthesized in the absence of cells. In some embodiments, the extracellular vesicles can be exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apoptotic bodies, oncosomes, exosomes, enveloped viruses, exosomes, or other very large extracellular vesicles.

[0092] In some embodiments, the oligonucleotides described herein are conjugated. In some embodiments, the oligonucleotides are conjugated to aptamers, peptides, antibodies, lipids, carbohydrates, or polymers. In some embodiments, the oligonucleotides are conjugated to aptamers, peptides, antibodies, lipids, carbohydrates, or polymers at the 5' end of the oligonucleotides. In some embodiments, the oligonucleotides are conjugated to aptamers, peptides, antibodies, lipids, carbohydrates, or polymers at the 3' end of the oligonucleotides. In some embodiments, the oligonucleotides are conjugated to aptamers, peptides, antibodies, lipids, carbohydrates, or polymers at any nucleic acid residue of the oligonucleotides. In some embodiments, the aptamers, peptides, antibodies, lipids, carbohydrates, or polymers conjugated to the oligonucleotides give therapeutic effects. For example, the peptides, antibodies, lipids, carbohydrates, or polymers conjugated to the oligonucleotides can be cytotoxic drugs or drugs for treating cancer. In some embodiments, the aptamers, peptides, antibodies, lipids, carbohydrates, or polymers conjugated to the oligonucleotides increase the efficiency of the oligonucleotides in combination with endogenous nucleic acids. In some embodiments, the aptamers, peptides, antibodies, lipids, carbohydrates, or polymers conjugated to the oligonucleotides give the oligonucleotides targeting specificity to specific types of cells (e.g., cancer cells, etc.). In some embodiments, the aptamer, peptide, antibody, lipid, carbohydrate or polymer conjugated to the oligonucleotide gives the oligonucleotide stability in vitro, in vitro or in vivo. For example, the oligonucleotide can be conjugated to polyethylene glycol (PEG) or an endosomal dissolution agent to reduce immunogenicity or degradation. In some embodiments, the aptamer, peptide, antibody, lipid, carbohydrate or polymer conjugated to the oligonucleotide promotes the oligonucleotide to enter the cell. In some embodiments, the aptamer, peptide, antibody, lipid, carbohydrate or polymer conjugated to the oligonucleotide promotes and releases the oligonucleotide into the cell. In some embodiments, the aptamer, peptide, antibody, lipid, carbohydrate or polymer conjugated to the oligonucleotide comprises at least one targeting moiety for targeting cells. Non-limiting examples of targeting moieties include signal peptides, chemokines, chemokine receptors, adhesion molecules, antigens or antibodies.

[0093] In an embodiment, the antisense oligonucleotides described herein are conjugated to an aptamer that targets pancreatic cells, e.g., as described in PCT / US2023 / 026980, which is hereby incorporated by reference in its entirety.

[0094] The linker used to conjugate the oligonucleotide to the aptamer, peptide, antibody, lipid or polymer can be any linker that connects a biomolecule. In some embodiments, the linker described herein is a cleavable linker or a non-cleavable linker. In some cases, the linker is a cleavable linker. In other cases, 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 a monomeric repeat unit produced by a polymerization process. In some embodiments, the linker comprises a peptide portion. In some cases, the peptide portion comprises at least 2, 3, 4, 5 or 6 amino acid residues. In some embodiments, the linker comprises a benzoic acid group or a derivative thereof. In some embodiments, the linker may comprise a nucleic acid linker, such as a DNA linker. In this case, the aptamer, peptide, antibody, lipid or polymer can be conjugated to one end of the nucleic acid linker or inserted into the nucleic acid base pairing of the nucleic acid linker. In some embodiments, the linker may be a peptide linker. The peptide linker can be flexible (e.g., a polyglycine linker) or rigid (e.g., an EAAAK repeat linker). In some embodiments, the peptide linker can be cleaved (e.g., a disulfide bond). In some embodiments, the linker comprises a polymer, such as PEG, polylactic acid (PLA), or polyacrylic acid (PAA).

[0095] In an embodiment, the melting temperature of an antisense oligonucleotide that hybridizes to its target sequence is at least about 35° C. The T of the oligonucleotide m T is the temperature at which 50% of the oligonucleotides form duplexes with their perfect complementary sequences and 50% of the oligonucleotides are free in solution. m It can be determined experimentally by measuring the change in absorbance of the oligonucleotide and its complementary sequence as a function of temperature. m Known publicly available T m In some embodiments, the T of an oligonucleotide that hybridizes to a target sequence is estimated using a calculator. 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 that hybridizes to a target sequence is m is about 35° C. to about 60° C. In some embodiments, the T of an oligonucleotide that hybridizes to a target sequence is m It is about 40°C to about 60°C, or about 50°C to about 60°C.

[0096] In some embodiments, the antisense oligonucleotide also includes a cell penetrating moiety, which in some embodiments is directly or indirectly conjugated to the 3' end of the oligonucleotide or is conjugated to the 3' end of the oligonucleotide, and is optionally conjugated by a joint (e.g., a polyethylene glycol joint or an alkyl joint). In some embodiments, the compound also includes a sterol conjugate (e.g., a cholesterol conjugate) or a fatty acid conjugate (such as a palmitoyl or stearoyl lipid conjugate), which is optionally conjugated to the 3' end of the antisense oligonucleotide. These parts can enhance cell penetration. Referring to US 9,012,225, which is hereby incorporated by reference in its entirety.

[0097] In some embodiments, the compound does not comprise any encapsulation or transfection reagents.

[0098] In some embodiments, the antisense oligonucleotide is encapsulated in a particle. In various embodiments, the particle is a liposome, a polymer nanoparticle or a lipid nanoparticle. Exemplary polymer nanoparticles can be formed by PLA, PLGA or their PEG copolymers. In some embodiments, the particle comprises poly-(β amino ester) polymers. In various embodiments, LNP comprises cationic lipids or ionizable lipids, neutral lipids, cholesterol or cholesterol moieties and pegylated lipids.

[0099] In some embodiments, the lipid nanoparticle (or LNP) comprises a structural lipid. Exemplary structural lipids can be selected from one or more of the following: cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatosterol, ursolic acid, and tocopherol (e.g., alpha-tocopherol). In some embodiments, the structural lipid is cholesterol.

[0100] In some embodiments, LNP comprises one or more phospholipids.Exemplary phospholipids are selected from cardiolipin, sterol-modified lipid (modified with the cholesterol moiety at the sn-2 carbon place attached to the glycerol backbone), mixed acylglycerophospholipids and symmetrical acylglycerophospholipids.The head group of acylglycerophospholipids includes for example phosphatidic acid, lysophosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphoinositol and phosphatidylserine. Exemplary phospholipids are selected from 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-diondecanoyl-sn-glycero-3-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-cholestylsuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lys PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diamidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

[0101] In some embodiments, the lipid nanoparticle composition further comprises one or more PEG lipids. PEG lipids are polyethylene glycol-modified lipids. Exemplary PEG lipids are selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. PEG lipids can be selected from PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-cholesterol, PEG tocopherol, or PEG-DSPE lipids.

[0102] Lipid particle formulations for use with embodiments of the present disclosure include those described in US 9,738,593, US 10,221,127, US 10,166,298, which are hereby incorporated by reference in their entireties. In some embodiments, the liposomes or nanoparticles further comprise a targeting moiety as described.

[0103] In other embodiments, the compound is formulated for parenteral administration.In some embodiments, the compound for parenteral administration comprises encapsulation in particles as described.

[0104] In some embodiments, disclosed herein are methods for modulating KRAS-mediated signaling pathways in cancer cells by treating or contacting the cancer cells with a composition comprising an antisense oligonucleotide, composition, or pharmaceutical composition described herein, thereby reducing the expression of KRAS or a mutant KRAS protein or mRNA in the cancer cells. In some embodiments, the mutant KRAS protein comprises a G12C mutation, a G12V mutation, a G12A mutation, or a G12D mutation.

[0105] In some embodiments, the present invention also discloses a method for treating a subject in need by administering a therapeutically effective amount of an oligonucleotide, composition, or pharmaceutical composition as described herein to a subject. In some embodiments, the method treats a subject by regulating the activity of gene expression or signaling pathway expression associated with a signaling pathway in the subject. In some embodiments, the method includes reducing gene expression by contacting a nucleic acid (e.g., endogenous mRNA) or a cell (e.g., a cancer cell) comprising the nucleic acid with an oligonucleotide as described herein. In some embodiments, the method includes reducing KRAS, mutated KRAS, or a combination of KRAS and mutated KRAS in a subject or cancer cell by contacting the mRNA of KRAS or mutated KRAS with an oligonucleotide as described herein, wherein the binding of the oligonucleotide to the mRNA recruits endogenous nucleases for degrading the mRNA. In some embodiments, the method includes reducing the expression of a signaling pathway such as a KRAS-mediated signaling pathway. In some embodiments, the method comprises decreasing the expression of genes in the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway, or the activity of these pathways.

[0106] In some embodiments, oligonucleotide, composition or pharmaceutical composition can be applied to subject alone (for example, independently).In some embodiments, oligonucleotide, composition or pharmaceutical composition is applied in combination with other agent.In some embodiments, other agent as used herein is applied alone.Oligonucleotide, composition or pharmaceutical composition and other agent can be applied 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; phenylethyl isothiocyanate; belinostat; berberine; vitexin; chrysin; bufalin; fisetin; fucoidan; gallic acid; gemcitabine; Guizhi Fuling Decoction; JOTO 1007; quercetin; rasfonin; 2,3,7,8-tetrachlorodibenzo-p-dioxin; triptolide; 4-hydroxybutenolide; or a combination thereof. The combination therapy can be administered on the same day or can be administered one or more days, weeks, months, or years apart.

[0107] In some embodiments, oligonucleotide, composition or pharmaceutical composition is a first-line treatment for a disease or illness. In some embodiments, oligonucleotide, composition or pharmaceutical composition is a second-line, third-line or fourth-line treatment. Generally speaking, methods disclosed herein include administering an oligonucleotide, composition or pharmaceutical composition by oral administration. However, in some cases, methods include administering an oligonucleotide, composition or pharmaceutical composition by intraperitoneal injection. In some cases, the method includes administering a pharmaceutical composition in the form of an anal suppository. In some cases, the method includes administering an oligonucleotide, composition or pharmaceutical composition by intravenous ("iv") administration. It is conceivable that oligonucleotide, composition or pharmaceutical composition disclosed herein can also be administered by other approaches, such as subcutaneous injection, intramuscular injection, intradermal injection, transdermal injection, transdermal administration, intranasal administration, intralymphatic injection, rectal administration, intragastric administration or any other suitable parenteral administration. In some embodiments, local delivery approaches closer to injury or inflammation sites are preferred compared to systemic approaches. The approach, dosage, time point and duration of administering therapeutic agents can be adjusted. In some embodiments, administration of the therapeutic agent is before or after the onset of one or both of the acute and chronic symptoms of the disease or disorder.

[0108] The appropriate dose and dosage for administration to a subject will be determined by factors including, but not limited to, the particular oligonucleotide, composition or pharmaceutical composition, the disease condition and its severity, the identity of the subject in need of treatment (e.g., weight, sex, age), and may be determined based on the particular circumstances surrounding the case, including, for example, the specific agent being administered, the route of administration, the condition being treated, and the subject being treated.

[0109] The effective dosage range can be adjusted depending on the subject's response to treatment. Some routes of administration will require higher concentrations of an effective amount of the therapeutic agent than other routes.

[0110] In some embodiments, administration of an oligonucleotide, composition, or pharmaceutical composition as described herein inhibits tumor growth by at least 10%, 15%, 20%, 30%, 40%, 50% or more. In some embodiments, the dosage of an oligonucleotide, composition, or pharmaceutical composition as described herein inhibits tumor growth by at least 10%, 15%, 20%, 30%, 40%, 50% or more. In some embodiments, the schedule of administration of an oligonucleotide, composition, or pharmaceutical composition as described herein inhibits tumor growth by at least 10%, 15%, 20%, 30%, 40%, 50% or more. In some embodiments, the dosage and schedule of administration of an oligonucleotide, composition, or pharmaceutical composition as described herein inhibits tumor growth by at least 10%, 15%, 20%, 30%, 40%, 50% or more.

[0111] In some embodiments, the dosage of an oligonucleotide, composition, or pharmaceutical composition described herein administered to a subject is sufficient to inhibit tumor growth. In some embodiments, the dosage and schedule of administration of an oligonucleotide, composition, or pharmaceutical composition described herein to a subject is sufficient to inhibit tumor growth. In some embodiments, the dosage and schedule of administration of an oligonucleotide, composition, or pharmaceutical composition described herein to a subject is sufficient to inhibit tumor growth.

[0112] In some embodiments, the disease or disorder described herein is cancer. In some embodiments, the cancer is associated with KRAS. In some embodiments, the cancer is associated with a mutated KRAS. In some embodiments, the cancer is associated with KRAS. In some embodiments, the cancer is associated with an abnormality in a KRAS-mediated signaling pathway. In some embodiments, the cancer is lung cancer, pancreatic cancer, or colon cancer. Other non-limiting examples of cancer may include acute lymphoblastic leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenal cancer, adrenocortical carcinoma, adult leukemia, AIDS-related lymphoma, amyloidosis, anal cancer, astrocytoma, ataxia-telangiectasia, atypical nevus syndrome, atypical malformation / rhabdoid tumor, basal cell carcinoma, bile duct cancer, Birt HoggDubé syndrome, bladder cancer, bone cancer, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid (gastrointestinal), cancer of unknown primary, cardiac (heart) tumor, cervical cancer, bile duct cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia, chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, neuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, bone fibrous histiocytoma, malignant and osteosarcoma, gallbladder cancer, stomach cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor (GIST) IST), germ cell tumors, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, HER2-positive breast cancer, histiocytosis, Langerhans cell, Hodgkin lymphoma, pharyngeal cancer, intraocular melanoma, islet cell tumor, juvenile polyposis syndrome, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cancer, liver cancer, lobular carcinoma, lung cancer (non-small cell and small cell), lymphoma, malignant fibrous histiocytoma and osteosarcoma, malignant glioma, melanoma, intraocular melanoma and meningioma, Merkel cell carcinoma, mesothelioma, malignant metastatic cancer, metastatic squamous neck cancer with occult primary cancer, midline tract cancer, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Plasma Cell Neoplasms, Mycosis Fungoides, Myelodysplastic Syndrome (MDS), Myeloproliferative Neoplasms, Chronic Nasal and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Neuroendocrine Tumors, Non-Hodgkin Lymphoma, Oral Cancer, Lip and Oral Cavity Cancer, Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Ovarian Germ Cell Tumors, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors, Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Peritoneal Cancer, Peutz-Jeghers Syndrome, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumors, Plasma Cell Neoplasms / Multiple Myeloma, Pleuropulmonary Bladder Cancer, Polycythemia Vera , pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, skin cancer, small intestine cancer, soft tissue sarcoma, solid tumors, squamous cell carcinoma of the skin, squamous cell carcinoma of the neck with occult primary metastasis, stomach cancer, T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma, thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, unusual childhood cancers, ureter and renal pelvis, transitional cell carcinoma, urethral cancer, uterine (endometrial) cancer, uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer, Wilms' tumor, or a combination thereof.

[0113] In some embodiments, the cancer is pancreatic cancer.

[0114] In some embodiments, pharmaceutical compositions comprising oligonucleotides or compounds as described herein are described herein. Pharmaceutical compositions used herein refer to mixtures of pharmaceutical compositions with other chemical ingredients (i.e., pharmaceutically acceptable inactive ingredients), such as carriers, excipients, adhesives, fillers, suspending agents, flavorings, sweeteners, disintegrants, dispersants, surfactants, lubricants, colorants, diluents, solubilizers, wetting agents, plasticizers, stabilizers, penetration enhancers, wetting agents, defoamers, antioxidants, preservatives, or one or more combinations thereof. Optionally, the compound includes two or more pharmaceutical compositions discussed herein. In the method for implementing the treatment or use provided herein, a therapeutically effective amount of a pharmaceutical composition as described herein is applied to a mammal suffering from a disease, condition, or illness to be treated, such as an inflammatory disease, fibrostenotic disease, and / or fibrotic disease, in the form of a pharmaceutical composition. In some embodiments, the mammal is a human. A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health status of the subject, the effectiveness of the pharmaceutical composition used, and other factors. Pharmaceutical compositions can be used alone or in combination with one or more pharmaceutical compositions as mixture components. The pharmaceutical compositions described herein comprise an oligonucleotide, a compound, a cell contacted with an oligonucleotide, or a cell contacted with a compound comprising an oligonucleotide, or a combination thereof.

[0115] The pharmaceutical formulations described herein are administered to a subject via an appropriate route of administration, including but not limited to intravenous, intratumoral, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal administration. 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 dissolve formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations.

[0116] Pharmaceutical compositions, including pharmaceutical compositions, are manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or compression processes.

[0117] The pharmaceutical composition may comprise at least a pharmaceutical composition as an active ingredient in the form of a free acid or free base or a pharmaceutically acceptable salt. 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 with the same type of activity. In some embodiments, the pharmaceutical composition is present in an unsolvated form or in a solvated form with a pharmaceutically acceptable solvent (such as water, ethanol, etc.). The solvated form of the pharmaceutical composition is also considered to be disclosed herein.

[0118] In some embodiments, the pharmaceutical compositions exist as enantiomers, diastereomers, or other stereoisomeric forms.The agents disclosed herein include all enantiomeric, diastereomeric, and epimeric forms and mixtures thereof.

[0119] The use of absolute or sequential terms such as “shall,” “will not,” “would,” “shall not,” “must,” “must not,” “first,” “first,” “next,” “then,” “before,” “after,” “last,” and “finally” are not meant to limit the scope of the embodiments disclosed herein, but rather serve as examples.

[0120] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, to the extent that the terms "including," "having," "has," "with," or variations thereof are used in the detailed description and / or claims, these terms are intended to be inclusive in a manner similar to the term "comprising."

[0121] As used herein, the phrases "at least one," "one or more," and "and / or" are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions "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" refers to A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together.

[0122] As used herein, "or" can mean "and," "or," or "and / or" and can be used exclusively or inclusively. For example, the term "A or B" can mean "A or B," "A but not B," "B but not A," and "A and B." In some embodiments, the context may dictate the specific meaning.

[0123] When referring to a number or a numerical range, the term "about" means that the number or numerical range mentioned is an approximation within the experimental variability (or within the statistical experimental error), and that the number or numerical range may vary, for example, 1% to 15% of the specified number or numerical range. In an example, the term "about" refers to ±10% of the specified number or value.

[0124] As used herein, the terms "increase," "increase," or "increase" generally refer to an increase in a statically significant amount. In some embodiments, the terms "increase" or "increase" mean an increase of at least 10% compared to a reference level, such as 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 up to and including an increase of 100% or any increase between 10-100%, compared to a reference level. 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.

[0125] The term "reduce", "reduce" or "reduce" is generally used to represent a statistically significant amount of reduction in the text. In some embodiments, "reduce" or "reduce" means to reduce by at least 10% compared to a reference level, for example, compared to a reference level, reduce by 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 up to and including reduction by 100% (for example, compared to a reference level, there is no level or level that cannot be detected) or any reduction between 10-100%. In the context of a marker or symptom, these terms refer to the statistically significant reduction of this level. Reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably reduced to an acceptable level in the normal range for the individuality without a given disease.

[0126] Example The following illustrative examples represent embodiments of the present disclosure.

[0127] Example 1. Exemplary KRAS antisense oligonucleotides and knockdown of KRAS mRNA Table 1. Exemplary KRAS antisense oligonucleotides

[0128] "+" indicates LNA, and can be optionally replaced by 2'-MOE or 2'-OMe or other bridging nucleotides such as cEt; “*” indicates phosphorothioate internucleotide linkage; “i2MOEr” indicates an internucleotide 2-methoxyethoxy-modified nucleotide; "i5HydMe" indicates an internucleotide 5-hydroxymethyl modified nucleotide.

[0129] Table 2. Additional antisense oligonucleotides

[0130] "+" indicates LNA, and can be optionally replaced by 2'-MOE or 2'-OMe or other bridging nucleotides such as cEt; “*” indicates phosphorothioate internucleotide linkage; "iMe" indicates an internucleotide 5-methyl modified nucleotide.

[0131] "3Me" indicates a 3' 5-methyl modified nucleotide.

[0132] Knockdown of KRAS mRNA Cell culture conditions and in vitro transfection Figure 1Depicts knockdown of mutant KRAS mRNA by KRAS G12C-specific antisense oligonucleotides (ASOs). The y-axis shows the percentage of KRAS mRNA knockdown relative to KRAS mRNA in cells treated with the non-KRAS targeting ASO control STN-030. The x-axis shows 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; g-STN-003. To avoid the TCC nucleotide at the 3' end, STN-001 has a base shift relative to STN-018; STN-002 is a 13-mer with a 3-8-2 wing-gap-wing configuration; STN-003 is a 13-mer with a 2-8-3 wing-gap-wing configuration. Mia PaCa-2 (ATCC) cells carrying the KRAS G12C mutation were plated in RPMI1640 containing 10% FBS and antibiotic-free in transparent flat-bottomed 96-well plates at a density of 20,000 cells per well. 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 supplemented with RPMI1640 + 10% FBS, penicillin / streptomycin, and incubated for another 48 hours. mRNA quantification was performed using QuantiGene (Thermo Fisher) according to the manufacturer's instructions. In order to normalize inter- and intra-experimental variations, STN-018 was selected as the benchmark ASO and included in all plates to normalize mRNA knockdown.

[0133] Example 2. Antisense oligonucleotide-mediated growth inhibition of cancer cells The following Figure 2 Table 3 depicts 3D growth inhibition of Mia PaCa-2 cells by G12C-specific ASOs. The y-axis shows the percentage of growth inhibition relative 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; k-STN-003.

[0134] Cells were plated at 800 cells per well in RPMI1640 containing 10% FBS in a clear 384-well plate (S-Bio, #MS-9384UZ) overnight and treated with 5 µM, 2.5 µM, 1.25 µM, 0.625 µM, 0.313 µM, 0.156 µM, or 0.078 µM ASO. 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. Figure 2 In the Figure 2, for each group of cells treated with a specific ASO, each individual bar from left to right depicts the highest to lowest concentration of the ASO used (two-fold dilutions from left to right). The ASOs used in this experiment exhibited significant growth inhibitory activity against cancer cell lines.

[0135] Table 3: 3D growth inhibition of the MIA PaCa-2 cell line by G12C-specific ASOs

[0136] "+" indicates LNA, and can be optionally replaced by 2'-MOE or 2'-OMe or other bridging nucleotides such as cEt; "*" indicates a phosphorothioate internucleotide linkage.

[0137] Example 3. Knockdown of mutant KRAS mRNA and KRAS pathway modulation cell lines Table 5: Evaluation of mutant-specific KRAS ASOs using different cell lines:

[0138] mRNA knockdown The ability of ASOs to knock down the desired mRNA was assessed as follows. Cells were plated at 20,000 cells per well in growth medium containing 10%-20% FBS and Pen / Strep 100 I.U / mL in a transparent flat-bottomed 96-well plate. Various concentrations of ASOs were added to the cells naked. The treated cells were incubated at 37°C for 96 hours. Pan KRAS ASO (STN-100019) was used as a positive control. mRNA quantification was performed using QuantiGene from Thermo Fisher according to its instructions. The results are shown in Tables 6-11 below.

[0139] Table 6: Knockdown of KRAS mRNA by G12V-modified specific sequences in Capan-1 and LCLC-97TM1 cell lines

[0140] "+" indicates LNA, and can be optionally replaced by 2'-MOE or 2'-OMe or other bridging nucleotides such as cEt; “*” indicates phosphorothioate internucleotide linkage; "iMe" indicates an internucleotide 5-methyl modified nucleotide.

[0141] "3Me" indicates a 3' 5-methyl modified nucleotide.

[0142] Table 7: Knockdown of KRAS mRNA by G12C-modified specific sequences in the Mia PaCa-2 cell line

[0143] "+" indicates LNA, and can be optionally replaced by 2'-MOE or 2'-OMe or other bridging nucleotides such as cEt; “*” indicates phosphorothioate internucleotide linkage; "i2MOEr" indicates an internucleotide 2-methoxyethoxy-modified nucleotide.

[0144] "i5HydMe" indicates an internucleotide 5-hydroxymethyl modified nucleotide.

[0145] "iMe" indicates an internucleotide 5-methyl modified nucleotide.

[0146] Table 8: KRAS mRNA knockdown by specific G12D-modified sequences in the PANC-1 cell line

[0147] "+" indicates LNA, and can be optionally replaced by 2'-MOE or 2'-OMe or other bridging nucleotides such as cEt; “*” indicates phosphorothioate internucleotide linkage; "iMe" indicates an internucleotide 5-methyl modified nucleotide.

[0148] "3Me" indicates a 3' 5-methyl modified nucleotide.

[0149] Inhibition of cell growth: The ability of ASOs to inhibit cell growth was assessed as follows. Cell lines were plated at 800 cells per well in clear 384-well plates (S-Bio, #MS-9384UZ) in growth medium containing 10%-20% FBS and 100 IU / mL Pen / Strep overnight. Cells were treated with varying concentrations of ASO. 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.

[0150] Table 9: 3D growth inhibition of specific sequences modified with G12V in CAPAN1 and LCLC97TM1 cell lines

[0151] "+" indicates LNA, and can be optionally replaced by 2'-MOE or 2'-OMe or other bridging nucleotides such as cEt; “*” indicates phosphorothioate internucleotide linkage; "iMe" indicates an internucleotide 5-methyl modified nucleotide.

[0152] "3Me" indicates a 3' 5-methyl modified nucleotide.

[0153] Table 10: 3D growth inhibition of specific sequences modified with G12C in the MIA PaCa-2 cell line

[0154] "+" indicates LNA, and can be optionally replaced by 2'-MOE or 2'-OMe or other bridging nucleotides such as cEt; “*” indicates phosphorothioate internucleotide linkage; “i2MOEr” indicates an internucleotide 2-methoxyethoxy-modified nucleotide; "i5HydMe" indicates an internucleotide 5-hydroxymethyl modified nucleotide.

[0155] "iMe" indicates an internucleotide 5-methyl modified nucleotide.

[0156] Table 11: 3D growth inhibition of specific sequences modified with G12D in the PANC-1 cell line

[0157] "+" indicates LNA, and can be optionally replaced by 2'-MOE or 2'-OMe or other bridging nucleotides such as cEt; “*” indicates phosphorothioate internucleotide linkage; "iMe" indicates an internucleotide 5-methyl modified nucleotide.

[0158] "3Me" indicates a 3' 5-methyl modified nucleotide.

[0159] Figure 3 Shown is Western blot analysis after knockdown of G12V KRAS with ASO in the LCLC-97TM1 cell line (day 4). Figure 3 Demonstrating KRAS protein knockdown and pathway modulation.

[0160] Table 4: Wild-type or mutant KRAS mRNA sequences

[0161] Although the aforementioned disclosure has been described in some detail for the purpose of clarity and understanding, it will be clear to those skilled in the art that various changes in form and detail can be made without departing from the true scope of the present disclosure by reading this disclosure. For example, all of the above-mentioned techniques and devices can 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, and the extent of the citation is as if each individual publication, patent, patent application and / or other document were specifically and individually 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 wherein 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. 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. 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. 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. The compound of claim 8, wherein the oligonucleotide has a nucleobase sequence selected from the group consisting of 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 6 DNA nucleotides sufficient to recruit RNase H.

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

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

13. The compound of any one of claims 10 to 12, wherein the antisense oligonucleotide is a gapmer having a 5' segment and a 3' segment, each of the 5' segment and the 3' segment being 2 to 6 nucleotides or 2 to 4 nucleotides, and wherein the 5' segment and the 3' segment do not contain DNA nucleotides.

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

15. The compound of claim 14, wherein one or more nucleotides of the 5' segment and the 3' segment comprise a 2'-0 substituent, optionally wherein all of the nucleotides of the 5' segment and the 3' segment comprise a 2'-0 substituent.

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 a bridged nucleotide having a 2' to 4' bridge.

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

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

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

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

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

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

23. 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 penetrating moiety.

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

26. A compound as claimed in claim 24 or claim 25, wherein the moiety comprises a sterol conjugate or a fatty acid conjugate, optionally a cholesteryl, palmitoyl or stearoyl conjugate.

27. 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 comprise any encapsulation or transfection reagents.

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

30. The compound of claim 29, wherein the particle is a liposome, a polymer 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 disease associated with abnormal expression of KRAS or associated with mutated KRAS, the method comprising administering to the subject an effective amount of the compound of any one of claims 1 to 32 or the pharmaceutical composition of claim 32.

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

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

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

37. 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 malignancy associated with an aberration in the KRAS-mediated signaling pathway is non-metastatic.

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

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

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

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

43. The method of claim 40, wherein the malignancy 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 cancer, 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.

Images