Compositions and methods for modulating KRAS expression

Abstract

Described herein are compositions for modulating gene expression. Also described herein are methods of using the compositions described herein for modulating gene expression.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 240,226, filed September 2, 2021, and U.S. Provisional Application No. 63 / 315,669, filed March 2, 2022, which are incorporated by reference in their entireties.

[0002] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated by reference herein to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that the publications and patents or patent applications incorporated by reference conflict with disclosure contained herein, the present specification is intended to supersede and / or take precedence over all such conflicting content. [Background technology]

[0003] Certain diseases or pathologies are caused by gene mutations or dysregulation of signaling pathways due to overexpression or underexpression of one or more genes that affect the signaling pathways.To treat such diseases or pathologies, one of the most sought-after therapeutic options involves direct editing of gene mutations or transcription / translation control using gene silencing means or methods.RNA-induced gene silencing controls the RNA expression of target genes in various aspects, including transcription inactivation, mRNA degradation, and transcription attenuation.Therefore, there is still a need for compositions and methods for effectively editing gene expression at the RNA level. Summary of the Invention

[0004] In some embodiments, the present disclosure provides compositions comprising antisense oligonucleotides capable of binding to KRAS mRNA. In some embodiments, the KRAS mRNA is a mutant KRAS mRNA. In some embodiments, the antisense oligonucleotide comprises a sequence that is at least 80%, 85%, or 90% identical to one of the following sequences: SEQ ID NOs: 100-556. In some embodiments, the antisense oligonucleotide comprises a sequence that is at least 80%, 85%, or 90% identical to any one of the following sequences: SEQ ID NOs: 24-43, 65-82, or 87. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:129, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:399, SEQ ID NO:400, SEQ ID NO:401, SEQ ID NO:402, SEQ ID NO:427, SEQ ID NO:428, SEQ ID NO:429, SEQ ID NO:430, SEQ ID NO:433, SEQ ID NO:434, SEQ ID NO:435, SEQ ID NO:436, SEQ ID NO:437, SEQ ID NO:438, SEQ ID NO:439, SEQ ID NO:440, SEQ ID NO:441, SEQ ID NO:494, SEQ ID NO:495, SEQ ID NO:496, SEQ ID NO:497, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:518, SEQ ID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to any one of SEQ ID NOs:18-20, SEQ ID NOs:24-43, SEQ ID NOs:65-82, or SEQ ID NO:87.In some embodiments, the antisense oligonucleotide comprises a length of 12 to 30 nucleotides. In some embodiments, the antisense oligonucleotide comprises a gap segment and a wing segment. In some embodiments, the antisense oligonucleotide comprises a 5'-wing segment and a 3'-wing segment. In some embodiments, each of the 5'-wing segment and the 3'-wing segment is 3 linked nucleotides. In some embodiments, the antisense oligonucleotide comprises at least one 2'-modified nucleoside, at least one modified internucleotide linkage, or at least one inverted abasic moiety. In some embodiments, at least one 2'-modified nucleotide comprises a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified nucleotide, a locked nucleic acid (LNA), a cEt (constrained ethyl) sugar, an ethylene-bridged nucleic acid (ENA), or a combination thereof. In some embodiments, at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some embodiments, the antisense oligonucleotide comprises phosphorodiamidate morpholino oligomer (PMO), locked nucleic acid (LNA), thiomorpholino, cEt (constrained ethyl) sugar, or a combination thereof.In some embodiments, the antisense oligonucleotide is conjugated with a peptide, an antibody, a lipid, a carbohydrate, an aptamer, or a polymer.In some embodiments, the antisense oligonucleotide is conjugated with a peptide, an antibody, a lipid, a carbohydrate, an aptamer, or a polymer via a linker.In some embodiments, the composition comprises a combination of an antisense oligonucleotide that specifically binds to KRAS mRNA and an antisense oligonucleotide that specifically binds to mutant KRAS mRNA. In some embodiments, the composition comprises an antisense oligonucleotide that can bind to both KRAS mRNA and mutant KRAS mRNA. In some embodiments, the composition further comprises an excipient. In some embodiments, the composition is formulated for parenteral or inhaled administration. In some embodiments, the mutant KRAS mRNA encodes a mutant KRAS protein that includes a G12C mutation, a G12V mutation, a G12A mutation, or a G12D mutation. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to any one of SEQ ID NOs: 19, 27, 28, 37, 44, or 65-81. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to any one of SEQ ID NOs: 19, 28, 44, 67, 72-77, or 79-81. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 19. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 28. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 44. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 67. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 72. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 73.In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 74. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 75. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 76. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 77. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 79. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 80. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 81. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is any one of SEQ ID NOs: 19, 27, 28, 37, 44, or 65-81. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is any one of SEQ ID NOs: 19, 28, 44, 67, 72-77, or 79-81. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence of SEQ ID NO: 19. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence of SEQ ID NO: 44. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence of SEQ ID NO: 67. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence of SEQ ID NO: 72. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence of SEQ ID NO: 73. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence of SEQ ID NO: 74.In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 75. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 76. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 77. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 79. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 80. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 81.

[0005] Described herein in some aspects is a method of modulating a KRAS-mediated signaling pathway in a cancer cell, comprising treating the cancer cell with a composition comprising an antisense oligonucleotide capable of binding to KRAS mRNA or mutant KRAS mRNA, thereby reducing expression of KRAS mRNA or mutant KRAS mRNA in the cancer cell. In some embodiments, the cancer cell is a lung cancer cell, a pancreatic cancer cell, or a colon cancer cell. In some embodiments, the antisense oligonucleotide comprises a sequence having at least 80%, 85%, or 90% similarity to one of the following sequences: SEQ ID NOs: 100-556. In some embodiments, the antisense oligonucleotide comprises a sequence having at least 80%, 85%, or 90% similarity to one of the following sequences: SEQ ID NOs: 24-43, 65-82, or 87. In some embodiments, the antisense oligonucleotides are selected from the group consisting of the following sequences: SEQ ID NO:129, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:399, SEQ ID NO:400, SEQ ID NO:401, SEQ ID NO:402, SEQ ID NO:427, SEQ ID NO:428, SEQ ID NO:429, SEQ ID NO:430, SEQ ID NO:433, SEQ ID NO:434, SEQ ID NO:435, SEQ ID NO:436, SEQ ID NO:437, SEQ ID NO:438, SEQ ID NO: No. 439, SEQ ID NO:440, SEQ ID NO:441, SEQ ID NO:494, SEQ ID NO:495, SEQ ID NO:496, SEQ ID NO:497, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:518, SEQ ID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20.In some embodiments, the composition comprises a combination of an antisense oligonucleotide that specifically binds to KRAS mRNA and an antisense oligonucleotide that specifically binds to mutant KRAS mRNA. In some embodiments, the composition comprises an antisense oligonucleotide that can bind to both KRAS mRNA and mutant KRAS mRNA. In some embodiments, the antisense oligonucleotide comprises at least one 2'-modified nucleoside, at least one modified internucleotide linkage, or at least one inverted abasic moiety. In some embodiments, the at least one 2' modified nucleotide comprises a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified nucleotide, or comprises a locked nucleic acid (LNA), a cEt (constrained ethyl) sugar or an ethylene-bridged nucleic acid (ENA), a thiomorpholino, or a combination thereof. In some embodiments, the expression of the KRAS protein, mutant KRAS protein, KRAS mRNA, or mutant KRAS mRNA is reduced by at least 30%, at least 40%, or at least 50% after treatment. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to any one of SEQ ID NOs: 19, 27, 28, 37, 44, or 65-81. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to any one of SEQ ID NOs: 19, 28, 44, 67, 72-77, or 79-81. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 19.In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:28. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:44. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:67. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:72. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:73. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:74. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:75. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 76. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 77. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 79. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 80. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 81. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is any one of SEQ ID NOs: 19, 27, 28, 37, 44, or 65-81. In some embodiments, the antisense oligonucleotide comprises a nucleic acid sequence that is any one of SEQ ID NOs: 19, 28, 44, 67, 72-77, or 79-81.In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 19. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 44. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 67. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 72. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 73. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 74. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 75. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 76. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 77. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 79. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 80. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 81.

[0006] In some embodiments, the present disclosure describes a method for treating cancer in a subject in need thereof, comprising administering to the subject a composition comprising the antisense oligonucleotide described herein, thereby treating the cancer in the subject. In some embodiments, the cancer is associated with an abnormality in the KRAS-mediated signaling pathway. In some embodiments, the cancer is lung cancer, pancreatic cancer, or colon cancer. In some embodiments, the composition is administered to the subject at a dose and schedule sufficient to increase the survival rate of the subject by at least 5%. In some embodiments, the composition is administered to the subject at a dose and schedule sufficient to inhibit tumor growth. In some embodiments, the cancer is associated with KRAS or mutant KRAS. In some embodiments, the mutant KRAS mRNA encodes a mutant KRAS protein comprising a G12C mutation, a G12V mutation, a G12A mutation, or a G12D mutation.

[0007] This patent application contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0008] [Figure 1] Illustrated is knockdown of KRAS mRNA encoding a mutant KRAS protein containing a G12C mutation mediated by an oligonucleotide described herein (eg, ASO SEQ ID NO: 19, 44, 28, 37, 67, or 80). [Diagram 2] Illustrated is knockdown of KRAS protein (G12C mutant or wild-type KRAS) and downstream biomarker (pERK, pS6 and cPARP) expression in two different cell lines (NCI-H358 and A375) mediated by the oligonucleotides described herein (e.g., ASO sequence number 28). [Diagram 3]1 illustrates knockdown of mutant KRAS protein and downstream biomarker (pERK, pS6 and cPARP) expression in NCI-H358 cell line mediated by oligonucleotides described herein (e.g., ASO sequence number 28 or ASO sequence number 67). [Figure 4] Illustrated is three-dimensional (3D) cell growth inhibition due to inhibition of expression of mutant KRAS by contacting cells harboring a KRAS mutation (NCI-H358) with an oligonucleotide described herein (e.g., ASO sequence numbers 19, 44, 28, 67, 72, 74, 75, 76, 77, 78, 79, 80 or 81). [Diagram 5] Illustrated is knockdown of KRAS mRNA encoding a mutant KRAS protein (NCI-H441) containing a G12V mutation mediated by oligonucleotides described herein (e.g., ASO sequence numbers 19, 44, 77, 78, 79, 80, 81, 28, 67, 37, 70 and 72). [Figure 6] 8 illustrates ASO sequence number 80-mediated knockdown of mutant KRAS protein and downstream biomarker (pERK, pAKT, and pS6) expression in LCLC97TM1 (cells harboring the G12V mutation) and wild-type KRAS (A375). [Figure 7] 1 illustrates ASO sequence number 81-mediated knockdown of mutant KRAS protein and downstream biomarker (pERK, pAKT, and pS6) expression in LCLC97TM1 (cells harboring the G12V mutation). [Figure 8] Illustrated is 3D cell growth inhibition by inhibiting expression of mutant KRAS by contacting cells harboring a KRAS mutation (LCLC97TM1 cells, NCI-H441 cells, or CFPAC-1 cells with a G12V mutation) with an oligonucleotide described herein (e.g., ASO sequence numbers 1, 19, 44, 28, 67, 72, 74, 75, 76, 77, 78, 79, 80, or 81). [Figure 9]Illustrated is 3D cell growth inhibition by inhibiting expression of mutant KRAS by contacting cells carrying a KRAS mutation (NCI-H2009 cells carrying a G12A mutation) with an oligonucleotide described herein (e.g., ASO sequence numbers 1, 19, 44, 28, 67, 72, 73, 74, 75, 76, 77, 79, 80 or 81). [Figure 10] Illustrated is knockdown of KRAS mRNA (Panc1 and AsPC1) encoding mutant KRAS proteins containing the G12D mutation mediated by oligonucleotides described herein (eg, ASO SEQ ID NOs: 19, 28, 37, 67, 78 and 81).

[0009] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description which sets forth illustrative embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] overview Described herein are compositions and methods for modulating the expression of a gene or signaling pathway. Also described herein are compositions and methods for treating a disease or condition by modulating the expression of a gene or signaling pathway associated with the disease or condition. In some embodiments, the composition comprises at least one oligonucleotide, which upon delivery into the cell binds to an endogenous nucleic acid, thereby degrading the target nucleic acid. In some embodiments, described herein are methods that utilize the compositions or oligonucleotides described herein. In some embodiments, the method treats a disease or condition by contacting a cell with an oligonucleotide to reduce the expression of a gene or signaling pathway associated with the disease or condition.

[0011] In some embodiments, the oligonucleotide is an antisense oligonucleotide, and the oligonucleotide is complementary to and binds to at least one endogenous nucleic acid (e.g., mRNA).In some embodiments, the binding of the oligonucleotide to the endogenous nucleic acid causes the endogenous nucleic acid to be degraded or the translation of the target protein derived from the endogenous nucleic acid to be blocked.Therefore, the expression of the gene encoded by the endogenous nucleic acid is reduced.For example, the binding of the oligonucleotide to the endogenous nucleic acid, including mRNA, can create a double-stranded nucleic acid molecule, which then recruits the endogenous nuclease that degrades the mRNA.

[0012] In some embodiments, the oligonucleotide comprises at least one gap segment. In some embodiments, the oligonucleotide comprises at least one wing segment. In some embodiments, the oligonucleotide comprises at least one gap segment adjacent to two wing segments. For example, the oligonucleotide comprises a gap segment adjacent to a 5'-wing segment and a 3'-wing segment. In some embodiments, the gap segment or the wing segment comprises at least one chemical modification.

[0013] In some embodiments, the gene regulated by the oligonucleotide is part of a signal transduction pathway. In some embodiments, the signal transduction pathway is the KRAS signal transduction pathway. In some embodiments, the KRAS signal transduction pathway includes the KRAS-RAF-MEK-ERK signal transduction pathway. In some embodiments, the KRAS signal transduction pathway includes the phosphoinositide 3-kinase (PI3K) signal transduction pathway, the mitogen-activated protein kinase (MAPK) signal transduction pathway, or the Ral guanine nucleotide exchange factor (Ral-GEF) signal transduction pathway. Thus, in some embodiments, the reduction in gene expression due to the binding of the oligonucleotide to the endogenous nucleic acid may further reduce the expression of the signal transduction pathway including the gene regulated by the oligonucleotide. In some embodiments, the reduction in the expression of the gene or signal transduction pathway provides a therapeutic effect for the treatment of the disease or condition. In some embodiments, the disease or condition is caused by an increase in the expression of the gene or signal transduction pathway. In some embodiments, the disease or condition described herein is caused by a genetic mutation associated with the gene or signal transduction pathway.

[0014] composition In some embodiments, the present invention describes a composition comprising at least one of the oligonucleotides described herein. In some embodiments, the composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more oligonucleotides. In some embodiments, the multiple oligonucleotides comprise the same or different nucleic acid sequences. In some embodiments, the oligonucleotides described herein are antisense oligonucleotides for targeting and binding to endogenous nucleic acids. In some embodiments, the binding of the oligonucleotide to the endogenous nucleic acid recruits endogenous nucleases that degrade the endogenous nucleic acid. In some embodiments, the degradation of the endogenous nucleic acid reduces the expression of the gene encoded by the endogenous nucleic acid. In some embodiments, the degradation of the endogenous nucleic acid can treat the disease or condition described herein.

[0015] In some embodiments, the oligonucleotide is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50 or more nucleobases in length. In some embodiments, the oligonucleotide is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length. In some embodiments, the oligonucleotide comprises 10 nucleobases. In some embodiments, the oligonucleotide comprises 11 nucleobases. In some embodiments, the oligonucleotide comprises 12 nucleobases. In some embodiments, the oligonucleotide comprises 13 nucleobases. In some embodiments, the oligonucleotide comprises 14 nucleobases. In some embodiments, the oligonucleotide comprises 15 nucleobases. In some embodiments, the oligonucleotide comprises 16 nucleobases. In some embodiments, the oligonucleotide comprises 17 nucleobases. In some embodiments, the oligonucleotide comprises 18 nucleobases. In some embodiments, the oligonucleotide comprises 19 nucleobases. In some embodiments, the oligonucleotide comprises 20 nucleobases.

[0016] In some embodiments, the oligonucleotide comprises at least one gap segment. In some embodiments, the gap segment comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, or more nucleobases. In some embodiments, the gap segment comprises at least 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleobases. In some embodiments, the gap segment comprises 4 nucleobases. In some embodiments, the gap segment comprises 5 nucleobases. In some embodiments, the gap segment comprises 6 nucleobases. In some embodiments, the gap segment comprises 7 nucleobases. In some embodiments, the gap segment comprises 8 nucleobases. In some embodiments, the gap segment comprises 9 nucleobases. In some embodiments, the gap segment comprises 10 nucleobases. In some embodiments, the gap segment comprises 11 nucleobases. In some embodiments, the gap segment comprises 12 nucleobases. In some embodiments, the gap segment comprises 13 nucleobases. In some embodiments, the gap segment comprises 14 nucleobases.

[0017] In some embodiments, the oligonucleotide comprises at least one wing segment. In some embodiments, at least one wing segment is a 5'-terminal wing segment, which is covalently attached to a gap segment at the 5'-end of the gap segment. In some embodiments, at least one wing segment is a 3'-terminal wing segment, which is covalently attached to a gap segment at the 3'-end of the gap segment. In some embodiments, a gap segment is adjacent to a wing segment at both the 5'-end and the 3'-end of the gap segment. In some embodiments, a wing segment comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleobases. In some embodiments, a wing segment comprises one nucleobase. In some embodiments, a wing segment comprises two nucleobases. In some embodiments, a wing segment comprises three nucleobases. In some embodiments, a wing segment comprises four nucleobases. In some embodiments, a wing segment comprises 5 nucleobases. In some embodiments, a wing segment comprises 6 nucleobases. In some embodiments, a wing segment comprises 7 nucleobases. In some embodiments, a wing segment comprises 8 nucleobases. In some embodiments, a wing segment comprises 9 nucleobases. In some embodiments, a wing segment comprises 10 nucleobases.

[0018] In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment followed by a gap segment followed by a 3'-terminal wing segment. In such sequences, the 5'-terminal wing segment comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleobases and the 3'-terminal wing segment comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleobases. In some embodiments, the 5'-terminal wing segment and the 3'-terminal wing segment comprise the same number of nucleobases. In some embodiments, the 5'-terminal wing segment and the 3'-terminal wing segment comprise different numbers of nucleobases. In some embodiments, the 5'-terminal wing segment comprises one nucleobase. In some embodiments, the 5'-terminal wing segment comprises two nucleobases. In some embodiments, the 5'-terminal wing segment comprises three nucleobases. In some embodiments, the 5'-terminal wing segment comprises 4 nucleobases. In some embodiments, the 5'-terminal wing segment comprises 5 nucleobases. In some embodiments, the 5'-terminal wing segment comprises 6 nucleobases. In some embodiments, the 5'-terminal wing segment comprises 7 nucleobases. In some embodiments, the 5'-terminal wing segment comprises 8 nucleobases. In some embodiments, the 5'-terminal wing segment comprises 9 nucleobases. In some embodiments, the 5'-terminal wing segment comprises 10 nucleobases. In some embodiments, the 3'-terminal wing segment comprises 1 nucleobase. In some embodiments, the 3'-terminal wing segment comprises 2 nucleobases. In some embodiments, the 3'-terminal wing segment comprises 3 nucleobases. In some embodiments, the 3'-terminal wing segment comprises 4 nucleobases. In some embodiments, the 3'-terminal wing segment comprises 5 nucleobases. In some embodiments, the 3'-terminal wing segment comprises 6 nucleobases. In some embodiments, the 3'-end wing segment comprises 7 nucleobases.In some embodiments, the 3'-terminal wing segment comprises 8 nucleobases. In some embodiments, the 3'-terminal wing segment comprises 9 nucleobases. In some embodiments, the 3'-terminal wing segment comprises 10 nucleobases.

[0019] In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising one nucleobase and a 3'-terminal wing segment comprising one nucleobase. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising two nucleobases and a 3'-terminal wing segment comprising two nucleobases. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising three nucleobases and a 3'-terminal wing segment comprising three nucleobases. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising four nucleobases and a 3'-terminal wing segment comprising four nucleobases. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising five nucleobases and a 3'-terminal wing segment comprising five nucleobases. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising six nucleobases and a 3'-terminal wing segment comprising six nucleobases. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising seven nucleobases and a 3'-terminal wing segment comprising seven nucleobases. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising 8 nucleobases and a 3'-terminal wing segment comprising 8 nucleobases. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising 9 nucleobases and a 3'-terminal wing segment comprising 9 nucleobases. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising 10 nucleobases and a 3'-terminal wing segment comprising 10 nucleobases.

[0020] In some embodiments, the oligonucleotide is an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide binds to a target nucleic acid. In some embodiments, the target nucleic acid is an endogenous nucleic acid. In some embodiments, the target nucleic acid includes nuclear RNA, cytoplasmic RNA, or mitochondrial RNA. In some embodiments, the target RNA includes intergenic DNA (including but not limited to heterochromatic DNA), messenger RNA (mRNA), pre-messenger RNA (pre-mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA sequence, isolated RNA sequence, sgRNA, oligonucleotide, nucleic acid probe, primer, snRNA, long non-coding RNA, small RNA, snoRNA, siRNA, miRNA, small RNA derived from tRNA (tsRNA), antisense RNA, shRNA, or RNA derived from small rDNA (srRNA). In some embodiments, the oligonucleotide includes a nucleic acid sequence that allows the oligonucleotide to bind to the target nucleic acid by base pairing, such as Watson-Crick base pairing. The compositions and methods provided herein can be used to modulate the expression of a gene or a signaling pathway. Modulation can refer to changing the expression of a gene or a portion thereof at one of a variety of stages in order to alleviate a disease or condition associated with the gene or a mutation in the gene. Modulation can affect the transcriptional level or can affect post-transcription. Modulating transcription can correct the aberrant expression of splice variants generated by gene mutations. In some cases, the compositions and methods provided herein can be used to control target gene translation. Modulation can refer to reducing or knocking down the expression of a gene or a portion thereof by decreasing the abundance of the transcript.The reduction in abundance of a transcript can be mediated by reducing the processing, splicing, turnover or stability of the transcript, or by reducing the accessibility of the transcript to translational machinery such as ribosomes. In some cases, the oligonucleotides described herein can promote knockdown. The knockdown can reduce the expression of the target RNA. In some cases, the knockdown can involve the regulation of mRNA. In some cases, the knockdown can occur without substantially regulating mRNA. In some instances, the knockdown can occur by targeting the untranslated region of the target RNA, such as the 3'UTR, 5'UTR, or both. In some cases, the knockdown can occur by targeting the coding region of the target RNA.

[0021] In some embodiments, the oligonucleotide is an antisense oligonucleotide for targeting and binding to any one of the genes described herein.In some embodiments, the gene(s) targeted and bound by the oligonucleotide is KRAS or mutant KRAS.In some embodiments, the mutant KRAS gene encodes a mutant KRAS protein comprising a G12C mutation.In some embodiments, the mutant KRAS gene encodes a mutant KRAS protein comprising a G12V mutation.In some embodiments, the mutant KRAS gene encodes a mutant KRAS protein comprising a G12A mutation.In some embodiments, the mutant KRAS gene encodes a mutant KRAS protein comprising a G12D mutation.

[0022] In some embodiments, the oligonucleotide targets and binds to KRAS mRNA or mutant KRAS mRNA. In some embodiments, the oligonucleotide comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of the nucleic acid sequences in Tables 7-9. In some embodiments, the oligonucleotide comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 100-556, 24-43, 65-82, or 87. In some embodiments, the oligonucleotide is SEQ ID NO:129, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:399, SEQ ID NO:400, SEQ ID NO:401, SEQ ID NO:402, SEQ ID NO:427, SEQ ID NO:428, SEQ ID NO:429, SEQ ID NO:430, SEQ ID NO:433, SEQ ID NO:434, SEQ ID NO:435, SEQ ID NO:436, SEQ ID NO:437, SEQ ID NO:438, SEQ ID NO:439, SEQ ID NO:440, The oligonucleotide comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NO:441, SEQ ID NO:494, SEQ ID NO:495, SEQ ID NO:496, SEQ ID NO:497, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:518, SEQ ID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20. In some embodiments, the oligonucleotide comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NOs:24-43, 65-82, or 87.

[0023] In some embodiments, the oligonucleotide comprises at least one gap segment. In some embodiments, at least one gap segment comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to at least a portion of any one of the nucleic acid sequences of SEQ ID NOs: 100-556, 24-43, 65-82, or 87. In some embodiments, at least one gap segment comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to at least 5, 6, 7, 8, 9, or 10 consecutive sequences of any one of the nucleic acid sequences of SEQ ID NOs: 100-556, 24-43, 65-82, or 87. In some embodiments, at least one gap segment is selected from the group consisting of SEQ ID NO:129, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:399, SEQ ID NO:400, SEQ ID NO:401, SEQ ID NO:402, SEQ ID NO:427, SEQ ID NO:428, SEQ ID NO:429, SEQ ID NO:430, SEQ ID NO:433, SEQ ID NO:434, SEQ ID NO:435, SEQ ID NO:436, SEQ ID NO:437, SEQ ID NO:438, SEQ ID NO:439, SEQ ID NO:440, SEQ ID NO:441, SEQ ID NO:442, SEQ ID NO:443, SEQ ID NO:444, SEQ ID NO:445, SEQ ID NO:446, SEQ ID NO:447, SEQ ID NO:448, SEQ ID NO:449, SEQ ID NO:450, SEQ ID NO:451, SEQ ID NO:452, SEQ ID NO:453, SEQ ID NO:454, SEQ ID NO:455, SEQ ID NO:456, 94, SEQ ID NO:495, SEQ ID NO:496, SEQ ID NO:497, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:518, SEQ ID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20, and / or at least 5, 6, 7, 8, 9, 10 contiguous sequences thereof.

[0024] In some embodiments, an oligonucleotide comprising any one of the nucleic acid sequences of SEQ ID NOs: 18-20 may bind or preferentially bind to a wild-type KRAS sequence. In some embodiments, an oligonucleotide comprising any one of the nucleic acid sequences of SEQ ID NOs: 24-33, 65-67, or 82 may bind or preferentially bind to a mutant KRAS sequence encoding a G12C mutation. In some embodiments, an oligonucleotide comprising any one of the nucleic acid sequences of SEQ ID NOs: 77-81 may bind or preferentially bind to a mutant KRAS sequence encoding a G12V mutation. In some embodiments, an oligonucleotide comprising any one of the nucleic acid sequences of SEQ ID NOs: 72-76 or 87 may bind or preferentially bind to a mutant KRAS sequence encoding a G12A mutation. In some embodiments, an oligonucleotide comprising any one of the nucleic acid sequences of SEQ ID NOs: 34-43 or 68-71 may bind or preferentially bind to a mutant KRAS sequence encoding a G12D mutation.

[0025] In some embodiments, the oligonucleotides described herein target and bind to endogenous nucleic acids encoding genes related to the KRAS-RAF-MEK-ERK signaling pathway. In some embodiments, the gene related to the KRAS-RAF-MEK-ERK signaling pathway is KRAS. In some embodiments, the gene related to the KRAS-RAF-MEK-ERK signaling pathway is mutated KRAS. In some embodiments, the oligonucleotides described herein regulate or affect the expression or activity of genes in or related to the KRAS-RAF-MEK-ERK signaling pathway. In some embodiments, the gene related to the KRAS-RAF-MEK-ERK signaling pathway is RAS. In some embodiments, the gene related to the KRAS-RAF-MEK-ERK signaling pathway is RAF. In some embodiments, the gene related to the KRAS-RAF-MEK-ERK signaling pathway is MEK. In some embodiments, the gene related to the KRAS-RAF-MEK-ERK signaling pathway is ERK.

[0026] In some embodiments, the oligonucleotides described herein target and bind to endogenous nucleic acids encoding genes associated with the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway.

[0027] In some embodiments, upon binding to an endogenous nucleic acid, the oligonucleotide forms a duplex with the endogenous nucleic acid and recruits an endogenous nuclease that degrades the endogenous nucleic acid. In some embodiments, the endogenous nuclease is a deoxyribonuclease. In some embodiments, the endogenous nuclease is a ribonuclease. In some embodiments, the ribonuclease is an endoribonuclease. In some embodiments, the endoribonuclease includes 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 includes RNase PH, II, R, D, or T. In some embodiments, the nuclease includes polynucleotide phosphorylase (PNPase), oligoribonuclease, exoribonuclease I, or exoribonuclease II. In some embodiments, the ribonuclease recruited by the oligonucleotide that binds to the endogenous nucleic acid is RNase H.

[0028] In some embodiments, the oligonucleotide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more chemical modifications. In some embodiments, the oligonucleotide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 at least one gap segment comprising 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more chemical modifications.In some embodiments, the oligonucleotide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 at least one wing segment comprising 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more chemical modifications. In some embodiments, the oligonucleotide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 at least one gap segment and at least one wing segment comprising 7, 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.

[0029] In some embodiments, the oligonucleotides described herein bind to an endogenous nucleic acid (e.g., mRNA) encoding KRAS, and the binding of the oligonucleotide to the endogenous nucleic acid of KRAS reduces the endogenous expression of KRAS in a cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the endogenous expression of KRAS not regulated by the oligonucleotide. In some embodiments, the oligonucleotides described herein bind to an endogenous nucleic acid (e.g., mRNA) encoding mutant KRAS, and the binding of the oligonucleotide to the endogenous nucleic acid of mutant KRAS reduces the endogenous expression of mutant KRAS in a cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the endogenous expression of mutant KRAS not regulated by the oligonucleotide. In some embodiments, the mutant KRAS endogenous nucleic acid encodes a mutant KRAS protein that includes a G12C mutation, a G12V mutation, a G12A mutation, or a G12D mutation.

[0030] In some embodiments, the oligonucleotides described herein bind to an endogenous nucleic acid (e.g., mRNA) encoding KRAS, and binding of the oligonucleotide to the endogenous KRAS nucleic acid reduces endogenous expression of the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway in a cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to endogenous expression of the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway that is not regulated by the oligonucleotide.

[0031] In some embodiments, the oligonucleotides described herein bind to an endogenous nucleic acid (e.g., mRNA) encoding a mutant KRAS, and binding of the oligonucleotide to the mutant KRAS endogenous nucleic acid reduces endogenous gene expression or activity in the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway in a cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to endogenous gene expression or activity in the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway that is not regulated by the oligonucleotide.

[0032] In some embodiments, the composition comprises at least two oligonucleotides, a first oligonucleotide binds to KRAS endogenous nucleic acid (e.g., KRAS mRNA), and a second oligonucleotide binds to mutant KRAS endogenous nucleic acid (e.g., mutant KRAS mRNA). In some embodiments, the mutant KRAS endogenous nucleic acid encodes a mutant KRAS protein comprising a G12C mutation, a G12V mutation, a G12A mutation, or a G12D mutation.

[0033] In some embodiments, binding of the oligonucleotide to both the KRAS endogenous nucleic acid and the mutant KRAS endogenous nucleic acid reduces endogenous gene expression or activity in the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway in a cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to endogenous gene expression or activity in the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway that is not regulated by the oligonucleotide.

[0034] In some embodiments, the composition is formulated for administration to a subject by a suitable route of administration, including but not limited to intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal routes of administration. Pharmaceutical formulations described herein include but are not limited to aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolving formulations, tablets, capsules, pills, delayed release formulations, sustained release formulations, pulsatile release formulations, multiparticulate formulations, and mixed formulations of immediate release formulations and controlled release formulations. In some embodiments, the composition is formulated into a dosage form. In some embodiments, the composition is formulated to include at least one excipient. In some embodiments, the excipient is a pharma- ceutically acceptable excipient.

[0035] In some embodiments, compositions comprising the oligonucleotides described herein treat a disease or condition by decreasing expression of a gene or signaling pathway associated with the disease or condition. In some embodiments, compositions comprising the oligonucleotides described herein treat a disease or condition by directly decreasing expression of a gene associated with the disease or condition. In some embodiments, compositions comprising the oligonucleotides described herein treat a disease or condition by decreasing gene expression as part of a signaling pathway described herein. In some embodiments, compositions comprising the oligonucleotides described herein treat a disease or condition by decreasing endogenous KRAS expression. In some embodiments, compositions comprising the oligonucleotides described herein treat a disease or condition by decreasing endogenous mutant KRAS expression. In some embodiments, compositions comprising the oligonucleotides described herein treat a disease or condition by decreasing both endogenous KRAS expression and mutant KRAS expression. In some embodiments, compositions comprising the oligonucleotides described herein treat a disease or condition by decreasing endogenous KRAS expression. In some embodiments, the composition comprising the oligonucleotide described herein treats disease or condition by reducing the expression or activity of endogenous KRAS-RAF-MEK-ERK signaling pathway, PI3K signaling pathway, MAPK signaling pathway, or Ral-GEF signaling pathway.In some embodiments, the disease or condition described herein is cancer.

[0036] chemical modification In some embodiments, the oligonucleotide described herein comprises at least one chemical modification.In some embodiments, the oligonucleotide is single-stranded.In some embodiments, the oligonucleotide is an antisense oligonucleotide.In some embodiments, the oligonucleotide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more chemical modifications.In some embodiments, the oligonucleotide has no structural features within the molecule. In some embodiments, the oligonucleotide comprises at least one gap segment comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more chemically modified nucleotides. In some embodiments, the oligonucleotide comprises at least one wing segment comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more chemically modified nucleotides. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more chemically modified nucleotides. In some embodiments, the oligonucleotide comprises a 3'-terminal wing segment comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more chemically modified nucleotides. In some embodiments, at least one wing segment is covalently fused to the 5'-terminus of the gap segment. In some embodiments, at least one wing segment is covalently fused to the 3'-terminus of the gap segment.

[0037] In some embodiments, the oligonucleotide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more chemically modified nucleotides at the 5'-end of the oligonucleotide. In some embodiments, the oligonucleotide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more chemically modified nucleotides at the 3'-end of the oligonucleotide. In some embodiments, the oligonucleotide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more chemically modified nucleotides at both the 5'-end and the 3'-end of the oligonucleotide. In some embodiments, the oligonucleotide comprises at least one chemical modification in the gap segment of the oligonucleotide. In some embodiments, the oligonucleotide comprises at least one chemical modification in the nucleotide base adjacent to the gap segment. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the bases or internucleotide linkages of the oligonucleotide comprise a modification. In some embodiments, the oligonucleotide comprises 100% modified nucleotide bases.

[0038] In some embodiments, the chemical modification may occur at the 3'OH group, 5'OH group, backbone, sugar moiety, or nucleotide base. The chemical modification may include a non-naturally occurring linker molecule at the interstrand or intrastrand crosslink. In one embodiment, the chemically modified nucleic acid includes one or more modifications of the 3'OH or 5'OH group, backbone, sugar moiety, or nucleotide base, or includes the addition of a non-naturally occurring linker molecule. In some embodiments, the chemically modified backbone includes a backbone other than a phosphodiester backbone. In some embodiments, the modified sugar includes a sugar other than deoxyribose (in modified DNA) or a sugar other than ribose (in modified RNA). In some embodiments, the modified base includes a base other than adenine, guanine, cytosine, thymine, or uracil. In some embodiments, the oligonucleotide includes at least one chemically modified base. In some examples, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more modified bases are included. In some cases, chemical modifications of the base moiety include natural and synthetic modifications of adenine, guanine, cytosine, thymine, or uracil, as well as natural and synthetic modifications of purine or pyrimidine bases.

[0039] In some embodiments, at least one chemical modification of the oligonucleotide includes any one of the following modifications, or any combination of the following modifications: 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-ON-methylacetamide). Modifications of one or both of the non-linking phosphate oxygens in a phosphodiester backbone linkage, modifications of one or more of the linking phosphate oxygens in a phosphodiester backbone linkage, modifications of the components of the ribose sugar, replacement of a phosphate moiety with a "dephospho" linker, modifications or substitutions of naturally occurring nucleobases, modifications of the ribose-phosphate backbone, modifications of the 5' end of a polynucleotide, modifications of the 3' end of a polynucleotide, modifications of the deoxyribose phosphate backbone, replacement of a phosphate group, modifications of the ribophosphate backbone, modifications of the sugar of a nucleotide, modifications of the base of a nucleotide, or stereochemically pure nucleotides. Non-limiting examples of chemical modifications to oligonucleotides include the following: modification of one or both of the non-linked or linking phosphate oxygens in the phosphodiester backbone linkages (e.g., sulfur (S), selenium (Se), BR3 (R can be, e.g., hydrogen, alkyl, or aryl), C (e.g., alkyl groups, aryl groups, and the like), H, NR2 (R can be, e.g., hydrogen, alkyl, or aryl, or R can be, e.g., alkyl or aryl), replacement of the phosphate moiety with a "dephospho" linker (e.g., methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo,methylenedimethylhydrazo, or methyleneoxymethylimino), modification or replacement of naturally occurring nucleobases with nucleic acid analogs, modification of the deoxyribose-phosphate or ribose-phosphate backbone (e.g., modifying the ribose-phosphate backbone to incorporate a phosphorothioate, phosphonothioacetate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphate, phosphonocarboxylate, phosphoramidate, alkyl or aryl phosphonate, phosphonoacetate, or phosphotriester), 5'-end modification of a nucleic acid sequence (e.g., a 5'-cap or 5'-cap-OH modification) or a 3'-end modification (a 3'-tail or 3'-end-OH modification), modification), replacement of the phosphate group with methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, or methyleneoxymethylimino, modification of the ribophosphate backbone to incorporate morpholino (phosphorodiamidate morpholino oligomers (PMOs)), thiomorpholino, cyclobutyl, pyrrolidine, or peptide nucleic acid (PNA) nucleoside surrogates, locked nucleic acid (LNA), non-locked nucleic acid (UNA), ethylene bridged nucleic acid (ENA), cEt (constrained modifying the sugar of a nucleotide to incorporate 2'-O-methyl, 2'-O-methoxy-ethyl (2'-MOE), 2'-fluoro, 2'-aminoethyl, 2'-deoxy-2'-fluoroarabinonucleic acid, 2'-deoxy, 2'-O-methyl, 3'-phosphorothioate, 3'-phosphonoacetate (PACE), or 3'-phosphonothioacetate (thioPACE));Modifications to the nucleotide base (A, T, C, G, or U), and stereochemically pure nucleotides (e.g., phosphorothioate S conformation or phosphorothioate R conformation).

[0040] In some embodiments, the chemical modification of the oligonucleotide comprises at least one substitution of one or both of the non-linking phosphate oxygen atoms in the phosphodiester backbone linkage of the oligonucleotide. In some embodiments, the chemical modification of the oligonucleotide comprises one or more substitutions of the linking phosphate oxygen atoms in the phosphodiester backbone linkage of the oligonucleotide. A non-limiting example of a chemical modification of a phosphate oxygen atom is a sulfur atom. In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification to the sugar of the nucleotide of the oligonucleotide. In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification to the sugar of the nucleotide of the oligonucleotide, the chemical modification comprising at least one locked nucleic acid (LNA). In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification to the sugar of the nucleotide of the oligonucleotide, the chemical modification comprising at least one non-locked nucleic acid (UNA). In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification to the sugar of the nucleotide of the oligonucleotide, the chemical modification comprising at least one ethylene-bridged nucleic acid (ENA). In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification to the sugar, comprising a modification of a component of the sugar, the sugar being a ribose sugar. In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification to the ribose sugar moiety of the nucleotide of the oligonucleotide, comprising a 2'-O-methyl group. In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification comprising a substitution of the phosphate moiety of the oligonucleotide with a dephosphoryl linker. In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification of the phosphate backbone of the oligonucleotide. In some embodiments, the oligonucleotide comprises a phosphothioate group. In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification comprising a modification to the base of the nucleotide of the oligonucleotide. In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification comprising a non-natural base of the nucleotide.In some embodiments, the chemical modification of the oligonucleotide comprises at least one chemical modification comprising a morpholino group (e.g., phosphorodiamidate morpholino oligomer, PMO), a cyclobutyl group, a pyrrolidine group, or a peptide nucleic acid (PNA) nucleoside surrogate. In some embodiments, the chemically modified form of the oligonucleotide comprises at least one chemical modification comprising at least one stereochemically pure nucleic acid. In some embodiments, the at least one chemical modification may be located proximal to the 5'-end of the oligonucleotide. In some embodiments, the at least one chemical modification may be located proximal to the 3'-end of the oligonucleotide. In some embodiments, the at least one chemical modification may be located proximal to both the 5'-end and the 3'-end of the oligonucleotide.

[0041] In some embodiments, the oligonucleotide comprises a backbone comprising multiple sugar and phosphate moieties covalently linked to each other. In some cases, the backbone of the oligonucleotide comprises a phosphodiester bond between the linkage of the first hydroxyl group of the phosphate group to the 5' carbon of the deoxyribose in DNA or ribose in RNA and the linkage of the second hydroxyl group to the 3' carbon of the deoxyribose in DNA or ribose in RNA.

[0042] In some embodiments, the backbone of the oligonucleotide may lack a 5' reducing hydroxyl, a 3' reducing hydroxyl, or both that may be exposed to solvent. In some embodiments, the backbone of the oligonucleotide may lack a 5' reducing hydroxyl, a 3' reducing hydroxyl, or both that may be exposed to nucleases. In some embodiments, the backbone of the oligonucleotide may lack a 5' reducing hydroxyl, a 3' reducing hydroxyl, or both that may be exposed to hydrolases. In some examples, the backbone of the oligonucleotide may be represented as a polynucleotide sequence in a circular two-dimensional format, where one nucleotide follows another. In some examples, the backbone of the oligonucleotide may be represented as a polynucleotide sequence in a circular two-dimensional format, where one nucleotide follows another. In some cases, the 5' hydroxyl, the 3' hydroxyl, or both are connected via phosphorus-oxygen bonds. In some cases, the 5' hydroxyl, the 3' hydroxyl, or both are modified to phosphoesters at phosphorus-containing moieties.

[0043] In some embodiments, the oligonucleotides described herein contain at least one chemical modification. The chemical modification can be a substitution, an insertion, a deletion, a chemical modification, a physical modification, a stabilization, a purification, or any combination thereof. In some cases, the modification is a chemical modification. Suitable chemical modifications include any one of the following: 5' adenylate, 5' guanosine-triphosphate cap, 5' N7-methylguanosine-triphosphate cap, 5' triphosphate cap, 3' phosphate, 3' thiophosphate, 5' phosphate, 5' thiophosphate, cis-sinthymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, d spacer, PC spacer, r spacer, spacer 18, spacer 9, 3'-3' modification, 5'-5' modification, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP. TEG, DNP-X, DOTA, dT-biotin, dual biotin, PC-biotin, psoralen C2, psoralen C6, TINA, 3'DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linker, 2' deoxyribonucleoside analog purine, 2' deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2'-O-methyl ribonucleoside analog, sugar modification analog, wobble / universal base, fluorescent dye label, 2' fluoro RNA, 2' O-methyl RNA, methyl phosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, LNA, cEt, pseudouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 2-O-methyl-phosphorothioate or combinations thereof.

[0044] In some cases, the modification can be permanent. In other cases, the modification can be transient. In some cases, multiple modifications are made to the oligonucleotide. The modification of the oligonucleotide can change the physiochemical properties of the nucleotide, such as the conformation, polarity, hydrophobicity, chemical reactivity, base pairing interaction, or any combination thereof. The chemical modification may also be a phosphorothioate substitution. In some cases, natural phosphodiester bonds may be susceptible to rapid degradation by cellular nucleases, and modifying the internucleotide linkages with phosphorothioate (PS) bond substitutions may make them more stable against hydrolysis by cellular degradation. The modification may increase the stability of the polynucleic acid. The modification may also increase biological activity. In some cases, phosphorothioate-enhanced RNA polynucleic acids may inhibit RNase A, RNase T1, calf serum nuclease, or any combination thereof. These properties may allow the use of PS-RNA polynucleic acids of interest in applications where exposure to nucleases is likely in vivo or in vitro. For example, phosphorothioate (PS) bonds may be introduced between the last 3-5 nucleotides at the 5'-end or 3'-end of the polynucleic acid, which may inhibit exonuclease degradation. In some cases, phosphorothioate bonds may be added throughout the polynucleic acid to reduce attack by endonucleases. In some embodiments, the oligonucleotides described herein contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 100 or more internucleotide linkages that contain PS linkages. In some embodiments, the oligonucleotides described herein contain only PS linkages as internucleotide linkage modifications. In some embodiments, all internucleotide linkages of the oligonucleotides described herein are fully PS-modified or contain phosphorothioate internucleotide linkages. In some embodiments, the oligonucleotides containing PS linkages as internucleotide linkage modifications contain a 5'-end wing segment that contains one nucleobase. In some embodiments, the oligonucleotides containing PS linkages as internucleotide linkage modifications contain a 5'-end wing segment that contains two nucleobases.In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 5'-end wing segment comprising 3 nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 5'-end wing segment comprising 4 nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 5'-end wing segment comprising 5 nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 5'-end wing segment comprising 6 nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 5'-end wing segment comprising 7 nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 5'-end wing segment comprising 8 nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 5'-end wing segment comprising 9 nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 5'-end wing segment comprising 10 nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 3'-terminal wing segment comprising one nucleobase. In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 3'-terminal wing segment comprising two nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 3'-terminal wing segment comprising three nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 3'-terminal wing segment comprising four nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 3'-terminal wing segment comprising five nucleobases.In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 3'-terminal wing segment comprising 6 nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 3'-terminal wing segment comprising 7 nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 3'-terminal wing segment comprising 8 nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 3'-terminal wing segment comprising 9 nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as the internucleotide linkage modification comprises a 3'-terminal wing segment comprising 10 nucleobases.

[0045] In some embodiments, the oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising one nucleobase and a 3'-terminal wing segment comprising one nucleobase. In some embodiments, the oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising two nucleobases and a 3'-terminal wing segment comprising two nucleobases. In some embodiments, the oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising three nucleobases and a 3'-terminal wing segment comprising three nucleobases. In some embodiments, the oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising four nucleobases and a 3'-terminal wing segment comprising four nucleobases. In some embodiments, the oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising five nucleobases and a 3'-terminal wing segment comprising five nucleobases. In some embodiments, the oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising six nucleobases and a 3'-terminal wing segment comprising six nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising 7 nucleobases and a 3'-terminal wing segment comprising 7 nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising 8 nucleobases and a 3'-terminal wing segment comprising 8 nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising 9 nucleobases and a 3'-terminal wing segment comprising 9 nucleobases. In some embodiments, the oligonucleotide comprising a PS linkage as an internucleotide linkage modification comprises a 5'-terminal wing segment comprising 10 nucleobases and a 3'-terminal wing segment comprising 10 nucleobases.

[0046] In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising one nucleobase, a gapmer, and a 3'-terminal wing segment comprising one nucleobase, and the internucleotide linkages of the oligonucleotide connecting the 5'-terminal wing segment, the gapmer, and the 3'-terminal wing segment comprise only PS linkages. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising two nucleobases, a gapmer, and a 3'-terminal wing segment comprising two nucleobases, and the internucleotide linkages of the oligonucleotide connecting the 5'-terminal wing segment, the gapmer, and the 3'-terminal wing segment comprise only PS linkages. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising three nucleobases, a gapmer, and a 3'-terminal wing segment comprising three nucleobases, and the internucleotide linkages of the oligonucleotide connecting the 5'-terminal wing segment, the gapmer, and the 3'-terminal wing segment comprise only PS linkages. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising 4 nucleobases, a gapmer, and a 3'-terminal wing segment comprising 4 nucleobases, and the internucleotide linkages of the oligonucleotide connecting the 5'-terminal wing segment, the gapmer, and the 3'-terminal wing segment comprise only PS linkages. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising 5 nucleobases, a gapmer, and a 3'-terminal wing segment comprising 5 nucleobases, and the internucleotide linkages of the oligonucleotide connecting the 5'-terminal wing segment, the gapmer, and the 3'-terminal wing segment comprise only PS linkages. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising 6 nucleobases, a gapmer, and a 3'-terminal wing segment comprising 6 nucleobases, and the internucleotide linkages of the oligonucleotide connecting the 5'-terminal wing segment, the gapmer, and the 3'-terminal wing segment comprise only PS linkages.In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment, a gapmer, and a 3'-terminal wing segment comprising 7 nucleobases, and the internucleotide linkages of the oligonucleotide connecting the 5'-terminal wing segment, the gapmer, and the 3'-terminal wing segment comprise only PS linkages. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment, a gapmer, and a 3'-terminal wing segment comprising 8 nucleobases, and the internucleotide linkages of the oligonucleotide connecting the 5'-terminal wing segment, the gapmer, and the 3'-terminal wing segment comprise only PS linkages. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment, a gapmer, and a 3'-terminal wing segment comprising 9 nucleobases, and the internucleotide linkages of the oligonucleotide connecting the 5'-terminal wing segment, the gapmer, and the 3'-terminal wing segment comprise only PS linkages. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment comprising 10 nucleobases, a gapmer, and a 3'-terminal wing segment comprising 10 nucleobases, and the internucleotide linkages of the oligonucleotide connecting the 5'-terminal wing segment, the gapmer, and the 3'-terminal wing segment comprise only PS linkages.

[0047] In some embodiments, the oligonucleotide comprising a 5'-terminal wing segment, a gapmer, a 3'-terminal wing segment, and a PS linkage as an internucleotide linkage comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NOs: 100-556. In some embodiments, the oligonucleotide comprising a 5'-terminal wing segment, a gapmer, a 3'-terminal wing segment, and a PS linkage as the internucleotide linkage is selected from the group consisting of SEQ ID NO:129, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:399, SEQ ID NO:400, SEQ ID NO:401, SEQ ID NO:402, SEQ ID NO:427, SEQ ID NO:428, SEQ ID NO:429, SEQ ID NO:430, SEQ ID NO:433, SEQ ID NO:434, SEQ ID NO:435, SEQ ID NO:436 , SEQ ID NO:437, SEQ ID NO:438, SEQ ID NO:439, SEQ ID NO:440, SEQ ID NO:441, SEQ ID NO:494, SEQ ID NO:495, SEQ ID NO:496, SEQ ID NO:497, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:518, SEQ ID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20.

[0048] In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment, a gapmer, a 3'-terminal wing segment, and a PS linkage as an internucleotide linkage, wherein the gapmer comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 100-556, 24-43, 65-82, or 87. In some embodiments, the oligonucleotide comprises a 5'-terminal wing segment, a gapmer, a 3'-terminal wing segment, and a PS linkage as the internucleotide linkage, the gapmer being selected from the group consisting of SEQ ID NO:129, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:399, SEQ ID NO:400, SEQ ID NO:401, SEQ ID NO:402, SEQ ID NO:427, SEQ ID NO:428, SEQ ID NO:429, SEQ ID NO:430, SEQ ID NO:433, SEQ ID NO:434, SEQ ID NO:435, SEQ ID NO: and / or SEQ ID NO:436, SEQ ID NO:437, SEQ ID NO:438, SEQ ID NO:439, SEQ ID NO:440, SEQ ID NO:441, SEQ ID NO:494, SEQ ID NO:495, SEQ ID NO:496, SEQ ID NO:497, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:518, SEQ ID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20.

[0049] In some instances, chemical modifications to enhance guide stability, synthesis, localization, intracellular retention, or extended half-life may not be genetically encoded. The oligonucleotides may be circular, substantially circular, or otherwise linked in a contiguous manner (e.g., arranged in a ring), and the oligonucleotides may also retain substantially similar secondary structures as substantially similar oligonucleotides that may not be circular or may not be ring-shaped.

[0050] Phosphate backbone modification In some embodiments, the chemical modification includes modification of one or both of the non-linking phosphate oxygens in the phosphodiester backbone bond, or modification of one or more linking phosphate oxygens in the phosphodiester backbone bond. As used herein, "alkyl" refers to a saturated hydrocarbon group that is straight or branched. Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl or isopropyl), butyl (e.g., n-butyl, isobutyl, or t-butyl), or pentyl (e.g., n-pentyl, isopentyl, or neopentyl). The alkyl group can contain 1 to about 20, 2 to about 20, 1 to about 12, 1 to about 8, 1 to about 6, 1 to about 4, or 1 to about 3 carbon atoms. As used herein, "aryl" refers to a monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) aromatic hydrocarbon, such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, or indenyl. In some embodiments, the aryl group has 6 to about 20 carbon atoms. As used herein, "alkenyl" refers to an aliphatic group containing at least one double bond. As used herein, "alkynyl" refers to a straight or branched hydrocarbon chain containing 2 to 12 carbon atoms and characterized by having one or more triple bonds. Examples of alkynyl groups include ethynyl, propargyl, or 3-hexynyl. "Arylalkyl" or "aralkyl" refers to an alkyl moiety in which an alkyl hydrogen atom is replaced with an aryl group. Aralkyl also includes groups in which more than one hydrogen atom is replaced by an aryl group. Examples of "arylalkyl" or "aralkyl" include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl. "Cycloalkyl" refers to a cyclic, bicyclic, tricyclic, or polycyclic non-aromatic hydrocarbon group having 3 to 12 carbons. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl. "Heterocyclyl" refers to a monovalent radical of a heterocyclic ring system. Representative heterocyclyls include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, and morpholinyl. "Heteroaryl" refers to a monovalent radical of an aromatic heterocyclic ring system. Examples of heteroaryl moieties include imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, indolyl, thiophenyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, quinolyl, and pteridinyl.

[0051] In some embodiments, the phosphate group of chemically modified nucleotides can be modified by replacing one or more oxygens with different substituents. In some embodiments, chemically modified nucleotides can include replacing unmodified phosphate moieties with modified phosphates as described herein. In some embodiments, the modification of the phosphate backbone can include modifications that produce either uncharged linkers or charged linkers with asymmetric charge distribution. Examples of modified phosphate groups can include phosphorothioates, phosphonothioacetates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (R can be, for example, hydrogen, alkyl, or aryl), C (e.g., alkyl, aryl, and the like), H, NR2 (R can be, for example, hydrogen, alkyl, or aryl, or R can be, for example, alkyl or aryl). The phosphorus atom in an unmodified phosphate group can be achiral. However, replacing one of the non-bridging oxygens with one of the above atoms or groups of atoms can make the phosphorus atom chiral. The phosphorus atom in such modified phosphate groups is an asymmetric center. The asymmetric center phosphorus atom can have either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). In some cases, the oligonucleotide comprises a stereochemically pure nucleotide, including a phosphorothioate S conformation or a phosphorothioate R conformation. In some embodiments, the chiral phosphate product is greater than 50%, 60%, 70%, 80%, 90% or more present as a diastereomer. In some embodiments, the chiral phosphate product is greater than 95% present as a diastereomer. In some embodiments, the chiral phosphate product is greater than 96% present as a diastereomer.In some embodiments, the chiral phosphate product is present as a diastereomer at greater than 97%. In some embodiments, the chiral phosphate product is present as a diastereomer at greater than 98%. In some embodiments, the chiral phosphate product is present as a diastereomer at greater than 99%. In some embodiments, the non-bridging oxygens of the phosphorodithioate can both be replaced with sulfur. The phosphorus center in the phosphorodithioate can be achiral, which prevents oligoribonucleotide diastereomers from forming. In some embodiments, the modification to one or both of the non-bridging oxygens can also include replacing the non-bridging oxygen with a group independently selected from S, Se, B, C, H, N, and OR (R can be, for example, alkyl or aryl). In some embodiments, the phosphate linker may also be modified by substituting the bridging oxygen (i.e., the oxygen that links the phosphate to the nucleoside) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates). Substitutions may occur at either or both of the linking oxygens.

[0052] In certain embodiments, the nucleic acid includes a linked nucleic acid. Nucleic acids can be linked to each other using any internucleic acid linkage. There are two main classes of internucleic acid linkage groups, defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleic acid linkage groups include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Representative non-phosphorus-containing internucleic acid linkage groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamates (-OC(O)(NH)-S-), siloxanes (-O-Si(H)2-O-), and N,N*-dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In certain embodiments, internucleic acid linking groups with chiral atoms, such as alkylphosphonates and phosphorothioates, can be prepared as racemic mixtures, as separate enantiomers. Non-natural nucleic acids can contain a single modification. Non-natural nucleic acids can contain multiple modifications within one moiety or between different moieties.

[0053] Phosphate backbone modifications to nucleic acids include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramidates (bridged or non-bridged), phosphotriesters, phosphorodithioates, phosphodithioates, and boranophosphates, which may be used in any combination. Other non-phosphate linkages may also be used.

[0054] In some embodiments, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoramidate, and phosphorodithioate internucleotide linkages) may confer immunomodulatory activity to the modified nucleic acid and / or enhance its stability in vivo.

[0055] In some examples, the phosphorus derivative (or modified phosphate group) is attached within a sugar or sugar analog moiety and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, or the like.

[0056] In some cases, backbone modifications include replacing phosphodiester linkages with alternative moieties such as anionic, neutral or cationic groups. Examples of such modifications include anionic internucleoside linkages, N3'→P5' phosphoramidate modifications, boranophosphate DNA, prooligonucleotides, neutral internucleoside linkages such as methylphosphonates, amide-linked DNA, methylene (methylimino) linkages, formacetal and thioformacetal linkages, sulfonyl-containing backbones, morpholino oligos, peptide nucleic acids (PNAs), and positively charged deoxyribonucleic guanidine (DNG) oligos. Modified nucleic acids can include chimeric or mixed backbones containing one or more modifications (e.g., combinations of phosphate linkages such as combinations of phosphodiester and phosphorothioate linkages).

[0057] Substituents for the phosphate include, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamic acid backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with a mixture of N, O, S and CH2 component moieties. It is also understood in the nucleotide substitution that both the sugar and phosphate moieties of the nucleotide can be replaced (PNAs), for example, with amide-type linkages (aminoethylglycine). Other types of molecules (conjugates) can also be linked to nucleotides or nucleotide analogs, for example, to enhance cellular uptake. Conjugates can be chemically linked to nucleotides or nucleotide analogs. Such conjugates include, but are not limited to, lipid moieties (cholesterol moieties, thioethers (e.g., hexyl-S-tritylthiol), thiocholesterol, aliphatic chains (e.g., dodecanediol or undecyl residues), phospholipids (e.g., di-hexadecyl-rac-glycerol or triethylammonium 1-di-O-hexadecyl-rac-glycero-SH-phosphonate, polyamines or polyethylene glycol chains, etc.), or adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterin moieties.

[0058] In some embodiments, the chemical modifications described herein include modifications of the phosphate backbone. In some embodiments, the oligonucleotides described herein include at least one chemically modified phosphate backbone. Exemplary chemical modifications of the phosphate group or phosphate backbone can include the replacement of one or more oxygens with different substituents. In addition, modified nucleotides present in the oligonucleotide can include the replacement of unmodified phosphate moieties with modified phosphates described herein. In some embodiments, the modifications of the phosphate backbone can include modifications that result in either uncharged linkers or charged linkers with asymmetric charge distribution. Exemplary modified phosphate groups can include phosphorothioates, phosphonothioacetates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (R can be, for example, hydrogen, alkyl, or aryl), C (e.g., alkyl, aryl, and the like), H, NR2 (R can be, for example, hydrogen, alkyl, or aryl), or (R can be, for example, alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygens with one of the above atoms or groups of atoms can make the phosphorus atom chiral. That is, the phosphorus atom in such modified phosphate groups is an asymmetric center. The asymmetric center phosphorus atom can have either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). In such cases, the chemically modified oligonucleotide can be stereochemically pure (e.g., S or R conformation). In some cases, the chemically modified oligonucleotide contains a stereochemically pure phosphate modification. For example, the chemically modified oligonucleotide comprises a phosphorothioate S conformation or a phosphorothioate R conformation.

[0059] Phosphorodithioates have both non-bridging oxygens replaced with sulfur. The phosphorus center in phosphorodithioates is achiral, which prevents oligoribonucleotide diastereomers from forming. In some embodiments, modifications to one or both non-bridging oxygens can also include replacement of the non-bridging oxygen with a group independently selected from S, Se, B, C, H, N, and OR (R can be, for example, alkyl or aryl).

[0060] The phosphate linker may also be modified by substituting the bridging oxygens (i.e., the oxygens that link the phosphate to the nucleoside) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates). Substitutions may occur at either or both of the linking oxygens.

[0061] Phosphate moiety replacement 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 linker that does not contain phosphorus. In some embodiments, the phosphate moiety can be replaced by a dephosphoryl linker. In some embodiments, the charged phosphate group can be replaced by a neutral group. In some cases, the phosphate group can be replaced by methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. In some embodiments, the nucleotide analogs described herein can also be modified at the phosphate group. Modified phosphate groups can include modifications in the linkage between two nucleotides with phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates including 3'-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. The phosphate linkage or modified phosphate linkage between two nucleotides can be via a 3'-5' linkage or a 2'-5' linkage, which linkage contains a reversal of direction, such as 3'-5' to 5'-3' or 2'-5' to 5'-2'.

[0062] Phosphate group replacement In some embodiments, the chemical modifications described herein include modifications by substitution of phosphate groups. In some embodiments, the oligonucleotides described herein include at least one chemical modification, including substitution or replacement of phosphate groups. Exemplary phosphate group substitutions can include non-phosphorus-containing linkers. In some embodiments, substitution or replacement of phosphate groups can include replacement of charged phosphate groups with neutral moieties. Exemplary moieties that can replace phosphate groups can include methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino.

[0063] Modification of the ribophosphate backbone In some embodiments, the chemical modifications described herein include modifying the ribophosphate backbone of the oligonucleotide. In some embodiments, the oligonucleotides described herein include at least one chemically modified ribophosphate backbone. Examples of chemically modified ribophosphate backbones include scaffolds that can mimic nucleic acids, in which the phosphate linker and ribose sugar are replaced with nuclease-resistant nucleosides or nucleotide surrogates. In some embodiments, the nucleobases can be linked by the surrogate backbone. Examples include morpholino, cyclobutyl, pyrrolidine, and peptide nucleic acid (PNA) nucleoside surrogates, such as phosphorodiamidate morpholino oligomers (PMOs).

[0064] Sugar modification In some embodiments, the chemical modifications described herein include sugar modifications. In some embodiments, the oligonucleotides described herein include at least one chemically modified sugar. Exemplary chemically modified sugars may include 2' hydroxyl groups (OH) modified or replaced with a number of different "oxy" or "deoxy" substituents. In some embodiments, modifications to the 2' hydroxyl group may enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion. The 2'-alkoxide may catalyze decomposition by intramolecular nucleophilic attack on the linker atom. Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, where "R" can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), polyethylene glycol (PEG), O(CH2CHO), and aryloxy groups. n and n is an integer from 0 to 20 (e.g., 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16, and 4 to 20). In some embodiments, "oxy"-2' hydroxyl group modifications include LNA (where the 2' hydroxyl can be connected to the 4' carbon of the same ribose sugar, e.g., by a Ci-6 alkylene or Cj-6 heteroalkylene bridge. Exemplary bridges can include methylene, propylene, ether, or amino bridges), O-amino (where amino can be, e.g., NH2 (alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino)) and aminoalkoxy, O(CH2) n-amino (wherein amino can be, for example, NH2 (alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino)). In some embodiments, the "oxy"-2' hydroxyl group modification can include a methoxyethyl group (MOE), (OCH2CHOCH3, e.g., a PEG derivative). In some cases, the deoxy modification can include hydrogen (i.e., a deoxyribose sugar, e.g., an overhanging portion of a partial dsRNA), halo (e.g., bromo, chloro, fluoro, or iodo), amino (wherein amino can be, for example, NH2 (alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid)), NH(CH2CH2NH) nExamples of the sugar group include CH2CH2-amino (wherein amino can be, for example, as described herein), NHC(O)R (wherein R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), cyano, mercapto, alkyl-thio-alkyl, thioalkoxy, and alkyl, cycloalkyl, aryl, alkenyl, and alkynyl, which can be optionally substituted with, for example, amino, as described herein. In some examples, the sugar group can also contain one or more carbons that have the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, modified nucleic acids can include, for example, nucleotides that contain arabinose as the sugar. Nucleotide "monomers" can have an alpha linkage at the Γ position of the sugar, e.g., alpha-nucleosides. Modified nucleic acids can also include "abasic" sugars that lack a nucleobase at C-. Also, the abasic sugar can be further modified at one or more of the sugar atoms. Modified nucleic acids can also include one or more L-form sugars, e.g., L-nucleosides. In some embodiments, the oligonucleotides described herein comprise a 5-membered ribose sugar group with oxygen.Exemplary modified nucleosides and modified nucleotides can include the substitution of oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene (e.g., methylene or ethylene), the addition of double bonds (e.g., to replace ribose with cyclopentenyl or cyclohexenyl), the ring contraction of ribose (e.g., to form 4-membered rings of cyclobutane or oxetane), the ring expansion of ribose (e.g., to form 6- or 7-membered rings with additional carbon or heteroatoms, such as anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino, which also have phosphoramidate backbone). In some embodiments, modified nucleotides can include polycyclic forms (e.g., tricyclic), as well as "non-locked" forms such as glycol nucleic acids (GNAs) (e.g., R-GNAs or S-GNAs, where the ribose is replaced with a glycol unit attached to a phosphodiester bond), threose nucleic acids, etc.In some embodiments, modification of the sugar of the oligonucleotide comprises modifying the oligonucleotide to contain a locked nucleic acid (LNA), a non-locked nucleic acid (UNA), an ethylene-bridged nucleic acid (ENA), a cEt (constrained ethyl) sugar, or a bridged nucleic acid (BNA).

[0065] Modification of ribose sugar components In some embodiments, the oligonucleotides described herein comprise at least one chemical modification of the ribose sugar moiety. In some embodiments, chemical modifications of the ribose sugar moiety can include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-fluoro, 2'-aminoethyl, 2'-deoxy-2'-fluoroarabinonucleic acid, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 3'-phosphorothioate, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), 3'-phosphonoacetate (PACE), or 3'-phosphonothioacetate (thioPACE). In some embodiments, the chemical modification of the ribose sugar moiety includes non-natural nucleic acids. In some examples, the non-natural nucleic acids include modifications at the 5' and 2' positions of the sugar ring, such as 5'-CH2-substituted 2'-O-protected nucleosides. In some cases, the non-natural nucleic acids include amide-linked nucleoside dimers that are prepared for incorporation into oligonucleotides, and the 3'-linked nucleosides (5' to 3') of the dimers include 2'-OCH3 and 5'-(S)-CH3. The non-natural nucleic acids can include 2'-substituted 5'-CH2 (or O) modified nucleosides. The non-natural nucleic acids can include 5'-methylene phosphonate DNA and RNA monomers and dimers. The non-natural nucleic acids can include 5'-phosphonate monomers with 2'-substitutions and other modified 5'-phosphonate monomers. The non-natural nucleic acids can include 5'-modified methylene phosphonate monomers. Non-naturally occurring nucleic acids can include 5' or 6'-phosphonate ribonucleoside analogs that contain a hydroxyl group at the 5' and / or 6' position. Non-naturally occurring nucleic acids can include 5'-phosphonate deoxyribonucleoside monomers and dimers that have a 5'-phosphate group.Non-natural nucleic acids can include nucleosides having a 6'-phosphonate group, where the 5' and / or 6' positions are unsubstituted or substituted with a thio-tert-butyl group (SC(CH3)3) (and its analogs), a methyleneamino group (CH2NH2) (and its analogs) or a cyano group (CN) (and its analogs).

[0066] In some aspects, the non-natural nucleic acids also include modifications of the sugar moiety. In some cases, the nucleic acid contains one or more nucleosides in which the sugar group has been modified. Such sugar-modified nucleosides may confer enhanced nuclease stability, improved binding affinity, or some other beneficial biological property. In certain embodiments, the nucleic acid includes a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and / or 2' substituents), bridging of two ring atoms to form bicyclic nucleic acids, S, N(R), or C(R1)(R2) (R=H, C1-C 12 and substitution of the ribosyl ring oxygen atom with an alkyl or protecting group, and combinations thereof.

[0067] In some examples, the oligonucleotides described herein include modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a sugar "analog" cyclopentyl group. The sugar can be in pyranosyl or furanosyl form. The sugar moiety can be a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, and the sugar can be attached to the respective heterocyclic base in either the [alpha] or [beta] anomeric configuration. Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy- or amino-RNA / DNA chimeras. For example, sugar modifications can include 2'-O-methyl-uridine or 2'-O-methyl-cytidine. Sugar modifications include 2'-O-alkyl substituted deoxyribonucleosides and 2'-O-ethylene glycol-like ribonucleosides.

[0068] Modifications to the sugar moiety include natural and non-natural modifications of the ribose and deoxyribose. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH, F, O-alkyl, S-alkyl, or N-alkyl, O-alkenyl, S-alkenyl, or N-alkenyl, O-alkynyl, S-alkynyl, or N-alkynyl, or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 Alkyl or C2-C 10 The 2' sugar modifications 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) nCH3)]2, where n and m are from 1 to about 10. Other chemical modifications at the 2' position include, but are not limited to, C1-C 10 These include lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups that improve the pharmacokinetic properties of an oligonucleotide, or groups that improve the pharmacodynamic properties of an oligonucleotide, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar in the 3' terminal nucleotide or 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Chemically modified sugars also include those that contain modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar. Examples of nucleic acids with modified sugar moieties include, but are not limited to, nucleic acids containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, and 2'-O(CH2)2OCH3 substituents. Substituents at the 2' position include allyl, amino, azido, thio, O-allyl, O-(C1-C2)-OCH3 ... 10 alkyl), OCF3, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), and O-CH2-C(=O)-N(R m )(R n ) can also be selected from each R m and R n are independently H or substituted or unsubstituted C1-C 10 It is an alkyl.

[0069] In certain embodiments, the nucleic acids described herein include one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acid includes a bridge between the 4' ribosyl ring atom and the 2' ribosyl ring atom. In certain embodiments, the nucleic acids provided herein include one or more bicyclic nucleic acids, where the bridge constitutes a 4' to 2' bicyclic nucleic acid. Examples of such 4' to 2' bicyclic nucleic acids include, but are not limited to, one of the following formulas: 4'-(CH2)-O-2' (LNA), 4'-(CH2)-S-2', 4'-(CH2)2-O-2' (ENA), 4'-CH(CH3)-O-2' and 4'-CH(CHOCH3)-O-2' and analogs thereof, 4'-C(CH3)(CH3)-O-2' and analogs thereof.

[0070] Modifications in the base of a nucleotide In some embodiments, the chemical modifications described herein include modifications of the base (e.g., nucleobase) of a nucleotide. Exemplary nucleobases can include adenine (A), thymine (T), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or substituted in the oligonucleotides described herein. The nucleobase of a nucleotide can be independently selected from a purine, a pyrimidine, a purine analog, or a pyrimidine analog. In some embodiments, the nucleobase can be a naturally occurring base or a synthetic base derivative.

[0071] In some embodiments, the chemical modification described herein comprises modification of uracil. In some embodiments, the oligonucleotide described herein comprises at least one chemically modified uracil. Exemplary chemically modified uracils include pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl ... -Methoxy-uridine, Uridine 5-oxyacetic acid, Uridine 5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine, 5-carboxyhydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine, 5-methoxycarbonylmethyl-2-thio-uridine, 5-aminomethyl-2-thio-uridine, 5-methylaminomethyl-uridine, 5-methylaminomethyl-2-thio-uridine, 5-methylaminomethyl-2-seleno-uridine, 5-carbamoylmethyl-uridine, 5-carboxymethylaminomethyl-uridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl- 4-thio-pseudouridine, 5-methyl-uridine, 1-methyl-pseudouridine, 5-methyl-2-thio-uridine, 1-methyl-4-thio-pseudouridine, 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 5,6-Dihydrouridine, 5-Methyl-dihydrouridine, 2-Thio-dihydrouridine, 2-Thio-dihydropseudouridine, 2-Methoxy-uridine, 2-Methoxy-4-thio-uridine, 4-Methoxy-pseudouridine, 4-Methoxy-2-thio-pseudouridine, N1-Methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine, 1-Methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thio-uridine, a-Thio-uridine, 2'-O-Methyl-uridine, 5,2'-O-Dimethyl-uridine, 2'-O-Methyl-pseudouridine Uridine, 2-thio-2'-O-methyl-uridine, 5-methoxycarbonylmethyl-2'-O-methyl-uridine, 5-carbamoylmethyl-2'-O-methyl-uridine, 5-carboxymethylaminomethyl-2'-O-methyl-uridine, 3,2'-O-dimethyl-uridine, 5-(isopentenylaminomethyl)-2'-O-methyl-uridine, 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, pyrazolo[3,4-d]pyrimidine, xanthine, and hypoxanthine.

[0072] In some embodiments, the chemical modifications described herein include modifications of cytosines. In some embodiments, the oligonucleotides described herein include at least one chemically modified cytosine. Exemplary chemically modified cytosines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5-methyl-cytidine, 5-halo-cytidine, 5-hydroxymethyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, lysine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysine, a-thio-cytidine, 2'-O-methyl-cytidine, 5,2'-O-dimethyl-cytidine, N4-acetyl-2'-O-methyl-cytidine, N4,2'-O-dimethyl-cytidine, 5-formyl-2'-O-methyl-cytidine, N4,N4,2'-O-trimethyl-cytidine, 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0073] In some embodiments, the chemical modifications described herein include modifications of adenines. In some embodiments, the oligonucleotides described herein include at least one chemically modified adenine.Exemplary chemically modified adenines include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, Purine, 1-methyl-adenosine, 2-methyl-adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6-isopentenyl-adenosine, 2-methylthio-N6-isopentenyl-adenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyl-adenosine, N6-threonylcarbamoyl-adenosine, N6-methionyl 2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6-dimethyl-adenosine, N6-hydroxynorvalylcarbamoyl-adenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2'-O-methyl-adenosine, N6-2'-O-dimethyl-adenosine, N6-methy Examples of such adenosine include 1-2'-deoxyadenosine, N6,N6,2'-O-trimethyl-adenosine, 1,2'-O-dimethyl-adenosine, 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0074] In some embodiments, the chemical modifications described herein include modifications of guanines. In some embodiments, the oligonucleotides described herein include at least one chemically modified guanine. Exemplary chemically modified guanosines include inosine, 1-methyl-inosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine, wyobutosine, peroxywyobutosine, hydroxywyobutosine, modified hydroxywyobutosine, 7-deaza-guanosine, queuosine, epoxyqueuosine, galactosyl-queuosine, mannosyl-queuosine, 7-cyano-7-deaza-guanosine, 7-aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2 ... Chil-guanosine, N2,N2-dimethyl-guanosine, N2,7-dimethyl-guanosine, N2,N2,7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methylthio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-O-methyl-guanosine, N2-methyl- 2'-O-methyl-guanosine, N2,N2-dimethyl-2'-O-methyl-guanosine, 1-methyl-2'-O-methyl-guanosine, N2,7-dimethyl-2'-O-methyl-guanosine, 2'-O-methyl-inosine, 1,2'-O-dimethyl-inosine, 6-O-phenyl-2'-deoxyinosine, 2'-O-ribosylguanosine, 1-thio-guanosine, 6-O-methylguanosine, O 6 -methyl-2'-deoxyguanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.

[0075] In some cases, chemical modification of oligonucleotide may include the introduction or substitution of nucleic acid analog or non-natural nucleic acid into oligonucleotide.In some embodiments, nucleic acid analog can be any one of the chemically modified nucleic acids described herein.All of them are expressly incorporated by reference in their entirety.The chemically modified nucleotides described herein may include variants of guanosine, uridine, adenosine, thymidine, and cytosine, including any naturally occurring or non-naturally occurring guanosine, uridine, adenosine, thymidine, or cytidine that is chemically modified, for example, by acetylation, methylation, hydroxylation. Exemplary chemically modified nucleotides include 1-methyl-adenosine, 1-methyl-guanosine, 1-methyl-inosine, 2,2-dimethyl-guanosine, 2,6-diaminopurine, 2'-amino-2'-deoxyadenosine, 2'-amino-2'-deoxycytidine, 2'-amino-2'-deoxyguanosine, 2'-amino-2'-deoxyuridine, 2-amino-6-chloropurine ribonuclease, and the like. 2'-aminopurine-riboside, 2'-ara adenosine, 2'-ara cytidine, 2'-ara uridine, 2'-azido-2'-deoxyadenosine, 2'-azido-2'-deoxycytidine, 2'-azido-2'-deoxyguanosine, 2'-azido-2'-deoxyuridine, 2-chloroadenosine, 2'-fluoro-2'-deoxyadenosine, 2'-fluoro-2'-deoxy Cytidine, 2'-fluoro-2'-deoxyguanosine, 2'-fluoro-2'-deoxyuridine, 2'-fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine, 2-methyl-thio-N6-isopentenyl-adenosine, 2'-O-methyl-2-aminoadenosine, 2'-O-methyl-2'-deoxyadenosine, 2'-O-methyl-2'-deoxycytidine, 2'- O-methyl-2'-deoxyguanosine, 2'-O-methyl-2'-deoxyuridine, 2'-O-methyl-5-methyluridine, 2'-O-methylinosine, 2'-O-methylpseudouridine, 2-thiocytidine, 2-thio-cytidine, 3-methyl-cytidine, 4-acetyl-cytidine, 4-thiouridine, 5-(carboxyhydroxymethyl)-uridine, 5,6-dihydrouridine,5-aminoallylcytidine, 5-aminoallyl-deoxyuridine, 5-bromouridine, 5-carboxymethylaminomethyl-2-thio-uracil, 5-carboxymethylaminomethyl-uracil, 5-chloro-ara-cytosine, 5-fluoro-uridine, 5-iodouridine, 5-methoxycarbonylmethyl-uridine, 5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-azacytidine, 6-azauridine, 6-chloro-7-deaza-guanosine, 6-chloropurine riboside, 6-mercapto-guanosine, 6-methyl-mercaptopurine riboside, 7-deaza-2'-deoxy-guanosine, 7-deazaadenosine, 7-methyl-guanosine, 8- Included are azaadenosine, 8-bromo-adenosine, 8-bromo-guanosine, 8-mercapto-guanosine, 8-oxoguanosine, benzimidazole-riboside, beta-D-mannosyl-queuosine, dihydro-uridine, inosine, N1-methyladenosine, N6-([6-aminohexyl]carbamoylmethyl)-adenosine, N6-isopentenyl-adenosine, N6-methyl-adenosine, N7-methyl-xanthosine, N-uracil-5-oxyacetic acid methyl ester, puromycin, queuosine, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, wybutoxosine, xanthosine, and xylo-adenosine. In some embodiments, the chemically modified nucleic acids described herein are 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluoro ... thymidine-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-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate The at least one chemically modified nucleotide selected from the group consisting of 5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, 6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, puromycin-5'-triphosphate, or xanthosine-5'-triphosphate. In some embodiments, the chemically modified nucleic acids described herein are selected from the group consisting of pyridin-4-one ribonucleosides, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudour ... Douridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine,The artificial nucleic acid described herein comprises at least one chemically modified nucleotide selected from dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In some embodiments, the artificial nucleic acid described herein comprises at least one chemically modified nucleotide selected from 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine. The compound comprises at least one chemically modified nucleotide selected from the group consisting of pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine. In some embodiments, the chemically modified nucleic acids described herein include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyl adenosine, N 6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine,and 2-methoxy-adenine. In other embodiments, the chemically modified nucleic acids described herein comprise at least one chemically modified nucleotide selected from inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. In certain embodiments, the chemically modified nucleic acids described herein are selected from the group consisting of 6-aza-cytidine, 2-thio-cytidine, alpha-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, alpha-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, 5-amino-cyt ... At least one chemically modified nucleotide selected from alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, alpha-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.

[0076] 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-propynyluracil and and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Certain non-naturally occurring nucleic acids include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6, and O-6 substituted purines, 5-aminopropyl adenine, 5-propynyl uracil, 5-propynyl cytosine, 5-methyl cytosine, those that enhance the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 2-aminopropyl adenine, 5-propynyl uracil, and 5-propynyl cytosine. Methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl (-C≡C-CH3) uracil, 5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, furansulfuric acid, fluorescein, fluorine-containing ... Phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamp, phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido [3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), those in which the purine or pyrimidine base is replaced by other heterocycles, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine , hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-iodouracil, 2-amino-adenine, 6-thio-guanine, 2-thio-thymine, 4-thio-thymine, 5-propynyl-uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, or 2-amino-2'-deoxyadenosine.

[0077] In some cases, the at least one chemical modification comprises a chemical modification of the 5' or 3' end, such as a 5' cap or 3' tail of the oligonucleotide. In some embodiments, the oligonucleotide comprises a chemical modification, including a 3' nucleotide, which may be stabilized against degradation, for example, by incorporating one or more of the modified nucleotides described herein. In this embodiment, uridine may be substituted with modified uridine, for example, 5-(2-amino)propyluridine and 5-bromouridine, or any of the modified uridines described herein, and adenosine and guanosine may be substituted with modified adenosine and guanosine, for example, modified at the 8 position (e.g., 8-bromoguanosine), or any of the modified adenosine or guanosine described herein. In some embodiments, deaza nucleotides (e.g., 7-deaza-adenosine) may be incorporated into the gRNA. In some embodiments, O-alkylated nucleotides and N-alkylated nucleotides (e.g., N6-methyladenosine) may be incorporated into the gRNA. In some embodiments, sugar-modified ribonucleotides may be incorporated, e.g., the 2'OH group is replaced with a group selected from H, -OR, -R (where R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), halo, -SH, -SR (where R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), amino (where amino can be, e.g., NH2 (alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid)), or cyano (-CN). In some embodiments, the phosphate backbone may be modified, e.g., with a phosphothioate group, as described herein.In some embodiments, the nucleotides in the overhang region of the gRNA can each independently be modified or unmodified nucleotides, including, but not limited to, 2'-sugar modifications, such as 2-F 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), or any combination thereof.

[0078] In some embodiments, the oligonucleotides containing at least one chemical modification, when bound to a target RNA, more specifically recruit endogenous nucleases that reduce expression of the target RNA compared to oligonucleotides that share the same nucleic acid sequence as the oligonucleotides containing at least one chemical modification but do not have the chemical modification. In some embodiments, the oligonucleotides containing at least one chemical modification more specifically recruit endogenous nucleases that reduce expression of the target RNA by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or more, compared to oligonucleotides that share the same nucleic acid sequence as the oligonucleotides containing at least one chemical modification but do not have the chemical modification.

[0079] In some embodiments, the oligonucleotide comprising at least one chemical modification has enhanced resistance to hydrolytic degradation compared to the oligonucleotide that shares the same nucleic acid sequence with the oligonucleotide comprising at least one chemical modification but does not have a chemical modification.In some embodiments, the oligonucleotide comprising at least one chemical modification has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, 1000-fold or more resistance to hydrolytic degradation compared to the oligonucleotide that shares the same nucleic acid sequence with the oligonucleotide comprising at least one chemical modification but does not have a chemical modification.

[0080] In some embodiments, the oligonucleotide comprising at least one chemical modification has enhanced resistance to degradation by nuclease digestion compared to the oligonucleotide that shares the same nucleic acid sequence with the oligonucleotide comprising at least one chemical modification but does not have a chemical modification.In some embodiments, the oligonucleotide comprising at least one chemical modification is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or more resistant to degradation by nuclease digestion compared to the oligonucleotide that shares the same nucleic acid sequence with the oligonucleotide comprising at least one chemical modification but does not have a chemical modification.

[0081] In some embodiments, the oligonucleotides containing at least one chemical modification induce less immunogenicity compared to the oligonucleotides that share the same nucleic acid sequence as the oligonucleotides containing at least one chemical modification but do not have the chemical modification. In some embodiments, the oligonucleotides containing at least one chemical modification are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or more less likely to induce immunogenicity compared to the oligonucleotides that share the same nucleic acid sequence as the oligonucleotides containing at least one chemical modification but do not have the chemical modification.

[0082] In some embodiments, the oligonucleotides containing at least one chemical modification induce a weaker innate immune response compared to oligonucleotides that share the same nucleic acid sequence as the oligonucleotides containing at least one chemical modification but do not have the chemical modification. In some embodiments, the oligonucleotides containing at least one chemical modification are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or more less likely to induce an innate immune response compared to an oligonucleotide that shares the same nucleic acid sequence as the oligonucleotides containing at least one chemical modification but does not have the chemical modification.

[0083] In some embodiments, the oligonucleotide comprising at least one chemical modification is less likely to induce off-target regulation of the target RNA when contacted with the target RNA, compared to the off-target regulation of the target RNA induced by the oligonucleotide that shares the same nucleic acid sequence as the oligonucleotide comprising at least one chemical modification but does not have the chemical modification.In some embodiments, the oligonucleotide comprising at least one chemical modification is less likely to induce off-target regulation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, 1000-fold or more, compared to the off-target regulation of the oligonucleotide that shares the same nucleic acid sequence as the oligonucleotide comprising at least one chemical modification but does not have the chemical modification.

[0084] Delivery method In some embodiments, the present specification describes a method for delivering the oligonucleotide described herein to a cell. In some embodiments, the method includes directly or indirectly delivering the oligonucleotide to a cell. In some embodiments, the method includes contacting the composition or oligonucleotide described herein with a cell. In some embodiments, the method includes expressing the composition or oligonucleotide described herein in a cell. In some embodiments, the oligonucleotide or the vector encoding the oligonucleotide can be delivered into the cell via any of the transfection methods described herein. In some embodiments, the oligonucleotide can be delivered into the cell via the use of an expression vector. In terms of the expression vector, the vector can be easily introduced into the cell described herein by any method in the art. For example, the expression vector can be transferred into the cell by physical, chemical, or biological means.

[0085] Physical methods for introducing oligonucleotides or vectors encoding oligonucleotides into cells can include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene gun, electroporation, and the like. Any method for generating cells containing vectors and / or exogenous nucleic acids is suitable for the methods herein. One method for introducing oligonucleotides or vectors encoding oligonucleotides into host cells is calcium phosphate transfection.

[0086] Chemical means for introducing oligonucleotides or vectors encoding 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 (SNAs), liposomes, or lipid nanoparticles). An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other state-of-the-art methods for delivering nucleic acids to targets are available, such as delivery of oligonucleotides or vectors encoding oligonucleotides using targeted nanoparticles or other suitable submicron-sized delivery systems.

[0087] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for introducing oligonucleotides or vectors encoding oligonucleotides into cells (in vitro, ex vivo, or in vivo). In another embodiment, the oligonucleotides or vectors encoding oligonucleotides can be associated with lipids. In some embodiments, the lipid-associated oligonucleotides or vectors encoding oligonucleotides are embedded in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, encapsulated within the liposome, complexed with the liposome, dispersed in a lipid-containing solution, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. The lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, in some embodiments, they exist in a bilayer structure, exist as micelles, or exist with a "collapsed" structure. Alternatively, they may simply be dispersed in solution and form aggregates that are not uniform in size or shape.In some embodiments, lipid is a fatty substance that is a lipid of natural origin or synthetic lipid.For example, lipid includes the lipid droplets that naturally occur in cytoplasm, and compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0088] Lipids suitable for use are obtained commercially. Stock solutions of lipids in chloroform or chloroform / methanol are often stored at about -20°C. Chloroform is used exclusively as a solvent because it evaporates more easily than methanol. "Liposome" is a general term that encompasses a variety of mono- and multi-lamellar lipid vehicles formed by the formation of closed lipid bilayers or aggregates. Liposomes are often characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement to form a closed structure, encapsulating water and dissolved solutes between the lipid bilayers. However, compositions with structures in solution that differ from the normal vesicular structure are also encompassed. For example, in some embodiments, the lipids assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0089] In some cases, non-viral delivery methods include lipofection, nucleofection, microinjection, particle bombardment, virosomes, liposomes, immunoliposomes, exosomes, polycation or lipid: cargo conjugates (or aggregates), naked polypeptides (e.g., recombinant polypeptides), naked DNA, artificial virions, and drug-enhanced uptake of polypeptides or DNA. In some embodiments, the delivery method includes conjugating or embedding the compositions 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, polyoxaesters, polyamide esters, polyoxaesters containing amine groups, and poly(anhydrides). Such synthetic polymers can be homopolymers or copolymers (e.g., random, block, segmented, graft) of multiple different monomers (e.g., two or more of lactic acid, lactide, glycolic acid, glycolide, epsilon-caprolactone, trimethylene carbonate, p-dioxanone, etc.). In one example, the scaffold can be composed of a polymer including glycolic acid and lactic acid, such as a 90 / 10 or 5 / 95 ratio of glycolic acid to lactic acid. Non-limiting examples of biocompatible, biodegradable polymers of natural origin can include glycoproteins, proteoglycans, polysaccharides, glycosaminoglycans (GAGs) and fragment(s) derived from these components, elastin, laminin, decorin, fibrinogen / fibrin, fibronectin, osteopontin, tenascin, hyaluronic acid, collagen, chondroitin sulfate, heparin, heparan sulfate, ORC, carboxymethylcellulose, and chitin.

[0090] In some cases, the oligonucleotide or vector encoding the oligonucleotide described herein can be packaged and delivered to cells via extracellular vesicles. The extracellular vesicles can be any membrane-bound particle. In some embodiments, the extracellular vesicles can be any membrane-bound particle secreted by at least one cell. In some examples, the extracellular vesicles can be any membrane-bound particle synthesized in vitro. In some examples, the extracellular vesicles can be any membrane-bound particle synthesized without cells. In some cases, the extracellular vesicles can be exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apoptosomes, oncosomes, exosomes, enveloped viruses, exomeres, or other extra-large extracellular vesicles.

[0091] In some cases, the oligonucleotide or the vector encoding the oligonucleotide described herein can be administered to a subject in need thereof by first introducing the oligonucleotide or the vector encoding the oligonucleotide into allogeneic or autologous cells, using transgenic cells.In some cases, the cell can be isolated.In some embodiments, the cell can be isolated from the subject.

[0092] In some embodiments, the oligonucleotides described herein are conjugated. In some embodiments, the oligonucleotides are conjugated with peptides, antibodies, lipids, carbohydrates, or polymers. In some embodiments, the oligonucleotides are conjugated with peptides, antibodies, lipids, carbohydrates, or polymers at the 5'-end of the oligonucleotide. In some embodiments, the oligonucleotides are conjugated with peptides, antibodies, lipids, carbohydrates, or polymers at the 3'-end of the oligonucleotide. In some embodiments, the oligonucleotides are conjugated with peptides, antibodies, lipids, carbohydrates, or polymers at any nucleic acid residue of the oligonucleotide. In some embodiments, the peptides, antibodies, lipids, carbohydrates, or polymers conjugated to the oligonucleotides provide a therapeutic effect. For example, the peptides, antibodies, lipids, carbohydrates, or polymers conjugated to the oligonucleotides can be cytotoxic agents or agents for treating cancer. In some embodiments, the peptides, antibodies, lipids, carbohydrates, or polymers conjugated to the oligonucleotides increase the binding efficiency of the oligonucleotides to endogenous nucleic acids. In some embodiments, the peptide, antibody, lipid, carbohydrate, or polymer conjugated to the oligonucleotide confers target specificity to a particular type of cell (such as, for example, cancer cells) to the oligonucleotide. In some embodiments, the peptide, antibody, lipid, carbohydrate, or polymer conjugated to the oligonucleotide confers stability to the oligonucleotide in vitro, ex vivo, or in vivo. For example, the oligonucleotide can be conjugated with polyethylene glycol (PEG) or endosomolytic drugs to reduce immunogenicity or degradation. In some embodiments, the peptide, antibody, lipid, carbohydrate, or polymer is conjugated to the oligonucleotide to facilitate the oligonucleotide's entry into cells.In some embodiments, peptide, antibody, lipid, carbohydrate or polymer is conjugated to oligonucleotide to facilitate the release of oligonucleotide in cells.In some embodiments, peptide, antibody, lipid, carbohydrate or polymer conjugated to oligonucleotide comprises at least one targeting moiety for targeting cells.Non-limiting examples of targeting moiety include signal transduction peptide, chemokine, chemokine receptor, adhesion molecule, antigen or antibody.

[0093] The linker for conjugating an oligonucleotide to a peptide, an antibody, a lipid, or a polymer can be any linker that connects biomolecules. In some embodiments, the linker described herein is a cleavable linker or a non-cleavable linker. In some examples, the linker is a cleavable linker. In other examples, the linker is a non-cleavable linker. In some cases, the linker is a non-polymeric linker. A non-polymeric linker refers to a linker that does not contain a repeating unit of a monomer generated by a polymerization process. In some embodiments, the linker comprises a peptide moiety. In some examples, the peptide moiety comprises at least 2, 3, 4, 5, or 6 or more amino acid residues. In some embodiments, the linker comprises a benzoic acid group or a derivative thereof. In some embodiments, the linker can comprise a nucleic acid linker, such as a DNA linker. In such cases, the peptide, antibody, lipid, or polymer can be conjugated to one end of the nucleic acid linker or inserted between the nucleic acid base pairs of the nucleic acid linker. In some embodiments, the linker can be a peptide linker. Peptide linkers can be flexible (e.g., polyglycine linkers) or rigid (e.g., EAAAK repeat linkers). In some embodiments, the peptide linker can be cleaved (e.g., disulfide bonds). In some embodiments, the linker comprises a polymer such as PEG, polylactic acid (PLA), or polyacrylic acid (PAA).

[0094] Treatment In some embodiments, disclosed herein is a method for reducing the expression of KRAS or mutant KRAS protein or KRAS or mutant KRAS mRNA in cancer cells by treating cancer cells with a composition comprising the antisense oligonucleotide, composition or pharmaceutical composition described herein or contacting the composition with cancer cells, thereby regulating the KRAS-mediated signal transduction pathway in cancer cells.In some embodiments, mutant KRAS protein comprises G12C mutation, G12V mutation, G12A mutation or G12D mutation.

[0095] Also disclosed herein, in some embodiments, is a method of treating a subject in need thereof by administering to the subject a therapeutically effective amount of an oligonucleotide, composition, or pharmaceutical composition described herein. In some embodiments, the method is a method of treating a subject by regulating gene expression or signaling pathway expression in the subject. In some embodiments, the method comprises reducing gene expression by contacting an endogenous nucleic acid (e.g., endogenous mRNA) with an oligonucleotide described herein. In some embodiments, the method comprises reducing KRAS, mutant KRAS, or a combination of KRAS and mutant KRAS in a subject or in a cancer cell by contacting KRAS mRNA or mutant KRAS mRNA with an oligonucleotide described herein, where binding of the oligonucleotide to the mRNA recruits an endogenous nuclease that degrades the mRNA. In some embodiments, the method comprises reducing expression of a signaling pathway, such as the KRAS-mediated signaling pathway. In some embodiments, the method comprises decreasing expression of a gene in, or activity of, the KRAS-RAF-MEK-ERK signaling pathway, the PI3K signaling pathway, the MAPK signaling pathway, or the Ral-GEF signaling pathway.

[0096] In some embodiments, the oligonucleotide, composition, or pharmaceutical composition can be administered alone to a subject (e.g., single treatment). In some embodiments, the oligonucleotide, composition, or pharmaceutical composition is administered in conjunction with an additional agent. In some cases, the additional agent used herein is administered alone. The oligonucleotide, composition, or pharmaceutical composition and the additional agent can be administered together or sequentially. Non-limiting examples of additional agents include N-(2-(4-(4-bis(2-chloroethyl)aminophenyl)butyryl)aminoethyl)-5-(4-amidinophenyl)-2-furancarboxamide hydrochloride, allyl isothiocyanate, benzyl isothiocyanate, phenethyl isothiocyanate, belinostat, berberine, casticin, chrysin, bufalin, fisetin, fucoidan, gallic acid, gemcitabine, keishibukuryogan, JOTO1007, quercetin, lasfonin, 2,3,7,8-tetrachlorodibenzodioxin, triptolide, 4-hydroxybutenolide, or combinations thereof. The combination therapy may be administered on the same day or may be administered one or more days, one or more weeks, one or more months, or one or more years apart.

[0097] In some embodiments, the oligonucleotide, composition, or pharmaceutical composition is a first-line treatment for a disease or condition. In some embodiments, the oligonucleotide, composition, or pharmaceutical composition is a second-line treatment, a third-line treatment, or a fourth-line treatment. In some embodiments, the oligonucleotide, composition, or pharmaceutical composition comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or more oligonucleotides. Generally, the methods disclosed herein include administering the oligonucleotide, composition, or pharmaceutical composition by oral administration. However, in some examples, the methods include administering the oligonucleotide, composition, or pharmaceutical composition by intraperitoneal injection. In some examples, the methods include administering the pharmaceutical composition in the form of a rectal suppository. In some examples, the methods include administering the oligonucleotide, composition, or pharmaceutical composition by intravenous ("iv") administration. It is contemplated that in some cases, the oligonucleotides, compositions, or pharmaceutical compositions disclosed herein may also be administered by other routes, 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, a local delivery route close to the site of injury or inflammation is preferred over a systemic route. The route, dosage, time of administration, and duration of administration of the therapeutic agent can be adjusted. In some embodiments, the therapeutic agent is administered before or after the onset of either or both of acute and chronic symptoms of a disease or condition.

[0098] The appropriate dose and dosage to be administered to a subject will be determined by factors including, but not limited to, the particular oligonucleotide, composition, or pharmaceutical composition, the disease state and its severity, the identity of the subject requiring treatment (e.g., weight, sex, age), and can be determined depending on the particular circumstances surrounding the situation (including, for example, the specific agent being administered, the route of administration, the condition being treated, and the subject being treated).

[0099] In some embodiments, the oligonucleotide, composition or pharmaceutical composition described herein is administered once every 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years or 5 years, or once every 10 years.The effective dosage range can be adjusted based on the subject's response to treatment. Some routes of administration may require higher concentrations of the effective amount of a therapeutic agent than others.

[0100] In some embodiments, administration of the oligonucleotide, composition, or pharmaceutical composition described herein increases the survival rate of the subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, or more. In some embodiments, the oligonucleotide, composition, or pharmaceutical composition described herein is administered at a dose that increases the survival rate of the subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, or more. In some embodiments, the oligonucleotide, composition, or pharmaceutical composition described herein is administered in a schedule that increases the survival rate of the subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, or more. In some embodiments, the oligonucleotides, compositions, or pharmaceutical compositions described herein are administered at a dose and schedule that increases the survival rate of a subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50% or more.

[0101] In some embodiments, administration of the oligonucleotide, composition, or pharmaceutical composition described herein inhibits tumor growth by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, or more. In some embodiments, the oligonucleotide, composition, or pharmaceutical composition described herein is administered at a dose that inhibits tumor growth by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, or more. In some embodiments, the oligonucleotide, composition, or pharmaceutical composition described herein is administered at a schedule that inhibits tumor growth by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, or more. In some embodiments, the oligonucleotides, compositions, or pharmaceutical compositions described herein are administered at a dose and schedule that inhibits tumor growth by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50% or more.

[0102] In some embodiments, the oligonucleotides, compositions, or pharmaceutical compositions described herein are administered to the subject at a dose sufficient to inhibit tumor growth. In some embodiments, the oligonucleotides, compositions, or pharmaceutical compositions described herein are administered to the subject on a schedule sufficient to inhibit tumor growth. In some embodiments, the oligonucleotides, compositions, or pharmaceutical compositions described herein are administered to the subject at a dose and schedule sufficient to inhibit tumor growth.

[0103] In certain embodiments, if the subject's condition does not improve, administration of the pharmaceutical composition is chronic, i.e., for an extended period of time, including throughout the subject's life, to alleviate symptoms of the subject's disease or condition, or in other cases, to control or limit symptoms of the subject's disease or condition, depending on the physician's judgment. In certain embodiments in which the subject's condition improves, the dose of the pharmaceutical composition administered may be temporarily reduced or temporarily discontinued for a period of time (i.e., a "drug holiday"). In certain embodiments, the length of the drug holiday period is between 2 days and 1 year, and by way of example only, is 2, 3, 4, 5, 6, 7, 10, 12, 15, 20, 28, or more than 28 days. Dose reductions during a drug holiday can be, by way of example only, 10% to 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%. In certain embodiments, the dose of the pharmaceutical composition administered can be temporarily reduced or temporarily suspended for a period of time (i.e., a "drug diversion"). In certain embodiments, the length of the diversion period of the pharmaceutical composition can be 2 days to 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, or more than 28 days. Dose reductions during diversions of the pharmaceutical composition can be, by way of example only, 10% to 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%. After an appropriate period of time has elapsed, a return to the normal dosing schedule is optionally performed.

[0104] In some embodiments, once the condition of the subject is improved, a maintenance dose is administered as necessary.Then, in certain embodiments, the dosage or frequency of administration, or both, can be reduced according to symptoms to a level at which the improvement of disease, disorder or condition is maintained.However, in certain embodiments, if symptoms recur at all, the subject needs to be treated intermittently on a long-term basis.

[0105] Toxicity and therapeutic efficacy of such treatment regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including but not limited to, LD50 and ED50 determinations. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. In certain embodiments, data obtained from cell culture assays and animal studies are used to establish a therapeutically effective daily dosage range and / or a therapeutically effective unit dosage for use in mammals, including humans. In some aspects, the daily dosage of the compositions described herein is within a range of circulating concentrations that includes the ED50 with minimal toxicity. In certain embodiments, the daily dosage range and / or unit dosage vary within this range depending on the dosage form employed and the route of administration utilized.

[0106] In some embodiments, the disease or condition described herein is cancer. In some embodiments, the cancer is associated with KRAS. In some embodiments, the cancer is associated with mutant KRAS. In some embodiments, the cancer is associated with KRAS. In some embodiments, the cancer is associated with abnormalities in KRAS-mediated signaling pathway. In some embodiments, the cancer is lung cancer, pancreatic cancer, or colon cancer. Other non-limiting examples of cancer include acute lymphocytic leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenal carcinoma, adrenal cortical carcinoma, adult leukemia, AIDS-related lymphoma, amyloidosis, anal cancer, astrocytoma, ataxia telangiectasia, atypical nevus syndrome, atypical teratoma / atypical rhabdomyosarcoma, basal cell tumor, cholangiocarcinoma, Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor (gastrointestinal), cancer of unknown primary, cardiac tumor (heart tumor), cervical cancer, bile duct cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia, chronic myeloid leukemia, chronic myeloid leukemia, chronic myelogenous ...leukemia), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, nasal neuroblastoma, Ewing's sarcoma, extracranial germ cell tumors, extragonadal germ cell tumors, eye tumors, fallopian tube cancer, fibrous histiocytoma of bone, malignant osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, HER2-positive breast cancer, histiocytosis, Langerhans cells, Hodgkin's lymphoma tumor, hypopharyngeal carcinoma, intraocular melanoma, islet cell tumor, juvenile polyposis syndrome, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lobular carcinoma, lung cancer (non-small cell and small cell), lymphoma, malignant fibrous histiocytoma and osteosarcoma of bone, malignant glioma, melanoma, intraocular melanoma, meningioma, Merkel cell carcinoma, mesothelioma, malignant metastatic carcinoma, primary occult metastatic squamous cell carcinoma of the neck, midline carcinoma, multiple endocrine neoplasia syndrome, multiple myeloma, plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome (MDS), bone Myeloproliferative neoplasms, chronic nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumors, non-Hodgkin's lymphoma, oral cavity cancer, lip and oral cavity cancer and oropharyngeal cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian germ cell tumors, pancreatic cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, peritoneal cancer, Peutz-Jeghers syndrome, pharyngeal cancer, pheochromocytoma, pituitary tumors, plasma cell neoplasms / multiple myeloma, pleuropulmonary blastoma, polycythemia vera, breast cancer during pregnancy, primary central nervous system (CNS) lymphoma, primary abdominal The cancer may include mesenteric cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer, small intestine cancer, soft tissue sarcoma, solid tumors, squamous cell carcinoma of the skin, primary occult squamous cell carcinoma of the cervix, metastatic gastric cancer, T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, rare cancers of childhood, ureter and renal pelvis, transitional cell carcinoma, urethral cancer, uterine cancer (endometrial cancer), uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer, Wilms' tumor, or a combination thereof.

[0107] Pharmaceutical Compositions In some embodiments, the present invention provides a pharmaceutical composition comprising the oligonucleotide or composition described herein. As used herein, a pharmaceutical composition refers to a mixture of a pharmaceutical composition and other chemical components (i.e., pharma- ceutically acceptable inactive components), such as carriers, excipients, binders, fillers, suspending agents, flavoring agents, sweeteners, disintegrants, dispersants, surfactants, lubricants, colorants, diluents, solubilizers, moistening agents, plasticizers, stabilizers, penetration enhancers, wetting agents, antifoaming agents, antioxidants, preservatives, or one or more combinations thereof. Optionally, the composition comprises two or more pharmaceutical compositions described herein. In practicing the treatment or use methods provided herein, a therapeutically effective amount of the pharmaceutical composition described herein is administered as a pharmaceutical composition to a mammal having a disease, disorder, or condition (e.g., an inflammatory disease, a fibrostenotic disease, and / or a fibrotic disease) to be treated. In some embodiments, the mammal is a human. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the pharmaceutical composition used, and other factors.The pharmaceutical composition can be used alone or can be used together with one or more pharmaceutical compositions as a component of a mixture.The pharmaceutical composition described herein includes oligonucleotide, composition, cell contacted with oligonucleotide or cell contacted with composition containing oligonucleotide, or combinations thereof.

[0108] The pharmaceutical formulations described herein are administered to a subject by a suitable route of administration, including but not limited to intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal routes of 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 dissolving formulations, tablets, capsules, pills, delayed release formulations, sustained release formulations, pulsatile release formulations, multiparticulate formulations, and mixed formulations of immediate release formulations and controlled release formulations.

[0109] The pharmaceutical compositions comprising the drug composition are manufactured in a conventional manner, such as, by way of example only, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compressing processes.

[0110] The pharmaceutical composition may comprise at least one pharmaceutical composition as an active ingredient in free acid or free base form, or in pharmaceutically acceptable salt form.In addition, the methods and pharmaceutical compositions described herein include the use of N-oxides (where appropriate), crystalline forms, amorphous phases, and active metabolites of these compounds with the same type of activity.In some embodiments, the pharmaceutical composition exists in a nonsolvated form or in a solvated form with a pharmaceutically acceptable solvent, such as water, ethanol, and the like.Solvated forms of the pharmaceutical composition are also considered to be disclosed herein.

[0111] In some embodiments, pharmaceutical compositions exist as tautomers.All tautomers are included within the scope of the drug presented herein.It should be understood that pharmaceutical compositions or their salts can exhibit the phenomenon of tautomerism, which means that two chemical compounds can be easily interconverted by exchanging hydrogen atoms between two atoms, one of which forms a covalent bond.Tautomers can be considered as different isomeric forms of the same compound, since they exist in a mobile equilibrium with each other.

[0112] In some embodiments, pharmaceutical compositions exist as enantiomers, diastereomers, or other stereoisomeric forms. The agents disclosed herein include all enantiomeric, diastereomeric, and epimeric forms, as well as mixtures thereof.

[0113] In some aspects, the pharmaceutical compositions described herein may be prepared as prodrugs. A "prodrug" refers to an agent that is converted to the parent drug in vivo. In some situations, prodrugs are often useful because they may be easier to administer than the parent drug. For example, a prodrug may be bioavailable by oral administration, whereas the parent drug is not. A prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. A non-limiting example of a prodrug is envisioned as the pharmaceutical composition described herein, which is administered as an ester ("prodrug") to facilitate delivery across cell membranes, where water solubility is unfavorable for transport, but then metabolically hydrolyzed to a carboxylic acid by active enzymes once inside the cell, where water solubility is favorable. A further example of a prodrug may be a short peptide (polyamino acid) bound to an acid group, which is metabolized to reveal the active moiety. In certain embodiments, the prodrug is chemically converted to the biologically, pharma- ceutical or therapeutically active form of the pharmaceutical composition when administered in vivo. In certain embodiments, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharma- ceutical or therapeutically active form of the pharmaceutical composition.

[0114] Prodrug forms of the pharmaceutical compositions, where the prodrug is metabolized in vivo to produce the agents described herein, are included within the scope of the claims. Prodrug forms of the pharmaceutical compositions, where the prodrug is metabolized in vivo to produce the agents described herein, are included within the scope of the claims. In some cases, some of the pharmaceutical compositions described herein may be prodrugs of another derivative or active compound. In some embodiments described herein, a hydrazone is metabolized in vivo to produce a pharmaceutical composition.

[0115] kit In some embodiments, kits are described herein for using the oligonucleotides, compositions, or pharmaceutical compositions described herein. In some embodiments, the kits disclosed herein can be used to treat a disease or condition in a subject. In some embodiments, the kits include a collection of materials or components separate from the oligonucleotides, compositions, or pharmaceutical compositions. In some embodiments, the kits include components for assaying and selecting suitable oligonucleotides for treating a disease or condition. In some embodiments, the kits include components for performing assays such as enzyme-linked immunosorbent assay (ELISA), Simoa (single-molecular array), PCR, or qPCR. The exact nature of the components that make up the kit depends on the intended purpose of the kit. For example, some embodiments are configured for the purpose of treating a disease or condition (e.g., cancer) disclosed herein in a subject. In some embodiments, the kits are specifically configured for the purpose of treating a mammalian subject. In some embodiments, the kits are specifically configured for the purpose of treating a human subject.

[0116] The kit may include instructions for use. In some embodiments, the kit includes instructions for administering the composition to a subject in need of administration. In some embodiments, the kit includes instructions for further manipulating the oligonucleotide. In some embodiments, the kit includes instructions for thawing the oligonucleotide, which may have been frozen or lyophilized during storage or transportation, or for otherwise restoring its biological activity. In some embodiments, the kit includes instructions for measuring the effectiveness of the kit for its intended purpose (e.g., therapeutic effectiveness when used to treat a subject).

[0117] Optionally, the kit also contains other useful components, such as diluents, buffers, pharma- ceutically acceptable carriers, syringes, catheters, applicators, pipettes or measuring tools, dressings or other useful tools. The materials or components assembled in the kit can be stored and provided to practitioners in any convenient and suitable manner that preserves their opera- bility and usefulness. For example, the oligonucleotide, composition or pharmaceutical composition can be in dissolved, anhydrous or lyophilized form. The components are usually packaged in suitable packaging material(s).

[0118] The use of absolute or chronological terms, such as "shall," "shall not," "shall not," "shall not," "must," "must," "first," "firstly," "next," "sequently," "before," "after," "lastly," and "finally" are not meant to be limiting of the scope of the embodiments disclosed herein but are exemplary.

[0119] 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. Furthermore, to the extent the terms "including," "include," "having," "has," "with," or variations thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a similar manner as the term "comprising."

[0120] As used herein, the terms "at least one," "one or more," and "and / or" are open-ended expressions that are conjunctive and disjunctive in operation. For example, each of the phrases "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" means A only, B only, C only, both A and B, both A and C, both B and C, or both A, B, and C.

[0121] As used herein, "or" can refer to "and," "or," or "and / or," and can be used both exclusive and inclusive. For example, the term "A or B" can refer to "A or B," "A but not B," "A but not B," and "A and B." In some cases, the context may dictate a particular meaning.

[0122] Any systems, methods, software, and platforms described herein are modular, and thus terms such as "first" and "second" do not necessarily imply a priority, order of importance, or order of action.

[0123] When referring to a number or range of numbers, the term "about" means that the number or range of numbers referred to is approximate within experimental variation (or within statistical experimental error) and that the number or range of numbers may vary, for example, by 1% to 15% of the stated number or range of numbers. As an example, the term "about" refers to ±10% of the stated number or value.

[0124] As used herein, the terms "increased", "increase" or "increase" are generally used to mean an increase of a statistically significant amount. In some embodiments, the term "increased" or "increase" means 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 an increase of up to 100% (including 100%), or any increase between 10 and 100% compared to a reference level, standard, or control. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more compared to a reference level.

[0125] As used herein, the terms "reduced", "reducing", or "reduction" are generally used to mean a statistically significant amount of reduction. In some embodiments, "reduced" or "reduction" means a decrease of at least 10% compared to a reference level, e.g., a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or a decrease of up to 100% (including 100%) (e.g., absent or undetectable levels compared to a reference level), or any decrease between 10 and 100%. In the context of a marker or symptom, these terms are intended to mean a statistically significant decrease in such levels. For example, the decrease can be at least 10%, at least 20%, at least 30%, at least 40% or more, preferably down to a level that is accepted as within the normal range for an individual without a given disease.

[0126] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided herein. The present invention has been described by the foregoing specification, but the description and examples of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. Furthermore, it is to be understood that all aspects of the present invention are not limited to the specific descriptions, configurations, or relative proportions shown herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives may be employed in the embodiments of the present invention described herein in the practice of the present invention. Thus, the present invention is also intended to cover any such alternatives, modifications, variations, or equivalents. It is intended that the claims define the scope of the present invention, and that methods and structures within the scope of the claims and their equivalents are thereby covered. JPEG2024532477000002.jpg226165JPEG2024532477000003.jpg239165JPEG202 4532477000004.jpg239165JPEG2024532477000005.jpg239165JPEG2024532477 000006.jpg239165JPEG2024532477000007.jpg239165JPEG2024532477000008. jpg239165JPEG2024532477000009.jpg239165JPEG2024532477000010.jpg23916 5JPEG2024532477000011.jpg239165JPEG2024532477000012.jpg239165JPEG20 24532477000013.jpg239165JPEG2024532477000014.jpg179165JPEG202453247 7000015.jpg226165JPEG2024532477000016.jpg166165JPEG2024532477000017 .jpg217165JPEG2024532477000018.jpg45165JPEG2024532477000019.jpg44165 EXAMPLES

[0127] The following illustrative examples are representative of embodiments of the stimulation actions, systems, and methods described herein, and are not meant to be limiting in any way.

[0128] Example 1. Knockdown of KRAS mRNA Cell culture conditions and in vitro transfection NCI-H358 cell lines carrying the KRAS G12C mutation were plated at a density of 20,000 cells per well in 96-well plates and treated with both 5 nM and 20 nM antisense oligonucleotides by transfection with Lipofectamine (Life Technology, USA). Transfection was performed according to the vendor's recommendations, with 0.3 μL Lipofectamine per well and incubated for 3 h. After 2 days, cells were harvested and subjected to Quantigene assays for relative quantification of mRNA (Life Technology, USA) according to the vendor's specifications. The catalog numbers of the KRAS probe and PPIB (reference gene for normalizing expression levels) probe are SA-50338 and SA-50155, respectively. Percentage reduction of mRNA relative to non-targeting control oligonucleotides was calculated and summarized in Table 1. JPEG2024532477000020.jpg133165

[0129] Example 2. Inhibition of pERK NCI-H358 cell lines carrying the KRAS G12C mutation were seeded at a density of 20,000 cells per well in 96-well plates and treated with both 5 nM and 20 nM antisense oligonucleotides by transfection with Lipofectamine (Life Technology, USA). Transfection was performed according to the vendor's recommendations and incubated with 0.3 μL Lipofectamine per well for 3 hours. After 4 days, cells were harvested and subjected to pERK AlphaLISA assay (Cat.ALSU-PERK-A10K, Perkin Elmer, USA). pERK inhibition for each treatment was calculated by normalizing with non-targeting control oligos and is summarized in the table below. JPEG2024532477000021.jpg236165JPEG2024532477000022.jpg105165

[0130] Example 3. Antisense oligonucleotide-mediated inhibition of proliferation of various cancer cells Various tumor cell lines carrying KRAS mutations (NCI-H358 and LCLC97TM1) and wild-type KRAS (NCI-H1975 and A375) were plated at a density of 800 cells per well in 384-well plates and treated with both 5uM and 1uM antisense oligonucleotides by co-incubation. After 7 days, cell viability was measured by CellTiter-Glo® 2.0 assay (Promega, USA) according to the vendor's protocol, and growth inhibition was calculated relative to non-targeting control oligos. The results are summarized in the table below. Pan KRAS ASO and KRAS mutation-matched ASO showed growth inhibition in NCI-H358 and LCLC97TM1 cells. KRAS ASO had limited effect on NCI-H1975 (EGFR mutant) and no or little effect on A375 (BRAF mutant). JPEG2024532477000023.jpg235165JPEG2024532477000024.jpg229165JPEG2024532477000025.jpg50165 JPEG2024532477000026.jpg240165JPEG2024532477000027.jpg234165JPEG2024532477000028.jpg56165 JPEG2024532477000029.jpg241165JPEG2024532477000030.jpg235165JPEG2024532477000031.jpg55165 JPEG2024532477000032.jpg236165JPEG2024532477000033.jpg230165JPEG2024532477000034.jpg51165

[0131] Example 4. Knockdown of KRAS G12C mutant mRNA and protein, modulation of KRAS pathway biomarkers and 3D growth inhibition by ASO treatment Tumor cells were treated with various ASOs for mRNA knockdown by Quantigene assay, analysis of KRAS protein and downstream biomarkers of KRAS pathway by Western, and cell growth inhibition by CellTiter-Glo® 2.0 assay. The procedures for mRNA knockdown and cell growth inhibition were as described in Example 1 and Example 3, respectively. The cells transfected by the method described in Example 1 were harvested 3 days after transfection and used to analyze proteins by Western blotting. Figure 1 illustrates the mRNA knockdown of mutant KRAS with G12C mutation (H358 cells) using ASO SEQ ID NO: 19, 44, 28, 37, 67, or 80. The mRNA knockdown of mutant KRAS with G12C mutation was more obvious when mediated by ASO SEQ ID NO: 28 and ASO SEQ ID NO: 67, as well as ASO targeting wild-type KRAS (ASO SEQ ID NO: 19 and ASO SEQ ID NO: 44). No significant mRNA knockdown was detected with the ASO designed to target KRAS G12D (ASO SEQ ID NO: 12) and the ASO targeting KRAS G12V (ASO SEQ ID NO: 80).

[0132] For analysis of protein levels, cells were seeded at 10,000 cells / well in 12-well plates and transfected with ASO / Lipofectamine (3ul Lipofectamine). Cells were harvested 3 days after transfection and protein expression was analyzed. The ASO used in the study was ASO SEQ ID NO:28 and the cell lines used were H358 (G12C, KRAS mutant heterozygous) and A375 (KRAS wild type). Figure 2 illustrates the protein expression knockdown of both KRAS and downstream proteins of the KRAS-RAF-MEK-ERK signaling pathway (pERK, a MAPK marker, and pS6, an ERK downstream marker). Figure 2 also illustrates the increase in apoptosis marker (cPARP) expression mediated by increasing amounts of ASO SEQ ID NO:28 used for transfection.

[0133] Figure 3 illustrates another experiment on protein expression knockdown of both KRAS and downstream proteins of the KRAS-RAF-MEK-ERK signaling pathway (pERK, a MAPK marker, and pS6, an ERK downstream marker). Cells were seeded at 100,000 cells / well in 6-well plates and transfected with ASO / Lipofectamine (3ul Lipofectamine). Cells were harvested 3 days after transfection and protein expression was analyzed. The ASOs used in the study were ASO SEQ ID NO:28 (G12C ASO, 16 mers) and ASO SEQ ID NO:67 (G12C ASO, 14 mers). The cell line used was H358 (G12C, KRAS mutant heterozygous). Protein expression of both KRAS and downstream proteins of the KRAS-RAF-MEK-ERK signaling pathway (pERK, a MAPK marker, and pS6, an ERK downstream marker) was dose-dependently decreased in cells transfected with ASO SEQ ID NO: 28 or ASO SEQ ID NO: 67. Figure 3 also illustrates the increase in apoptosis marker (cPARP) expression mediated by increasing amounts of ASO (ASO SEQ ID NO: 28 and ASO SEQ ID NO: 67) used for transfection.

[0134] Figure 4 illustrates 3D cell growth inhibition due to inhibition of mutant KRAS expression by contacting cells carrying a KRAS mutation (H358 cells with a KRAS G12C mutation) with oligonucleotides described herein. 3D growth of H358 cells (with a G12C KRAS mutation) was measured at 7 days (left) and 13 days (right) after ASO treatment.

[0135] Example 5. KRAS G12V mutant mRNA and protein knockdown, modulation of KRAS pathway biomarkers and 3D growth inhibition by ASO treatment All procedures were performed in accordance with the methods described in Example 4. The cell lines used were NCI-H441 and LCLC97TM1 (KRAS G12V), and A375 (KRAS wild type).

[0136] Figure 5 illustrates mRNA knockdown of mutant KRAS (NCI-H441 cells) with the G12V mutation using the ASOs shown herein. Figure 6 illustrates KRAS protein knockdown (mediated by ASO SEQ ID NO: 80) for protein expression of both G12V mutant KRAS and downstream proteins of the KRAS-RAF-MEK-ERK signaling pathway (pERK and pAKT, both MAPK markers, and pS6, an ERK downstream marker). Figure 7 illustrates KRAS protein knockdown (mediated by ASO SEQ ID NO: 81) for protein expression of both G12V mutant KRAS and downstream proteins of the KRAS-RAF-MEK-ERK signaling pathway (pERK and pAKT, both MAPK markers, and pS6, an ERK downstream marker). FIG. 8 illustrates 3D cell growth inhibition resulting from inhibition of expression of mutant KRAS by contacting cells carrying a KRAS mutation (LCLC97TM1 cells, NCI-H441 cells, or CFPAC-1 cells) with the oligonucleotides described herein.

[0137] Example 6. Growth inhibition of KRAS G12A tumor cells by ASO treatment To inhibit cell proliferation, cells were treated as described in Example 3. NCI-H2009 was used as a KRAS G12A mutant tumor cell line. The results are illustrated in Figure 9. Figure 9 illustrates 3D cell proliferation inhibition due to the inhibition of expression of mutant KRAS by contacting cells carrying KRAS mutation (NCI-H2009 cells) with the oligonucleotides described herein. 3D proliferation was measured on day 7 (left) or day 13 (right) after ASO treatment.

[0138] Example 7. Knockdown of mutant KRAS (G12D mutation) mRNA by ASO treatment KRAS G12D tumor cells were treated with ASOs and KRAS mRNA was detected by Quantigene assay 48 hours after transfection according to the procedures in Example 1. Figure 10 illustrates mRNA knockdown of mutant KRAS (Panc1 and AsPC1 cells) carrying the G12D mutation using the ASOs presented herein.

[0139] Although the foregoing disclosure has been described in some detail for purposes of clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail may be made without departing from the true scope of the disclosure. For example, all of the techniques and devices described above may be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document was individually and separately indicated to be incorporated by reference for all purposes.

Claims

1. A composition comprising an antisense oligonucleotide capable of binding to KRAS mRNA.

2. The composition of claim 1 , wherein the KRAS mRNA is a mutant KRAS mRNA.

3. 3. The composition of claim 1 or 2, wherein the antisense oligonucleotide comprises a sequence that is at least 80%, 85%, or 90% identical to one of the following sequences: SEQ ID NOs: 100-556.

4. 3. The composition of claim 1 or 2, wherein the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to any one of the following sequences: SEQ ID NOs: 24-43, 65-82, or 87.

5. The antisense oligonucleotide has the following sequence: SEQ ID NO:129, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:399, SEQ ID NO:400, SEQ ID NO:401, SEQ ID NO:402, SEQ ID NO:427, SEQ ID NO:428, SEQ ID NO:429, SEQ ID NO:430, SEQ ID NO:433, SEQ ID NO:434, SEQ ID NO:435, SEQ ID NO:436, SEQ ID NO:437, SEQ ID NO:438 , SEQ ID NO:439, SEQ ID NO:440, SEQ ID NO:441, SEQ ID NO:494, SEQ ID NO:495, SEQ ID NO:496, SEQ ID NO:497, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:518, SEQ ID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20 3. The composition of claim 1 or 2, comprising a sequence that is at least 80%, 85%, or 90% identical to any one of:

6. 3. The composition of claim 1 or 2, wherein the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to any one of SEQ ID NOs:18-20, SEQ ID NOs:24-43, SEQ ID NOs:65-82, or SEQ ID NO:

87.

7. The composition of claim 1 or 2, wherein the antisense oligonucleotide is 12 to 30 nucleotides in length.

8. The composition of claim 1 or 2, wherein the antisense oligonucleotide comprises a gap segment and a wing segment.

9. The composition of claim 8, wherein the antisense oligonucleotide comprises a 5'-wing segment and a 3'-wing segment.

10. 10. The composition of claim 9, wherein each of the 5'-wing segment and the 3'-wing segment is a 3-linked nucleotide.

11. 3. The composition of claim 1 or 2, wherein the antisense oligonucleotide comprises at least one 2'-modified nucleoside, at least one modified internucleotide linkage, or at least one inverted abasic moiety.

12. 12. The composition of claim 11, wherein the at least one 2'-modified nucleotide comprises a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamide (2'-O-NMA) modified nucleotide, locked nucleic acid (LNA), cEt (constrained ethyl) sugar, ethylene-bridged nucleic acid (ENA), or a combination thereof.

13. 12. The composition of claim 11, wherein the at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage.

14. 3. The composition of claim 1 or 2, wherein the antisense oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO), a locked nucleic acid (LNA), a thiomorpholino, a cEt (constrained ethyl) sugar, or a combination thereof.

15. The composition of claim 1 or 2, wherein the antisense oligonucleotide is conjugated to a peptide, antibody, lipid, carbohydrate, aptamer, or polymer.

16. The composition of claim 15 , wherein the antisense oligonucleotide is conjugated to a peptide, antibody, lipid, carbohydrate, aptamer, or polymer via a linker.

17. The composition of claim 2 , wherein the composition comprises a combination of an antisense oligonucleotide that specifically binds to the KRAS mRNA and an antisense oligonucleotide that specifically binds to the mutant KRAS mRNA.

18. The composition of claim 2 , wherein the composition comprises an antisense oligonucleotide capable of binding to both KRAS mRNA and mutant KRAS mRNA.

19. The composition of claim 1 or 2, wherein the composition further comprises an excipient.

20. The composition of claim 1 or 2, wherein the composition is formulated for parenteral or inhaled administration.

21. The composition of claim 2 , wherein the mutant KRAS mRNA encodes a mutant KRAS protein comprising a G12C mutation, a G12V mutation, a G12A mutation, or a G12D mutation.

22. 3. The composition of claim 1 or 2, wherein the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to any one of SEQ ID NOs: 19, 27, 28, 37, 44, or 65-81.

23. 23. The composition of claim 22, wherein the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to any one of SEQ ID NOs: 19, 28, 44, 67, 72-77, or 79-81.

24. The composition of claim 1 or 2, wherein the antisense oligonucleotide comprises a nucleic acid sequence that is any one of SEQ ID NOs: 19, 27, 28, 37, 44, or 65-81.

25. A composition for modulating a KRAS-mediated signaling pathway in cancer cells, the composition comprising an antisense oligonucleotide capable of binding to KRAS mRNA or a mutant KRAS mRNA.

26. 26. The composition of claim 25, wherein the cancer cells are lung cancer cells, pancreatic cancer cells, or colon cancer cells.

27. 27. The composition of claim 25 or 26, wherein the antisense oligonucleotide comprises a sequence having at least 80%, 85%, or 90% similarity to one of the following sequences: SEQ ID NOs: 100-556.

28. 27. The composition of claim 25 or 26, wherein the antisense oligonucleotide comprises a sequence having at least 80%, 85%, or 90% similarity to one of the following sequences: SEQ ID NOs: 24-43, 65-82, or 87.

29. The antisense oligonucleotide has the following sequence: SEQ ID NO:129, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:399, SEQ ID NO:400, SEQ ID NO:401, SEQ ID NO:402, SEQ ID NO:427, SEQ ID NO:428, SEQ ID NO:429, SEQ ID NO:430, SEQ ID NO:433, SEQ ID NO:434, SEQ ID NO:435, SEQ ID NO:436, SEQ ID NO:437, SEQ ID NO:438 , SEQ ID NO:439, SEQ ID NO:440, SEQ ID NO:441, SEQ ID NO:494, SEQ ID NO:495, SEQ ID NO:496, SEQ ID NO:497, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:518, SEQ ID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20 27. The composition of claim 25 or 26, comprising a sequence having at least 80%, 85%, or 90% similarity to one of:

30. 27. The composition of claim 25 or 26, wherein the composition comprises a combination of an antisense oligonucleotide that specifically binds to the KRAS mRNA and an antisense oligonucleotide that specifically binds to the mutant KRAS mRNA.

31. 27. The composition of claim 25 or 26, wherein the composition comprises an antisense oligonucleotide capable of binding to both KRAS mRNA and mutant KRAS mRNA.

32. 27. The composition of claim 25 or 26, wherein the antisense oligonucleotide comprises at least one 2'-modified nucleoside, at least one modified internucleotide linkage, or at least one inverted abasic moiety.

33. 33. The composition of claim 32, wherein the at least one 2'-modified nucleotide comprises a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamide (2'-O-NMA) modified nucleotide; a locked nucleic acid (LNA), a cEt (constrained ethyl) sugar or ethylene-bridged nucleic acid (ENA), a thiomorpholino, or a combination thereof.

34. 27. The composition of claim 25 or 26, wherein the modulation comprises a decrease in expression of KRAS protein, mutant KRAS protein, KRAS mRNA, or mutant KRAS mRNA by at least 30%, at least 40%, or at least 50%.

35. 27. The composition of claim 25 or 26, wherein the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to any one of SEQ ID NOs: 19, 27, 28, 37, 44, or 65-81.

36. 36. The composition of claim 35, wherein the antisense oligonucleotide comprises a nucleic acid sequence that is at least 80%, 85%, or 90% identical to any one of SEQ ID NOs: 19, 28, 44, 67, 72-77, or 79-81.

37. 37. The composition of claim 36, wherein the antisense oligonucleotide comprises a nucleic acid sequence that is any one of SEQ ID NOs: 19, 27, 28, 37, 44, or 65-81.

38. 38. The composition of claim 37, wherein the antisense oligonucleotide comprises a nucleic acid sequence that is any one of SEQ ID NOs: 19, 28, 44, 67, 72-77, or 79-81.

39. 3. The composition of claim 1 or 2 for the treatment of cancer in a subject in need thereof.

40. 40. The composition of claim 39, wherein the cancer is associated with an abnormality in the KRAS-mediated signaling pathway.

41. 40. The composition of claim 39, wherein the cancer is lung cancer, pancreatic cancer, or colon cancer.

42. 40. The composition of claim 39, wherein the composition is administered to the subject at a dose and on a schedule sufficient to increase the subject's survival rate by at least 5%.

43. 40. The composition of claim 39, wherein the composition is administered to the subject at a dose and on a schedule sufficient to inhibit growth of the tumor.

44. 40. The composition of claim 39, wherein the cancer is associated with KRAS or mutated KRAS.

45. 45. The composition of claim 44, wherein the mutant KRAS mRNA encodes a mutant KRAS protein comprising a G12C mutation, a G12V mutation, a G12A mutation, or a G12D mutation.

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