CN118251494A - Compositions and methods for modulating KRAS expression - Google Patents

Compositions and methods for modulating KRAS expression Download PDF

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CN118251494A
CN118251494A CN202280072704.2A CN202280072704A CN118251494A CN 118251494 A CN118251494 A CN 118251494A CN 202280072704 A CN202280072704 A CN 202280072704A CN 118251494 A CN118251494 A CN 118251494A
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seq
aspects
composition
oligonucleotide
nucleic acid
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陈力山
靳博涵
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Microelectronics Inc
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Microelectronics Inc
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Priority claimed from PCT/US2022/042393 external-priority patent/WO2023034537A1/en
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Abstract

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

Description

Compositions and methods for modulating KRAS expression
Cross reference
The present application claims the benefit of U.S. provisional application serial No. 63/240,226 filed on month 9 of 2021 and U.S. provisional application serial No. 63/315,669 filed on month 3 of 2022, the disclosures of which are hereby incorporated by reference in their entireties.
Incorporated by reference
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. If publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.
Background
Certain diseases or conditions are caused by mutations in genes or deregulation of signaling pathways due to over-or under-expression of one or more genes that affect the signaling pathway. To treat such diseases or conditions, one of the most popular treatment options involves direct editing of the gene mutations, or transcriptional/translational regulation using gene silencing tools or methods. RNA-induced gene silencing controls RNA expression of a target gene from a number of aspects, including transcriptional inactivation, mRNA degradation, transcriptional attenuation. Thus, there remains a need for compositions and methods for efficiently editing gene expression at the RNA level.
Disclosure of Invention
In some aspects, described herein are compositions comprising antisense oligonucleotides capable of binding to KRAS mRNA. In some embodiments, the KRAS mRNA is a mutated 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 NO. 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: 24-43, 65-82 or 87. In some embodiments, the antisense oligonucleotide comprises 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 that is at least 80%, 85% or 90% identical to any of the following sequences. 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, 24-43, 65-82 or 87. In some embodiments, antisense oligonucleotides comprise a length of 12-30 nucleotides. In some embodiments, the antisense oligonucleotide comprises a gap segment and a wing segment. In some embodiments, the antisense oligonucleotide comprises a5 '-wing segment and a 3' -wing segment. In some embodiments, each of the 5 '-wing segment and the 3' -wing segment is three 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: comprising 2 '-O-methyl, 2' -O-methoxyethyl (2 '-O-MOE), 2' -O-aminopropyl, 2 '-deoxy-2' -fluoro, 2 '-O-aminopropyl (2' -O-AP), 2 '-O-dimethylaminoethyl (2' -O-DMAOE), 2 '-O-dimethylaminopropyl (2' -O-DMAP), 2 '-O-dimethylaminoethoxyethyl (2' -O-DMAEOE) or 2 '-O-N-methylacetamido (2' -O-NMA) modified nucleotide, locked Nucleic Acid (LNA), restricted ethyl (cEt) sugar, ethylene Nucleic Acid (ENA), or combinations thereof. In some embodiments, the 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, a restricted ethyl (cEt) sugar, or a combination thereof. In some embodiments, the antisense oligonucleotide is conjugated to a peptide, antibody, lipid, carbohydrate, aptamer, or polymer. In some embodiments, the antisense oligonucleotide is conjugated to a peptide, antibody, lipid, carbohydrate, aptamer, or polymer through a linker. In some embodiments, the composition comprises a combination of antisense oligonucleotides that specifically bind to KRAS mRNA and antisense oligonucleotides that specifically bind to mutated KRAS mRNA. In some embodiments, the composition comprises an antisense oligonucleotide capable of binding to KRAS mRNA and mutated KRAS mRNA. In some embodiments, the composition further comprises an excipient. In some embodiments, the composition is formulated for parenteral or inhalation administration. In some embodiments, the mutated KRAS mRNA encodes a mutated KRAS protein comprising 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 of 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 of 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.
In some aspects, described herein is a method of modulating a KRAS-mediated signaling pathway in a cancer cell, the method comprising: treating the cancer cell with a composition comprising an antisense oligonucleotide capable of binding to KRAS mRNA or mutated KRAS mRNA, thereby reducing expression of KRAS mRNA or mutated 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 NO. 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: 24-43, 65-82 or 87. In some embodiments, the antisense oligonucleotide comprises 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 that has at least 80%, 85% or 90% similarity to one of the following sequences. In some embodiments, the composition comprises a combination of antisense oligonucleotides that specifically bind to KRAS mRNA and antisense oligonucleotides that specifically bind to mutated KRAS mRNA. In some embodiments, the composition comprises an antisense oligonucleotide capable of binding to KRAS mRNA and mutated 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, at least one 2' modified nucleotide: nucleotides comprising 2 '-O-methyl, 2' -O-methoxyethyl (2 '-O-MOE), 2' -O-aminopropyl, 2 '-deoxy-2' -fluoro, 2 '-O-aminopropyl (2' -O-AP), 2 '-O-dimethylaminoethyl (2' -O-DMAOE), 2 '-O-dimethylaminopropyl (2' -O-DMAP), 2 '-O-dimethylaminoethoxyethyl (2' -O-DMAEOE) or 2 '-O-N-methylacetamido (2' -O-NMA) modification: comprising a Locked Nucleic Acid (LNA), Locked Nucleic Acid (LNA), restricted ethyl (cEt) sugar or Ethylene Nucleic Acid (ENA), thiomorpholino, or combinations thereof. In some embodiments, the expression of KRAS protein, mutated KRAS protein, KRAS mRNA or mutated KRAS mRNA is reduced by at least 30%, at least 40%, 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 of 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 of 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.
In some aspects, described herein is a method of treating cancer in a subject in need thereof, the method comprising: administering to the subject a composition comprising an antisense oligonucleotide described herein, thereby treating cancer in the subject. In some embodiments, the cancer is associated with an abnormality in a KRAS-mediated signaling pathway. In some embodiments, the cancer is lung cancer, pancreatic cancer, or colon cancer. 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 mutated KRAS. In some embodiments, the mutated KRAS mRNA encodes a mutated KRAS protein comprising a G12C mutation, a G12V mutation, a G12A mutation, or a G12D mutation.
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FIG. 1 shows the knock-down of KRAS mRNA encoding a mutant KRAS protein comprising a G12C mutation mediated by an oligonucleotide described herein (e.g., ASO SEQ ID NO:19, 44, 28, 37, 67 or 80).
FIG. 2 shows knockdown of KRAS protein (G12C mutant or wild-type KRAS) and downstream biomarker (pERK, pS6 and cPARP) expression mediated by oligonucleotides described herein (e.g., ASO SEQ ID NO: 28) in two different cell lines (NCI-H358 and A375).
FIG. 3 shows knockdown of mutant KRAS proteins and downstream biomarker (pERK, pS6 and cPARP) expression in NCI-H358 cell lines mediated by oligonucleotides described herein (e.g., ASO SEQ ID NO:28 or ASO SEQ ID NO: 67).
FIG. 4 shows three-dimensional (3D) cell proliferation inhibition due to inhibition of mutated KRAS expression by contacting cells harboring the KRAS mutation (NCI-H358) with an oligonucleotide described herein (e.g., ASO SEQ ID NO:19, 44, 28, 67, 72, 74, 75, 76, 77, 78, 79, 80, or 81).
FIG. 5 shows the knockdown of KRAS mRNA encoding a mutant KRAS protein (NCI-H441) comprising a G12V mutation mediated by the oligonucleotides described herein (e.g., ASO SEQ ID NOS: 19, 44, 77, 78, 79, 80, 81, 28, 67, 37, 70 and 72).
FIG. 6 shows the knockdown of mutant KRAS protein and downstream biomarker (pERK, pAKT and pS 6) expression in LCLC97TM1 (cells harboring the G12V mutation) and wild-type KRAS (A375) mediated by ASO SEQ ID NO: 80.
FIG. 7 shows the knockdown of mutant KRAS protein and downstream biomarker (pERK, pAKT and pS 6) expression in LCLC97TM1 (cells harboring the G12V mutation) mediated by ASO SEQ ID NO: 81.
FIG. 8 shows inhibition of 3D cell proliferation by contacting KRAS mutant-bearing cells (LCLC 97TM1 cells, NCI-H441 cells, or CFPAC-1 cells having a G12V mutation) with an oligonucleotide described herein (e.g., ASO SEQ ID NO:1, 19, 44, 28, 67, 72, 74, 75, 76, 77, 78, 79, 80, or 81).
Fig. 9 shows inhibition of 3D cell proliferation by contacting a cell harboring a KRAS mutation (NCI-H2009 cell harboring a G12A mutation) with an oligonucleotide described herein (e.g., ASO SEQ ID NOs: 1, 19, 44, 28, 67, 72, 73, 74, 75, 76, 77, 79, 80, or 81) to inhibit mutated KRAS expression.
FIG. 10 shows the knockdown of KRAS mRNA encoding mutant KRAS proteins comprising G12D mutations (Panc 1 and AsPC 1) mediated by the oligonucleotides described herein (e.g., ASO SEQ ID NOs: 19, 28, 37, 67, 78 and 81).
The novel features of the 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 that sets forth illustrative embodiments.
Detailed Description
Overview of the invention
Described herein are compositions and methods for modulating expression of a gene or signaling pathway. Also described herein are compositions and methods for treating a disease or disorder by modulating expression of a gene or signaling pathway associated with the disease or disorder. In some aspects, the composition comprises at least one oligonucleotide that binds to an endogenous nucleic acid after delivery into a cell, resulting in degradation of the target nucleic acid. In some aspects, described herein are methods of using the compositions or oligonucleotides described herein. In some aspects, the methods treat a disease or disorder by contacting a cell with an oligonucleotide to reduce expression of a gene or signaling pathway associated with the disease or disorder.
In some aspects, the oligonucleotide is an antisense oligonucleotide, wherein the oligonucleotide is complementary to and binds to at least one endogenous nucleic acid (e.g., mRNA). In some aspects, binding of the oligonucleotide to the endogenous nucleic acid results in degradation of the endogenous nucleic acid or blocks translation of the target protein from the endogenous nucleic acid, thereby reducing expression of a gene encoded by the endogenous nucleic acid. For example, binding of an oligonucleotide to an endogenous nucleic acid comprising mRNA produces a duplex nucleic acid molecule, which can then recruit endogenous nucleases to degrade the mRNA.
In some aspects, the oligonucleotide comprises at least one gap segment. In some aspects, the oligonucleotide comprises at least one wing segment. In some aspects, the oligonucleotide comprises at least one gap segment flanked by two wing segments. For example, an oligonucleotide comprises a gap segment flanked by a 5 '-wing segment and a 3' -wing segment. In some aspects, the gap segment or wing segment comprises at least one chemical modification.
In some aspects, the gene regulated by the oligonucleotide is part of a signaling pathway. In some aspects, the signaling pathway is a KRAS signaling pathway. In some aspects, the KRAS signaling pathway comprises a KRAS-RAF-MEK-ERK signaling pathway. In some aspects, the KRAS signaling pathway comprises a phosphoinositide 3-kinase (PI 3K) signaling pathway, a Mitogen Activated Protein Kinase (MAPK) signaling pathway, or a Ral guanine nucleotide exchange factor (Ral-GEF) signaling pathway. Thus, in some aspects, a decrease in gene expression due to binding of an oligonucleotide to an endogenous nucleic acid can further decrease signaling pathway expression comprising a gene regulated by the oligonucleotide. In some aspects, decreasing gene or signaling pathway expression results in a therapeutic effect in treating a disease or disorder. In some aspects, the disease or condition is caused by increased expression of a gene or signaling pathway. In some aspects, the disease or condition described herein is caused by a mutation in a gene associated with a gene or signaling pathway.
Composition and method for producing the same
In some aspects, described herein are compositions comprising at least one oligonucleotide described herein. In some aspects, the composition comprises at least two, three, four, five, six, seven, eight, nine, ten, or more oligonucleotides. In some aspects, the oligonucleotides comprise the same or different nucleic acid sequences. In some aspects, the oligonucleotides described herein are antisense oligonucleotides for targeting and binding to endogenous nucleic acids. In some aspects, binding of the oligonucleotide to the endogenous nucleic acid recruits an endogenous nuclease to degrade the endogenous nucleic acid. In some aspects, degradation of the endogenous nucleic acid reduces expression of a gene encoded by the endogenous nucleic acid. In some aspects, degradation of endogenous nucleic acids can treat a disease or condition described herein.
In some aspects, the oligonucleotide comprises a length of at least five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, or more nucleobases. In some aspects, the oligonucleotide comprises a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases. In some aspects, the oligonucleotide comprises 10 nucleobases. In some aspects, the oligonucleotide comprises 11 nucleobases. In some aspects, the oligonucleotide comprises 12 nucleobases. In some aspects, the oligonucleotide comprises 13 nucleobases. In some aspects, the oligonucleotide comprises 14 nucleobases. In some aspects, the oligonucleotide comprises 15 nucleobases. In some aspects, the oligonucleotide comprises 16 nucleobases. In some aspects, the oligonucleotide comprises 17 nucleobases. In some aspects, the oligonucleotide comprises 18 nucleobases. In some aspects, the oligonucleotide comprises 19 nucleobases. In some aspects, the oligonucleotide comprises 20 nucleobases.
In some aspects, the oligonucleotide comprises at least one gap segment. In some aspects, the gap segment comprises at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, or more nucleobases. In some aspects, the gap segment comprises at least 1, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleobases. In some aspects, the gap segment comprises 4 nucleobases. In some aspects, the gap segment comprises 5 nucleobases. In some aspects, the gap segment comprises 6 nucleobases. In some aspects, the gap segment comprises 7 nucleobases. In some aspects, the gap segment comprises 8 nucleobases. In some aspects, the gap segment comprises 9 nucleobases. In some aspects, the gap segment comprises 10 nucleobases. In some aspects, the gap segment comprises 11 nucleobases. In some aspects, the gap segment comprises 12 nucleobases. In some aspects, the gap segment comprises 13 nucleobases. In some aspects, the gap segment comprises 14 nucleobases.
In some aspects, the oligonucleotide comprises at least one wing segment. In some aspects, at least one wing segment is a 5 '-end wing segment that is covalently attached to the gap segment at the 5' end of the gap segment. In some aspects, at least one wing segment is a 3 '-end wing segment that is covalently linked to the gap segment at the 3' end of the gap segment. In some aspects, the gap section flanks the wing sections at the 5 'end and the 3' end of the gap section. In some aspects, a wing segment comprises at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, or more nucleobases. In some aspects, the wing segment comprises 1 nucleobase. In some aspects, the wing segment comprises 2 nucleobases. In some aspects, the wing segment comprises 3 nucleobases. In some aspects, the wing segment comprises 4 nucleobases. In some aspects, the wing segment comprises 5 nucleobases. In some aspects, the wing segment comprises 6 nucleobases. In some aspects, the wing segment comprises 7 nucleobases. In some aspects, the wing segment comprises 8 nucleobases. In some aspects, the wing segment comprises 9 nucleobases. In some aspects, the wing segment comprises 10 nucleobases.
In some aspects, the oligonucleotide comprises a 5 'terminal wing segment, followed by a gap segment, followed by a 3' terminal wing segment. In this arrangement, the 5 'terminal wing segment comprises one, two, three, four, five, six, seven, eight, nine, 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 aspects, the 5 'terminal wing segment and the 3' terminal wing segment comprise the same number of nucleobases. In some aspects, the 5 'terminal wing segment and the 3' terminal wing segment comprise different numbers of nucleobases. In some aspects, the 5' terminal wing segment comprises 1 nucleobase. In some aspects, the 5' terminal wing segment comprises 2 nucleobases. In some aspects, the 5' terminal wing segment comprises 3 nucleobases. In some aspects, the 5' terminal wing segment comprises 4 nucleobases. In some aspects, the 5' terminal wing segment comprises 5 nucleobases. In some aspects, the 5' terminal wing segment comprises 6 nucleobases. In some aspects, the 5' terminal wing segment comprises 7 nucleobases. In some aspects, the 5' terminal wing segment comprises 8 nucleobases. In some aspects, the 5' terminal wing segment comprises 9 nucleobases. In some aspects, the 5' terminal wing segment comprises 10 nucleobases. In some aspects, the 3' -terminal wing segment comprises 1 nucleobase. In some aspects, the 3' -terminal wing segment comprises 2 nucleobases. In some aspects, the 3' -terminal wing segment comprises 3 nucleobases. In some aspects, the 3' -terminal wing segment comprises 4 nucleobases. In some aspects, the 3' -terminal wing segment comprises 5 nucleobases. In some aspects, the 3' -terminal wing segment comprises 6 nucleobases. In some aspects, the 3' -terminal wing segment comprises 7 nucleobases. In some aspects, the 3' -terminal wing segment comprises 8 nucleobases. In some aspects, the 3' -terminal wing segment comprises 9 nucleobases. In some aspects, the 3' -terminal wing segment comprises 10 nucleobases.
In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 1 nucleobase and a 3' flanking region comprising 1 nucleobase. In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 2 nucleobases and a 3' flanking region comprising 2 nucleobases. In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 3 nucleobases and a 3' flanking region comprising 3 nucleobases. In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 4 nucleobases and a 3' flanking region comprising 4 nucleobases. In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 5 nucleobases and a 3' flanking region comprising 5 nucleobases. In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 6 nucleobases and a 3' flanking region comprising 6 nucleobases. In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 7 nucleobases and a 3' flanking region comprising 7 nucleobases. In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 8 nucleobases and a 3' flanking region comprising 8 nucleobases. In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 9 nucleobases and a 3' flanking region comprising 9 nucleobases. In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 10 nucleobases and a 3' flanking region comprising 10 nucleobases.
In some aspects, the oligonucleotide is an antisense oligonucleotide. In some aspects, antisense oligonucleotides bind to target nucleic acids. In some aspects, the target nucleic acid is an endogenous nucleic acid. In some aspects, the target nucleic acid comprises nuclear RNA, cytoplasmic RNA, or mitochondrial RNA. In some aspects, the target RNA comprises intergenic DNA (including, but not limited to, heterochromatin DNA), messenger RNA (mRNA), pre-messenger RNA (pre-mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of sequences, isolated RNA of sequences, sgrnas, oligonucleotides, nucleic acid probes, primers, snrnas, long non-coding RNAs, small RNAs of small RNA, snoRNA, siRNA, miRNA, tRNA origin (tsrnas), antisense RNAs, shRNA, or small rDNA-derived RNAs (srrrna). In some aspects, an oligonucleotide comprises a nucleic acid sequence that allows the oligonucleotide to bind to a target nucleic acid by base pairing, e.g., watson Crick base pairing. The compositions and methods provided herein are useful for modulating expression of a gene or signaling pathway. Modulation may refer to altering the expression of a gene or portion thereof at one of various stages to alleviate a disease or condition associated with the gene or mutation of the gene. Modulation may be mediated at the transcriptional or post-transcriptional level. Modulation of transcription corrects aberrant expression of splice variants resulting from the genetic mutation. In some cases, the compositions and methods provided herein can be used to modulate gene translation of a target. Modulation may refer to reducing or knocking down expression of a gene or portion thereof by reducing the abundance of a transcript. A decrease in transcript abundance may be achieved by decreasing transcript processing, splicing, turnover, or stability; or by reducing the accessibility of transcripts by translation mechanisms such as ribosomes. In some cases, the oligonucleotides described herein can facilitate knockdown. Knock-down can reduce expression of the target RNA. In some cases, knockdown may be accompanied by modulation of mRNA. In some cases, knockdown may occur with little modulation of mRNA. In some cases, the knockdown can occur by targeting an untranslated region of the target RNA (e.g., the 3'utr, the 5' utr, or both). In some cases, the knockdown can occur by targeting the coding region of the target RNA.
In some aspects, the oligonucleotide is an antisense oligonucleotide for targeting and binding any of the genes described herein. In some aspects, the one or more genes targeted and bound by the oligonucleotide is KRAS or mutated KRAS. In some embodiments, the mutated KRAS gene encodes a mutated KRAS protein comprising a G12C mutation. In some embodiments, the mutated KRAS gene encodes a mutated KRAS protein comprising a G12V mutation. In some embodiments, the mutated KRAS gene encodes a mutated KRAS protein comprising a G12A mutation. In some embodiments, the mutated KRAS gene encodes a mutated KRAS protein comprising a G12D mutation.
In some aspects, the oligonucleotide targets and binds to mRNA of KRAS or mutated mRNA of KRAS. In some aspects, 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 of tables 7-9. In some aspects, 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 aspects, the oligonucleotide comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to either 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 aspects, 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.
In some aspects, the oligonucleotide comprises at least one gap segment. In some aspects, 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 aspects, 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, 10 consecutive sequences of any of the nucleic acid sequences of SEQ ID NOs 100-556, 24-43, 65-82 or 87. In some aspects, 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 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 and/or at least 5, 6, 7, 8, 9, 10 consecutive sequences.
In some aspects, an oligonucleotide comprising the nucleic acid sequence of any one of SEQ ID NOS.18-20 may bind or preferentially bind to a wild type KRAS sequence. In some embodiments, oligonucleotides comprising the nucleic acid sequence of any one of SEQ ID NOS.24-33, 65-67 or 82 may bind or preferentially bind to mutant KRAS sequences encoding G12C mutations. In some embodiments, an oligonucleotide comprising the nucleic acid sequence of any one of SEQ ID NOS: 77-81 may bind or preferentially bind to a mutant KRAS sequence encoding a G12V mutation. In some embodiments, oligonucleotides comprising the nucleic acid sequence of any one of SEQ ID NOS: 72-76 or 87 may bind or preferentially bind to mutant KRAS sequences encoding G12A mutations. In some embodiments, oligonucleotides comprising the nucleic acid sequence of any one of SEQ ID NOS: 34-43 or 68-71 may bind or preferentially bind to mutant KRAS sequences encoding G12D mutations.
In some aspects, the oligonucleotides described herein target and bind to an endogenous nucleic acid encoding a gene associated with the KRAS-RAF-MEK-ERK signaling pathway. In some aspects, the gene associated with the KRAS-RAF-MEK-ERK signaling pathway is KRAS. In some aspects, the gene associated with the KRAS-RAF-MEK-ERK signaling pathway is mutated KRAS. In some aspects, the oligonucleotides described herein modulate or affect the expression or activity of a gene in or associated with the KRAS-RAF-MEK-ERK signaling pathway. In some aspects, the gene associated with the KRAS-RAF-MEK-ERK signaling pathway is RAS. In some aspects, the gene associated with the KRAS-RAF-MEK-ERK signaling pathway is RAF. In some aspects, the gene associated with the KRAS-RAF-MEK-ERK signaling pathway is MEK. In some aspects, the gene associated with the KRAS-RAF-MEK-ERK signaling pathway is ERK.
In some aspects, the oligonucleotides described herein target and bind to an endogenous nucleic acid encoding a gene associated with the PI3K signaling pathway, MAPK signaling pathway, or Ral-GEF signaling pathway.
In some aspects, the oligonucleotide, upon binding to an endogenous nucleic acid, forms a duplex with the endogenous nucleic acid and recruits an endogenous nuclease for degrading the endogenous nucleic acid. In some aspects, the endogenous nuclease is a deoxyribonuclease. In some aspects, the endogenous nuclease is a ribonuclease. In some aspects, the ribonuclease is an endoribonuclease. In some aspects, the endoribonuclease comprises an endoribonuclease or RNase a, P, H, I, III, T, T2, U2, V1, phyM, or V. In some aspects, the ribonuclease is a riboexonuclease. In some aspects, the riboexonuclease comprises RNase PH, II, R, D, or T. In some aspects, the nuclease comprises a polynucleotide phosphorylase (PNPase), oligoribonuclease, ribonuclease I, or ribonuclease II. In some aspects, the ribonuclease recruited by the oligonucleotide bound to the endogenous nucleic acid is RNase H.
In some aspects, the oligonucleotide comprises at least one, two, three, four, five, six, seven, eight, nine 、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100 or more chemical modifications. In some aspects, the oligonucleotide comprises at least one gap segment comprising at least one, two, three, four, five, six, seven, eight, nine 、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100 or more chemical modifications. In some aspects, the oligonucleotide comprises at least one wing segment comprising at least one, two, three, four, five, six, seven, eight, nine 、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、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 aspects, the oligonucleotide comprises at least one gap segment and at least one wing segment comprising at least one, two, three, four, five, six, seven, eight, nine, 、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100 or more chemical modifications.
In some aspects, an oligonucleotide described herein binds to an endogenous nucleic acid (e.g., mRNA) encoding KRAS, wherein binding of the oligonucleotide to the KRAS endogenous nucleic acid reduces endogenous expression of KRAS in the cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as compared to endogenous expression of KRAS not regulated by the oligonucleotide. In some aspects, an oligonucleotide described herein binds to an endogenous nucleic acid (e.g., mRNA) encoding a mutated KRAS, wherein binding of the oligonucleotide to the mutated KRAS endogenous nucleic acid reduces endogenous expression of the mutated KRAS in a cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to endogenous expression of the mutated KRAS not regulated by the oligonucleotide. 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.
In some aspects, an oligonucleotide described herein binds to an endogenous nucleic acid (e.g., mRNA) encoding KRAS, wherein binding of the oligonucleotide to the KRAS endogenous nucleic acid reduces endogenous expression of the KRAS-RAF-MEK-ERK signaling pathway, PI3K signaling pathway, MAPK signaling pathway, or Ral-GEF signaling pathway in the cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as compared to endogenous expression of the KRAS-RAF-MEK-ERK signaling pathway, PI3K signaling pathway, MAPK signaling pathway, or Ral-GEF signaling pathway that is not modulated by the oligonucleotide.
In some aspects, an oligonucleotide described herein binds to an endogenous nucleic acid (e.g., mRNA) encoding a mutated KRAS, wherein binding of the oligonucleotide to the mutated KRAS endogenous nucleic acid reduces endogenous expression or activity of a KRAS-RAF-MEK-ERK signaling pathway, PI3K signaling pathway, MAPK signaling pathway, or Ral-GEF signaling pathway gene in a cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as compared to endogenous expression or activity of a KRAS-RAF-MEK-ERK signaling pathway, PI3K signaling pathway, MAPK signaling pathway, or Ral-GEF signaling pathway gene not modulated by the oligonucleotide.
In some aspects, the composition comprises at least two oligonucleotides, wherein a first oligonucleotide binds to a KRAS endogenous nucleic acid (e.g., KRAS mRNA) and a second oligonucleotide binds to a mutated KRAS endogenous nucleic acid (e.g., mutated 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.
In some aspects, binding of the oligonucleotide to both KRAS and the mutated KRAS endogenous nucleic acid reduces the endogenous expression or activity of a KRAS-RAF-MEK-ERK signaling pathway, PI3K signaling pathway, MAPK signaling pathway, or Ral-GEF signaling pathway gene in the cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as compared to the endogenous expression or activity of a KRAS-RAF-MEK-ERK signaling pathway, PI3K signaling pathway, MAPK signaling pathway, or Ral-GEF signaling pathway gene not modulated by the oligonucleotide.
In some aspects, the compositions are formulated for administration to a subject by suitable routes of administration including, but not limited to, intravenous, intra-arterial, 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, liposome dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast-dissolving formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations. In some aspects, the composition is formulated into a dosage form. In some aspects, the composition is formulated to include at least one excipient. In some aspects, the excipient is a pharmaceutically acceptable excipient.
In some aspects, a composition comprising an oligonucleotide described herein treats a disease or disorder by reducing expression of a gene or signaling pathway associated with the disease or disorder. In some aspects, a composition comprising an oligonucleotide described herein treats a disease or disorder described herein by directly reducing gene expression associated with the disease or disorder described herein. In some aspects, a composition comprising an oligonucleotide treats a disease or disorder by reducing gene expression as part of a signaling pathway described herein. In some aspects, a composition comprising an oligonucleotide described herein treats a disease or disorder by reducing endogenous KRAS expression. In some aspects, a composition comprising an oligonucleotide described herein treats a disease or disorder by reducing KRAS expression of an endogenous mutation. In some aspects, a composition comprising an oligonucleotide described herein treats a disease or disorder by reducing both endogenous KRAS and mutated KRAS expression. In some aspects, a composition comprising an oligonucleotide described herein treats a disease or disorder by reducing endogenous KRAS expression. In some aspects, a composition comprising an oligonucleotide described herein treats a disease or disorder by reducing expression or activity of an endogenous KRAS-RAF-MEK-ERK signaling pathway, PI3K signaling pathway, MAPK signaling pathway, or Ral-GEF signaling pathway. In some aspects, the disease or condition described herein is cancer.
Chemical modification
In some aspects, described herein are oligonucleotides comprising at least one chemical modification. In some aspects, the oligonucleotide is single stranded. In some aspects, the oligonucleotide is an antisense oligonucleotide. In some aspects, the oligonucleotide comprises at least one, two, three, four, five, six, seven, eight, nine 、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50, or more chemical modifications. In some aspects, the oligonucleotide does not have an intramolecular structural feature. In some aspects, the oligonucleotide comprises at least one gap segment comprising at least one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, or more chemically modified nucleotides. In some aspects, the oligonucleotide comprises at least one wing segment comprising at least one, two, three, four, five, six, seven, eight, nine, ten, or more chemically modified nucleotides. In some aspects, the oligonucleotide comprises a 5' terminal wing segment comprising at least one, two, three, four, five, six, seven, eight, nine, ten, or more chemically modified nucleotides. In some aspects, the oligonucleotide comprises a 3' terminal wing segment comprising at least one, two, three, four, five, six, seven, eight, nine, ten, or more chemically modified nucleotides. In some aspects, at least one wing segment is covalently fused to the 5' end of the gap segment. In some aspects, at least one wing segment is covalently fused to the 3' end of the gap segment.
In some aspects, the oligonucleotide comprises at least one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more chemically modified nucleotides at the 5' end of the oligonucleotide. In some aspects, the oligonucleotide comprises at least one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more chemically modified nucleotides at the 3' end of the oligonucleotide. In some aspects, the oligonucleotide comprises at least one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more chemically modified nucleotides at the 5 'and 3' ends of the oligonucleotide. In some aspects, the oligonucleotide comprises at least one chemical modification in a gap segment of the oligonucleotide. In some aspects, the oligonucleotide comprises at least one chemical modification in the nucleotide base adjacent to the gap segment. In some aspects, 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 aspects, the oligonucleotide comprises 100% modified nucleotide bases.
In some aspects, the chemical modification can occur at a 3'oh group, a 5' oh group, at the backbone, at the sugar component, or at the nucleotide base. Chemical modifications may include inter-or intra-chain cross-linking of non-naturally occurring linker molecules. In one aspect, the chemically modified nucleic acid comprises a modification of one or more of a 3'oh or 5' oh group, a backbone, a sugar component, or a nucleotide base, or the addition of a non-naturally occurring linker molecule. In some aspects, the chemically modified backbone comprises a backbone other than a phosphodiester backbone. In some aspects, the modified sugar comprises a sugar other than deoxyribose (in modified DNA) or other than ribose (modified RNA). In some aspects, the modified base comprises a base other than adenine, guanine, cytosine, thymine, or uracil. In some aspects, the oligonucleotide comprises at least one chemically modified base. In some cases, at least one, two, three, four, five, six, seven, eight, nine, 10, 15, 20, or more modified bases are included. In some cases, chemical modifications to the base moiety include natural and synthetic modifications of adenine, guanine, cytosine, thymine, or uracil, and purine or pyrimidine bases.
In some aspects, the at least one chemical modification of the oligonucleotide comprises a modification of any one or any combination of the following: a 2' modified nucleotide comprising 2' -O-methyl, 2' -O-methoxyethyl (2 ' -O-MOE), 2' -O-aminopropyl, 2' -deoxy-2 ' -fluoro, 2' -O-aminopropyl (2 ' -O-AP), 2' -O-dimethylaminoethyl (2 ' -O-DMAOE), 2' -O-dimethylaminopropyl (2 ' -O-DMAP), 2' -O-dimethylaminoethoxyethyl (2 ' -O-DMAEOE) or 2' -O-N-methylacetamido (2 ' -O-NMA); Modification of one or both of the phosphate diester backbone linkages to a non-linked oxygen phosphate; modification of one or more of the phosphodiester backbone linkages to an oxygen phosphate; modification of ribose components; replacing the phosphate moiety with a "dephosphorylation" linker; modification or substitution of naturally occurring nucleobases; modification of ribose-phosphate backbone; modification of the 5' end of the polynucleotide; modification of the 3' end of the polynucleotide; modification of deoxyribose phosphate backbone; substitution of a phosphate group; modification of ribose phosphate backbone; modification of nucleotide sugars; modification of nucleotide bases; or a stereopure nucleotide. Non-limiting examples of chemical modifications to the oligonucleotides may include: modification of one or both of the non-linked or linked oxygen phosphates in the phosphodiester backbone linkages (e.g., sulfur (S), selenium (Se), BR3 (where R may be, for example, hydrogen, alkyl, or aryl), C (e.g., alkyl, aryl, etc.), H, NR2, where R may be, for example, hydrogen, alkyl, or aryl, or where R may be, for example, alkyl or aryl); Replacement of the phosphate moiety with a "dephosphoric" linker (e.g., replacement with a methyl phosphonate, hydroxyamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thiomethylal, methylal, oxime, methyleneimino, methylenehydrazine, methylenedimethylhydrazine, or methylenemethyleneimino); modifying or replacing naturally occurring nucleobases with nucleic acid analogs; modification of the deoxyribose-phosphate or ribose-phosphate backbone (e.g., modification of the ribose-phosphate backbone to incorporate phosphorothioates, phosphonothioates, phosphoroselenos, boranophosphates, hydrogen phosphonates, phosphonocarboxylates, phosphoramidates, alkyl or aryl phosphonates, phosphonoacetates or phosphotriesters); Modification of the 5 'end (e.g., modification of the 5' cap or 5 'cap-OH) or the 3' end (modification of the 3 'tail or 3' end-OH) of the nucleic acid sequence; substitution of the phosphate group with methylphosphonate, hydroxyamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, oxirane linker, sulfonate, sulfonamide, thiomethylal, methylal, oxime, methyleneimino, methylenemethylimino, methylenehydrazino, methylenedimethylimino, or methylenemethyleneimino; modifying the ribophosphate backbone to incorporate morpholino (phosphorodiamidate morpholino oligomer PMO), thiomorpholino, cyclobutyl, pyrrolidine or Peptide Nucleic Acid (PNA) nucleoside substitutes; Modifying the sugar of the nucleotide to incorporate Locked Nucleic Acid (LNA), unlocked Nucleic Acid (UNA), ethylene Nucleic Acid (ENA), limited ethyl (cEt) sugar or Bridging Nucleic Acid (BNA); modification of ribose moiety (e.g., 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 (thioppace)); modification of the nucleotide (A, T, C, G or U) base; And sterically pure nucleotides (e.g., the S-conformation of phosphorothioate or the R-conformation of phosphorothioate).
In some aspects, the chemical modification of the oligonucleotide includes at least one substitution of one or two non-linking phosphate oxygen atoms in the phosphodiester backbone linkage of the oligonucleotide. In some aspects, at least one chemical modification of the oligonucleotide includes substitution of one or more of the phosphodiester backbone linkages of the oligonucleotide to an oxygen atom of a phosphate. A non-limiting example of a chemical modification of an oxygen atom of phosphoric acid is a sulfur atom. In some aspects, the chemical modification of the oligonucleotide comprises at least one chemical modification to a sugar of a nucleotide of the oligonucleotide. In some aspects, the chemical modification of the oligonucleotide comprises at least one chemical modification of a sugar of the nucleotide, wherein the chemical modification comprises at least one Locked Nucleic Acid (LNA). In some aspects, the chemical modification of the oligonucleotide comprises at least one chemical modification of a sugar of a nucleotide of the oligonucleotide comprising at least one Unlocked Nucleic Acid (UNA). In some aspects, the chemical modification of the oligonucleotide comprises at least one chemical modification of a sugar of a nucleotide of the oligonucleotide comprising at least one Ethylene Nucleic Acid (ENA). In some aspects, the chemical modification of the oligonucleotide comprises at least one chemical modification to a sugar, including a modification to a component of the sugar, wherein the sugar is a ribose sugar. In some aspects, the chemical modification of the oligonucleotide comprises at least one chemical modification of the ribose sugar component of a nucleotide of the oligonucleotide comprising a 2' -O-methyl group. In some aspects, the chemical modification of the oligonucleotide comprises at least one chemical modification comprising replacing a phosphate moiety of the oligonucleotide with a dephosphorylation linker. In some aspects, the chemical modification of the oligonucleotide comprises at least one chemical modification of the phosphate backbone of the oligonucleotide. In some aspects, the oligonucleotide comprises a phosphorothioate group. In some aspects, the chemical modification of the oligonucleotide comprises at least one chemical modification comprising modification of a base of a nucleotide of the oligonucleotide. In some aspects, the chemical modification of the oligonucleotide comprises chemical modification of at least one unnatural base comprising a nucleotide. In some aspects, the chemical modification of the oligonucleotide comprises at least one chemical modification comprising a morpholino group (e.g., phosphorodiamidate morpholino oligomer, PMO), cyclobutyl, pyrrolidinyl, or Peptide Nucleic Acid (PNA) nucleoside substitute. In some aspects, the chemical modification of the oligonucleotide comprises at least one chemical modification comprising at least one stereotactic nucleic acid. In some aspects, at least one chemical modification may be located near the 5' end of the oligonucleotide. In some aspects, at least one chemical modification may be located near the 3' end of the oligonucleotide. In some aspects, at least one chemical modification may be located near the 5 'and 3' ends of the oligonucleotide.
In some aspects, an oligonucleotide comprises a backbone comprising a plurality of sugar and phosphate moieties covalently linked together. In some cases, the backbone of the oligonucleotide comprises a phosphodiester linkage between a first hydroxyl group in a phosphate group on the 5 'carbon of deoxyribose in DNA or ribose in RNA and a second hydroxyl group on the 3' carbon of deoxyribose in DNA or ribose in RNA.
In some aspects, the backbone of the oligonucleotide may lack a 5 'reduced hydroxyl group, a 3' reduced hydroxyl group, or both that are capable of being exposed to a solvent. In some aspects, the backbone of the oligonucleotide may lack a 5 'reduced hydroxyl group, a 3' reduced hydroxyl group, or both that are capable of being exposed to the nuclease. In some aspects, the backbone of the oligonucleotide may lack a 5 'reduced hydroxyl group, a 3' reduced hydroxyl group, or both that are capable of being exposed to a hydrolase. In some cases, the backbone of an oligonucleotide may be represented as a circular two-dimensional format of polynucleotide sequences, one nucleotide following the other. In some cases, the backbone of an oligonucleotide may be represented as a polynucleotide sequence in circular two-dimensional format, one nucleotide following the other. In some cases, the 5 'hydroxyl group, the 3' hydroxyl group, or both are linked by a phosphorus-oxygen bond. In some cases, the 5 'hydroxyl group, the 3' hydroxyl group, or both are modified to a phosphate ester having a phosphorus-containing moiety.
In some aspects, an oligonucleotide described herein comprises at least one chemical modification. The chemical modification may be a substitution, insertion, deletion, chemical modification, physical modification, stabilization, purification, or any combination thereof. In some cases, the modification is a chemical modification. Suitable chemical modifications include any of the following: 5 'adenylate, 5' guanosine-triphosphate cap, 5 'N7-methylguanosine-triphosphate cap, 5' triphosphate cap, 3 'phosphate, 3' thiophosphoric acid, 5 'phosphate, 5' thiophosphoric acid, cis-Syn thymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, d spacer, PC spacer, r spacer, spacer 18, spacer 9,3'-3' modification, 5'-5' modification, abasic, acridine, azobenzene, biotin BB, biotin TEG, cholesterol TEG, desthiobiotin TEG, DNP-X, DOTA, dT-biotin, biphytin, PC biotin, psoralen C2, psoralen C6, TINA, 3 'DABCCYL black hole quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, a carboxyl linker, a thiol linker, a 2' deoxyribonucleoside analog purine, a 2 'deoxyribonucleoside analog pyrimidine, a ribonucleoside analog, a 2' -O-methyl ribonucleoside analog, a sugar modified analog, a wobble/universal base, a fluorescent dye tag, a 2 'fluoro RNA, a 2' O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphorothioate DNA, phosphorothioate RNA, UNA, LNA, cEt, pseudouridine-5 '-triphosphate, 5-methylcytidine-5' -triphosphate, 2-O-methyl-phosphorothioate, or any combination thereof.
In some cases, the modification may be permanent. In some cases, the modification may be temporary. In some cases, the oligonucleotide is modified multiple times. Oligonucleotide modifications may alter the physiochemical properties of the nucleotides, such as their conformation, polarity, hydrophobicity, chemical reactivity, base pairing interactions, or any combination thereof.
The chemical modification may also be a phosphorothioate substitute. In some cases, natural phosphodiester bonds may be susceptible to rapid degradation by cellular nucleases; modification of internucleotide linkages using Phosphorothioate (PS) linkage substitutes can be more stable to hydrolysis by cellular degradation. Modification may increase the stability of the polynucleic acid. Modifications may also enhance biological activity. In some cases, the phosphorothioate-enhanced RNA polynucleic acids may inhibit RNase a, RNase T1, calf serum nucleases or any combination thereof. These properties make PS-RNA polynucleic acids useful in applications where exposure to nucleases is highly likely in vivo or in vitro. For example, phosphorothioate (PS) linkages may be introduced between the last 3-5 nucleotides of the 5 'or 3' end of the polynucleic acid, which may inhibit exonuclease degradation. In some cases phosphorothioate linkages may be added throughout the polynucleic acid to reduce endonuclease attack. In some aspects, an oligonucleotide described herein comprises at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 100, or more internucleotide linkages comprising a PS bond. In some aspects, the oligonucleotides described herein comprise only PS linkages as internucleotide linkage modifications. In some aspects, all internucleotide linkages of the oligonucleotides described herein are fully PS modified or comprise phosphorothioate internucleotide linkages. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' flanking region comprising 1 nucleobase. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' flanking region comprising 2 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' flanking region comprising 3 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' flanking region comprising 4 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' terminal wing segment comprising 5 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' flanking region comprising 6 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' flanking region comprising 7 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' flanking region comprising 8 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' flanking region comprising 9 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5' flanking region comprising 10 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' terminal wing segment comprising 1 nucleobase. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' terminal wing segment comprising 2 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' terminal wing segment comprising 3 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' terminal wing segment comprising 4 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' terminal wing segment comprising 5 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' terminal wing segment comprising 6 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' terminal wing segment comprising 7 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' terminal wing segment comprising 8 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' terminal wing segment comprising 9 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 3' terminal wing segment comprising 10 nucleobases.
In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5 'terminal wing segment comprising 1 nucleobase and a 3' terminal wing segment comprising 1 nucleobase. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5 'terminal wing segment comprising 2 nucleobases and a 3' terminal wing segment comprising 2 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5 'terminal wing segment comprising 3 nucleobases and a 3' terminal wing segment comprising 3 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5 'terminal wing segment comprising 4 nucleobases and a 3' terminal wing segment comprising 4 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5 'terminal wing segment comprising 5 nucleobases and a 3' terminal wing segment comprising 5 nucleobases. In some aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5 'terminal wing segment comprising 6 nucleobases and a 3' terminal wing segment comprising 6 nucleobases. In some aspects, an oligonucleotide comprising a PS bond 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 aspects, an oligonucleotide comprising a PS bond 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 aspects, an oligonucleotide comprising a PS bond 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 aspects, an oligonucleotide comprising a PS bond as an internucleotide linkage modification comprises a 5 'terminal wing segment comprising 10 nucleobases and a 3' terminal wing segment comprising 10 nucleobases.
In some aspects, the oligonucleotide comprises a 5 'terminal wing segment comprising 1 nucleobase, a gap mer, and a 3' terminal wing segment comprising 1 nucleobase, wherein the internucleotide linkages of the oligonucleotide connecting the 5 'terminal wing segment, the gap mer, and the 3' terminal wing segment comprise only PS linkages. In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 2 nucleobases, a gap mer, and a 3' flanking region comprising 2 nucleobases, wherein the internucleotide linkages of the oligonucleotide connecting the 5 'flanking region, the gap mer, and the 3' flanking region comprise only PS linkages. In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 3 nucleobases, a gap mer, and a 3' flanking region comprising 3 nucleobases, wherein the internucleotide linkages of the oligonucleotide connecting the 5 'flanking region, the gap mer, and the 3' flanking region comprise only PS linkages. In some aspects, the oligonucleotide comprises a 5 'terminal wing segment comprising 4 nucleobases, a gap mer, and a 3' terminal wing segment comprising 4 nucleobases, wherein the internucleotide linkages of the oligonucleotide connecting the 5 'terminal wing segment, the gap mer, and the 3' terminal wing segment comprise only PS linkages. In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 5 nucleobases, a gap mer, and a 3' flanking region comprising 5 nucleobases, wherein the internucleotide linkages of the oligonucleotide connecting the 5 'flanking region, the gap mer, and the 3' flanking region comprise only PS linkages. In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 6 nucleobases, a gap mer, and a 3' flanking region comprising 6 nucleobases, wherein the internucleotide linkages of the oligonucleotide connecting the 5 'flanking region, the gap mer, and the 3' flanking region comprise only PS linkages. In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 7 nucleobases, a gap mer, and a 3' flanking region comprising 7 nucleobases, wherein the internucleotide linkages of the oligonucleotide connecting the 5 'flanking region, the gap mer, and the 3' flanking region comprise only PS linkages. In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 8 nucleobases, a gap mer, and a 3' flanking region comprising 8 nucleobases, wherein the internucleotide linkages of the oligonucleotide connecting the 5 'flanking region, the gap mer, and the 3' flanking region comprise only PS linkages. In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 9 nucleobases, a gap mer, and a 3' flanking region comprising 9 nucleobases, wherein the internucleotide linkages of the oligonucleotide connecting the 5 'flanking region, the gap mer, and the 3' flanking region comprise only PS linkages. In some aspects, the oligonucleotide comprises a 5 'flanking region comprising 10 nucleobases, a gap mer, and a 3' flanking region comprising 10 nucleobases, wherein the internucleotide linkages of the oligonucleotide connecting the 5 'flanking region, the gap mer, and the 3' flanking region comprise only PS linkages.
In some aspects, an oligonucleotide comprising a 5 'terminal wing segment, a gap mer, a 3' terminal wing segment, and a PS linkage as an internucleotide linkage comprises a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO. 100-556. In some aspects, an oligonucleotide comprising a 5 'terminal wing segment, a gap mer, a 3' terminal wing segment, and a PS bond as an internucleotide linkage comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID 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 aspects, the oligonucleotide comprises a 5 'terminal wing segment, a gap mer, a 3' terminal wing segment, and a PS linkage as internucleotide linkages, wherein the gap mer comprises a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any of SEQ ID NOs 100-556, 24-43, 65-82, or 87. In some aspects, the oligonucleotide comprises a 5 'terminal wing segment, a gap mer, a 3' terminal wing segment, and a PS bond as internucleotide linkages, wherein the gap mer comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to 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 cases, chemical modifications to enhance guided stability, synthesis, localization, intracellular retention, or extended half-life may not be genetically encodable. The oligonucleotides may be circular, substantially circular, or otherwise linked in a continuous manner (e.g., may be arranged as a circle), or may retain a substantially similar secondary structure as a substantially similar oligonucleotide that may or may not be circular.
Modification of the phosphate backbone
In some aspects, the chemical modification comprises modification of one or both of the non-linked phosphate oxygen linkages or modification of one or more of the linked phosphate oxygen linkages of the phosphate diester backbone linkages. As used herein, "alkyl" means a straight or branched saturated hydrocarbon group. Exemplary alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl or isopropyl), butyl (e.g., n-butyl, isobutyl or tert-butyl), or pentyl (e.g., n-pentyl, isopentyl or neopentyl). The alkyl group can contain from 1 to about 20, 2 to about 20, 1 to about 12, 1 to about 8,1 to about 6, 1 to about 4, or 1 to about 3 carbon atoms. As used herein, "aryl" refers to a monocyclic or polycyclic (e.g., having 2,3, or 4 fused rings) aromatic hydrocarbon, such as phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, or indenyl. In some aspects, aryl groups have 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, characterized by having one or more triple bonds. Examples of alkynyl groups may include ethynyl, propargyl or 3-hexynyl. "arylalkyl"
Or "aralkyl" refers to an alkyl moiety in which an alkyl hydrogen atom is replaced with an aryl group. Aralkyl groups include groups in which more than one hydrogen atom is replaced with an aryl group. Examples of "arylalkyl" or "aralkyl" include benzyl, 2-phenethyl, 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 group of a heterocyclic system. Representative heterocyclyl groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidinonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl, dioxolanyl, diazanylA group, an oxazepinyl group, a thiazepinyl group, and a morpholinyl group. "heteroaryl" refers to a monovalent group of a heteroaromatic ring system. Examples of heteroaryl moieties may include imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, indolyl, thiophenylpyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, quinolinyl, and pteridinyl.
In some aspects, the phosphate group of a chemically modified nucleotide may be modified by replacing one or more oxygens with different substituents. In some aspects, chemically modified nucleotides may include replacing an unmodified phosphate moiety with a modified phosphate as described herein. In some aspects, modification of the phosphate backbone may include modification of the charged linker resulting in an uncharged linker or having an asymmetric charge distribution. Examples of modified phosphate groups may include phosphorothioates, phosphonothioates, selenophosphate, borane phosphates, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates and phosphotriesters. In some aspects, one of the non-bridging oxygen phosphate atoms in the phosphate backbone moiety may be replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (where R may be, for example, hydrogen, alkyl, or aryl), C (e.g., alkyl, aryl, etc.), H, NR2 (where R may be, for example, hydrogen, alkyl, or aryl), or (where R may be, for example, alkyl or aryl). The phosphorus atom in the unmodified phosphate group may be achiral. However, substitution of one of the above atoms or groups of atoms for one of the non-bridging oxygens may impart chirality to the phosphorus atom. The phosphorus atom in the phosphate group modified in this way is a stereocenter. The stereocomphosporous atom may have an "R" configuration (herein Rp) or an "S" configuration (herein Sp). In some cases, the oligonucleotide comprises a sterically pure nucleotide comprising an S-conformation of a phosphorothioate or an R-conformation of a phosphorothioate. In some aspects, the chiral phosphate product is present in a diastereomeric excess of 50%, 60%, 70%, 80%, 90% or more. In some aspects, the chiral phosphoric acid product is present in 95% diastereomeric excess. In some aspects, the chiral phosphoric acid product is present in a diastereomeric excess of 96%. In some aspects, the chiral phosphate product is present in a diastereomeric excess of 97%. In some aspects, the chiral phosphoric acid product is present in a diastereomeric excess of 98%. In some aspects, the chiral phosphoric acid product is present in a diastereomeric excess of 99%. In some aspects, both of the non-bridging oxygens of the dithiophosphate may be replaced with sulfur. The phosphorus center in the dithiophosphate may be achiral, which prevents the formation of oligoribonucleotide diastereomers. In some aspects, the modification of one OR both of the non-bridging oxygens may further comprise replacing the non-bridging oxygens with a group independently selected from S, se, B, C, H, N and OR (R may be, for example, alkyl OR aryl). In some aspects, the phosphate linker can also be modified by replacing the bridging oxygen (i.e., the oxygen linking the phosphate to the nucleoside) with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate), and carbon (bridged methylphosphonate). Replacement may occur on one or both of the linking oxygens.
In certain embodiments, the nucleic acid comprises a linked nucleic acid. The nucleic acids may be linked together using any internucleotide linkage. The linking group between two broad classes of nucleic acids is defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing nucleic acid linkages include, but are not limited to, phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (p=s). Representative phosphorus-free internucleotide linkages include, but are not limited to, methyleneimino (-CH 2-N(CH3)-O-CH2 -), thiodiester (-O-C (O) -S-), thiocarbamate (-O-C (O) (NH) -S-); siloxane (-O-Si (H) 2 -O-); n, N-dimethylhydrazine (-CH 2-N(CH3)-N(CH3)). In certain embodiments, the internucleotide linkages having chiral atoms can be prepared as racemic mixtures as individual enantiomers, such as alkylphosphonates and phosphorothioates. The non-natural nucleic acid may contain a single modification. The non-natural nucleic acid may contain multiple modifications within one portion or between different portions.
Backbone phosphate modifications to nucleic acids include, but are not limited to, methylphosphonate, phosphorothioate, phosphoramidate (bridged or unbridged), phosphotriester, phosphorodithioate, and borane phosphate, and may be used in any combination. Other non-phosphate linkages may also be used.
In some aspects, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoramidate, and phosphorodithioate internucleotide linkages) may confer immunomodulatory activity on the modified nucleic acids and/or enhance their in vivo stability.
In some cases, the phosphorus derivative (or modified phosphate group) is attached to the sugar or sugar analog moiety and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, or the like.
In some cases, backbone modification includes replacing the phosphodiester linkage with a replacement moiety, such as an anionic, neutral, or cationic group. Examples of such modifications include: anionic internucleoside linkages; n3 'to P5' phosphoramidate modification; borane phosphate DNA; a precursor oligonucleotide; neutral internucleoside linkages, such as methylphosphonate; amide linked DNA; methylene (methylimino) linkages; methylal and thiomethylal linkages; a sulfonyl-containing backbone; morpholino oligonucleotides; peptide Nucleic Acid (PNA); and positively charged Deoxyriboguanidine (DNG) oligonucleotides. The modified nucleic acid may comprise a chimeric or mixed backbone comprising one or more modifications, such as a combination of phosphate linkages, e.g., a combination of phosphodiester and phosphorothioate linkages.
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 having the following: morpholino linkages (formed in part from the sugar moiety of the nucleoside); a siloxane backbone; sulfide, sulfoxide, and sulfone backbones; formylacetyl and thioacetylacetyl backbones; methylene formylacetyl and thioformylacetyl backbones; an olefin-containing backbone; a sulfamate backbone; methylene imino and methylene hydrazino backbones; sulfonate and sulfonamide backbones; an amide backbone; and other backbones with mixed N, O, S and CH 2 moieties. It is also understood that in nucleotide substitutions, both the sugar and phosphate moieties of the nucleotide may be substituted with, for example, an amide linkage (aminoethylglycine) (PNA). Other types of molecules (conjugates) can also be attached to the nucleotide or nucleotide analog to enhance, for example, cellular uptake. The conjugate may be chemically linked to a nucleotide or nucleotide analogue. Such conjugates include, but are not limited to, lipid moieties such as cholesterol moieties, thioether such as hexyl-S-trityl thiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as di-hexadecyl-racemic glycerol or triethylammonium l-di-O-hexadecyl-racemic-glycerol-S-H-phosphonate, polyamine or polyethylene glycol chains, or adamantaneacetic acid, palmitoyl moieties, or octadecylamine or hexylamino-carbonyl-oxy cholesterol moieties.
In some aspects, the chemical modifications described herein include modifications of the phosphate backbone. In some aspects, the oligonucleotides described herein comprise at least one chemically modified phosphate backbone. Exemplary chemical modifications of the phosphate group or backbone may include substitution of one or more of the oxygen with different substituents. Furthermore, the modified nucleotide present in the oligonucleotide may comprise replacing the unmodified phosphate moiety with a modified phosphate as described herein. In some aspects, modification of the phosphate backbone may include modification of the charged linker resulting in an uncharged linker or having an asymmetric charge distribution. Examples of modified phosphate groups may include phosphorothioates, phosphonothioates, selenophosphate, borane phosphates, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates and phosphotriesters. In some aspects, one of the non-bridging oxygen phosphate atoms in the phosphate backbone moiety may be replaced by any of the following groups: sulfur (S), selenium (Se), BR 3 (where R may be, for example, hydrogen, alkyl, or aryl), C (e.g., alkyl, aryl, etc.), H, NR 2 (where R may be, for example, hydrogen, alkyl, or aryl), or (where R may be, for example, alkyl or aryl). The phosphorus atoms in the unmodified phosphate groups are achiral. However, substitution of one of the non-bridging oxygens with one of the above atoms or groups of atoms may impart chirality to the phosphorus atom; that is, the phosphorus atom in the phosphate group modified in this way is a stereocenter. The stereocomphosporous atom may have an "R" configuration (herein Rp) or an "S" configuration (herein Sp). In this case, the chemically modified oligonucleotide may be sterically pure (e.g., S or R conformation). In some cases, the chemically modified oligonucleotide comprises a sterically pure phosphate modification. For example, a chemically modified oligonucleotide includes an S conformation of a phosphorothioate or an R conformation of a phosphorothioate.
Dithiophosphate has two non-bridging oxygens replaced with sulfur. The phosphorus center in the dithiophosphate is achiral, which prevents the formation of oligoribonucleotide diastereomers. In some aspects, the modification of one OR both of the non-bridging oxygens may further comprise replacing the non-bridging oxygens with a group independently selected from S, se, B, C, H, N and OR (R may be, for example, alkyl OR aryl).
The phosphate linker can also be modified by replacing the bridging oxygen (i.e., the oxygen that links the phosphate to the nucleoside) with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate), and carbon (bridged methylphosphonate). Substitution may occur on either or both of the linking oxygens.
Replacement of the phosphate moiety
In some aspects, at least one phosphate group of the oligonucleotide may be chemically modified. In some aspects, the phosphate group can be replaced with a non-phosphorus containing linker. In some aspects, the phosphate moiety may be replaced with a dephosphorylation linker. In some aspects, the charged phosphate groups may be replaced with neutral groups. In some cases, the phosphate group may be replaced with a methyl phosphonate, hydroxyamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thiomethylal, methylal, oxime, methyleneimino, methylenehydrazine, methylenedimethylhydrazine, and methylenemethyleneimino. In some aspects, the nucleotide analogs described herein may also be modified at the phosphate group. Modified phosphate groups may include modification at the linkage between two nucleotides with phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates (including phosphonic acid 3 'alkylene esters and chiral phosphonates), phosphonites, phosphoramidates (e.g., 3' -phosphoramidates and aminoalkyl phosphoramidates), phosphorothioates, phosphorothioate alkyl phosphates, phosphorothioate alkyl phosphotriesters and borane phosphates. The phosphate or modified phosphate linkage between two nucleotides may be through a 3'-5' linkage or a 2'-5' linkage, and the linkage comprises a reverse polarity, e.g., 3'-5' to 5'-3' or 2'-5' to 5'-2'.
Substitution of phosphate groups
In some aspects, the chemical modifications described herein include modifications by replacing phosphate groups. In some aspects, the oligonucleotides described herein comprise at least one chemical modification comprising a phosphate group substitution or replacement. Exemplary phosphate group substitutions may include a non-phosphorus containing linker. In some aspects, the phosphate group substitution or replacement may include replacing a charged phosphate group with a neutral moiety. Exemplary moieties of the replaceable phosphate groups may include methyl phosphonate, hydroxyamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thiomethylal, methylal, oxime, methyleneimino, methylenehydrazine, methylenedimethylhydrazine, and methylenemethyleneimino.
Modification of ribose phosphate backbone
In some aspects, the chemical modifications described herein include modification of the ribophosphate backbone of the oligonucleotide. In some aspects, the oligonucleotides described herein comprise at least one chemically modified ribose phosphate backbone. Exemplary chemically modified ribose phosphate backbones can include scaffolds that can mimic nucleic acids, as well as scaffolds in which the phosphate linker and ribose sugar are replaced with nuclease resistant nucleosides or nucleotide substitutes. In some aspects, nucleobases can be tethered by a surrogate backbone. Examples may include morpholinos, such as Phosphorodiamidate Morpholino Oligomers (PMOs), cyclobutyls, pyrrolidines, and Peptide Nucleic Acid (PNA) nucleoside substitutes.
Modification of sugar
In some aspects, the chemical modifications described herein include modification of a sugar. In some aspects, the oligonucleotides described herein comprise at least one chemically modified sugar. Exemplary chemically modified sugars can include 2' hydroxyl (OH) groups modified or replaced with a number of different "oxy" or "deoxy" substituents. In some aspects, modification of the 2 'hydroxyl group may enhance the stability of the nucleic acid, as the hydroxyl group is no longer able to be deprotonated to form a 2' -alkoxide ion. The 2' -alkoxide may catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom. Examples of "oxy" -2' hydroxyl modifications may include alkoxy OR aryloxy (OR, where "R" may be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, OR sugar); Polyethylene glycol (PEG), O (CH 2CH2O)nCH2CH2 OR), where R may be, for example, H OR optionally substituted alkyl, and n may be an integer from 0 to 20 (e.g., 0 to 4, 0 to 8,0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16, and 4 to 20). In some aspects, "oxy" -2' hydroxyl modification may include (LNA, where 2' hydroxyl may be attached to the 4' carbon of the same ribose sugar, for example, through a Ci- 6 alkylene OR Cj-6 heteroalkylene bridge, where exemplary bridges may include methylene, propylene, ether, OR amino bridges; O-amino (where amino may be, for example, NH 2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine or polyamino) and aminoalkoxy, O (CH 2)n -amino (where amino may be, for example, NH 2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine or polyamino). In some aspects, the "oxy" -2' hydroxyl modification may include Methoxyethyl (MOE) (OCH 2CH2OCH3, e.g., PEG derivative). In some cases, the deoxy modification can include hydrogen (i.e., deoxyribose sugar, e.g., at a protruding portion of a dsRNA); halogen (e.g., bromine, chlorine, fluorine, or iodine); amino (where amino may be, for example, NH 2; Alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH (CH 2CH2NH)nCH2CH2 -amino (where amino may be, for example, as described herein), NHC (O) R (where R may be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), cyano, mercapto, alkyl-thio-alkyl, thioalkoxy, and alkyl, cycloalkyl, aryl, alkenyl, and alkynyl groups, which may be optionally substituted with amino as described herein. In some cases, the glycosyl may also contain one or more carbons having a stereochemical configuration opposite the corresponding carbon in ribose. Thus, a modified nucleic acid may include a nucleotide containing, for example, arabinose as a sugar. The nucleotide "monomer" may have an alpha linkage at the Γ position on the sugar, such as an alpha-nucleoside. Modified nucleic acids may also include "abasic" sugars, which lack nucleobases at C-. The abasic sugar may also be further modified at one or more of the constituent sugar atoms. The modified nucleic acid may also include one or more L-form sugars, such as L-nucleosides. In some aspects, the oligonucleotides described herein include glycosylribose, which is a5 membered ring with oxygen. Exemplary modified nucleosides and modified nucleotides can include substitution of oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene groups, such as methylene or ethylene); adding a double bond (e.g., replacing ribose with cyclopentenyl or cyclohexenyl); ring shrinkage of ribose (e.g., 4 membered rings forming cyclobutane or oxetane); the ring of ribose expands (e.g., forming a 6 or 7 membered ring with additional carbon or heteroatoms, such as anhydrohexitol, arabitol, mannitol, cyclohexyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). In some aspects, the modified nucleotide may include a polycyclic form (e.g., a tricyclic; And "unlocked" forms, such as a Glycol Nucleic Acid (GNA) (e.g., R-GNA or S-GNA, wherein ribose is replaced with a glycol unit attached to a phosphodiester linkage), threose nucleic acid. In some aspects, the modification of the sugar of the oligonucleotide comprises modifying the oligonucleotide to include Locked Nucleic Acid (LNA), unlocked Nucleic Acid (UNA), ethylene Nucleic Acid (ENA), a limited ethyl (cEt) sugar, or a Bridging Nucleic Acid (BNA).
Modification of ribose sugar component
In some aspects, the oligonucleotides described herein comprise chemical modifications of at least one ribose sugar component. In some aspects, the chemical modification of the ribose sugar component may include 2 '-O-methyl, 2' -O-methoxyethyl (2 '-O-MOE), 2' -fluoro, 2 '-aminoethyl, 2' -deoxy-2 '-fluoroarabinoacid, 2' -deoxy-2 '-fluoro, 2' -O-methyl, 3 '-phosphorothioate, 2' -O-aminopropyl (2 '-O-AP), 2' -O-dimethylaminoethyl (2 '-O-DMAOE), 2' -O-dimethylaminopropyl (2 '-O-DMAP), 2' -O-dimethylaminoethoxyethyl (2 '-O-DMAEO), 2' -O-DMAEO, 2 '-O-N-methylacetamido (2' -O-NMA), 3 '-Phosphonoacetate (PACE) or 3' -phosphonothioacetate (thioPACE). In some aspects, the chemical modification of the ribose sugar component comprises a non-natural nucleic acid. In some cases, the non-natural nucleic acid includes modifications at the 5 '-and 2' -positions of the sugar ring, such as 5'-CH 2 -substituted 2' -O-protected nucleosides. In some cases, the non-natural nucleic acid includes amide linked nucleoside dimers that have been prepared for incorporation of oligonucleotides, wherein the 3' linked nucleosides (5 ' to 3 ') in the dimers comprise 2' -OCH 3 and 5' - (S) -CH 3. The non-natural nucleic acid may comprise a2 '-substituted 5' -CH 2 (or O) modified nucleoside. Non-natural nucleic acids can include 5' -methylenephosphonate DNA and RNA monomers and dimers. The non-natural nucleic acids can include 5' -phosphonate monomers having 2' -substitution and other modified 5' -phosphonate monomers. The non-natural nucleic acid may include a 5' -substituted methylene phosphonate monomer. The non-natural nucleic acid may include 5 'or 6' -phosphoribosyl analogs that contain hydroxyl groups at the 5 'and/or 6' -positions. The non-natural nucleic acids can include 5 '-phosphonate deoxyribonucleoside monomers and dimers having 5' -phosphate groups. The non-natural nucleic acid may include nucleosides having a 6' -phosphonate group in which the 5' or/and 6' -position is unsubstituted or substituted with thio-tert-butyl (SC (CH 3)3) (and analogs thereof), methyleneamino (CH 2NH2) (and analogs thereof) or Cyano (CN) (and analogs thereof).
In some aspects, the non-natural nucleic acid further comprises modification of the sugar moiety. In some cases, the nucleic acid contains one or more nucleosides, wherein the glycosyl has been modified. Such sugar modified nucleosides can confer enhanced nuclease stability, increased binding affinity, or some other beneficial biological property. In certain embodiments, the nucleic acid comprises 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 two ring atoms to form a bicyclic nucleic acid; substitution of a ribosyl epoxy atom with S, N (R), or C (R 1)(R2)(R＝H、C1-C12 alkyl or a protecting group), and combinations thereof.
In some cases, the oligonucleotides described herein comprise a modified sugar or sugar analog. Thus, in addition to ribose and deoxyribose, the sugar moiety may be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a sugar "analog" cyclopentyl group. The sugar may be in the form of a pyranosyl or furanosyl group. The sugar moiety may be a furanoside of ribose, deoxyribose, arabinose, or 2' -O-alkylribose, and the sugar may be attached to the respective heterocyclic base in an [ 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, the sugar modification may include 2 '-O-methyl-uridine or 2' -O-methyl-cytidine. Sugar modifications include 2 '-O-alkyl substituted deoxyribonucleosides and 2' -O-ethyleneglycol-like ribonucleosides.
Modifications to the sugar moiety include natural modifications of ribose and deoxyribose and non-natural modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; f, performing the process; o-, S-or N-alkyl; o-, S-or N-alkenyl; o-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl is, Alkenyl and alkynyl groups may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl groups. 2' sugar modifications also include, but are not limited to -O[(CH2)nO]mCH3、-O(CH2)nOCH3、-O(CH2)nNH2、-O(CH2)nCH3、-O(CH2)nONH2 and-O (CH 2)nON[(CH2)n CH3)]2, where n and m are from 1 to about 10. Other chemical modifications at the 2' position include, but are not limited to: c 1 -C 10 lower alkyl, substituted lower alkyl, alkylaryl, arylalkyl, O-alkylaryl, O-arylalkyl 、SH、SCH3、OCN、Cl、Br、CN、CF3、OCF3、SOCH3、SO2 CH3、ONO2、NO2、N3、NH2、 heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of the oligonucleotide or groups for improving the pharmacodynamic properties of the oligonucleotide and other substituents with similar properties. Similar modifications can also be made at other positions of the sugar, particularly at the 3 'position of the sugar and at the 5' position of the 5 'terminal nucleotide on the 3' terminal nucleotide or in the 2'-5' linked oligonucleotide. Chemically modified saccharides also include saccharides that contain modifications in the bridging epoxy, such as CH 2 and S. Nucleotide sugar analogs may also have sugar mimics, such as cyclobutyl moieties in place of the pentofuranosyl sugar. Examples of nucleic acids having modified sugar moieties include, but are not limited to, nucleic acids comprising 5 '-vinyl, 5' -methyl (R or S), 4'-S, 2' -F, 2'-OCH 3, and 2' -O (CH 2)2OCH3 substituents). The substituents at the 2' position may also be selected from allyl, amino, azido, thio, O-allyl, O- (C 1-C1O alkyl )、OCF3、O(CH2)2SCH3、O(CH2)2-O-N(Rm)(Rn),, and O-CH 2-C(＝O)-N(Rm)(Rn), where each R m and R n is independently H or a substituted or unsubstituted C 1-C10 alkyl.
In certain embodiments, the nucleic acids described herein comprise one or more double-loop nucleic acids. In some of these embodiments, the bicyclic nucleic acid comprises a bridge between the 4 'and 2' ribosyl ring atoms. In certain embodiments, the nucleic acids provided herein include one or more bicyclic nucleic acids, wherein bridging includes 4 'to 2' bicyclic nucleic acids. Examples of such 4 'to 2' bicyclic nucleic acids include, but are not limited to, one of formulas ：4'-(CH2)-O-2'(LNA);4'-(CH2)-S-2';4'-(CH2)2-O-2'(ENA);4'-CH(CH3)-O-2' and 4'-CH (CH 2OCH3) -O-2' and analogs thereof; 4'-C (CH 3)(CH3) -O-2' and analogues thereof.
Modification of nucleotide bases
In some aspects, the chemical modifications described herein include modifications of nucleotide bases (e.g., nucleobases). Exemplary nucleobases can include adenine (a), thymine (T), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or replaced with oligonucleotides as described herein. The nucleobases of the nucleotides may be independently selected from purines, pyrimidines, purine or pyrimidine analogues. In some aspects, a nucleobase can be a naturally occurring base or a synthetic derivative of a base.
In some aspects, the chemical modifications described herein include modifying uracil. In some aspects, the oligonucleotides described herein comprise at least one chemically modified uracil. Exemplary chemically modified uracils may include pseudouridine, pyridin-4-riboketoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine, 5-methoxy-uridine, uridine 5-oxyacetic acid 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-2-thio-uridine, 5-methylaminomethyl-2-seleno-uridine, 5-carbamoylmethyl-uridine, 5-carboxymethyl aminomethyl-2-thio-uridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurine methyl-uridine, 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uridine, l-taurine methyl-4-thio-pseudouridine, 5-methyl-uridine, 1-methyl-pseudouridine, 5-methyl-2-thio-uridine, l-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, dihydrobaicalin, dihydropseudouridine, 5, 6-dihydrouridine, 5-methyldihydrouridine, 2-thiodihydrouridine, 2-thiodihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 2-thiodihydrouridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3- (3-amino-3-carboxypropyl) uridine, 1-methyl-3- (3-amino-3-carboxypropyl-pseudouridine, 5- (isopentenylaminomethyl) uridine, 5- (isopentenylaminomethyl) 2-thiouridine, a-thiouridine, 2 '-O-methyl-uridine, 5,2' -O-dimethyl-uridine, 2 '-O-methyl-pseudouridine, 2-thio-2' -O-methyl-uridine, 5-methoxycarbonylmethyl-2 '-O-methyl-uridine, 5-carbamoylmethyl-2' -O-methyl-uridine, 5-carboxymethyl aminomethyl-2 '-O-methyl-uridine, 3,2' -O-dimethyl uridine, 5- (isopentenyl aminomethyl) -2 '-O-methyl uridine, l-thiouridine, deoxythymidine, 2' -F-ara-uridine, 2 '-F-uridine, 2' -OH-ara-uridine, 5- (2-carboxymethoxyvinyl) uridine, 5- [3- (l-E-acrylamido) uridine, pyrazolo [3,4-d ] pyrimidine, xanthine and hypoxanthine.
In some aspects, the chemical modifications described herein include modifying cytosine. In some aspects, an oligonucleotide described herein comprises at least one chemically modified cytosine. Exemplary chemically modified cytosines may include 5-aza-cytidine, 6-aza-cytidine, pseudoiso-cytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5-halo-cytidine, 5-hydroxymethyl-cytidine, 1-methyl-pseudoiso-cytidine, pyrrolo-cytidine, pyrrole-pseudoiso-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoiso-cytidine, 4-thio-1-methyl-pseudoiso-cytidine, 4-thio-l-methyl-1-deaza-pseudoiso-cytidine, 1-methyl-l-deaza-pseudoiso-cytidine, zebulin, 5-aza-brin, 5-methyl-zebulin, 5-aza-2-thio-zebrin, 2-thio-bunin, 2-methoxy-cytidine, 2-thio-methyl-cytidine, 2-methoxy-2-methyl-cytidine, 4-thio-1-methyl-pseudoiso-cytidine, 4-methyl-cytidine, 2' -O-methyl-cytidine, 2' -methoxy-2-O-methyl-cytidine, 2' -O-dimethylcytidine, 5-formyl-2 ' -O-methyl-cytidine, N4,2' -O-trimethyl-cytidine, 1-thio-cytidine, 2' -F-cytarabine, 2' -F-cytidine, and 2' -OH-cytarabine.
In some aspects, the chemical modifications described herein include modification of adenine. In some aspects, an oligonucleotide described herein comprises at least one chemically modified adenine. Exemplary chemically modified adenine may include 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diamino-adenine, 1-methyl-adenine, 2-methyl-adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6-isopentenyl-adenosine, N6- (cis-hydroxyisopentenyl) adenosine, N6- (cis-hydroxyisoenyl) adenosine, 2-methylthio-N-6- (cis-hydroxypentenyl) adenosine, N-6-hydroxypentenyl-N-6-carbamoyl-adenine, threo-6-carbamoyl-N-amino-6-carbamoyl-N-6-methyl-adenine, threo-amino-N-6-carbamoyl-N-6-methyl-adenine, threo-methyl-N6-carbamoyl-N6-methyl-adenine N6, N6-dimethyl-adenosine, N6-hydroxy-N-valyl-adenosine, 2-methylsulfanyl-N6-hydroxy-N-valyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenine, 2-methylsulfanyl-adenine, 2-methoxy-adenine, a-thio-adenosine, 2' -O-methyl-adenosine, N6,2' -O-dimethyl-adenosine, N6-methyl-2 ' -deoxyadenosine, N6,2' -O-trimethyl-adenosine, l,2' -O-dimethyl-adenosine, 2' -O-ribosyl (phosphate) (Ar (p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2' -F-arabino-adenosine, 2' -F-adenosine, 2' -OH-arabino-adenosine and N6- (19-amino-pentaoxacyclododecyl) -adenosine.
In some aspects, the chemical modifications described herein include modification of guanine. In some aspects, the oligonucleotides described herein comprise at least one chemically modified guanine. Exemplary chemically modified guanines may include inosine, 1-methyl-inosine, hupeside, methyl-hupeside, 4-desmethyl-hupeside, isonicotin, weibutoxin peroxide, hydroxyweibutoxin, non-deiodinated hydroxyweibutose, 7-deaza-guanosine, quinine Wu Gan, epoxy-quinine, galactosyl-quinine Wu Gan, mannosyl-quinine Wu Gan, 7-cyano-7-deaza-guanosine, 7-aminomethyl-7-deaza-guanosine, gulin, 7-deaza-8-aza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2, exemplary chemically modified guanines of N2-dimethyl-guanosine, N2, 7-dimethyl-guanosine, N2, 7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methylthio-guanosine, N2-methyl-6-thio-guanosine may include inosine, 1-methyl-inosine, hurusoside, methyl hurusoside, 4-desmethyl-hurusoside, isoliquiritigenin, weitutin, weitutututututin peroxide, weitutututututin, hydroxyweibutoxin, non-deiodinated hydroxyweibutose, 7-deaza-guanosine, quinine Wu Gan, epoxy-quinine, galactosyl-quinine Wu Gan, mannosyl-quinine Wu Gan, 7-cyano-7-deazaguanosine, 7-aminomethyl-7-deazaguanosine, gulurin, 7-deaza-8-aza guanosine, 6-thio-7-deazaguanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2, N2-dimethyl-guanosine, N2, 7-dimethyl-guanosine, N2, N2, 7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methylsulfanyl-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, l-methyl-2' -O-methyl-guanosine, N2, 7-dimethyl-2 '-O-methyl-guanosine, 2' -O-methyl-inosine, l,2 '-O-dimethyl-inosine, 6-O-phenyl-2' -deoxyinosine, 2 '-O-ribosyl-guanosine, 1-thio-guanosine, 6-O-methyl-guanosine, O 6 -methyl-2' -deoxyguanosine, 2 '-F-cytarabine, and 2' -F-guanosine.
In some cases, chemical modification of an oligonucleotide may include introducing or substituting a nucleic acid analog or non-natural nucleic acid into the oligonucleotide. In some aspects, the nucleic acid analog can be any of the chemically modified nucleic acids described herein. All of which are expressly incorporated by reference in their entirety. Chemically modified nucleotides as 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 has been chemically altered (e.g., by acetylation, methylation, hydroxylation). Exemplary chemically modified nucleotides may include 1-methyl-adenosine, 1-methyl-guanosine, 1-methyl-inosine, 2-dimethyl-guanosine, 2, 6-diaminopurine, 2' -amino-2 ' -deoxyadenosine, 2' -amino-2 ' -deoxycytidine, 2' -amino-2 ' -deoxyguanosine, 2' -amino-2 ' -deoxyuridine, 2-amino-6-chloropurine nucleoside, 2-aminopurine nucleoside, 2' -arabinoside, 2' -arabinosyl cytidine, 2' -arabinoside, 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 ' -deoxycytidine, 2' -fluoro-2 ' -deoxyguanosine, 2' -fluoro-2 ' -deoxyuridine, 2' -fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine, 2-methyl-thio-N6-isopentenyl-adenosine, 2' -O-methyl-2-amino-adenosine, 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-methyl inosine, 2' -O-methyl pseudouridine, 2-thiocytidine, 3-methylcytidine, 4-acetylcytidine, 4-thiouridine, 5- (carboxymethyl) -uridine, 5, 6-dihydrouridine, 5-aminoallylcytidine, 5-aminoalldeoxyuridine, 5-bromouridine, 5-carboxymethyl aminomethyl-2-thiouracil, 5-carboxymethyl aminomethyluracil, 5-chloro-cytarabine, 5-fluorouridine, 5-iodouridine, 5-methoxycarbonylmethyl-uridine, 5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-azacytidine, 6-azauridine, 6-chloro-7-deaza-guanosine, 6-chloropurine nucleoside, 6-mercapto-guanosine, 6-methyl-mercaptopurine-nucleoside, 7-deaza-2' -deoxyguanosine, 7-deaza-adenosine, 7-methylguanosine, 8-aza-adenosine, 8-bromoadenosine, 8-bromoguanosine, 8-thioguanosine, 8-oxo-guanosine, benzimidazole nucleoside, beta-D-mannosyl-braided-glycoside, dihydro-uridine, inosine, N1-methyl-adenosine, N6- ([ 6-aminohexyl ] carbamoylmethyl) -adenosine, N6-isopentenyl-adenosine, N6-methyl-adenosine, N7-methyl-xanthosine, N-uracil-5-hydroxy-acetic acid methyl ester, puromycin, pigtail glycoside, uracil-5-glycolic acid methyl ester, weibutoxin, xanthosine and xyloside. In some aspects, the chemically modified nucleic acids described herein comprise at least one chemically modified nucleotide selected from the group consisting of: 2-amino-6-chloropurine nucleoside-5 '-triphosphate, 2-aminopurine-nucleoside-5' -triphosphate, 2-aminoadenosine-5 '-triphosphate, 2' -amino-2 '-deoxycytidine triphosphate, 2-thiocytidine-5' -triphosphate, 2-thiouridine-5 '-triphosphate, 2' -fluorothymidine-5 '-triphosphate, 2' -O-methyl-inosine-5 '-triphosphate, 4-thiouridine-5' -triphosphate, 5-aminoallyl cytidine-5 '-triphosphate, 5-aminoallyl uridine-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 nucleoside-5 ' -triphosphate, 7-deazaguanosine-5 ' -triphosphate, 8-azaadenosine-5 ' -triphosphate, 8-azido adenosine-5 ' -triphosphate, benzoimidazole nucleoside-5 ' -triphosphate, N1-methyladenosine-5 ' -triphosphate, N1-methylguanosine-5 ' -triphosphate, N6-methyladenosine-5 ' -triphosphate, 6-methylguanosine-5 ' -triphosphate, pseudouridine-5 ' -triphosphate, puromycin-5 ' -triphosphate or xanthosine-5 ' -triphosphate. In some aspects, the chemically modified nucleic acids described herein comprise at least one chemically modified nucleotide selected from the group consisting of: pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl uridine, 1-carboxymethyl pseudouridine, 5-propynyluridine, 1-propynylpseudouridine, 5-taurine methyluridine, 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thiouridine, 1-taurine methyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine and 4-methoxy-2-thio-pseudouridine. In some aspects, the artificial nucleic acids described herein comprise at least one chemically modified nucleotide selected from the group consisting of: 5-azacytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methylpseudoisocytidine, pyrrolocytidine, pyrrolopyrrolocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, zebulin, 5-aza-zebulin, 5-methyl-zebulin, 5-aza-2-thio-zebulin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine. In some aspects, the chemically modified nucleic acids described herein comprise at least one chemically modified nucleotide selected from the group consisting of: 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyladenosine, N6-isopentenyl adenosine, N6- (cis-hydroxyisopentenyl) adenosine, 2-methylsulfanyl-N6- (cis-hydroxyisopentenyl) adenosine, N6-glycyl formyl adenosine, N6-threonyl formyl adenosine, 2-methylthio-N6-threonyl formyl adenosine, N6-dimethyl adenosine, 7-methyl adenine, 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 the group consisting of: inosine, 1-methyl-inosine, huacoside, huai Dinggan, 7-deaza-guanosine, 7-deaza-8-aza-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-dimethyl guanosine, 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, a chemically modified nucleic acid described herein comprises at least one chemically modified nucleotide 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-dihydro-uridine, alpha-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolocytidine, inosine, 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, pseudoisocytosine, 6-chloro-purine, N6-methyl-adenosine, alpha-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.
Modified bases of unnatural 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 cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), and, 4-thiouracil, 8-halogeno, 8-amino, 8-mercapto, 8-sulfanyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogeno, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Certain non-natural nucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, 2-aminopropyladenines, 5-propynyluracils, 5-methylcytosines, those that increase duplex formation stability, universal nucleic acids, hydrophobic nucleic acids, hybrid nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracils and 5-propynyluracils. 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkyl derivatives of adenine and guanine, 2-propyl, other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl (-C.ident.C-CH 3) uracil, 5-propynyl cytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, in particular 5-bromo, 5-trifluoromethyl, other 5-substituted adenines 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 pyrimidine, Phenoxazine cytidine ([ 5,4-b ] [ l,4] benzoxazin-2 (3H) -one), phenothiazine cytidine (1H-pyrimidine [5,4-b ] [ l,4] benzothiazin-2 (3H) -one), G-clamp, phenoxazine cytidine (e.g., 9- (2-aminoethoxy) -H-pyrimidinyl [5,4-b ] [ l,4] benzoxazin-2 (3H) -one), carbazole cytidine (2H-pyrimidinyl [4,5-b ] indol-2-one), pyrido-indole cytidine (H-pyrido [3',2':4,5] pyrrolo [2,3-d ] pyrimidin-2-one), compounds in which the purine or pyrimidine base is substituted with other heterocycles, 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluorouracil, 5, 6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil, 2-aminoadenine, 6-thioguanine, 2-thiothymine, 4-thiothymine, 5-propynyluracil, 4-thiouracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2 '-deoxyuridine or 2-amino-2' -deoxyadenosine.
In some cases, the at least one chemical modification includes chemically modifying the 5 'or 3' end of the oligonucleotide, e.g., a 5 'cap or 3' tail. In some aspects, the oligonucleotides comprise chemical modifications comprising 3' nucleotides, which may be stabilized against degradation, for example, by incorporation of one or more modified nucleotides described herein. In this embodiment, uridine may be replaced with modified uridine such as 5- (2-amino) propyluridine and 5-bromouridine or any of the modified uridine described herein; adenosine and guanosine may be replaced by modified adenosine and guanosine, for example with a modification at position 8, for example 8-bromoguanosine, or with any of the modified adenosine or guanosine described herein. In some aspects, a deaza nucleotide, such as 7-deaza-adenosine, can be incorporated into the gRNA. In some aspects, O-and N-alkylated nucleotides, such as N6-methyladenosine, can be incorporated into the gRNA. In some aspects, sugar-modified ribonucleotides can be incorporated, for example, wherein the 2' OH-group is selected from H, -OR, -R (wherein R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, OR sugar), halogen, -SH, -SR (wherein R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, OR sugar), amino (wherein amino can be, for example, NH 2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, OR amino acid); or cyano (-CN) groups. In some aspects, the phosphate backbone can be modified as described herein, for example with phosphorothioate groups. In some aspects, the nucleotides in the gRNA overhang may each independently be modified or unmodified nucleotides, including but not limited to 2' -sugar modifications, such as 2-F2 ' -O-methyl, thymidine (T), 2' -O-methoxyethyl-5-methyluridine (Teo), 2' -O-methoxyethyl adenosine (Aeo), 2' -O-methoxyethyl-5-methylcytidine (m 5 Ceo), or any combination thereof.
In some aspects, an oligonucleotide comprising at least one chemical modification, upon binding to a target RNA, more specifically recruits an endogenous nuclease to reduce expression of the target RNA than an oligonucleotide sharing the same nucleic acid sequence but without any chemical modification, wherein the oligonucleotide comprises at least one chemical modification. In some aspects, an 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 specificity in recruiting an endogenous nuclease to reduce expression of a target RNA as compared to an oligonucleotide sharing the same nucleic acid sequence but without any chemical modification, wherein the oligonucleotide comprises at least one chemical modification.
In some aspects, an oligonucleotide comprising at least one chemical modification has increased resistance to degradation by hydrolysis as compared to an oligonucleotide sharing the same nucleic acid sequence but without any chemical modification, wherein the oligonucleotide comprises at least one chemical modification. In some aspects, an 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 hydrolysis as compared to an oligonucleotide sharing the same nucleic acid sequence but without any chemical modification, wherein the oligonucleotide comprises at least one chemical modification.
In some aspects, an oligonucleotide comprising at least one chemical modification is increased in resistance to degradation by nuclease digestion as compared to an oligonucleotide sharing the same nucleic acid sequence but without any chemical modification, wherein the oligonucleotide comprises at least one chemical modification. In some aspects, an 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 as compared to an oligonucleotide sharing the same nucleic acid sequence but without any chemical modification, wherein the oligonucleotide comprises at least one chemical modification.
In some aspects, an oligonucleotide comprising at least one chemical modification induces less immunogenicity than an oligonucleotide sharing the same nucleic acid sequence but without any chemical modification, wherein the oligonucleotide comprises at least one chemical modification. In some aspects, an 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 less likelihood of inducing immunogenicity than an oligonucleotide sharing the same nucleic acid sequence but without any chemical modification, wherein the oligonucleotide comprises at least one chemical modification.
In some aspects, an oligonucleotide comprising at least one chemical modification induces less innate immune response relative to an oligonucleotide sharing the same nucleic acid sequence but without any chemical modification, wherein the oligonucleotide comprises at least one chemical modification. In some aspects, an 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 less likelihood of inducing an innate immune response as compared to an innate immune response induced by an oligonucleotide sharing the same nucleic acid sequence but without any chemical modification, wherein the oligonucleotide comprises at least one chemical modification.
In some aspects, an oligonucleotide comprising at least one chemical modification is less likely to induce off-target modulation of a target RNA when contacted with the target RNA than off-target modulation of the target RNA induced by an oligonucleotide sharing the same nucleic acid sequence but without any chemical modification, wherein the oligonucleotide comprises at least one chemical modification. In some aspects, an 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 less likelihood of inducing off-target modulation than an oligonucleotide that shares the same nucleic acid sequence but does not have any chemical modification, wherein the oligonucleotide comprises at least one chemical modification.
Delivery method
In some aspects, described herein are methods of delivering an oligonucleotide described herein to a cell. In some aspects, the method comprises delivering the oligonucleotide directly or indirectly to the cell. In some aspects, the method comprises contacting the cell with a composition or oligonucleotide described herein. In some aspects, the method comprises expressing a composition or oligonucleotide described herein in a cell. In some aspects, the oligonucleotide or vector encoding the oligonucleotide may be delivered into the cell by any transfection method described herein. In some aspects, the oligonucleotides may be delivered into the cells by using an expression vector. In the case of expression vectors, the vectors may be readily introduced into the cells described herein by any method in the art. For example, the expression vector may be transferred into the cell by physical, chemical or biological means.
Physical methods for introducing the oligonucleotide or vector encoding the oligonucleotide into the cell may include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene gun, electroporation, and the like. Methods for producing cells comprising a vector and/or an exogenous nucleic acid are suitable for use in the methods herein. One method of introducing an oligonucleotide or a vector encoding an oligonucleotide into a host cell is calcium phosphate transfection.
Chemical means for introducing the oligonucleotide or vector encoding the oligonucleotide into the cell may 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 an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle). Other most advanced methods of nucleic acid targeted delivery are available, such as delivering oligonucleotides or vectors encoding oligonucleotides with targeted nanoparticles or other suitable sub-micron sized delivery systems.
In the case of non-viral delivery systems, an exemplary delivery vehicle is a liposome. It is contemplated that the oligonucleotide or vector encoding the oligonucleotide is introduced into the cell (in vitro, ex vivo or in vivo) using a lipid formulation. In another aspect, the oligonucleotide or vector encoding the oligonucleotide may be conjugated to a lipid. In some aspects, the lipid-bound oligonucleotide or oligonucleotide-encoding carrier is encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule that binds to the liposome and oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing the lipid, mixed with the lipid, combined with the lipid, contained in the lipid as a suspension, contained or complexed with the micelle, or otherwise bound to the lipid. The lipid, lipid/DNA or lipid/expression vector-related composition is not limited to any particular structure in solution. For example, in some aspects they exist in a bilayer structure, such as micelles, or have a "collapsed" structure. Or they may simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which in some aspects are naturally occurring or synthetic lipids. For example, lipids include fat droplets naturally occurring in the cytoplasm as well as a class of compounds containing long chain aliphatic hydrocarbons and derivatives thereof, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
Lipids suitable for use are obtained from commercial sources. The lipid stock solution in chloroform or chloroform/methanol is typically stored at about-20 ℃. Chloroform is used as the only solvent because it evaporates more readily than methanol. "liposome" is a generic term that encompasses various unilamellar and multilamellar lipid carriers formed by the formation of a closed lipid bilayer or aggregate. Liposomes are generally characterized as having a vesicle structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. Phospholipids spontaneously form when suspended in excess aqueous solution. The lipid components self-rearrange before forming a closed structure and entrap water and dissolved solutes between the lipid bilayers. However, compositions having a structure in solution that is different from the normal vesicle structure are also included. For example, in some aspects, the lipid exhibits a micelle structure or exists only as heterogeneous aggregates of lipid molecules. Liposamine-nucleic acid complexes are also contemplated.
In some cases, non-viral delivery methods include lipofection, nuclear transfection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, exosomes, polycations or lipids: cargo conjugates (or aggregates), naked polypeptides (e.g., recombinant polypeptides), naked DNA, artificial viral particles, and drug-enhanced polypeptide or DNA uptake. In some aspects, the delivery methods comprise conjugating or encapsulating a composition or oligonucleotide described herein with at least one polymer, such as a natural polymer or a synthetic material. The polymer may be biocompatible or biodegradable. Non-limiting examples of suitable biocompatible, biodegradable synthetic polymers may include aliphatic polyesters, poly (amino acids), co-poly (ether-esters), polyalkylene oxalates, polyamides, poly (iminocarbonates), polyorthoesters, polyesteramides, amine group-containing polyoxoesters, and poly (anhydrides). Such synthetic polymers may be homopolymers or copolymers (e.g., random, block, segmented, grafted) of a variety of different monomers, such as two or more of lactic acid, lactide, glycolic acid, glycolide, epsilon-caprolactone, trimethylene carbonate, p-dioxanone, and the like. In one example, the scaffold may be composed of a polymer comprising glycolic acid and lactic acid, such as those having a ratio of glycolic acid to lactic acid of 90/10 or 5/95. Non-limiting examples of naturally occurring biocompatible, biodegradable polymers can include glycoproteins, proteoglycans, polysaccharides, glycosaminoglycans (GAGs) and fragments derived from these components, elastin, laminin, decrorin, fibrinogen/fibrin, fibronectin, osteopontin, tenascin, hyaluronic acid, collagen, chondroitin sulfate, heparin, heparan sulfate, ORC, carboxymethyl cellulose, and chitin.
In some cases, the oligonucleotides described herein or vectors encoding the oligonucleotides can be packaged and delivered to cells by extracellular vesicles. The extracellular vesicles may be any membrane-bound particles. In some aspects, the extracellular vesicles may be any membrane-bound particles secreted by at least one cell. In some cases, the extracellular vesicles may be any membrane-bound particles synthesized in vitro. In some cases, the extracellular vesicles may be any membrane-bound particles synthesized in the absence of cells. In some cases, the extracellular vesicles may be exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, cancer bodies, exosomes, enveloped viruses, exosomes, or other very large extracellular vesicles.
In some cases, the oligonucleotides or vectors encoding oligonucleotides described herein may be administered to a subject in need thereof by using transgenic cells produced by first introducing the oligonucleotides or vectors encoding oligonucleotides into allogeneic or autologous cells. In some cases, the cells may be isolated. In some aspects, cells may be isolated from a subject.
In some aspects, the oligonucleotides described herein are conjugated. In some aspects, the oligonucleotide is conjugated to a peptide, antibody, lipid, carbohydrate, or polymer. In some aspects, the oligonucleotide is conjugated to a peptide, antibody, lipid, carbohydrate, or polymer at the 5' end of the oligonucleotide. In some aspects, the oligonucleotide is conjugated to a peptide, antibody, lipid, carbohydrate, or polymer at the 3' end of the oligonucleotide. In some aspects, the oligonucleotide is conjugated to a peptide, antibody, lipid, carbohydrate, or polymer at any nucleic acid residue of the oligonucleotide. In some aspects, a peptide, antibody, lipid, carbohydrate, or polymer conjugated to an oligonucleotide imparts a therapeutic effect. For example, the peptide, antibody, lipid, carbohydrate or polymer conjugated to the oligonucleotide may be a cytotoxic drug or a drug for the treatment of cancer. In some aspects, a peptide, antibody, lipid, carbohydrate, or polymer conjugated to an oligonucleotide increases the efficiency of binding of the oligonucleotide to endogenous nucleic acid. In some aspects, a peptide, antibody, lipid, carbohydrate, or polymer conjugated to an oligonucleotide confers targeted specificity to a particular type of cell (e.g., cancer cell, etc.). In some aspects, a peptide, antibody, lipid, carbohydrate, or polymer conjugated to an oligonucleotide imparts stability to the oligonucleotide in vitro, ex vivo, or in vivo. For example, the oligonucleotides may be conjugated with polyethylene glycol (PEG) or endosomolytic agents to reduce immunogenicity or degradation. In some aspects, a peptide, antibody, lipid, carbohydrate, or polymer conjugated to an oligonucleotide facilitates entry of the oligonucleotide into a cell. In some aspects, a peptide, antibody, lipid, carbohydrate, or polymer conjugated to an oligonucleotide facilitates and releases the oligonucleotide into a cell. In some aspects, the peptide, antibody, lipid, carbohydrate, or polymer conjugated to the oligonucleotide comprises at least one targeting moiety for targeting a cell. Non-limiting examples of targeting moieties include signal peptides, chemokines, chemokine receptors, adhesion molecules, antigens, or antibodies.
The linker used to conjugate the oligonucleotide to the peptide, antibody, lipid or polymer may be any linker that links to the biomolecule. In some aspects, the linkers described herein are cleavable linkers or non-cleavable linkers. In some cases, the joint is a cleavable joint. In other cases, the joint is a non-cleavable joint. In some cases, the linker is a non-polymeric linker. Non-polymeric linkers refer to linkers that do not contain monomeric repeat units resulting from the polymerization process. In some aspects, the linker comprises a peptide moiety. In some cases, the peptide moiety comprises at least 2,3, 4, 5, or 6 or more amino acid residues. In some aspects, the linker comprises a benzoic acid group or derivative thereof. In some aspects, the linker may comprise a nucleic acid linker, such as a DNA linker. In this case, the peptide, antibody, lipid or polymer may be conjugated on one end of the nucleic acid linker or inserted into a nucleic acid base pairing of the nucleic acid linker. In some aspects, the linker may be a peptide linker. The peptide linker can be flexible (e.g., a poly glycine linker) or rigid (e.g., EAAAK repeat linker). In some aspects, the peptide linker may be cleaved (e.g., disulfide bond). In some aspects, the linker comprises a polymer, such as PEG, polylactic acid (PLA), or polyacrylic acid (PAA).
Therapeutic method
In some aspects, disclosed herein are methods of modulating a KRAS-mediated signaling pathway in a cancer cell by treating or contacting the cancer cell with a composition comprising an antisense oligonucleotide, composition or pharmaceutical composition described herein, thereby reducing expression of KRAS or mutated KRAS protein or mRNA in the cancer cell. In some embodiments, the mutated KRAS protein comprises a G12C mutation, a G12V mutation, a G12A mutation, or a G12D mutation.
In some aspects, also disclosed herein are methods of treating a subject in need thereof by administering to the subject a therapeutically effective amount of an oligonucleotide, composition, or pharmaceutical composition described herein. In some aspects, the methods treat a subject by modulating gene expression or signaling pathway expression in the subject. In some aspects, the methods comprise reducing gene expression by contacting an endogenous nucleic acid (e.g., endogenous mRNA) with an oligonucleotide described herein. In some aspects, the methods comprise reducing KRAS, mutated KRAS, or a combination of KRAS and mutated KRAS in a subject or cancer cell by contacting mRNA of KRAS or mutated KRAS with an oligonucleotide described herein, wherein binding of the oligonucleotide to the mRNA recruits an endogenous nuclease for degrading the mRNA. In some aspects, the method comprises reducing expression of a signaling pathway, such as a KRAS-mediated signaling pathway. In some aspects, the methods comprise reducing gene expression or activity of a KRAS-RAF-MEK-ERK signaling pathway, PI3K signaling pathway, MAPK signaling pathway, or Ral-GEF signaling pathway.
In some aspects, the oligonucleotide, composition, or pharmaceutical composition may be administered to a subject alone (e.g., as a stand-alone treatment). In some aspects, the oligonucleotide, composition, or pharmaceutical composition is administered in combination with an additional agent. In some cases, the additional agents used herein are administered alone. The oligonucleotide, composition or pharmaceutical composition and the additional agent may be administered together or sequentially. Non-limiting examples of additional agents include N- (2- (4- (4-bis (2-chloroethyl) aminophenyl) butanoyl) aminoethyl) -5- (4-carbamimidophenyl) -2-furancarboxamide hydrochloride; allyl isothiocyanate; benzyl isothiocyanate; phenethyl isothiocyanate; bei Linuo he; berberine; ricin; chrysin; bufalin; fisetin; fucoidin; gallic acid; gemcitabine; ramulus Cinnamomi Poria decoction; JOTO1007; quercetin; lasfroning; 2,3,7, 8-tetrachlorodibenzodioxin; triptolide; 4-hydroxy butenolide; or a combination thereof. The combination therapy may be administered within the same day, or may be administered one or more days, weeks, months, or years apart.
In some aspects, the oligonucleotide, composition or pharmaceutical composition is a first line treatment of a disease or disorder. In some aspects, the oligonucleotide, composition or pharmaceutical composition is a two-wire, three-wire or four-wire treatment. In some aspects, the oligonucleotide, composition, or pharmaceutical composition comprises at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, or more oligonucleotides. In general, the methods disclosed herein comprise administering the oligonucleotide, composition, or pharmaceutical composition by oral administration. However, in some cases, the method comprises administering the oligonucleotide, composition, or pharmaceutical composition by intraperitoneal injection. In some cases, the method comprises administering the pharmaceutical composition in the form of an anal suppository. In some cases, the method comprises administering the oligonucleotide, composition, or pharmaceutical composition by intravenous ("i.v.") administration. It is envisioned that the oligonucleotides, compositions, or pharmaceutical compositions disclosed herein may also be administered by other routes, such as subcutaneous injection, intramuscular injection, intradermal injection, transdermal administration, intranasal administration, intralymphatic injection, rectal administration, intragastric administration, or any other suitable parenteral administration. In some aspects, a local delivery route closer to the site of injury or inflammation is preferred over the systemic route. The route, dosage, point of time and duration of administration of the therapeutic agent may be adjusted. In some aspects, the administration of the therapeutic agent is before or after the onset of one or both of the acute and chronic symptoms of the disease or disorder.
Suitable dosages and dosages to be administered to a subject are determined by factors including, but not limited to, the particular oligonucleotide, composition or pharmaceutical composition, the disease condition and severity thereof, the identity of the subject in need of treatment (e.g., body weight, sex, age), and may be determined according to the particular circumstances surrounding the case, including, for example, the particular agent being administered, the route of administration, the condition being treated, and the subject being treated.
In some aspects, the oligonucleotide, composition, or pharmaceutical composition described herein is administered once per 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 10 times. The effective dosage range can be adjusted according to the subject's response to treatment. Some routes of administration will require a higher concentration of the effective amount of therapeutic agent than other routes.
In some aspects, administration of an oligonucleotide, composition, or pharmaceutical composition described herein increases survival of a subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50% or more. In some aspects, the administration of an oligonucleotide, composition, or pharmaceutical composition described herein increases survival of a subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50% or more. In some aspects, the administration schedule of an oligonucleotide, composition, or pharmaceutical composition described herein increases survival of a subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50% or more. In some aspects, the administration doses and schedules of the oligonucleotides, compositions, or pharmaceutical compositions described herein increase 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.
In some aspects, administration of an 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 aspects, an 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 aspects, the administration schedule of an 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 aspects, the administration doses and schedules of the oligonucleotides, compositions, or pharmaceutical compositions described herein result in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50% or more inhibition of tumor growth.
In some aspects, an oligonucleotide, composition, or pharmaceutical composition described herein is administered to a subject in a dose sufficient to inhibit tumor growth. In some aspects, the oligonucleotide, composition, or pharmaceutical composition described herein is administered to a subject at a schedule sufficient to inhibit tumor growth. In some aspects, the oligonucleotide, composition, or pharmaceutical composition described herein is administered to a subject at a dose and schedule sufficient to inhibit tumor growth.
In certain embodiments, when the condition of the subject is not improved, the administration of the pharmaceutical composition is administered chronically, i.e., for a longer period of time, at the discretion of the physician, including throughout the life of the subject, in order to improve or otherwise control or limit symptoms of the disease or disorder in the subject. In certain embodiments, wherein the status of the subject does improve, the dose of the administered pharmaceutical composition may be temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). In specific embodiments, the length of the drug holiday is between 2 days and 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. For example only, the dose reduction during drug holidays is 10% -100%, including for 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 may be temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug transfer"). In specific embodiments, the length of transfer of the pharmaceutical composition is between 2 days and 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. By way of example only, the dose reduction during transfer of the pharmaceutical composition is 10% -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 a suitable length of time, the normal dosing regimen is optionally restored.
In some aspects, once the condition of the subject has improved, a maintenance dose is administered if needed. Subsequently, in specific embodiments, the dosage or frequency of administration, or both, is reduced to a level that maintains an improved disease, disorder, or condition, depending on the symptoms. However, in certain embodiments, once symptoms recur, the subject requires long-term intermittent treatment.
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, determination of the LD50 and the ED 50. The dose ratio between toxicity and therapeutic effect is the therapeutic index and it can be expressed as the LD50/ED50 ratio. In certain embodiments, the data obtained from cell culture assays and animal studies are used in formulating a range of therapeutically effective daily doses and/or therapeutically effective unit doses for use in mammals, including humans. In some aspects, the daily dosage amount of the compositions described herein is within a circulating concentration range with minimal toxicity, including the ED 50. In certain embodiments, the daily dose range and/or unit dose amount varies within this range, depending on the dosage form employed and the route of administration used.
In some aspects, the disease or condition described herein is cancer. In some aspects, the cancer is associated with KRAS. In some aspects, the cancer is associated with mutated KRAS. In some aspects, the cancer is associated with KRAS. In some aspects, the cancer is associated with an abnormality in a KRAS-mediated signaling pathway. In some aspects, the cancer is lung cancer, pancreatic cancer, or colon cancer. other non-limiting examples of cancers may include acute lymphoblastic leukemia, acute Lymphoblastic Leukemia (ALL), acute Myeloid Leukemia (AML), adenoid cystic carcinoma, adrenal carcinoma, adrenocortical carcinoma, adult leukemia, AIDS-related lymphoma, amyloidosis, anal carcinoma, astrocytoma, ataxia telangiectasia, atypical nevus syndrome, atypical malformation/rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, birt Hogg Dube syndrome, bladder carcinoma, bone carcinoma, brain tumor, breast carcinoma, bronchial tumor, burkitt lymphoma, carcinoid (gastrointestinal tract), primary unknown carcinoma, cancer, Heart (heart) tumors, cervical cancer, cholangiocarcinomas, chordoma, chronic Lymphocytic Leukemia (CLL), chronic myelogenous leukemia, chronic myeloproliferative tumors, colorectal cancer, craniopharyngeal tube tumors, cutaneous T-cell lymphomas, ductal carcinoma, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, neuroblastoma, ewing sarcoma, extracranial germ cell tumors, extragonadal germ cell tumors, eye cancer, fallopian tube cancer, bone fibroblastic tumor, malignant and osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor (GIST), germ cell tumors, gestational trophoblastosis, Hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, HER2 positive breast cancer, histiocytosis, langerhans cells, hodgkin's lymphoma, pharyngeal cancer, intraocular melanoma, islet cell tumor, juvenile polyposis syndrome, kaposi's sarcoma, renal cancer, langerhans cell histiocytosis, laryngeal cancer, leukemia, lip cavity cancer, liver cancer, lobular cancer, lung cancer (non-small cells and small cells), lymphoma, malignant fibrous bone histiocytoma and osteosarcoma, malignant glioma, melanoma, intraocular melanoma and meningioma, merck's cell carcinoma, mesothelioma, malignant metastatic cancer, metastatic squamous neck cancer with latent primary carcinoma, In-bundle cancer, multiple endocrine tumor syndrome, multiple myeloma, plasma cell tumor, mycosis fungoides, myelodysplastic syndrome (MDS), myeloproliferative neoplasms, chronic nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, neuroendocrine tumor, non-hodgkin's lymphoma, oral cancer, lip and oral cancer and oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic neuroendocrine tumor, papillomatosis, paraganglioma, paranasal and nasal cavity cancer, parathyroid cancer, penile cancer, peritoneal cancer, peutz-Jeghers syndrome, pharyngeal cancer, pheochromocytoma, Pituitary tumor, plasma cell tumor/multiple myeloma, pleural-lung blastoma, polycythemia vera, pregnancy and breast cancer, primary Central Nervous System (CNS) lymphoma, primary peritoneal carcinoma, prostate cancer, rectal cancer, recurrent cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma, szechuril syndrome, skin cancer, small intestine cancer, soft tissue sarcoma, solid tumor, skin squamous cell carcinoma, neck squamous cell carcinoma with occult primary metastasis, gastric cancer, T-cell lymphoma, testicular cancer, laryngeal carcinoma, thymoma, thymus cancer, thyroid cancer, renal pelvis and ureter transitional cell carcinoma, Unusual childhood cancers, ureters and renal pelvis, transitional cell carcinoma, urinary tract cancer, uterine (endometrial) cancer, uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer, wilms' cell tumor, or combinations thereof.
Pharmaceutical composition
In some aspects, described herein are pharmaceutical compositions comprising the oligonucleotides or compositions described herein. As used herein, a pharmaceutical composition refers to a mixture of the pharmaceutical composition with other chemical ingredients (i.e., pharmaceutically acceptable inactive ingredients) such as carriers, excipients, binders, fillers, suspending agents, flavoring agents, sweeteners, disintegrants, dispersing agents, surfactants, lubricants, colorants, diluents, solubilizers, wetting agents, plasticizers, stabilizers, permeation enhancers, wetting agents, defoamers, antioxidants, preservatives, or one or more combinations thereof. Optionally, the composition comprises two or more pharmaceutical compositions discussed herein. In practicing the methods of treatment or use provided herein, a therapeutically effective amount of a pharmaceutical composition described herein is administered in the form of a pharmaceutical composition to a mammal having a disease, disorder or condition to be treated, such as an inflammatory disease, a fibrotic disease, and/or a fibrotic disease. In some aspects, 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 efficacy of the pharmaceutical composition employed, and other factors. The pharmaceutical compositions may be used alone or in combination with one or more pharmaceutical compositions as a component of a mixture. The pharmaceutical compositions described herein comprise an oligonucleotide, a composition, a cell contacted with an oligonucleotide, or a cell contacted with a composition comprising an oligonucleotide, or a combination thereof.
The pharmaceutical formulations described herein are administered to a subject by suitable routes of administration including, but not limited to, intravenous, intra-arterial, 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, liposome dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast-dissolving formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations.
Pharmaceutical compositions, including pharmaceutical compositions, are manufactured in a conventional manner, such as, by way of example only, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compressing methods.
The pharmaceutical composition may comprise at least the pharmaceutical composition in free acid or free base form or in pharmaceutically acceptable salt form as active ingredient. In addition, the methods and pharmaceutical compositions described herein include the use of N-oxides (if appropriate), crystalline forms, amorphous phases, and active metabolites of these compounds having the same type of activity. In some aspects, the pharmaceutical composition is present in unsolvated form or in solvated form with pharmaceutically acceptable solvents (such as water, ethanol, and the like). Solvated forms of the pharmaceutical compositions are also believed to be disclosed herein.
In some aspects, the pharmaceutical composition exists as a tautomer. All tautomers are included within the scope of the agents presented herein. Thus, it will be appreciated that the pharmaceutical composition or salt thereof may exhibit tautomerism whereby the two compounds are able to readily interconvert by exchanging a hydrogen atom between the two atoms to form a covalent bond with either atom. Tautomeric compounds can be considered as different isomeric forms of the same compound because of their flow equilibrium with each other.
In some aspects, the pharmaceutical composition exists as an enantiomer, diastereomer, or other stereoisomeric form. The agents disclosed herein include all enantiomeric, diastereoisomeric and epimeric forms and mixtures thereof.
In some aspects, the pharmaceutical compositions described herein may be prepared as prodrugs. "prodrug" refers to an agent that is converted in vivo to the parent drug. Prodrugs are generally useful because, in some cases, they are easier to administer than the parent drug. For example, they may be orally available for bioavailability, whereas the parent is not. Prodrugs may also have improved solubility in pharmaceutical compositions compared to the parent drug. Examples of prodrugs are, but are not limited to, pharmaceutical compositions described herein, which are administered as esters ("prodrugs") to facilitate transport across cell membranes, where water solubility is detrimental to fluidity, but which are then metabolically hydrolyzed to carboxylic acids, i.e., active enzymes, which are advantageous once inside the cell. Another example of a prodrug may be a short peptide (polyamino acid) bonded to an acid group, where the peptide is metabolized to reveal the active moiety. In certain embodiments, the prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the pharmaceutical composition when administered in vivo. In certain embodiments, the prodrug is enzymatically metabolized to the biologically, pharmaceutically or therapeutically active form of the pharmaceutical composition by one or more steps or processes.
Prodrug forms of the pharmaceutical compositions are included within the scope of the claims, wherein the prodrugs are metabolized in vivo to produce the agents described herein. Prodrug forms of the pharmaceutical compositions described herein, wherein the prodrugs are metabolized in vivo to produce the agents described herein are included within the scope of the claims. In some cases, some pharmaceutical compositions described herein may be a prodrug of another derivative or active compound. In some embodiments described herein, the hydrazone is metabolized in vivo to produce a pharmaceutical composition.
Medicine box
In some aspects, described herein are kits for using the oligonucleotides, compositions, or pharmaceutical compositions described herein. In some aspects, the kits disclosed herein can be used to treat a disease or disorder in a subject. In some aspects, the kit comprises a collection of materials or components other than the oligonucleotide, composition, or pharmaceutical composition. In some aspects, the kit comprises components for determining and selecting suitable oligonucleotides for treating a disease or disorder. In some aspects, the kit comprises components for performing an assay, such as an enzyme-linked immunosorbent assay (ELISA), single molecule array (Simoa), PCR, or qPCR. The exact nature of the components configured in the kit depends on its intended purpose. For example, some embodiments are configured for treating a disease or disorder disclosed herein (e.g., cancer) in a subject. In some aspects, the kit is specifically configured for treating a mammalian subject. In some aspects, the kit is specifically configured for treating a human subject.
Instructions for use may be included in the kit. In some aspects, the kit comprises instructions for administering the composition to a subject in need thereof. In some aspects, the kit comprises instructions for further engineering the oligonucleotide. In some aspects, the kit comprises instructions to thaw or otherwise restore the biological activity of the oligonucleotides, which may be cryopreserved or lyophilized during storage or transport. In some aspects, the kit comprises instructions for measuring efficacy for its intended purpose (e.g., therapeutic efficacy if used to treat a subject).
Optionally, the kit also contains other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials or other useful personal belongings. The materials or components assembled in the kit may be provided to the operator for preservation in any convenient and suitable manner that maintains its operability and utility. For example, the oligonucleotide, composition or pharmaceutical composition may be in dissolved, dehydrated or lyophilized form. The components are typically contained in a suitable packaging material.
The use of absolute or sequential terms such as "to," "not," "to," "must," "first," "next," "then," "preceding," "following," "last," and "last" are not meant to limit the scope of the embodiments disclosed herein, but are by way of example.
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 that the terms "includes," including, "" has, "" having, "or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term" comprising.
As used herein, the phrases "at least one," "one or more," and/or "are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions "at least one of A, B and C", "at least one of A, B or C", "one or more of A, B and C", "one or more of A, B or C" and "A, B and/or C" means a alone, B alone, C, A and B together, a and C together, B and C together, or A, B and C together.
As used herein, "or" may refer to "and," "or" and/or "and may be used exclusively or inclusively. For example, the term "a or B" may refer to "a or B", "a but not B", "B but not a", and "a and B". In some cases, the context may determine a particular meaning.
Any of the systems, methods, software, and platforms described herein are modular. Thus, terms such as "first" and "second" do not necessarily imply a priority, order of importance, or order of acts.
When referring to a number or range of values, the term "about" means that the number or range of values referred to is an approximation within experimental variability (or within statistical experimental error), and that the number or range of values may vary, for example, from 1% to 15% of the specified number or range of values. In an example, the term "about" refers to ± 10% of a specified number or value.
The terms "increase", "increase" or "increase" as used herein generally mean a static significant amount of increase. In some aspects, the term "increase" or "increase" means an increase of at least 10% compared to a reference level, e.g., an increase of at least about 10%, at least about 20% or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including any increase between 100% or 10-100% compared to a reference level, standard or control. Other examples of "increasing" include increasing by 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 as compared to a reference level.
The terms "reduce", "decrease" or "decrease" are generally used herein to mean a decrease by a statistically significant amount. In some aspects, "reduce" or "reduce" means at least 10% reduction from a reference level, e.g., at least about 20% or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including 100% reduction (e.g., no or undetectable levels as compared to the reference level) or any reduction between 10-100%. In the context of markers or symptoms, these terms refer to a statistically significant reduction in such levels. The reduction may be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and preferably to a level acceptable within the normal range for individuals without the given disease.
While preferred embodiments of the present invention have been shown and described herein, such embodiments are provided by way of example only, as will be apparent to those skilled in the art. It is not intended that the invention be limited to the specific examples provided within the specification. While the invention has been described with reference to the above description, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it is to be understood that all aspects of the invention are not limited to the specific descriptions, constructions, or relative proportions set forth herein depending on various conditions and variables. It should be understood that various alternatives to the inventive aspects described herein may be employed in practicing the invention. Accordingly, it is intended that the present invention also cover any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
TABLE 7 exemplary KRAS antisense oligonucleotides
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TABLE 8 exemplary selected KRAS antisense oligonucleotides
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TABLE 9 exemplary KRAS antisense oligonucleotides targeting mutated KRAS
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TABLE 10 negative control sequences used in the application
SEQ ID NO Nucleic acid sequences
SEQ ID NO:1 CACGTCTATACACCAC
SEQ ID NO:17 TTTCTGAGGATTATCG
Examples
The following illustrative examples represent embodiments of the stimuli, systems, and methods described herein, and are not meant to be limiting in any way.
Example 1 knock-down of KRAS mRNA
Cell culture conditions and in vitro transfection
The NCI-H358 cell line with KRAS G12C mutation was seeded at a density of 20,000 cells per well in 96-well plates and treated with 5nM and 20nM antisense oligonucleotides by transfection with Lipofectamine (Life Technology, USA). Transfection was performed according to the supplier's recommendations, with 0.3 μl Lipofectamine added per well and incubated for 3 hours. After 2 days, cells were harvested and Quantigene assays were performed for relative mRNA quantitative analysis (Life Technology, USA) and following the instructions of the suppliers. The catalogue numbers of KRAS and PPIB (reference gene for normalized expression) probes are SA-50338 and SA-50155, respectively. The percent reduction in mRNA for the non-targeted control oligonucleotides was calculated and summarized in table 1.
TABLE 1 knock-down of KRAS mRNA by antisense oligonucleotides
EXAMPLE 2 inhibition of pERK
The NCI-H358 cell line with KRAS G12C mutation was seeded at a density of 20,000 cells per well in 96-well plates and treated with 5nM and 20nM antisense oligonucleotides by transfection with Lipofectamine (Life Technology, USA). Transfection was performed according to the supplier's recommendations, with 0.3 μl Lipofectamine added per well and incubated for 3 hours. After 4 days, cells were harvested and PERK ALPHALISA assays were performed (Cat. ALSU-PERK-A10K, PERKIN ELMER, USA). pERK inhibition was calculated for each treatment by normalization with non-targeted control oligonucleotides and is summarized in the table below.
TABLE 2 pERK inhibition induced by KRAS knockdown
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Example 3 antisense oligonucleotide mediated growth inhibition of various cancer cells
Various tumor cell lines carrying KRAS mutations (NCI-H358 and LCLC 97. TM.1) and wild-type KRAS (NCI-H1975 and A375) were plated in 384-well plates at a density of 800 cells per well and treated with 5uM and 1uM antisense oligonucleotides by co-incubation. After 7 days, according to the supplier protocol2.0 Assay (Promega, USA) cell viability was measured and growth inhibition was calculated against non-targeted control oligonucleotides. The results are summarized in the following table: pan KRAS ASO and KRAS mutation matched ASO exhibited growth inhibition in NCI-H358 and LCLC97TM1 cells. KRAS ASO has limited effect on NCI-H1975 (EGFR mutant) and no or little effect on a375 (BRAF mutant).
TABLE 3 antisense oligonucleotide mediated growth inhibition in bronchioloalveolar carcinoma
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TABLE 4 antisense oligonucleotide mediated growth inhibition in lung large cell carcinoma
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TABLE 5 antisense oligonucleotide mediated growth inhibition in lung adenocarcinoma cells
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TABLE 6 antisense oligonucleotide mediated growth inhibition in melanoma cells
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Example 4 mRNA and protein knockdown, KRAS G12C mutation by ASO treatment, KRAS pathway biomarker modulation and 3D growth inhibition
Tumor cells were treated with various ASOs, mRNA knockdown by Quantigene assay, KRAS protein and KRAS pathway downstream biomarker analysis by Western blot, and by2.0 Assay cell growth inhibition was performed. mRNA knockdown and cell growth inhibition procedures followed the methods described in example 1 and example 3, respectively. Western blotting was performed using cells transfected using the method described in example 1, and harvested 3 days after transfection for protein analysis. FIG. 1 shows mRNA knockdown of mutant KRAS (H358 cells) with G12C mutation using ASO SEQ ID NO:19, 44, 28, 37, 67 or 80. mRNA knockdown of mutant KRAS with G12C mutation was more pronounced when mediated by ASO SEQ ID NO:28 and ASO SEQ ID NO:67, similar to that of targeting wild-type KRAS (ASO SEQ ID NO:19 and ASO SEQ ID NO: 44). NO significant mRNA knockdown was detected by ASOs designed for targeting KRAS G12D (ASO SEQ ID NO: 12) and targeting KRAS G12V (ASO SEQ ID NO: 80).
For protein level analysis, cells were seeded at 10,000 cells/well in 12-well plates and transfected with ASO/Lipofectamine (3 ul Lipofectamine). Cells were harvested 3 days after transfection for protein expression analysis. The ASO used in the study was ASO SEQ ID NO:28, and the cell lines used were H358 (G12C, heterozygous for KRAS mutation) and A375 (KRAS wild type). FIG. 2 shows knockdown of protein expression of both KRAS and KRAS-RAF-MEK-ERK signaling pathway downstream proteins (pERK, MAPK markers; and pS6, ERK downstream markers). FIG. 2 also shows the increased expression of apoptosis markers (cPARP) mediated by increased amounts of ASO SEQ ID NO:28 for transfection.
FIG. 3 shows another experiment of knockdown of protein expression of both KRAS and KRAS-RAF-MEK-ERK signaling pathway downstream proteins (pERK, MAPK markers; and pS6, downstream markers of ERK). Cells were seeded at 100,000 cells/well in 6-well plates and transfected with ASO/Lipofectamine (3 ul Lipofectamine). Cells were harvested 3 days after transfection for protein expression analysis. ASOs used in the study were ASO SEQ ID NO:28 (G12C ASO,16 mer) and ASO SEQ ID NO:67 (G12C ASO,14 mer). The cell line used was H358 (G12C, heterozygous for KRAS mutation). In cells transfected in a dose-dependent manner with ASO SEQ ID NO:28 or ASO SEQ ID NO:67, protein expression was reduced for both KRAS and proteins downstream of the KRAS-RAF-MEK-ERK signaling pathway (pERK, MAPK markers; and downstream markers of pS6, ERK). FIG. 3 also shows increased expression of apoptosis markers (cPARP) mediated by increased amounts of ASO (ASO SEQ ID NO:28 and ASO SEQ ID NO: 67) for transfection.
Figure 4 shows 3D cell proliferation inhibition due to inhibition of expression of mutated KRAS by contacting cells bearing a KRAS mutation (H358 cells with a KRAS G12C mutation) with an oligonucleotide as described herein. 3D growth of H358 cells (with G12C KRAS mutations) 7 days (left) and 13 days (right) after ASO treatment was measured.
Example 5 mRNA and protein knockdown of KRAS G12V mutations by ASO treatment, KRAS pathway biomarker modulation and 3D growth inhibition
All procedures followed the procedure described in example 4. The cell lines used were NCI-H441 and LCLC97TM1 (KRAS G12V) and A375 (KRAS wild type).
FIG. 5 shows mRNA knockdown of mutant KRAS (NCI-H441 cells) with a G12V mutation using ASO as described herein. FIG. 6 shows KRAS protein knockdown (mediated by ASO SEQ ID NO: 80) of protein expression of both the G12V mutant KRAS and the proteins downstream of the KRAS-RAF-MEK-ERK signaling pathway (pERK and pAKT, both MAPK markers; and pS6, downstream markers of ERK). FIG. 7 shows KRAS protein knockdown (mediated by ASO SEQ ID NO: 81) of protein expression of both the G12V mutant KRAS and the protein downstream of the KRAS-RAF-MEK-ERK signaling pathway (pERK and pAKT, both MAPK markers; and pS6, downstream markers of ERK). FIG. 8 shows the inhibition of 3D cell proliferation due to inhibition of expression of mutant KRAS by contacting cells carrying KRAS mutations (LCLC 97TM1 cells, NCI-H441 cells or CFPAC-1 cells) with the oligonucleotides described herein.
EXAMPLE 6 KRAS G12A tumor cell growth inhibition by ASO treatment
For cell growth inhibition, cells were treated as described in example 3. NCI-H2009 was used as a KRAS G12A mutated tumor cell line. The results are shown in fig. 9. Figure 9 shows the inhibition of 3D cell proliferation due to inhibition of expression of mutated KRAS by contacting cells bearing a KRAS mutation (NCI-H2009 cells) with the oligonucleotides described herein. 3D growth was measured 7 days (left) or 13 days (right) after ASO treatment.
Example 7 mutant KRAS (G12D mutant) mRNA knock-down by ASO treatment
KRAS G12D tumor cells were treated with ASO and KRAS mRNA was detected by Quantigene assay 48 hours after transfection following the procedure in example 1. FIG. 10 shows mRNA knockdown of mutant KRAS (Panc 1 and AsPC1 cells) with G12D mutation with ASO as described herein.
Although the foregoing disclosure has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure. For example, all of the techniques and apparatus described above may be used in various combinations. All publications, patents, patent applications, and/or other documents cited in this disclosure are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent application, and/or other document was specifically and individually indicated to be incorporated by reference for all purposes.

Claims (45)

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 mutated KRAS mRNA.
3. The composition of claim 1 or claim 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 NO. 100-556.
4. The composition of claim 1 or claim 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: 24-43, 65-82 or 87.
5. The composition of claim 1 or claim 2, wherein the antisense oligonucleotide comprises a sequence that is at least 80%, 85% or 90% identical to any one 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: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.
6. The composition of any one of claims 1-3, 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, 24-43, 65-82 or 87.
7. The composition of any one of claims 1-6, wherein the antisense oligonucleotide comprises a length of 12-30 nucleotides.
8. The composition of any one of claims 1-7, 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. The composition of claim 9, wherein each of the 5 '-wing segment and the 3' -wing segment is three linked nucleotides.
11. The composition of any one of claims 1-10, 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. The composition of claim 11, wherein the at least one 2' modified nucleotide: comprising 2 '-O-methyl, 2' -O-methoxyethyl (2 '-O-MOE), 2' -O-aminopropyl, 2 '-deoxy-2' -fluoro, 2 '-O-aminopropyl (2' -O-AP), 2 '-O-dimethylaminoethyl (2' -O-DMAOE), 2 '-O-dimethylaminopropyl (2' -O-DMAP), 2 '-O-dimethylaminoethoxyethyl (2' -O-DMAEOEE) or 2 '-O-N-methylacetamido (2' -O-NMA) modified nucleotides, locked Nucleic Acids (LNA), restricted ethyl (cEt) sugars, vinylnucleic acids (ENA), or combinations thereof.
13. The composition of claim 11, wherein the at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage.
14. The composition of any one of claims 1-13, wherein the antisense oligonucleotide comprises a Phosphorodiamidate Morpholino Oligomer (PMO), locked Nucleic Acid (LNA), thiomorpholino, a restricted ethyl (cEt) sugar, or a combination thereof.
15. The composition of any one of claims 1-14, 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 through 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 mutated KRAS mRNA.
18. The composition of claim 2, wherein the composition comprises an antisense oligonucleotide capable of binding to both KRAS mRNA and mutated KRAS mRNA.
19. The composition of any one of claims 1-18, wherein the composition further comprises an excipient.
20. The composition of any one of claims 1-18, wherein the composition is formulated for parenteral or inhalation administration.
21. The composition of claim 2, wherein the mutated KRAS mRNA encodes a mutated KRAS protein comprising a G12C mutation, a G12V mutation, a G12A mutation, or a G12D mutation.
22. The composition of any one of claims 1-21, 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. 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 any one of claims 1-21, wherein the antisense oligonucleotide comprises a nucleic acid sequence of any one of SEQ ID NOs 19, 27, 28, 37, 44, or 65-81.
25. A method of modulating a KRAS-mediated signaling pathway in a cancer cell, the method comprising: treating the cancer cell with a composition comprising an antisense oligonucleotide capable of binding to KRAS mRNA or mutated KRAS mRNA, thereby reducing expression of KRAS mRNA or mutated KRAS mRNA in the cancer cell.
26. The method of claim 25, wherein the cancer cell is a lung cancer cell, a pancreatic cancer cell, or a colon cancer cell.
27. The method 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 NO. 100-556.
28. The method 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: 24-43, 65-82 or 87.
29. The method 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 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.
30. The method of any one of claims 25-29, 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 mutated KRAS mRNA.
31. The method of any one of claims 25-29, wherein the composition comprises an antisense oligonucleotide capable of binding to both KRAS mRNA and mutated KRAS mRNA.
32. The method of any one of claims 25-31, 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. The method of claim 32, wherein the at least one 2' modified nucleotide: nucleotides comprising 2 '-O-methyl, 2' -O-methoxyethyl (2 '-O-MOE), 2' -O-aminopropyl, 2 '-deoxy-2' -fluoro, 2 '-O-aminopropyl (2' -O-AP), 2 '-O-dimethylaminoethyl (2' -O-DMAOE), 2 '-O-dimethylaminopropyl (2' -O-DMAP), 2 '-O-dimethylaminoethoxyethyl (2' -O-DMAEOE) or 2 '-O-N-methylacetamido (2' -O-NMA) modification; comprising Locked Nucleic Acid (LNA), restricted ethyl (cEt) sugar or Ethylene Nucleic Acid (ENA), thiomorpholino, or a combination thereof.
34. The method of any one of claims 25-33, wherein the expression of KRAS protein, mutated KRAS protein, KRAS mRNA, or mutated KRAS mRNA is reduced by at least 30%, at least 40%, at least 50% after the treatment.
35. The method of any one of claims 25, 26 or 30-34, 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. The method 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. The method of any one of claims 36, wherein the antisense oligonucleotide comprises a nucleic acid sequence of any one of SEQ ID NOs 19, 27, 28, 37, 44 or 65-81.
38. The method of claim 37, wherein the antisense oligonucleotide comprises a nucleic acid sequence of any one of SEQ ID NOs 19, 28, 44, 67, 72-77 or 79-81.
39. A method of treating cancer in a subject in need thereof, the method comprising: administering the composition of any one of claims 1-24 to the subject, thereby treating the cancer in the subject.
40. The method of claim 39, wherein the cancer is associated with an abnormality in a KRAS-mediated signaling pathway.
41. The method of claim 39, wherein the cancer is lung cancer, pancreatic cancer, or colon cancer.
42. The method of claim 39, wherein 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%.
43. The method of claim 39, wherein the composition is administered to the subject at a dose and schedule sufficient to inhibit tumor growth.
44. The method of claim 39, wherein the cancer is associated with KRAS or mutated KRAS.
45. The method of claim 44, wherein the mutated KRAS mRNA encodes a mutated KRAS protein comprising a G12C mutation, a G12V mutation, a G12A mutation, or a G12D mutation.
CN202280072704.2A 2021-09-02 2022-09-01 Compositions and methods for modulating KRAS expression Pending CN118251494A (en)

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