Tunable reversir (TM) compounds

Abstract

To provide agents that modulate the pharmacological activity of siRNAs.SOLUTION: The present invention provides a tunable REVERSIR compound comprising 8 or 9 modified nucleotides, wherein at least three of the modified nucleotides are high affinity monomers and one of the high affinity monomers is base paired with the 6th nucleotide from the 5'-end of the target strand of the siRNA.SELECTED DRAWING: Figure 1

Description

Technical field

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 546,779, filed August 17, 2017 under 35 U.S.C. §119(e), the contents of which are incorporated herein by reference. incorporated herein by.

[0002] The present invention generally relates to oligomeric compounds (oligomers) that target siRNA (e.g., conjugated or unconjugated siRNA) in vivo, thereby improving the pharmacology of RNAi pharmacology and, therefore, siRNA-based therapeutics in vivo. The present invention relates to oligomeric compounds (oligomers) that offer a method of individualized control of therapeutic activity and / or side effects. The present invention also generally relates to methods and systems for use in evaluating the efficacy and safety of pharmaceutical compositions for use in treating or preventing disease. [Background technology]

[0003] In recent years, safety and efficacy data from several clinical trials have provided increasing evidence of the therapeutic potential of RNA interference (RNAi). Small interfering RNA (siRNA) molecules that use the endogenous RNAi pathway potently and specifically suppress mRNA, thereby preventing the formation of disease-causing proteins or proteins involved in disease pathways. Targeted delivery of RNAi therapeutics to hepatocytes is achieved by conjugating chemically modified siRNAs to trivalent N-acetylgalactosamine (GalNAc) ligands, which induce asialoglycoprotein receptor (ASGPR)-mediated tissue specificity. promote target uptake. The development of GalNAc-siRNA conjugates with enhanced stabilization chemistry has resulted in substantial improvements in potency and persistence. In preclinical animal models and humans, GalNAc-siRNA conjugates demonstrated significant duration of action, with potent silencing sustained for several months after a single subcutaneous administration. Along with their long duration of action, RNAi therapeutics can benefit from technologies that allow rapid reversal of silencing activity, thus providing personalized control over RNAi pharmacology, i.e., personalized precision medicine. provide the desired properties. [Summary of the invention] [Means to solve the problem]

[0004] The inventors surprisingly found that adjustment of appropriate levels of the regulatable REVERSIR agent was required to effectively restore the RNAi activity of the siRNA compound after readministration of the same siRNA compound. Tunable persistence of REVERSIR action is achieved by optimization of design parameters that enable efficient recovery of RNAi pharmacodynamics after re-administration of the same siRNA, thereby modulating the therapeutic efficacy of siRNA therapeutics in vivo. Provides control over dissociation and association rates. The tunable persistence of tunable REVERSIR agents can be extended to the control of siRNA effects in designing clinical trials with siRNA therapeutics in humans.

[0005] In some embodiments, the invention includes a modified oligonucleotide comprised of 8 to 10 linked nucleotides (e.g., 8, 9 or 10) and an siRNA compound (e.g., conjugated or unconjugated siRNA). A tunable REVERSIR compound is provided having a nucleobase sequence substantially complementary to at least one strand of. In some embodiments, the regulatable REVERSIR compound comprises a modified oligonucleotide that is comprised of 8 to 10 (e.g., 8, 9, or 10) linking nucleotides and that is substantially equal to the antisense strand of the siRNA compound. It has a nucleobase sequence complementary to . In some embodiments, the regulatable REVERSIR compound comprises a modified oligonucleotide comprised of 8 to 10 (e.g., 8, 9, or 10) linking nucleotides and substantially equal to the sense strand of the siRNA compound. have complementary nucleobase sequences.

[0006] In some such embodiments, the modified oligonucleotide is a single-stranded oligonucleotide and / or is at least 90% complementary to at least one strand of the siRNA. In some embodiments, the modified oligonucleotide is a single-stranded oligonucleotide and / or is at least 90% complementary to the antisense strand of the siRNA. In some embodiments, the modified oligonucleotide is a single-stranded oligonucleotide and / or is at least 90% complementary to the sense strand of the siRNA.

[0007] In some embodiments, the regulatable REVERSIR compound is fully complementary to at least one strand of the conjugated or unconjugated siRNA. In some embodiments, the regulatory REVERSIR compound is fully complementary to the antisense strand of the siRNA. In some embodiments, the regulatory REVERSIR compound is fully complementary to the sense strand of the siRNA.

[0008] In some embodiments, the regulatable REVERSIR compound comprises at least one modified internucleotide or intersugar linkage. In some such embodiments, at least one (eg, including 1, 2, 3, 4, 5, 6, and all) internucleotide linkages are phosphorothioate internucleotide linkages.

[0009] In some embodiments, the regulatable REVERSIR compound includes at least one nucleotide that includes a modified sugar. In certain embodiments, the modified sugar is a bicyclic sugar or sugar that includes 2'-O-methyl or 2'-O-methoxyethyl.

[0010] In some embodiments, the tunable REVERSIR compound comprises one or more (e.g., including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) locked nucleic acid (LNA) monomers. including.

[0011] In some embodiments, each nucleotide in the tunable REVERSIR compound is a 2' modified nucleotide, and the tunable REVERSIR compound has at least one (e.g., 1, 2 or 3) G-clamp nucleobases. including.

[0012] In some embodiments, the regulatable REVERSIR compounds of the invention further include at least one nucleotide that includes a modified nucleobase. In some such embodiments, the modified nucleobase is 5-methylcytosine, 5-methyluracil or 2,6-diaminopurine.

[0013] In some embodiments, tunable REVERSIR compounds include at least one modification. In some such embodiments, the tunable REVERSIR compounds include one or more nucleotide modifications and / or one or more binding modifications. In some embodiments, the tunable REVERSIR compound comprises one or more modifications selected from sugar modifications, linkage modifications, nucleobase modifications, conjugates (eg, ligands), and any combinations thereof.

[0014] In some embodiments, the invention provides tunable REVERSIR compounds that include 8 or 9 modified nucleotides.

[0015] In some embodiments, the present invention provides a tunable REVERSIR compound comprised of 8 or 9 modified nucleotides and a DNA nucleotide linker at the 3'-end of the tunable REVERSIR compound that connects the molecule to the 3'-GalNAc ligand. provide a compound.

[0016] In some embodiments, the regulatable REVERSIR compound comprising at least three (3 or 4) LNA nucleotides and one LNA nucleotide base pairs with the sixth nucleotide from the 5'-end of the target strand of the siRNA. match.

[0017] In some embodiments, the tunable REVERSIR of the invention further comprises no more than three or four phosphorothioate (PS) backbone modifications.

[0018] In some embodiments, a modulatory REVERSIR compound modulates the RISC pathway. In some embodiments, modulatory REVERSIR compounds inhibit the RISC pathway.

[0019] In some embodiments, the invention provides a composition comprising a tunable REVERSIR compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.

[0020] In some embodiments, the activity of siRNA is neutralized by the oligomer-tunable REVERSIR compounds of the invention. In some embodiments, at some point after the administration of the oligomer-tunable REVERSIR compound, the effect of the oligomer-tunable REVERSIR compound to reduce the activity of the siRNA is counteracted by administration of the same siRNA.

[0021] In some embodiments, the action of the regulatable REVERSIR compound allows for drug-on and drug-off control of the therapeutic action of the siRNA agent, in which case the sequential drug-on and drug-off action of the siRNA is controlled by the therapeutic action of the siRNA. This can be accomplished by sequential administration of regulated REVERSIR compounds after and / or before administration.

[0022] In some embodiments, the invention provides methods of inhibiting RNAi activity of conjugated or unconjugated siRNA in a cell. The method generally includes the step of inhibiting RNAi activity in a cell by contacting the cell with a regulatory REVERSIR compound of the invention. In some such embodiments, the cell is in vivo. In some embodiments, the cells are in vitro. In some embodiments, the cells are ex vivo. In some embodiments, the cell is within the subject. In some further embodiments of this, the cells are of animal origin. In some embodiments, the animal is a human.

[0023] In some embodiments, the invention provides a method that includes contacting a cell with a conjugated or unconjugated siRNA; detecting RNAi activity; and contacting a cell with a modulatory REVERSIR compound. In some embodiments, the method of detecting RNAi activity includes measuring the amount of target mRNA present, the amount of target protein present, and / or the activity of the target protein. In some embodiments, such methods include detecting regulatory REVERSIR activity by contacting the cell with a regulatory REVERSIR compound and then measuring RNAi activity. In some such embodiments, the cells are in vivo. In some embodiments, the cell is in an animal. In some embodiments, the animal is a human.

[0024] In some embodiments, the present invention provides a method of ameliorating side effects of siRNA therapeutics, which includes the steps of: contacting a cell with a conjugated or unconjugated siRNA; detecting the side effect; contacting a compound to ameliorate side effects of siRNA, endogenous, exogenous microRNA, or a combination thereof.

[0025] In some embodiments, the invention provides a method of treating a patient, which comprises: administering to the patient a conjugated or unconjugated siRNA; monitoring the patient for siRNA activity; If so, include administering a modulating REVERSIR compound. In some such embodiments, monitoring siRNA activity comprises measuring the amount of target mRNA present, measuring the amount of target protein present, and / or measuring the activity of the target protein. . In some embodiments, such methods include detecting regulatory REVERSIR activity by measuring siRNA activity after administration of a regulatory REVERSIR compound. In some embodiments, the patient is a mammal. In some embodiments, the patient is a human.

[0026] In some embodiments, the invention provides a method of treating a patient, comprising: administering conjugated or unconjugated siRNA to the patient; monitoring the patient for one or more side effects; and 1 If one or more of the side effects reaches an undesirable level, administering a modulating REVERSIR compound. In some embodiments, the patient is a mammal. In some embodiments, the patient is a human.

[0027] In some embodiments, the structure and properties of the tunable REVERSIR compound are designed such that the tunable REVERSIR compound achieves maximal inhibition or reversal of siRNA activity in vivo. For example, maximal inhibition or reversal of siRNA activity can be achieved when the mRNA level is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, up to 100%, and at any time between 5% and 100%. occurs when the value decreases to an integer of .

[0028] In some embodiments, the structure and properties of the tunable REVERSIR compound are designed such that the tunable REVERSIR compound achieves sustained control of inhibition or reversal of siRNA activity in vivo. In some embodiments, persistence is controlled using tunable REVERSIR and requires designing tunable REVERSIR compounds with low phosphorothioate internucleotide linkages and strategic placement of LNA modifications within the oligonucleotide linkages. .

[0029] In some embodiments, the regulatable REVERSIR compound comprises 5, 4 or no more than 3 phosphorothioates and comprises at least three (3 or 4) LNA nucleotides, and one of the LNA nucleotides is an siRNA. base pairs with the 6th nucleotide from the 5'-end of the target strand.

[0030] In some embodiments, the structure and properties of the tunable REVERSIR compounds are designed such that the tunable REVERSIR compounds enable effective re-administration of siRNA and recovery of RNAi in vivo.

[0031] The present invention also generally relates to methods of demonstrating the efficacy and safety of pharmaceutical compositions for use in the treatment or prevention of diseases, such as diseases caused by aberrant expression of target genes. In particular, the present invention provides a means to improve efficacy in situations where pharmaceutical compositions such as siRNA have sustained pharmacodynamics (PD) and long-term safety and efficacy are desirable but not feasible in open-label, single-arm clinical trials. and improved methods for demonstrating safety. The present invention also relates to situations where recruitment of patients is difficult and few subjects receive a placebo or less active therapeutic agent, so detection of therapeutic efficacy and safety may be hampered, such as in rare disease situations.

[0032] Accordingly, the present invention provides various methods and systems for evaluating the efficacy and safety of pharmaceutical compositions for use in the treatment or prevention of disease, and the randomized treatment discontinuation method includes: (1) treating all subjects with the pharmaceutical composition for a first treatment period; (2) deriving mRNA levels and / or physiological outcome measures for all subjects; (3) separating responsive members of the treated subject from non-responsive members; (4) randomizing and stratifying the responder members into at least two further subgroups; (5) subsequently treating members of one subgroup in (4) with a pharmaceutical composition and treating members of the other subgroup with a REVERSIR compound of claim 1 for a second treatment period; (6) deriving mRNA levels and / or physiological outcome measures for these subgroups; (7) comparing the outcome in (6) with the outcome in (2); and (8) Using the comparison in (7) to derive efficacy and safety measures for the pharmaceutical composition. including.

[0033] In some embodiments, the present invention provides various methods and systems for evaluating the efficacy and safety of pharmaceutical compositions for use in treating or preventing disease, and crossover methods include: (1) stratifying the target group into at least two subgroups; (2) treating members of one subgroup with a pharmaceutical composition and treating members of a second subgroup with a blinded placebo for a first treatment period; (3) deriving mRNA levels and / or biomarkers and / or physiological outcome measures for these subgroups; (4) treating members of the treated subgroup with the modulatory REVERSIR compound and treating members of the other blinded placebo subgroup with the pharmaceutical composition for a second treatment period; (5) deriving mRNA levels and / or physiological outcome measures for these subgroups; (6) comparing the outcome in (5) with the outcome in (3); (7) Using the comparison in (6) to derive efficacy and safety measures for the pharmaceutical composition. including. [Brief explanation of the drawing]

[0034]

Figure 1

Figure 2

[0035] It is to be understood that both the foregoing summary and the following detailed description are intended to be illustrative and explanatory and not to limit the invention, as defined in the claims. In this specification, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "comprising" and other forms such as "including" and "included" is also non-limiting. Additionally, terms such as "element" or "component" encompass both elements and components containing one unit and elements and components containing two or more subunits, unless otherwise specified.

[0036] The section headings used herein are for organizational purposes only and should not be construed as limitations on the subject matter described. All documents or portions of documents cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby incorporated by reference in their entirety for all purposes. shall be.

[0037] In some embodiments, the invention provides tunable REVERSIR compounds comprising 8 or 9 modified nucleotides, wherein at least three of the modified nucleotides are high affinity monomers; One base pairs with the 6th nucleotide from the 5'-end of the target strand of the siRNA.

[0038] In some embodiments, the high affinity monomer is LNA.

[0039] In some embodiments, the regulatable REVERSIR compound comprises 3 or 4 LNA nucleotides.

[0040] In some embodiments, the regulatable REVERSIR compound is a single-stranded oligonucleotide that has at least 90% complementarity to the antisense strand.

[0041] In some embodiments, the regulatable REVERSIR compound is fully complementary to the antisense strand.

[0042] In some embodiments, the regulatable REVERSIR compound comprises at least one modified internucleotide linkage.

[0043] In some embodiments, the tunable REVERSIR compound comprises an internucleotide linkage that is a phosphorothioate.

[0044] In some embodiments, tunable REVERSIR compounds contain no more than three or four phosphorothioate modifications.

[0045] In some embodiments, a tunable REVERSIR compound is conjugated to a ligand.

[0046] In some embodiments, the tunable REVERSIR compound is [ka] Contains a ligand that is

[0047] In some embodiments, the tunable REVERSIR compound includes a ligand conjugated to the 3'-terminus of the compound.

[0048] In some embodiments, the regulatory REVERSIR compound comprises a deoxy sugar that is 2'-deoxyribose.

[0049] In some embodiments, the invention provides a kit comprising a tunable REVERSIR compound comprising 8 or 9 modified nucleotides, wherein at least three of the modified nucleotides are high affinity monomers and One of the affinity monomers base pairs with the 6th nucleotide from the 5'-end of the target strand of the siRNA.

[0050] In some embodiments, the invention provides kits comprising siRNA and a tunable REVERSIR compound comprising 8 or 9 modified nucleotides, wherein at least three of the modified nucleotides are high affinity monomers. and one of the high affinity monomers base pairs with the 6th nucleotide from the 5'-end of the target strand of the siRNA.

[0051] In some embodiments, the invention provides a method or system for evaluating the efficacy and safety of a pharmaceutical composition for use in treating or preventing a disease, the method comprising: (1) treating all subjects with the pharmaceutical composition for a first treatment period; (2) deriving mRNA levels and / or physiological outcome measures for all subjects; (3) separating responsive members of the treated subject from non-responsive members; (4) randomizing and stratifying the responder members into at least two further subgroups; (5) subsequently treating members of one subgroup in (4) with a pharmaceutical composition and treating members of the other subgroup with a REVERSIR compound of claim 1 for a second treatment period; (6) deriving mRNA levels and / or physiological outcome measures for these subgroups; (7) comparing the outcome in (6) with the outcome in (2); and (8) Using the comparison in (7) to derive efficacy and safety measures for the pharmaceutical composition. including.

[0052] In some embodiments, the invention provides a method or system for evaluating the efficacy and safety of a pharmaceutical composition for use in treating or preventing a disease, the method comprising: (1) treating all subjects with the pharmaceutical composition for a first treatment period; (2) deriving mRNA levels and / or physiological outcome measures for all subjects; (3) separating responsive members of the treated subject from non-responsive members; (4) randomizing and stratifying the responder members into at least two further subgroups; (5) subsequently treating members of one subgroup in (4) with a pharmaceutical composition and treating members of the other subgroup with a REVERSIR compound of claim 1 for a second treatment period; (6) deriving mRNA levels and / or physiological outcome measures for these subgroups; (7) comparing the outcome in (6) with the outcome in (2); and (8) Using the comparison in (7) to derive efficacy and safety measures for the pharmaceutical composition. In this case, subjects are divided into four subgroups, following the Williams design and randomization in crossover clinical trials (Journal of Statistical Software, V29, February 2009).

[0053] In one example, the number of treatments during the study is 4, and the following sequence of treatments is performed.

[0054]

table 1

[0055] where Drug 1A includes the first dose of the pharmaceutical composition, Drug 2B includes the second dose of the pharmaceutical composition, and ActCtrl is the initial dose of the pharmaceutical composition, active control drug or other standard of care drug. (i.e., other treatments available for the disease), and the placebo includes the group containing the REVERSIR treatment.

[0056] In some embodiments, the invention provides a method or system for evaluating the efficacy and safety of a pharmaceutical composition for use in treating or preventing a disease, where the disease is characterized by an abnormality in a target gene. caused by expression.

[0057] In some embodiments, the invention provides a method or system for evaluating the efficacy and safety of a pharmaceutical composition for use in treating or preventing a disease, wherein the pharmaceutical composition comprises an oligonucleotide. include.

[0058] In some embodiments, the invention provides a method or system for evaluating the efficacy and safety of a pharmaceutical composition for use in treating or preventing a disease, wherein the oligonucleotide is an antisense or siRNA. It is.

[0059] In some embodiments, the invention provides a method or system for evaluating the efficacy and safety of a pharmaceutical composition for use in treating or preventing a disease, wherein the oligonucleotide is a sustainable drug. provided mechanics.

[0060] In some embodiments, the invention provides a method or system for evaluating the efficacy and safety of a pharmaceutical composition for use in treating or preventing a disease, wherein the modulatory REVERSIR compound is used to homogenize and enable randomized treatment withdrawal of members.

[0061] In some embodiments, the modulatory REVERSIR compounds of the invention induce washout of the treated subpopulation.

[0062] In some embodiments, the tunable REVERSIR compounds of the invention reset the baseline of RNAi acidity.

[0063] In some embodiments, siRNA can be readministered at least 1, 2, 3, or 4 weeks after regulated REVERSIR treatment.

[0064] In some embodiments, the target gene is TTR, AGT, ALAS-1, Factor XI, Factor XII, CC5, CC3, GO1, AT3, AAT, Eg5, PCSK9, TPX2, ApoB, SAA, RSV, PDGFβ gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1 / 2 gene, PCNA (p21) gene, MYB gene, JUN gene, FOS gene, BCL -2 gene, cyclin D gene, VEGF gene, EGFR gene, cyclin A gene, cyclin E gene, WNT-I gene, β-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2 / Neu gene, topoisomerase I gene, topoisomerase IIα gene, mutation in p73 gene, mutation in p21 (WAF1 / CIP1) gene, mutation in p27 (KIP1) gene, mutation in PPM1D gene, RAS gene mutations in the caveolin I gene, mutations in the MIB I gene, mutations in the MTAI gene, mutations in the M68 gene, mutations in the tumor suppressor gene, and mutations in the p53 tumor suppressor gene. selected from the group consisting of mutations.

[0065] In some embodiments, the second period is until completion of the open label study.

[0066] In some embodiments, the first period is about 1 month, 2 months or 3 months.

[0067] In some embodiments, the third period is until completion of the clinical trial.

[0068] In some embodiments, the method or system constitutes a clinical trial or a system for conducting a clinical trial to validate a pharmaceutical agent. In some embodiments, the method or system is for evaluating a treatment regimen using a pharmaceutical agent for its effectiveness.

[0069] definition Unless specific definitions are provided, the names used in connection with analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, and the procedures and techniques described herein, are those known in the art. It is well known and commonly used. Standard techniques can be used for chemical synthesis and chemical analysis. Some such techniques and procedures are described, for example, in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994; “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 18th edition, 1990; and “Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratory Manual,” 2nd Edition , Cold Spring Harbor Laboratory Press, 1989, which are incorporated herein by reference for all purposes. To the extent permitted, all patents, applications, published applications and other publications and other data mentioned in this disclosure are incorporated herein by reference in their entirety.

[0070] Unless otherwise indicated, the terms below have the meanings given below.

[0071] As used herein, the term "adjustable REVERSIR" means after about 8, 9, 10, 11, 12, 13, 14 or 15 days, about 50%, 60%, 70%, 80%, 90% refers to an oligomeric REVERSIR compound that neutralizes the activity of siRNA at a protein recovery level of % or full recovery. In one embodiment, the modulatory REVERSIR compound neutralizes the activity of the siRNA after about 8, 9, 10, 11, 12, 13, 14 or 15 days with full recovery levels of protein. In one embodiment, the modulatory REVERSIR compound neutralizes the activity of the siRNA with a protein recovery level of about 70% after about 9, 10, 11 or 12 days.

[0072] In some embodiments, at some point after the time of administration of the oligomer-tunable REVERSIR compound, the effect of the oligomer-tunable REVERSIR compound to reduce the activity of siRNA is after about 15 to 45 days, preferably about 37 to 42 days. neutralized by administration of the same siRNA.

[0073] As used herein, the term "nucleoside" refers to glycosylamines, including nucleobases and sugars. Nucleosides include, but are not limited to, naturally occurring nucleosides, abasic nucleosides, modified nucleosides, and nucleosides with simulated bases and / or sugar groups.

[0074] As used herein, the term "nucleotide" refers to a glycosomine that includes a nucleobase and a sugar to which a phosphate group is covalently attached. Nucleotides can be modified with any of a variety of substituents.

[0075] As used herein, the term "nucleobase" refers to the base portion of a nucleoside or nucleotide. A nucleobase can include any atom or group of atoms that can hydrogen bond to a base of another nucleic acid.

[0076] As used herein, the term "heterocyclic base moiety" refers to a nucleobase that includes a heterocycle.

[0077] As used herein, the term "oligomeric compound" refers to a polymeric structure that includes two or more substructures and is capable of hybridizing with a region of a nucleic acid molecule. In some embodiments, oligomeric compounds are oligonucleotides. In some embodiments, oligomeric compounds are oligonucleotides. In some embodiments, the oligomeric compound is an antisense compound. In some embodiments, the oligomeric compound is a tunable REVERSIR compound. In some embodiments, the oligomeric compound includes a conjugate group.

[0078] As used herein, "oligonucleoside" refers to oligonucleotides in which the internucleoside linkages do not contain phosphorus atoms.

[0079] As used herein, the term "oligonucleotide" refers to an oligomeric compound that includes multiple linked nucleotides. In some embodiments, one or more nucleotides of the oligonucleotide are modified. In some embodiments, the oligonucleotide comprises ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, oligonucleotides are composed of natural and / or non-natural nucleobases, sugars and covalent internucleotide linkages, and may further include non-nucleic acid conjugates.

[0080] As used herein, the term "internucleoside linkage" refers to a non-phosphorus covalent bond between adjacent nucleosides.

[0081] As used herein, the term "internucleotide bond" refers to a covalent phosphorus bond between adjacent nucleotides.

[0082] As used herein, the term "naturally occurring internucleotide linkage" refers to a 3'-5' phosphodiester linkage.

[0083] As used herein, the terms "detecting siRNA activity" or "measuring siRNA activity" mean that a test to detect or measure siRNA activity is performed on a particular sample, and a test on a control sample is performed on a particular sample. means to be compared with Such detection and / or measurement may include a value of zero. Therefore, even if the test for detecting siRNA activity reveals that there is no siRNA activity (zero siRNA activity), the "step of detecting siRNA activity" is still performed.

[0084] As used herein, the term "control sample" refers to a sample that has not been contacted with a reporter oligomer compound.

[0085] As used herein, the term "motif" refers to a pattern of unmodified and modified nucleotides in an oligomeric compound.

[0086] As used herein, the term "regulatory REVERSIR compound" refers to an oligomeric compound that is complementary to and capable of hybridizing with at least one strand of a conjugated or unconjugated siRNA. Without limitation, modulatory REVERSIR compounds can not only block unintended target PD effects, but also any potential off-target activity that may occur with conjugated or unconjugated siRNA.

[0087] As used herein, the term "regulatory REVERSIR activity" refers to either the intensity or duration of any siRNA activity that can be attributed to hybridization of a regulatable REVERSIR compound with one of the strands of the siRNA. Refers to reduction.

[0088] As used herein, the term "mixed backbone oligomeric compound" refers to an oligomeric compound in which at least one internucleotide linkage of the oligomeric compound differs from at least one other internucleotide linkage of the oligomeric compound.

[0089] As used herein, the term "target protein" refers to the protein whose modulation is desired.

[0090] As used herein, the term "target gene" refers to a gene encoding a target protein.

[0091] As used herein, the term "target nucleic acid" refers to any nucleic acid molecule whose expression or activity can be modulated by a conjugated or unconjugated siRNA compound. Target nucleic acids include, but are not limited to, RNA transcribed from DNA encoding the target protein (including, but not limited to, pre-mRNA and mRNA or portions thereof), and cDNA and miRNA obtained from such RNA. For example, a target nucleic acid can be a cellular gene (or an mRNA transcribed from a gene) whose expression is associated with a nucleic acid molecule derived from a particular disorder or disease state or infectious agent.

[0092] As used herein, the term "target siRNA" refers to an siRNA compound that is targeted by a regulatable REVERSIR compound.

[0093] As used herein, the term "targeting" or "targeted" refers to the association of the antisense strand of the siRNA to a particular target nucleic acid molecule or to a particular region of nucleotides within a target nucleic acid molecule.

[0094] As used herein, the term "nucleobase complementarity" refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In some embodiments, a complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a specific position of an antisense compound can form a hydrogen bond with a nucleobase at a specific position of a target nucleic acid, the position of the hydrogen bond between the oligonucleotide and the target nucleic acid is determined by that nucleobase pair. considered complementary.

[0095] As used herein, the term "non-complementary nucleobases" refers to a pair of nucleobases that do not form hydrogen bonds with each other or otherwise do not support hybridization.

[0096] As used herein, the term "complementary" refers to the ability of an oligomeric compound to hybridize with another oligomeric compound or nucleic acid through nucleobase complementarity. In some embodiments, the oligomeric compound and its target are such that a sufficient number of corresponding positions within each molecule are occupied by nucleobases capable of binding to each other such that a stable association between the antisense compound and the target occurs. are complementary to each other when they enable Those skilled in the art will recognize that mismatches can be included without eliminating the ability of the oligomeric compound to maintain association. Accordingly, oligomeric compounds (e.g., regulatable REVERSIR compounds, siRNAs, etc.) that can contain up to about 20% of nucleotides that are mismatched (i.e., not complementary nucleobases to the corresponding nucleotides of the target) are described herein. be done. Preferably, the regulatable REVERSIR compounds and oligomeric compounds such as siRNAs contain no more than about 15% mismatches, more preferably no more than about 10%, most preferably no more than 5% mismatches, or no mismatches at all. The remaining nucleotides are complementary nucleobases or otherwise do not disrupt hybridization (eg, universal bases). Those skilled in the art will appreciate that the compounds provided herein can have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the target nucleic acid. It will be appreciated that % or 100% complementary.

[0097] As used herein, "hybridization" refers to the pairing of complementary oligomeric compounds (eg, the antisense strand of an siRNA and its target nucleic acid or regulatory REVERSIR to the target siRNA). Although not limited to a particular mechanism, the most common mechanisms of pairing involve hydrogen bonding between complementary nucleotides or nucleotide bases (nucleobases), which may be of the Watson-Crick type, Hoogsteen type (Hoogsteen) type or reverse Hoogsteen type hydrogen bonding. For example, the natural base adenine is a complementary nucleobase to the natural nucleobases thymidine and uracil, which pair through the formation of hydrogen bonds. The natural base guanine is a nucleobase that is complementary to the natural bases cytosine and 5-methylcytosine. Hybridization can occur under a variety of circumstances.

[0098] As used herein, the term "specifically hybridizes" refers to the ability of an oligomeric compound to hybridize to one nucleic acid site with higher affinity than to other nucleic acid sites. Point. In some embodiments, the siRNA antisense specifically hybridizes to more than one target site.

[0099] As used herein, "design" or "designed" refers to the process of designing oligomeric compounds that specifically hybridize with selected nucleic acid molecules.

[0100] As used herein, the term "modulation" refers to a perturbation of function or activity as compared to the level of function or activity prior to modulation. For example, modulation includes a change in gene expression, either an increase (stimulation or induction) or a decrease (inhibition or repression). As another example, modulating expression can include perturbing splice site selection of pre-mRNA processing.

[0101] As used herein, the term "expression" refers to all functions and steps by which the encoded information of a gene is converted into a structure that exists and operates within a cell. Such structures include, but are not limited to, products of transcription and translation.

[0102] As used herein, "variant" refers to another RNA transcript that can be produced from the same genomic region of DNA. Variants include, but are not limited to, "pre-mRNA variants," which are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA by their initiation. They differ either in position or end position and include both intronic and exonic sequences. Additionally, some variants may have, but are not limited to, alternative splice sites or alternative start and stop codons.

[0103] As used herein, a "high affinity modified monomer" has at least one modified nucleobase, internucleotide linkage, or sugar moiety as compared to a naturally occurring monomer, and this modification Refers to monomers that increase the affinity of antisense compounds, including high-affinity modified monomers, for target nucleic acids. High affinity modifications include, but are not limited to, monomers containing 2'-modified sugars (eg, nucleosides and nucleotides).

[0104] As used herein, the term "2'-modified" or "2'-substituted" refers to a sugar containing a substituent at the 2' position other than H or OH. 2'-modified monomers include, but are not limited to, 2'-substituents such as allyl, amino, azido, thio, O-allyl, O-C 1 ~C 10 Alkyl, -OCF3, O-(CH 2 ) 2 -O-CH 3 ,2’-O(CH 2 ) 2 SCH 3 ,O-(CH 2 ) 2 -O-N(Rm)(Rn) or O-CH 2 -C(=O)-N(Rm)(Rn), where each Rm and Rn is independently H or a substituted or unsubstituted C 1 ~C 10 It is an alkyl. In some embodiments, the oligomeric compound has the formula 2'-O(CH 2 ) n Includes 2' modified monomers without H (where n is 1-6). In some embodiments, the oligomeric compound has the formula 2'-OCH 3 Contains no 2' modified monomers. In some embodiments, the oligomeric compound has the above formula or, in another embodiment, 2'-O(CH 2 ) 2 OCH 3 Contains no 2' modified monomers.

[0105] As used herein, a "locked nucleic acid" or "LNA" or "locked nucleoside" or "locked nucleotide" means that the furanose portion of the nucleoside contains a bridge connecting two carbon atoms on the furanose ring. , refers to a nucleoside or nucleotide thereby forming a bicyclic system. Locked nucleic acids are also called bicyclic nucleic acids (BNA).

[0106] As used herein, unless otherwise indicated, the term "methyleneoxy LNA" alone refers to β-D-methyleneoxy LNA.

[0107] As used herein, the term "MOE" refers to a 2'-O-methoxyethyl substituent.

[0108] As used herein, the term "gapmer" refers to a chimeric oligomeric compound that includes a central region (the "gap") and regions on either side of the central region (the "wings"), where: The gap includes at least one modification that is different from each wing. Such modifications include nucleobase, monomer linkage and sugar modifications as well as the absence of modification (unmodified). Thus, in some embodiments, the nucleotide linkages in each wing are different than the nucleotide linkages within the gap. In some embodiments, each wing includes nucleotides with a high affinity modification and the gap includes nucleotides without that modification. In some embodiments, the nucleotides within the gap and the nucleotides within the wings all include high affinity modifications, but the high affinity modifications within the gap are different from the high affinity modifications within the wings. In some embodiments, the modifications within the wings are the same as each other. In some embodiments, the modifications within the wings are different from each other. In some embodiments, the nucleotides within the gap are unmodified and the nucleotides within the wings are modified. In some embodiments, the modifications within each wing are the same. In some embodiments, the modifications within one wing are different from the modifications within the other wing. In some embodiments, the oligomeric compound is a gapmer having a 2'-deoxynucleotide in the gap and a nucleotide containing a high affinity modification in the wing.

[0109] As used herein, the term "prodrug" refers to an inactive form (i.e., a drug) that is converted within the body or its cells by the action of endogenous enzymes or other chemicals and / or conditions. Refers to therapeutic agents prepared in active form.

[0110] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of an active compound that retains the desired biological activity of the active compound and does not impart undesirable toxicological effects.

[0111] As used herein, the term "cap structure" or "terminal cap moiety" refers to a chemical modification incorporated at either terminus of an antisense compound.

[0112] As used herein, the term "prevention" refers to delaying or forestalling the onset or development of a condition or disease over a period of hours to days, preferably weeks to months.

[0113] As used herein, the term "improvement" refers to a reduction in at least one activity or one indicator of severity of a condition or disease. The severity of an indicator can be determined by subjective or objective measures well known to those skilled in the art.

[0114] As used herein, the term "treatment" refers to administering a composition of the invention to effect a change or amelioration of a disease or condition. Prevention, amelioration and / or treatment may require the administration of multiple doses at regular intervals or prior to the onset of the condition or disease in order to alter the course of the disease or condition. Additionally, single agents may be used sequentially or simultaneously in each individual for the prevention, amelioration and treatment of a condition or disease.

[0115] As used herein, the term "pharmaceutical agent" refers to a substance that provides a therapeutic benefit when administered to a subject. In some embodiments, the pharmaceutical agent is an active pharmaceutical agent. In some embodiments, the pharmaceutical agent is a prodrug.

[0116] As used herein, the term "therapeutically effective amount" refers to the amount of a pharmaceutical agent that provides a therapeutic benefit to an animal.

[0117] As used herein, the term "administering" means providing a pharmaceutical agent to an animal and includes, but is not limited to, administration by a health care professional and self-administration.

[0118] As used herein, the term "co-administer" means providing two or more pharmaceutical agents to an animal. In some embodiments, two or more such pharmaceutical agents are administered together. In some embodiments, such two or more pharmaceutical agents are administered separately. In some embodiments, two or more such pharmaceutical agents are administered simultaneously. In some embodiments, such two or more pharmaceutical agents are administered at different times. In some embodiments, such two or more pharmaceutical agents are administered by the same route of administration. In some embodiments, such two or more pharmaceutical agents are administered by different routes of administration. In some embodiments, two or more such pharmaceutical agents are contained in the same pharmaceutical formulation. In some embodiments, such two or more pharmaceutical agents are in the form of separate pharmaceutical formulations.

[0119] As used herein, the term "pharmaceutical composition" refers to a mixture of substances suitable for administration to an individual. For example, a pharmaceutical composition can include an antisense oligonucleotide and a sterile aqueous solution. In some embodiments, a pharmaceutical composition includes a pharmaceutical agent and a diluent and / or carrier.

[0120] As used herein, the term "in vitro" refers to events that occur not within an organism (eg, an animal or plant), but in an artificial environment, such as a test tube or reaction vessel, in a cell culture. As used herein, the term "ex vivo" refers to cells that are removed from a living organism and cultured outside the organism (eg, in a test tube). As used herein, the term "in vivo" refers to events that occur within an organism (eg, an animal, plant, and / or microorganism).

[0121] As used herein, the term "subject" or "patient" refers to any living organism to which the compositions disclosed herein can be administered, e.g., for experimental, diagnostic, and / or therapeutic purposes. refers to Typical subjects include animals (eg, mammals such as mice, rats, rabbits, non-human primates and humans) and / or plants. Usually the animal is a vertebrate such as a primate, rodent, livestock or sport animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys and macaques, such as rhesus monkeys (Rhesus). Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and sport animals include cattle, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, canine species such as dogs, foxes, wolves, avian species such as chickens, emus, ostriches and fish. , such as trout, catfish and salmon. Patients or subjects include any of the subsets listed above, such as all of the foregoing, but exclude one or more groups or species such as humans, primates or rodents. In some embodiments of the aspects described herein, the subject is a mammal, eg, a primate, eg, a human. The terms "subject" or "patient" are used interchangeably herein. The subject can be either male (male) or female (female).

[0122] Preferably the subject is a mammal. The mammal can be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples. Non-human mammals can be advantageously used as animal model subjects for human diseases and disorders. Additionally, the compounds, compositions and methods described herein can also be used in livestock and / or pets.

[0123] In one embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or surrogate animal as a disease model. This term does not imply a particular age or gender. Thus, adult and newborn subjects as well as fetuses, whether male (male) or female (female), are intended to be included. Examples of subjects include humans, dogs, cats, cows, goats, and mice. The term subject is also intended to include transgenic species. In some embodiments, the subject may be of European descent. In some embodiments, the subject may be African American. In some embodiments, the subject may be of Asian descent.

[0124] In jurisdictions that prohibit the patenting of methods performed on the human body, "administration" of a composition to a human subject means will be limited to prescribing controlled substances for self-administration (e.g., injection, insertion, etc.). The broadest reasonable interpretation consistent with the laws or regulations defining patentable subject matter is intended. In jurisdictions that do not prohibit patenting of methods performed on the human body, "administration" of a composition includes both methods performed on the human body and the activities described above.

[0125] As used herein, the term "parenteral administration" refers to administration by injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous, intravenous or intramuscular administration.

[0126] As used herein, the term "subcutaneous administration" refers to administration just below the skin. "Subcutaneous administration" means administration intravenously.

[0127] As used herein, the term "dose" refers to a specified amount of a pharmaceutical agent delivered in a single administration. In some embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in some embodiments where subcutaneous administration is desired, the desired dose requires an amount that cannot be readily delivered by a single injection. In such embodiments, more than one injection may be used to achieve the desired dose. In some embodiments, one dose may be administered in two or more injections to minimize injection site reactions in an individual.

[0128] As used herein, the term "unit dosage form" refers to the form in which the pharmaceutical agent is provided. In some embodiments, the unit dosage form is a vial containing lyophilized antisense oligonucleotide. In some embodiments, the unit dosage form is a vial containing reconstituted antisense oligonucleotide.

[0129] As used herein, the term "active pharmaceutical ingredient" refers to a substance in a pharmaceutical composition that confers a desired effect.

[0130] As used herein, the term "side effect" refers to physiological reactions that are believed to result from treatment other than the desired effect. In some embodiments, side effects include, but are not limited to, injection site reactions, liver function test abnormalities, renal function abnormalities, hepatotoxicity, central nervous system abnormalities, and myopathy. For example, elevated levels of aminotransferases in serum may indicate hepatotoxicity or abnormal liver function. For example, increased bilirubin may indicate hepatotoxicity or abnormal liver function.

[0131] As used herein, the term "alkyl," as used herein, refers to a saturated straight or branched hydrocarbon radical containing up to 24 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl, and the like. Alkyl groups generally contain 1 to about 24 carbon atoms, more typically 1 to about 12 carbon atoms (C1-C12 alkyl), and more preferably 1 to about 6 carbon atoms. The term "lower alkyl" as used herein contains 1 to about 6 carbon atoms. Alkyl groups as used herein may optionally include one or more additional substituents.

[0132] As used herein, the term "alkenyl," as used herein, refers to a straight or branched hydrocarbon containing up to 24 carbon atoms and having at least one carbon-carbon double bond. Refers to radicals. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene, and the like. Alkenyl groups generally contain from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms, with 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally contain one or more additional substituents.

[0133] As used herein, the term "alkynyl," as used herein, refers to a straight or branched hydrocarbon radical containing up to 24 carbon atoms and having at least one carbon-carbon triple bond. refers to Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups generally contain from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms, with 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more additional substituents.

[0134] As used herein, the term "aminoalkyl," as used herein, refers to an amino-substituted alkyl radical. The term is meant to include C1-C12 alkyl groups having an amino substituent at any position, where the alkyl group associates the aminoalkyl group with the parent molecule. The alkyl and / or amino portions of the aminoalkyl group can be further substituted with substituents.

[0135] As used herein, the term "aliphatic," as used herein, includes up to 24 carbon atoms and the saturation between any two carbons is a single, double, or triple bond. refers to a straight-chain or branched hydrocarbon radical. Aliphatic groups preferably contain 1 to about 24 carbon atoms, more typically 1 to about 12 carbon atoms, and more preferably 1 to about 6 carbon atoms. Straight or branched chains of aliphatic groups may be interrupted by one or more heteroatoms including nitrogen, oxygen, sulfur and phosphorus. Such heteroatom-cleaved aliphatic groups include, but are not limited to, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include other substituents.

[0136] As used herein, the term "cycloaliphatic" or "alicyclyl" refers to a ring system in which the rings are aliphatic. The ring system may include one or more rings, at least one of which is aliphatic. Preferred alicyclic compounds include rings having from about 5 to about 9 carbon atoms in the ring. As used herein, cycloaliphatic may optionally include other substituents. As used herein, the term "alkoxy," as used herein, refers to a radical formed between an alkyl group and an oxygen atom, where the oxygen atom separates the alkoxy group from the parent molecule. Used to connect to. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy, and the like. Alkoxy groups as used herein may optionally include other substituents. As used herein, the terms "halo" and "halogen," as used herein, refer to atoms selected from fluorine, bromine, and iodine.

[0137] As used herein, the terms "aryl" and "aromatic", as used herein, refer to mono- or polycyclic carbocyclic radicals having one or more aromatic rings. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl, and the like. Preferred aryl ring systems have about 5 to about 20 carbon atoms in the ring or rings. Aryl groups as used herein may optionally include other substituents.

[0138] As used herein, the terms "aralkyl" and "arylalkyl," as used herein, refer to a radical formed between an alkyl group and an aryl group, where the alkyl group is Used to attach an aralkyl group to a parent molecule. Examples include, but are not limited to, benzyl, phenethyl, and the like. Aralkyl groups as used herein may optionally include another substituent attached to the alkyl, aryl, or both groups forming the radical group.

[0139] As used herein, the term "heterocyclic radical," as used herein, contains at least one heteroatom, is unsaturated, partially saturated, or fully saturated, and thus represents a heteroaryl group. Refers to a radical monocyclic or polycyclic ring system containing. Heterocyclic is also meant to include fused ring systems, where one or more of the fused rings contains at least one heteroatom and the other ring contains one or more heteroatoms. optionally free of heteroatoms. Heterocyclic groups typically contain at least one atom selected from sulfur, nitrogen or oxygen. Examples of heterocyclic groups include [1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, Examples include tetrahydrofuryl. Heterocyclic groups as used herein may optionally include other substituents. As used herein, the terms "heteroaryl" and "heteroaromatic," as used herein, mean a single ring in which at least one of the rings is aromatic and contains one or more heteroatoms. Refers to radicals containing cyclic or polycyclic aromatic rings, ring systems, or fused ring systems. Heteroaryl is also meant to include fused ring systems, including systems in which one or more of the fused rings does not contain a heteroatom. A heteroaryl group typically contains one ring atom selected from sulfur, nitrogen or oxygen. Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, Examples include quinoxalinyl. A heteroaryl radical can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or a heteroatom. Heteroaryl groups as used herein may optionally include other substituents.

[0140] As used herein, the term "heteroarylalkyl," as used herein, refers to a heteroaryl as previously defined having an alkyl radical capable of attaching the heteroarylalkyl group to the parent molecule. Refers to the base. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl, naphthyridinylpropyl, and the like. As used herein, a heteroarylalkyl group can optionally include another substituent on one or both of the heteroaryl or alkyl moieties.

[0141] As used herein, the term "monocyclic or polycyclic system" means any ring system that is monocyclic or polycyclic with fused or linked rings, as used in the present invention. and includes single or mixed ring systems selected from aliphatic, cycloaliphatic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic, heteroarylalkyl. Such monocyclic and polycyclic structures can be homogeneous or include rings with varying degrees of saturation, such as fully saturated, partially saturated, or fully unsaturated. Each ring contains ring atoms selected from C, N, O and S, such that rings containing only one C ring atom may be present in heterocycles as well as mixed motifs, e.g. one ring contains only carbon atoms. to form a benzimidazole in which the fused ring has two nitrogen atoms. Monocyclic or polycyclic structures can be further substituted with substituents such as, for example, phthalimide having two =O groups attached to one of the rings. In another aspect, the monocyclic or polycyclic structure can be attached to the parent molecule directly through a ring atom, through a substituent, or through a bifunctional linking molecule.

[0142] As used herein, the term "acyl," as used herein, refers to a radical formed by the removal of a hydroxyl group from an organic acid, which has the general formula -C(O)-X where X is typically aliphatic, cycloaliphatic or aromatic. Examples include aliphatic carbonyl, aromatic carbonyl, aliphatic sulfonyl, aromatic sulfinyl, aliphatic sulfinyl, aromatic phosphate, aliphatic phosphate, and the like. Acyl groups as used herein may optionally include other substituents.

[0143] As used herein, the term "hydrocarbyl" includes groups containing C, O and H. Straight chain, branched and cyclic groups with any degree of saturation are included. Such hydrocarbyl groups may contain one or more heteroatoms selected from N, O and S and may be mono- or polysubstituted with one or more substituents.

[0144] As used herein, the terms "substituent" and "substituent group," as used herein, mean , other groups or groups commonly added to the parent compound. Substituents can be either protected or unprotected and can be attached to one available site or to many available sites within the parent compound. Substituents can also be further substituted with other substituents and can be attached to the parent compound directly or through a linking group such as an alkyl or hydrocarbyl group. Such groups include, but are not limited to, Halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (-C(O)Raa), carboxyl (-C(O)O-Raa), aliphatic group, alicyclic group, alkoxy, substituted oxo (-O-Raa) ), aryl, aralkyl, heterocyclic, heteroaryl, heteroarylalkyl, amino (-NRbbRcc), imino (=NRbb), amide (-C(O)N-RbbRcc or -N(Rbb)C(O)Raa ), azide (-N3), nitro (-NO2), cyano (-CN), carbamide (-OC(O)NRbbRcc or -N(Rbb)C(O)ORaa), ureido (-N(Rbb)C( O)NRbbRcc), thioureido (-N(Rbb)C(S)NRbbRcc), guanidinyl (-N(Rbb)C(=NRbb)NRbbRcc), amidinyl (-C(=NRbb)-NRbbRcc or -N(Rbb) C(NRbb)Raa), thiol (-SRbb), sulfinyl (-S(O)Rbb), sulfonyl (-S(O)2Rbb), sulfonamidyl (-S(O)2NRbbRcc or -N(Rbb)S (O)2Rbb) and conjugate groups, each Raa, Rbb and Rcc independently being H, an optionally linked chemical functionality or another substituent; a preferred list is non-limiting. includes H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, cycloaliphatic, heterocyclic and heteroarylalkyl.

[0145] The regulatable REVERSIR compounds disclosed herein are particularly effective in reducing the activity of siRNA. For example, the regulatable REVERSIR compounds disclosed herein can increase the activity of siRNA by 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 at least about 95%, or at least about 97%, or at least about 99%, or up to 100% (i.e., a non-existent level compared to a reference sample), or 50-100% compared to a reference level. can result in a reduction in any of the ranges. The reference level can be the siRNA activity in the absence of a modulatory REVERSIR compound.

[0146] In some embodiments, the regulated REVERSIR compounds described herein are administered within less than 7 days (e.g., 6 days, 5 days, 4 days, 3 days, 2 days, or within one day), the activity of the siRNA can be reduced by at least about 75%, such as 80%, 85%, 90%, 95% or more, including more complete reduction or inhibition of siRNA activity.

[0147] In some embodiments, the regulatory REVERSIR compound is capable of completely reducing the activity of siRNA within 4 days of administration or use of the regulatory REVERSIR compound. A complete reduction in the activity of the siRNA means a reduction in the activity of the siRNA by at least 80% relative to the reference level.

[0148] oligomeric compounds In some embodiments, the siRNA and / or the regulatable REVERSIR compound is an oligomeric compound. In some embodiments, it is desirable to chemically modify oligomeric compounds, such as siRNA and / or regulatable REVERSIR compounds, as compared to naturally occurring oligomers such as DNA or RNA. Some such modifications alter the activity of oligomeric compounds. Some such chemical modifications, for example, increase the affinity of the siRNA for the target nucleic acid or the affinity of the regulated REVERSIR for the target siRNA, increase its resistance to one or more nucleases, and / or increase the affinity of the oligomeric compound. Activity can be modified by modifying pharmacokinetics or tissue distribution. In some cases, the use of chemistry that increases the affinity of the oligomeric compound for the target may allow shorter oligomeric compound usage.

[0149] monomer In some embodiments, oligomeric compounds include one or more modified monomers. In some such embodiments, the oligomeric compound includes one or more high affinity monomers. In some embodiments, such high affinity monomers are selected from monomers containing 2'-modified sugars (e.g., nucleosides and nucleotides), such as, but not limited to, BNA and 2' - Monomers with substituents (e.g. nucleosides and nucleotides), e.g. allyl, amino, azide, thio, O-allyl, O-C 1 ~C 10 Alkyl, -OCF 3 -, O-(CH 2 ) 2 -O-CH3, 2’-O(CH 2 ) 2 SCH 3 ,O-(CH 2 ) 2 -O-N(Rm)(Rn) or O-CH 2 -C(=O)-N(Rm)(Rn), where each Rm and Rn is independently H or substituted or unsubstituted C 1 ~C 10 It is an alkyl.

[0150] In some embodiments, the regulatable REVERSIR compounds and oligomeric compounds of the invention, such as, but not limited to, siRNA, include one or more high affinity monomers.

[0151] In some embodiments, oligomeric compounds such as, but not limited to, the regulatable REVERSIR compounds and siRNAs of the invention contain one or more β-D-methyleneoxy (4'-CH 2 -O-2’) Contains LNA monomer.

[0152] In some embodiments, oligomeric compounds such as, but not limited to, regulatable REVERSIR compounds and siRNAs of the present invention contain one or more α-D-methyleneoxy (4'-CH 2 -O-2’) Contains LNA monomer.

[0153] In some embodiments, oligomeric compounds such as, but not limited to, regulatable REVERSIR compounds and siRNAs of the invention include one or more (S)-cEt monomers.

[0154] In some embodiments, the oligomeric compounds, such as, but not limited to, the regulatable REVERSIR compounds and siRNAs of the present invention, include one or more high affinity monomers, with the proviso that the oligomeric compounds include 2'-O( CH 2 ) n Does not contain nucleotides containing H (n is 1 to 6).

[0155] In some embodiments, oligomeric compounds such as, but not limited to, regulatable REVERSIR compounds and siRNAs include one or more high affinity monomers, with the proviso that the oligomeric compounds include 2'-OCH 3 or 2’-O(CH 2 ) 2 OCH 3 Contains no nucleotides.

[0156] In some embodiments, one or more oligomeric compounds, such as, but not limited to, regulatable REVERSIR compounds and siRNAs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15 or more), with the proviso that the oligomeric compound contains α-L-methyleneoxy (4'-CH 2 -O-2’) Does not include LNA.

[0157] In some embodiments, oligomeric compounds, such as, but not limited to, regulatable REVERSIR compounds and siRNA, include one or more high affinity monomers, with the proviso that the oligomeric compounds include β-D-methyleneoxy (4 '-CH 2 -O-2’) Does not include LNA.

[0158] In some embodiments, oligomeric compounds, such as, but not limited to, regulatable REVERSIR compounds and siRNA, include one or more high affinity monomers, with the proviso that the oligomeric compounds include α-L-methyleneoxy (4 '-CH 2 -O-2’)LNA or β-D-methyleneoxy(4’-CH 2 -O-2’) Does not include LNA.

[0159] specific nucleobases The naturally occurring base portion of the nucleoside is typically a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. For nucleosides containing pentofuranosyl sugars, the phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, these phosphate groups covalently link adjacent nucleosides to each other to form linear polymeric compounds. Within oligonucleotides, phosphate groups generally refer to those that form the internucleoside or internucleotide backbone of the oligonucleotide. The naturally occurring bond or backbone of RNA and DNA is the 3'-5' phosphodiester bond.

[0160] In addition to the "unmodified" or "natural" nucleobases such as the purine nucleobases adenine (A) and guanine (G) and the pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U), those skilled in the art will appreciate that Many well-known modified nucleobases or nucleobase mimetics can be used in the compounds described herein. Unmodified or naturally occurring nucleobases can be modified or substituted to provide oligonucleotides with improved properties. For example, nuclease-resistant oligonucleotides can be synthesized using any one of these bases or synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine or tubercidine) and the oligomer modifications described herein. , can be prepared. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be used. When a natural base is replaced by a non-natural and / or universal base, the nucleotide is said herein to contain a modified nucleobase and / or a nucleobase modification. Modified nucleobases and / or nucleobase modifications also include natural, non-natural and universal bases that include conjugated moieties, such as the ligands described herein. Preferred conjugate moieties for conjugation with nucleobases include cationic amino groups, which can be conjugated with the nucleobase via a linker with a suitable alkyl, alkenyl or amide bond.

[0161] The oligomeric compounds described herein can also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). include. Exemplary modified nucleobases include, but are not limited to, inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidin, 2-(halo)adenine, 2(alkyl)adenine, 2-(propyl)adenine, 2-(amino) )adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6 -(isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8 -(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N 6 -(isopentyl)adenine, N 6 -(methyl)adenine, N 6 ,N 6-(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7- (deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-( thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl) Cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl) )cytosine, 6-(azo)cytosine, N 4 -(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2- (thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4- (dithio)uracil, 5-(methylaminomethylethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-( Alylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl) Uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2 -(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3 -(Methyl)uracil, 5-uracil (i.e. pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil , 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio) )pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)-pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1-( Aminocarbonylethylenyl)-4-(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(amino alkylaminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio )-pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazine, 1,3- (Diaza)-2-(oxo)-phentiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phentiazin-1-yl, 7-substituted 1,3-(diaza) -2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-( Diaza)-2-(oxo)-phentiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phentiazin-1-yl, 7-(aminoalkylhydroxy)- 1,3-(Diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazine- 1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phentiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio )-3-(aza)-phentiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2- (oxo)-phentiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phentiazin-1-yl, 1,3,5- (Triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularin, tubercidin, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl , nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrylyl, 5-(methyl)isocarbostyrylyl, 3-(methyl)-7-(propynyl)isocarbostyrylyl, 7 -(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbostyryl, 7-(propynyl)iso Carbostyryl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl , pyrenyl, stibenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidine, N 2 -substituted purine, N 6 -substituted purine, O 6 -substituted purines, substituted 1,2,4-triazoles, pyrrolo-pyrimidin-2-one-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-substituted-6-phenyl-pyrrolo -pyrimidin-2-one-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3 -yl, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl , bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl , 2-oxo-pyridopyrimidin-3-yl, or any O-alkylated or N-alkylated derivatives thereof. Alternatively, substituted or modified analogs of any of the bases listed above and "universal bases" may be used.

[0162] As used herein, universal nucleobase refers to all four naturally occurring nucleobases without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of the oligonucleoside duplex. Can base pair. Some exemplary universal nucleic acids include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazole, 4-methyl Benzimidazle, 3-methylisocarbostyrylyl, 5-methylisocarbostyrylyl, 3-methyl-7-propynylisocarbostyrylyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9-Methyl-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrylyl, 7-propynylisocarbostyrylyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6- Mention may be made of dimethylindolyl, phenyl, naphthalenyl, anthracenyl, phenathracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl and structural derivatives thereof (see, for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0163] Further nucleobases are those disclosed in U.S. Patent No. 3,687,808; those disclosed in International Application PCT / U.S. Patent Application No. 09 / 038425 filed March 26, 2009; Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990; English et al., Angewandte Chemie, International Edition, 1991, 30, 613. ; Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijin, p. Ed. Wiley-VCH, 2008; and Sanghvi, Y. S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Examples include those disclosed by Lebleu, B., Eds., CRC Press, 1993. The contents of all these documents are incorporated herein by reference.

[0164] In some embodiments, the modified nucleobase is a nucleobase that is significantly similar in structure to the parent nucleobase, such as, for example, 7-deazapurine, 5-methylcytosine or G-clamp. In some embodiments, the nucleobase mimetic comprises a more complex structure, such as, for example, a tricyclic phenoxazine nucleobase mimetic. Methods for preparing the modified nucleobases described above are well known to those skilled in the art.

[0165] In some embodiments, the tunable REVERSIR compound is [ka] (n is 0, 1, 2, 3, 4, 5 or 6) at least one (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) G-clamp nucleobases selected from.

[0166] specific sugar Oligomeric compounds provided in the invention can include one or more monomers, such as nucleosides or nucleotides, with modified sugar moieties. For example, the furanosyl sugar ring of a nucleoside can be modified in a number of ways, including, but not limited to, addition of substituents, locked nucleic acids or bicyclic nucleic acids by bridging two non-geminal ring atoms. formation, etc. In some embodiments, the oligomeric compound includes one or more monomers that are LNAs.

[0167] In some embodiments of locked nucleic acids, the 2' position of furnaosyl is -[C(R1)(R2)] n -, -[C(R1)(R2)] n -O-, -[C(R1)(R2)] n -N(R1)-, -[C(R1)(R2)] n -N(R1)-O-, -[C(R1R2)] n -O-N(R1)-, -C(R1)=C(R2)-O-, -C(R1)=N-, -C(R1)=N-O-, -C(=NR1)-, -C( =NR1)-O-, -C(=O)-, -C(=O)O-, -C(=S)-, -C(=S)O-, -C(=S)S-, -O-, -Si(R1)2-, -S(=O) X linked to the 4' position by a linker independently selected from - and -N(R1)-; During the ceremony, x is 0, 1 or 2; n is 1, 2, 3 or 4; Each R1 and R2 is independently H, protecting group, hydroxyl, C1~C12 alkyl, substituted C1~C12 alkyl, C2~C12 alkenyl, substituted C2~C12 alkenyl, C2~C12 alkynyl, substituted C2~C12 alkynyl, C5~C20 aryl, substituted C5~C20 aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C5~C7 alicyclic radical, substituted C5~C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1) or sulfoxyl (S(=O)-J1); and Each J1 and J2 independently represents H, C1~C12 alkyl, substituted C1~C12 alkyl, C2~C12 alkenyl, substituted C2~C12 alkenyl, C2~C12 alkynyl, substituted C2~C12 alkynyl, C5~C20 aryl, substituted C5~C20 aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1~C12 aminoalkyl, substituted C1-C12 aminoalkyl or protecting group.

[0168] In one embodiment, each of the linkers of the LNA compound is independently -[C(R1)(R2)] n -, -[C(R1)(R2)] n -O-, -C(R1R2)-N(R1)-O- or -C(R1R2)-O-N(R1)-. In another embodiment, each of said linkers is independently 4'-CH 2 -2’, 4’-(CH 2 ) 2 -2’, 4’-(CH 2 ) 3 -2’, 4’-CH 2 -O-2’, 4’-(CH 2 ) 2 -O-2’, 4’-CH 2 -O-N(R1)-2’ and 4’-CH 2 -N(R1)-O-2'-, where each R1 is independently H, a protecting group, or C1-C12 alkyl.

[0169] Several LNAs have been prepared and disclosed in the patent and scientific literature (Singh et al., Chem.Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630 ;Wahlestedt et al.,Proc.Natl.Acad.Sci.U.S.A.,2000,97,5633-5638;Kumar et al.,Bioorg.Med.Chem.Lett.,1998,8,2219-2222;International Publication No. 94 / 14226 pamphlet; 2005 / 021570 pamphlet; Singh et al., J.Org.Chem., 1998, 63, 10035-10039; Specification No. 7,053,207; Specification No. 6,268,490; Specification No. 6,770,748; Specification No. 6,794,499; Specification No. 7,034,133; and Specification No. 6,525,191; and U.S. Patent Application Publication No. 2004- Specification No. 0171570; Specification No. 2004-0219565; Specification No. 2004-0014959; Specification No. 2003-0207841; Specification No. 2004-0143114; and Specification No. 20030082807. It will be done.

[0170] Furthermore, the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring, thereby making methyleneoxy (4'-CH 2 -O-2') bond to form a bicyclic sugar moiety has also been described (Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2,558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; also U.S. Patent Nos. 6,268,490 and 6,670,461. (see also). The linkage is a methylene (-CH 2 -) group, in which case this bicyclic moiety has the term methyleneoxy (4’-CH 2 -O-2’)LNA is used; if there is an ethylene group in this position, the term ethyleneoxy (4’-CH 2 CH 2 -O-2')LNA is used (Singh et al., Chem.Commun., 1998, 4, 455-456; Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). Methyleneoxy(4’-CH 2 -O-2')LNA and other bicyclic sugar analogs have very high double helix thermostability (Tm=+3~+10°C) with complementary DNA and RNA, 3'-exonuclease Shows stability against degradation and good solubility. Potent and non-toxic antisense oligonucleotides containing BNA have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638).

[0171] Methyleneoxy (4’-CH 2 -O-2’)LNA isomer is α-L-methyleneoxy(4’-CH 2 -O-2')LNA, which has been shown to have excellent stability against 3'-exonucleases. α-L-methyleneoxy(4’-CH 2-O-2')LNA was incorporated into antisense gapmers and chimeras, which showed potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0172] Methyleneoxy(4’-CH 2 -O-2') The synthesis and preparation of LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil have been described, along with their oligomerization and nucleic acid recognition properties (Koshkin et al., Tetrahedron, 1998,54,3607-3630). BNA and its preparation are also described in WO 98 / 39352 and WO 99 / 14226.

[0173] In addition, methyleneoxy (4’-CH 2 -O-2’) analogue of LNA, phosphorothioate-methyleneoxy(4’-CH 2 -O-2')LNA and 2'-thio-LNA have also been prepared (Kumar et al., Bioorg.Med.Chem.Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs containing oligodeoxynucleotide double helices as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99 / 14226). Furthermore, the synthesis of a novel conformationally fixed high affinity oligonucleotide analog, 2'-amino-LNA, has also been described in the art (Singh et al., J.Org.Chem., 1998,63,10035-10039). Furthermore, 2'-amino- and 2'-methylamino-LNAs have been prepared and the thermostability of their double helices with complementary RNA and DNA strands has been previously reported.

[0174] Modified sugar moieties are well known and can be used to modify, typically increase, the affinity of an antisense compound for its target and / or increase nuclease resistance. A representative list of preferred modified sugars includes, but is not limited to, methyleneoxy (4'-CH 2 -O-2’)LNA and ethyleneoxy(4’-(CH 2 ) 2 -O-2' bridge) Bicyclic modified sugars such as ENA; substituted sugars, especially 2'-F, 2'-OCH 3 or 2’-O(CH 2 ) 2 -OCH 3 2'-substituted sugars with substituents; as well as 4'-thio modified sugars. Sugars can also be substituted with sugar mimetic groups, among others. Methods for preparing modified sugars are well known to those skilled in the art. Some representative patents and publications teaching the preparation of such modified sugars include, but are not limited to, U.S. Pat. No. 4,981,957; U.S. Pat. No. 5,118,800; U.S. Pat. No. 5,393,878; No. 5,446,137; No. 5,466,786; No. 5,514,785; No. 5,519,134; No. 5,567,811; No. 5,576,427 Specification: No. 5,591,722; No. 5,597,909; No. 5,610,300; No. 5,627,053; No. 5,639,873; No. 5,646,265; No. 5,658,873 Specification No. 5,670,633; Specification No. 5,792,747; Specification No. 5,700,920; Specification No. 6,531,584; and Specification No. 6,600,032; and International Publication No. 2005 / 121371 pamphlet. .

[0175] Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g. R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethylene glycol (PEG), O(CH 2 CH 2 O) n CH 2 CH 2 O-AMINE or O-(CH 2 ) n AMINE(n=1~10, AMINE=NH 2 ;alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine or polyamino); and O-CH 2 CH 2 (NCH 2 CH 2 NMe 2 ) 2 can be mentioned.

[0176] "Deoxy" modifications include hydrogen (i.e. deoxyribose sugars, particularly associated with protruding single strands); halo (e.g. fluoro); amino (e.g. NH 2 ;alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino or amino acid);NH(CH 2 CH 2 N.H.) n CH 2 CH 2 -AMINE(AMINE=NH 2 ;alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diarylamino);-NHC(O)R(R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar);cyano;mercapto ;alkyl-thio-alkyl; thioalkoxy; thioalkyl; alkyl; cycloalkyl; aryl; alkenyl and alkynyl, which can be optionally substituted, for example with an amino function.

[0177] Other suitable 2'-modifications, such as modified MOEs, are described in US Patent Application Publication No. 20130130378, the contents of which are incorporated herein by reference.

[0178] Modifications at the 2' position may occur in the arabinose configuration. The term "arabinose configuration" refers to the configuration of a substituent on the C2' of the ribose in the same configuration as the 2'-OH in arabinose.

[0179] A sugar can contain two different modifications, such as a gem modification, on the same carbon of the sugar. The sugar group can also contain one or more carbons with the opposite stereochemical configuration than the corresponding one in the ribose. Thus, the oligomeric compound may contain one or more monomers containing, for example, arabinose as sugar. The monomer may have an α-linkage, eg, an α-nucleotide, in position 1 of the sugar. The monomers may also have an opposite configuration at the 4' position, eg, C5' and H4' or the substituents replacing them are exchanged. If C5' and H4' or the substituents replacing them are exchanged, the sugar is said to be 4'-modified.

[0180] Additionally, oligomeric compounds may also include abasic sugars, ie, sugars that lack a nucleobase at C-1' or have another chemical group in place of the nucleobase at C1'. See, eg, US Pat. No. 5,998,203, the contents of which are incorporated herein by reference in their entirety. These abasic sugars may also contain modifications on one or more constituent sugar atoms. Oligomeric compounds can also contain one or more sugars that are L isomers, eg, L-nucleosides. Additionally, modifications to the sugar groups include sulfur, optionally substituted nitrogen or CH 2 It may also include substitution of 4'-O by groups. In some embodiments, the linkage between C1' and the nucleobase is in the alpha configuration.

[0181] Sugar modification is the absence of a C-C bond between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4') and / or Acyclic nucleotides may also be included in which at least one of the ribose carbons or oxygens (eg, C1', C2', C3', C4' or O4'), independently or in combination, is not present in the nucleotide. In some embodiments, the acyclic nucleotide is [ka] , where B is a modified or unmodified nucleobase, and R 1 and R 2 are independently H, halogen, OR 3 or alkyl ;R 3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar).

[0182] In some embodiments, the sugar modifications include 2'-H, 2'-O-Me (2'-O-methyl), 2'-O-MOE (2-O-methoxyethyl), 2'-F , 2'-O-[2-(methylamino)-2-oxoethyl](2'-O-NMA), 2'-S-methyl, 2'-O-CH 2 -(4’-C)(LNA), 2’-O-CH 2 CH 2-(4'-C)(ENA), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethyl aminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) and gem2'-OMe / 2'F with 2'-O-Me in the arabinose configuration selected from the group consisting of.

[0183] If a particular nucleotide is linked to the next nucleotide via its 2' position, then the sugar modifications described herein are linked to the next nucleotide via its 2' position, e.g. It should be understood that the nucleotide can be placed at the 3' position of the sugar. Modifications at the 3' position may be present in the xylose configuration. The term "xylose configuration" refers to the configuration of the substituent on the C3' of the ribose in the same configuration as the 3'-OH of the xylose sugar.

[0184] The hydrogen bonded to C4' and / or C1' can be substituted by linear or branched, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, Alkyl, alkenyl and alkynyl skeletons are O, S, S(O), SO 2 , N(R'), C(O), N(R')C(O)O, OC(O)N(R'), CH(Z'), phosphorus-containing bond, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted heterocyclic or optionally substituted cycloalkyl, where R' is hydrogen, acyl or optionally substituted aliphatic, Z' is OR 11 ,COR 11 , CO 2 R 11 , [ka] ,NR 21 R 31 ,CONR 21 R 31 ,CON(H)NR 21 R 31 ,ONR 21 R 31 , CON(H)N=CR 41 R 51 , N(R 21 )C(=NR 31 )NR 21 R 31 , N(R 21 )C(O)NR 21 R 31 , N(R 21 )C(S)NR 21 R 31 ,OC(O)NR 21 R 31 , SC(O)NR 21 R 31 , N(R 21 )C(S)OR 11 , N(R 21 )C(O)OR 11 , N(R 21 )C(O)SR 11 , N(R 21 )N=CR 41 R 51 ,ON=CR 41 R 51 , S.O. 2 R 11 ,SOR 11 , S.R. 11 and substituted or unsubstituted heterocyclic; 21 and R 31 for each occurrence independently hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 ,COR 11 , CO 2 R 11 or NR 11 R 11 ’ or R 21 and R 31 together with the atoms to which they are attached form a heterocycle;R 41 and R 51 for each occurrence independently hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 ,COR 11 Or CO 2 R 11 or NR 11 R 11 ’ and;R 11 and R 11 ' is independently hydrogen, aliphatic, substituted aliphatic, aryl, heteroaryl or heterocyclic. In some embodiments, the hydrogen attached to C4' of the 5' terminal nucleotide is substituted.

[0185] In some embodiments, C4' and C5' together form an optionally substituted heterocycle, which includes at least one -PX(Y)-, where X is , H, OH, OM, SH, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted alkylamino or optionally substituted dialkylamino where M is, for each occurrence independently, an alkyl metal or a transition metal with a total charge of +1; Y is O, S or NR'; where R' is Hydrogen, optionally substituted aliphatic. Preferably, this modification is at the 5-terminus of the oligonucleotide.

[0186] In some embodiments, the LNA has the formula: [ka] comprising a bicyclic nucleotide having During the ceremony, Bx is a heterocyclic base moiety; T1 is H or a hydroxyl protecting group; T2 is H, a hydroxyl protecting group or a reactive phosphorus group; Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, or substituted amide.

[0187] In one embodiment, each substituted group is independently halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN (each J1, J2 and J3 are independently H or C1-C6 alkyl, and X is O, S or NJ1) Single or multiple substitutions.

[0188] In some such embodiments, each substituted group is independently halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1 and NJ3C(=X)NJ1J2 (each J1, J2 and J3 are independently mono- or polysubstituted with substituents independently selected from H, C1-C6 alkyl or substituted C1-C6 alkyl, and X is O or NJ1 .

[0189] In some embodiments, the Z group is C1-C6 alkyl substituted with one or more Xx, where each Xx is independently OJ1, NJ1J2, SJ1, N3, OC(=X )J1, OC (= It is. In another embodiment, the Z group is C1-C6 alkyl substituted with one or more Xx, where each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3-), substituted alkoxy or azide.

[0190] In some embodiments, the Z group is -CH2Xx, where Xx is OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 or CN (each J1, J2 and J3 is independently H or C1-C6 alkyl, and X is O, S or NJ1). In another embodiment, the Z group is -CH2Xx, where Xx is halo (eg, fluoro), hydroxyl, alkoxy (eg, CH3-), or azido.

[0191] In some embodiments, the Z group has the (R)-configuration: [ka] It is.

[0192] In some embodiments, the Z group has the (S)-configuration: [ka] It is.

[0193] In some embodiments, each T1 and T2 is a hydroxyl protecting group. A preferred list of hydroxyl protecting groups is benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthine-9-yl( pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). In some embodiments, T1 is a hydroxyl protecting group selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldimethylsilyl and dimethoxytrityl; more preferred hydroxyl protecting groups are 4,4'- T1 is dimethoxytrityl.

[0194] In some embodiments, T2 is a reactive phosphorus group, and preferred reactive phosphorus groups include diisopropylcyanoethoxyphosphoramidite and H-phosphate. In some embodiments, T1 is 4,4'-dimethoxytrityl and T2 is diisopropylcyanoethoxyphosphoramidite.

[0195] In some embodiments, the oligomeric compound has the formula: [ka] Or expression: [ka] Or expression: [ka] at least one monomer of During the ceremony, Bx is a heterocyclic base moiety; T3 is H, a hydroxyl protecting group, a linked conjugate group or an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomer subunit or oligomeric compound; T4 is H, a hydroxyl protecting group, a linked conjugate group or an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomer subunit or oligomeric compound; wherein at least one of T3 and T4 is an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomer subunit or oligomeric compound; Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, or substituted amide.

[0196] In one embodiment, each substituted group is independently halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN (each J1, J2 and J3 are independently H or C1-C6 alkyl, and X is O, S or NJ1) , mono- or polysubstituted.

[0197] In one embodiment, each of the substituted groups is independently halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1 and NJ3C(=X)NJ1J2 (each J1, J2 and J3 are independently H or C1-C6 alkyl, and X is O or NJ1).

[0198] In some such embodiments, at least one Z is C1-C6 alkyl or substituted C1-C6 alkyl. In some embodiments, each Z is independently C1-C6 alkyl or substituted C1-C6 alkyl. In some embodiments, at least one Z is C1-C6 alkyl. In some embodiments, each Z is independently C1-C6 alkyl. In some embodiments, at least one Z is methyl. In some embodiments, each Z is methyl. In some embodiments, at least one Z is ethyl. In some embodiments, each Z is ethyl. In some embodiments, at least one Z is substituted C1-C6 alkyl. In some embodiments, each Z is independently substituted C1-C6 alkyl. In some embodiments, at least one Z is substituted methyl. In some embodiments, each Z is substituted methyl. In some embodiments, at least one Z is substituted ethyl. In some embodiments, each Z is substituted ethyl.

[0199] In some embodiments, at least one substituent is C1-C6 alkoxy (eg, at least one Z is C1-C6 alkyl substituted with one or more C1-C6 alkoxy). In another embodiment, each substituent is independently C1-C6 alkoxy (e.g., each Z is independently C1-C6 alkyl substituted with one or more C1-C6 alkoxy) .

[0200] In some embodiments, at least one C1-C6 alkoxy substituent is CH3O- (e.g., at least one Z is CH 3 OCH 2 -is). In another embodiment, each C1-C6 alkoxy substituent is CH 3 O- (e.g., each Z is CH 3 OCH 2 -is).

[0201] In some embodiments, at least one substituent is halogen (eg, at least one Z is C1-C6 alkyl substituted with one or more halogens). In some embodiments, each substituent is independently a halogen (eg, each Z is independently a C1-C6 alkyl substituted with one or more halogens). In some embodiments, at least one substituent is fluoro (e.g., at least one Z is CH 2 FCH 2 -,CHF 2 CH 2 -or CF 3 CH 2 -is). In some embodiments, each halo substituent is fluoro (e.g., each Z is CH 2 FCH 2 -,CHF 2 CH 2 -or CF 3 CH 2 -is).

[0202] In some embodiments, at least one substituent is hydroxyl (eg, at least one Z is C1-C6 alkyl substituted with one or more hydroxyls). In some embodiments, each substituent is independently a hydroxyl (eg, each Z is independently a C1-C6 alkyl substituted with one or more hydroxyl). In some embodiments, at least one Z is HOCH 2 -is. In another embodiment, each Z is HOCH 2 -is.

[0203] In some embodiments, at least one Z is CH 3 -, CH 3 CH 2 -, CH 2 OCH 3 -, CH 2 F- or HOCH 2 -is. In some embodiments, each Z is CH 3 -, CH 3 CH 2 -, CH 2 OCH 3 -, CH 2 F- or HOCH 2 -is.

[0204] In some embodiments, at least one Z group is C1-C6 alkyl substituted with one or more Xx, where each Xx is independently OJ1, NJ1J2, SJ1, N3, OC (=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 or CN; each J1, J2 and J3 is independently H or C1~C6 alkyl, and X is O, S Or NJ1. In another embodiment, at least one Z group is C1-C6 alkyl substituted with one or more Xx, where each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (eg CH3O-) or azide.

[0205] In some embodiments, each Z group is independently C1-C6 alkyl substituted with one or more Xx, where each Xx is independently OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 or CN; each J1, J2 and J3 is independently H or C1~C6 alkyl; X is O, S or NJ1. In another embodiment, each Z group is independently C1-C6 alkyl substituted with one or more Xx, where each Xx is independently halo (e.g., fluoro), hydroxyl, Alkoxy (eg CH3O-) or azide.

[0206] In some embodiments, at least one Z group is -CH 2 Xx, where each Xx is OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 or CN; each J1, J2 and J3 are independently H or C1-C6 alkyl, and X is O, S or NJ1. In some embodiments, at least one Z group is -CH 2 Xx, where each Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH 3 O-) or azide.

[0207] In some embodiments, each Z group is independently -CH 2Xx, where each Xx is independently OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 or CN; each J1, J2 and J3 are independently H or C1-C6 alkyl, and X is O, S or NJ1. In another embodiment, each Z group is independently -CH 2 Xx, where each Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH 3 O-) or azide.

[0208] In some embodiments, at least one Z group is CH 3 -is. In another embodiment, each Z group is CH 3 -is.

[0209] In some embodiments, the Z group of at least one monomer has the formula: [ka] Or expression: [ka] Or expression: [ka] The (R)-configuration represented by

[0210] In some embodiments, the Z group of each monomer of the above formula is in the (R)-configuration.

[0211] In some embodiments, the Z group of at least one monomer has the formula: [ka] Or expression: [ka] Or expression: [ka] This is the (S)-configuration represented by.

[0212] In some embodiments, the Z group of each monomer of the above formula is in the (S)-configuration.

[0213] In some embodiments, T3 is H or a hydroxyl protecting group. In some embodiments, T4 is H or a hydroxyl protecting group. In another embodiment, T3 is a nucleoside, nucleotide or internucleoside linking group attached to a monomer subunit. In some embodiments, T4 is a nucleoside, a nucleotide, or an internucleoside linking group attached to a monomer subunit. In some embodiments, T3 is an oligonucleoside or an internucleoside linking group linked to an oligonucleotide. In some embodiments, T4 is an oligonucleoside or an internucleoside linking group linked to an oligonucleotide. In some embodiments, T3 is an internucleoside linking group linked to an oligomeric compound. In some embodiments, T4 is an internucleoside linking group linked to an oligomeric compound. In some embodiments, at least one of T3 and T4 is an internucleotide linking group selected from phosphodiester or phosphorothioate.

[0214] In some embodiments, the oligomeric compound has the formula: [ka] formula: [ka] formula: [ka] at least one region of at least two consecutive monomers.

[0215] In some such embodiments, the LNA includes, but is not limited to, [ka] As shown in (A) α-L-methyleneoxy(4'-CH2-O-2')LNA, (B) β-D-methyleneoxy(4'-CH2-O-2')LNA, ( C) ethyleneoxy(4'-(CH2)2-O-2')LNA, (D) aminooxy(4'-CH2-O-N(R)-2')LNA and (E) oxyamino(4'- CH2-N(R)-O-2')LNA is mentioned.

[0216] In some embodiments, the oligomeric compound comprises at least two regions of at least two contiguous monomers of the above formula. In some embodiments, the oligomeric compound comprises a gap oligomeric compound. In some embodiments, the oligomeric compound comprises at least one region of about 8 to about 14 contiguous β-D-2'-deoxyribofuranosyl nucleosides. In some embodiments, the oligomeric compound comprises at least one region of about 9 to about 12 contiguous β-D-2'-deoxyribofuranosyl nucleosides.

[0217] In some embodiments, the oligomeric compound has the formula: [ka] (wherein Bx is a heterocyclic base moiety) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) S-cEt monomers.

[0218] In some embodiments, the oligomeric compound, such as a tunable REVERSIR compound, is [ka] (In the formula, B is A-001 to A-026, and n is 0 to 6 (for example, 1, 2, 3, 4, 5, or 6)) at least one (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) nucleotides selected from.

[0219] In some embodiments, the monomer includes a sugar mimetic. In some such embodiments, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization with the target of choice. Representative examples of sugar mimetics include, but are not limited to, cyclohexenyl or morpholino. Representative examples of mimetics of sugar-internucleoside linkage combinations include, but are not limited to, peptide nucleic acids (PNAs) and morpholino groups linked by uncharged achiral linkages. In some cases, mimetics are used in place of nucleobases. Representative nucleobase mimetics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res. 2000, 28:2911-14 , incorporated herein by reference). Methods for the synthesis of sugars, nucleosides, nucleotides and nucleobase mimetics are well known to those skilled in the art.

[0220] In some embodiments, the tunable REVERSIR compound comprises at least one monomer that is an LNA and at least one G-clamp nucleobase. For example, a tunable REVERSIR compound may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more monomers that are LNA; , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more G-clamp nucleobases.

[0221] In some embodiments, the tunable REVERSIR compound is at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more). peptide nucleic acid monomers). In some embodiments, the tunable REVERSIR compound comprises at least one monomer that is LNA and at least one monomer that is PNA. For example, a tunable REVERSIR compound can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more monomers that are LNA and PNA. Contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more monomers.

[0222] In some embodiments, the tunable REVERSIR compound comprises at least one PNA and at least one G-clamp nucleobase. For example, tunable REVERSIR compounds may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more PNAs and 1, 2, 3, Contains 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more G-clamp nucleobases.

[0223] In some embodiments, the tunable REVERSIR compound comprises at least one LNA, at least one PNA, and at least one G-clamp nucleobase. For example, a tunable REVERSIR compound may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more LNA monomers; 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more PNA and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or more G-clamp nucleobases.

[0224] monomer linkage Described herein are linking groups that link monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) together, thereby forming oligomeric compounds. Such linking groups are also called intersugar bonds. Two major classes of linking groups are distinguished by the presence or absence of a phosphorus atom. Representative phosphorus-containing linkages include, but are not limited to, phosphodiester (P=O), phosphorotriester, methyl phonsphonate, phosphoramidate, and phosphorothioate (P=S). Representative phosphorus-containing bonds include, but are not limited to, methylene methylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thiocarbamate (-O-C( O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). It will be done. Oligomeric compounds with non-phosphorous binding groups are called oligonucleosides. Compared to natural phosphodiester linkages, modified linkages can be used to modify, typically increase, the nuclease resistance of oligomeric compounds. In some embodiments, bonds with chiral atoms can be prepared as individual enantiomers and in racemic mixtures. Representative chiral linkages include, but are not limited to, alkyl phosphates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known to those skilled in the art.

[0225] The phosphate group in the linking group can be modified by replacing one of the oxygens with a different substituent. One result of this modification may be increased resistance of the oligonucleotide to nucleic acid degradation. Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphates, hydrogen phosphates, phosphoramidates, alkyl or aryl phosphates, and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the linkage is S, Se, BR 3 (R is hydrogen, alkyl, aryl), C (i.e. alkyl, aryl, etc.), H, NR 2 (R is hydrogen, optionally substituted alkyl, aryl) or OR (R is optionally substituted alkyl or aryl). The phosphorus atom in the unmodified phosphate group is achiral. However, the replacement of one of the non-bridging oxygen atoms by one of the above atoms or groups renders the phosphorus atom chiral. In other words, the phosphorus atom in the phosphate group modified in this way is a stereocenter. Stereogenic phosphorus atoms can have either the "R" configuration (herein Rp) or the "S" configuration (herein Sp).

[0226] Phosphorothioates have both non-bridging oxygen atoms replaced by sulfur. The phosphorus center in the phosphorothioate is achiral, eliminating the formation of oligonucleotide diastereomers. Thus, without intending to be bound by any particular theory, modifications to both non-bridging oxygen atoms preclude chiral centers, e.g. phosphorothioate formation, since they cannot generate diastereomeric mixtures. May be desirable. Thus, a non-bridging oxygen atom can be independently either O, S, Se, B, C, H, N or OR (R is alkyl or aryl).

[0227] Phosphate linkers can also be modified by replacement of the bridging oxygen (i.e., the oxygen that links the phosphate to the monomer sugar) by nitrogen (bridged phosphoroamidate), sulfur (bridged phosphorothioate), and carbon (bridged methylene phosphonate). You can also. This substitution can be performed on either or both of the linking oxygens. When the bridging oxygen is the 3'-oxygen of the nucleoside, substitution by carbon is preferred. When the bridging oxygen is the 5'-oxygen of the nucleoside, substitution by nitrogen is preferred.

[0228] A modified phosphate bond in which at least one of the oxygens linked to the phosphate is substituted or the phosphate group is replaced by a non-phosphoric group is referred to as a "non-phosphodiester intersugar linkage" or "non-phosphodiester intersugar linkage". Also called a linker.

[0229] In some embodiments, a phosphate group can be replaced by a non-phosphorus-containing connector, such as a dephospholinker. Dephospholinkers are also referred to herein as non-phosphodiester linkers. Without intending to be bound by any particular theory, it is believed that because the charged phosphodiester group is the reactive center of nucleic acid degradation, its replacement by a neutral structural mimic confers enhanced nuclease stability. . Again, without intending to be bound by any particular theory, in some embodiments it may be desirable to introduce modifications that replace charged phosphate groups with neutral moieties.

[0230] Examples of moieties that can be substituted for phosphate groups include, but are not limited to: amides (e.g., amido-3(3'-CH 2 -C(=O)-N(H)-5’) and amide-(3’-CH 2 -N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylic acid ester, thioether, ethylene oxide linker, sulfide, sulfonic acid salts, sulfonamides, sulfonic acid esters, thioformacetals (3'-S-CH 2 -O-5’), formacetal (3’-O-CH 2 -O-5’), oxime, methyleneimino, methylenecarbonylamino, methylenemethylimino (MMI, 3’-CH 2 -N(CH 3 )-O-5'), methylene hydrazo, methylene dimethyl hydrazo, methyleneoxymethylimino, ether (C3'-O-C5'), thioether (C3'-S-C5'), thioacetamide (C3'- N(H)-C(=O)-CH 2 -S-5’, C3’-O-P(O)-O-SS-C5’, C3’-CH 2 -NH-NH-C5’,3’-NHP(O)(OCH 3 )-O-5’ and 3’-NHP(O)(OCH 3 )-O-5’ and mixed N, O, S and CH 2 A nonionic bond involving constituent parts. See, eg, Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65). Preferred embodiments include methylene methylimino (MMI), methylene carbonylamino, amide, carbamate and ethylene oxide linkers.

[0231] Those skilled in the art will appreciate that in many cases modification of a non-bridging oxygen atom can result in enhanced cleavage of the intersugar bond by the adjacent 2'-OH. , it is well recognized that modification of the 2'-OH, for example, may require modifications that do not participate in the cleavage of adjacent intersugar bonds, e.g. arabinose sugars, 2'-O-alkyl, 2'-F, LNA and ENA. ing.

[0232] Preferred non-phosphodiester intersugar linkages include phosphorothioates, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. Phosphorothioates containing Sp isomers in enantiomeric excess of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% Phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkyl-phosphonates (e.g. methyl-phosphonates), selenophosphates, phosphoroamidates (e.g. methyl-phosphonates), phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkyl-phosphonates (e.g. methyl-phosphonates), selenophosphates, phosphoroamidates (e.g. methyl-phosphonates), containing or greater enantiomeric excess of the Rp isomer Examples include N-alkylphosphoroamidates) as well as boranophosphonates.

[0233] In some embodiments, the oligomeric compound, e.g., a regulatable REVERSIR compound or siRNA, has at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15 or more and less than each numerical value inclusive) or non-phosphodiester bonds. In one embodiment, the oligomeric compound, e.g., a regulatable REVERSIR compound or siRNA, has at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15 or more and less than each numerical value inclusive).

[0234] In some embodiments, all internucleotide linkages in the reverser compound are phosphorothioate (PS) internucleotide linkages. In some embodiments, a tunable REVERSIR compound comprises at least one phosphorothioate (PS) internucleotide linkage, but not all internucleotide linkages in the tunable REVERSIR compound are phosphorothioate linkages. In other words, in some embodiments less than 100% (e.g., 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40% or less) of the internucleotide linkages are phosphorothioate linkages.

[0235] In some embodiments, a tunable REVERSIR compound comprises at least one phosphorothioate internucleotide linkage and at least one internucleoside or internucleotide linkage that is not a phosphorothioate. For example, a tunable REVERSIR compound includes at least one phosphorothioate internucleotide linkage and at least one phosphodiester internucleotide linkage. In some embodiments, the non-phosphorothioate internucleotide linkage is between the terminus and the penultimate nucleotide.

[0236] In some embodiments, the internucleotide bond between the nucleobase at the 3' end of the tunable REVERSIR compound and the remaining tunable REVERSIR compound is a phosphodiester bond. In some embodiments, all internucleotide linkages in the reverser compound, except for the internucleotide linkages between the 3'-terminal nucleoside of the regulatable REVERSIR compound and the remaining regulatable REVERSIR compound, It is a phosphorothioate.

[0237] Oligomeric compounds can also be constructed in which phosphate linkers and sugars are replaced with nuclease-resistant nucleosides, nucleotides, or nucleotide surrogates. Without wishing to be bound by any particular theory, it is believed that the repeatedly charged backbone weakens binding to proteins (eg, nucleases) that recognize polyanions. Again, while not intending to be bound by any particular theory, in some embodiments it may be desirable to introduce modifications in which the base is tethered by a neutral surrogate backbone. Examples include morpholino, cyclobutyl, pyrrolidine, peptide nucleic acid (PNA), aminoethylglycyl (aegPNA) and backbone extended pyrrolidine PNA (bepPNA) nucleoside surrogates. A preferred surrogate is a PNA surrogate.

[0238] The oligomeric compounds described herein contain one or more asymmetric centers, thus giving rise to enantiomers, diastereomers, and other configurations, which with respect to absolute stereochemistry, such as sugar anomers, may be defined as (R) or (S) or as (D) or (L) in the case of amino acids and the like. Antisense compounds described herein include all such possible isomers as well as their racemic and optionally pure forms.

[0239] terminal modification The ends of oligomeric compounds can be modified. Such modifications are possible at one or both ends. For example, the 3' and / or 5' end of the oligonucleotide can be labeled with a moiety such as a fluorophore (e.g. pyrene, TAMRA, fluorescein, Cy3 or Cy5 dye) or a protecting group (e.g. sulfur, silicon, boron or ester-based). can be conjugated with other functional molecular entities such as The functional molecular entity can be attached to the sugar via a phosphate group and / or a linker. The terminal atom of the linker may be attached to or substituted for the bonding atom of the phosphate group or the C-3' or C-5'O, N, S or C group of the sugar. Alternatively, the linker can be attached to or replace the terminal atom of the nucleotide surrogate (eg, PNA).

[0240] When a linker / phosphate-functional molecular entity-linker / phosphate array is placed between two strands of a double-stranded oligomeric compound, this array may replace the hairpin loop in a hairpin-type oligomeric compound. .

[0241] Terminal modifications useful in modulating activity include modification of the 5' end of the oligomeric compound with phosphate or phosphate analogs. In some embodiments, the 5' end of the oligomeric compound is phosphorylated or includes a phosphoryl analog. Exemplary 5' phosphate modifications include those that are compatible with RISC-mediated gene silencing. Modifications at the 5' end may also be useful in stimulating or inhibiting a subject's immune system. In some embodiments, the 5' end of the oligomeric compound is modified [ka] where W, X and Y are each independently O, OR (R is hydrogen, alkyl, aryl), S, Se, BR 3 (R is hydrogen, alkyl, aryl), BH 3 - , C (i.e. alkyl group, aryl group, etc.), H, NR 2 (R is hydrogen, alkyl, aryl) or OR (R is hydrogen, alkyl, or aryl); A and Z are each independently selected from, for each occurrence, absent, O, S, CH 2 , NR (R is hydrogen, alkyl, aryl) or optionally substituted alkylene, where the alkylene backbone is internally and / or terminally comprised of O, S, SS and NR (R is hydrogen, alkyl, aryl). , hydrogen, alkyl, aryl); n is 0 to 2. In some embodiments, n is 1 or 2. It is understood that A replaces the oxygen linked to the 5' carbon of the sugar. When n is 0, W and Y, together with the P to which they are attached, can form an optionally substituted 5- to 8-membered heterocyclic compound, where W and Y are , each independently O, S, NR' or alkylene. Preferably, the heterocyclic ring is substituted with aryl or heteroaryl. In some embodiments, one or both hydrogens on C5' of the 5' terminal nucleotide are replaced with a halogen, eg, F.

[0242] Exemplary 5'-modifications include, but are not limited to, 5'-monophosphate ((HO) 2 (O)p-O-5’);5’-diphosphate ((HO) 2 (O)P-O-P(HO)(O)-O-5’);5’-triphosphate((HO) 2 (O)P-O-(HO)(O)P-O-P(HO)(O)-O-5');5'-Monothiophosphate (phosphorothioate; (HO)2(S)P-O-5');5'- Monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); 5'-α -Thiotriphosphate;5'-β-thiotriphosphate;5'-γ-thiotriphosphate;5'-phosphoroamidate ((HO) 2 (O)P-NH-5’, (HO)(NH 2 )(O)P-O-5'). Other 5'-modifications include 5'-alkyl phosphates (R(OH)(O)P-O-5', R=alkyl, e.g. methyl, ethyl, isopropyl, propyl, etc.), 5'-alkyl ether phosphones Acid salt (R(OH)(O)P-O-5', R=alkyl ether, e.g. methoxymethyl (CH 2 OMe), ethoxymethyl, etc.). Other exemplary 5'-modifications include those where Z is alkyl, optionally substituted at least once, such as ((HO) 2 (X)P-O[-(CH 2 ) a -O-P(X)(OH)-O] b -5’, ((HO)2(X)P-O[-(CH 2 ) a -P(X)(OH)-O] b -5’, ((HO)2(X)P-[-(CH 2 ) a -O-P(X)(OH)-O] b -5’;Dialkyl-terminated phosphate and phosphate mimetics;HO[-(CH 2 ) a -O-P(X)(OH)-O] b -5’, H 2 N[-(CH 2 ) a -O-P(X)(OH)-O] b -5’, H[-(CH 2 ) a -O-P(X)(OH)-O] b -5’, Me 2 N[-(CH 2 ) a -O-P(X)(OH)-O] b -5’, HO[-(CH 2 ) a -P(X)(OH)-O] b -5’, H 2 N[-(CH 2 ) a -P(X)(OH)-O] b -5’, H[-(CH 2 ) a -P(X)(OH)-O] b -5’, Me 2 N[-(CH 2 ) a -P(X)(OH)-O] b -5', where a and b are each independently from 1 to 10. Other embodiments include BH 3 , B.H. 3 - and / or substitution of oxygen and / or sulfur by Se.

[0243] Terminal modifications may also be useful in monitoring distribution; in such cases, preferred groups for attachment include fluorophores, such as fluorescein, or Alexa dyes, such as Alexa 488. Terminal modifications may also be useful to enhance uptake; useful modifications for this include targeting ligands. Additionally, terminal modifications may be useful in cross-linking the oligonucleotide to another moiety; useful modifications for this include mitomycin C, psoralen and their derivatives.

[0244] oligomeric compounds In some embodiments, provided herein are oligomeric compounds having reactive phosphorus groups useful in forming bonds such as, for example, phosphodiester and phosphorothioate internucleotide bonds. The method of preparation and / or purification of the precursor or oligomeric compound is not intended to limit the compositions or methods described herein. Methods for the synthesis and purification of oligomeric compounds such as DNA, RNA, oligonucleotides, oligonucleosides and antisense compounds are well known to those skilled in the art.

[0245] Generally, oligomeric compounds include multiple monomeric subunits linked together by linking groups. Non-limiting examples of oligomeric compounds include primers, probes, antisense compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternative splicers, and siRNA. Thus, these compounds can be introduced in single-stranded, double-stranded, circular, branched or hairpin forms, which may contain structural elements such as internal or terminal bulges or loops. can. The oligomeric double-stranded compound is a double-stranded compound that hybridizes to form a double-stranded compound or has sufficient self-complementarity to allow hybridization and formation of a fully or partially double-stranded compound. It can be either single stranded.

[0246] In some embodiments, the invention provides chimeric oligomeric compounds. In some such embodiments, the chimeric oligomeric compound is a chimeric oligonucleotide. In some such embodiments, the chimeric oligonucleotides include various modified nucleotides. In some embodiments, the chimeric oligonucleotide is a mixed backbone antisense oligonucleotide.

[0247] Generally, chimeric oligomeric compounds have modified nucleosides that can be in isolated positions or grouped together in regions that define a particular motif. Any combination of modifications and / or mimetics may include the chimeric oligomeric compounds described herein.

[0248] In some embodiments, chimeric oligomeric compounds typically include at least one region that has been modified to confer increased resistance to nuclease degradation, enhanced cellular uptake, and / or increased binding affinity for a target nucleic acid. include. In some embodiments, another region of the oligomeric compound can serve as a substrate for an enzyme capable of cleaving RNA:DNA or RNA:RNA hybrids.

[0249] In some embodiments, the chimeric oligomeric compound is a gapmer. In some embodiments, a mixed backbone oligomeric compound has one type of internucleotide linkage in one or both wings and another type of internucleoside linkage within the gap. In some such embodiments, the mixed backbone oligonucleotide has phosphodiester linkages in the wings and phosphorothioate linkages in the gaps. In some embodiments, the internucleotide linkages within the wings are different from the internucleotide linkages within the gaps, and the internucleotide linkages bridging the wings and gaps are the same as the internucleotide linkages within the wings. In some embodiments, the internucleotide linkages in the wings are different than the internucleotide linkages in the gaps, and the internucleotide linkages bridging the wings and gaps are the same as the internucleoside linkages in the gaps.

[0250] In some embodiments, the invention provides oligomeric compounds that include siRNAs of any length in a variety of length ranges and regulatable REVERSIR compounds. In some embodiments, the invention provides oligomeric compounds comprised of X-Y linked oligonucleotides, where X and Y are each independently 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 , 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50; where X<Y. For example, in some embodiments, the present application describes 18, 8~19, 8~20, 8~21, 8~22, 8~23, 8~24, 8~25, 8~26, 8~27, 8~28, 8~29, 8~30, 9~10, 9~11, 9~12, 9~13, 9~14, 9~15, 9~16, 9~17, 9~18, 9~19, 9~20, 9~21, 9~ 22, 9~23, 9~24, 9~25, 9~26, 9~27, 9~28, 9~29, 9~30, 10~11, 10~12, 10~13, 10~14, 10~15, 10~16, 10~17, 10~18, 10~19, 10~20, 10~21, 10~22, 10~23, 10~24, 10~25, 10~26, 10~ 27, 10~28, 10~29, 10~30, 11~12, 11~13, 11~14, 11~15, 11~16, 11~17, 11~18, 11~19, 11~20, 11~21, 11~22, 11~23, 11~24, 11~25, 11~26, 11~27, 11~28, 11~29, 11~30, 12~13, 12~14, 12~ 15, 12~16, 12~17, 12~18, 12~19, 12~20, 12~21, 12~22, 12~23, 12~24, 12~25, 12~26, 12~27, 12~28, 12~29, 12~30, 13~14, 13~15, 13~16, 13~17, 13~18, 13~19, 13~20, 13~21, 13~22, 13~ 23, 13~24, 13~25, 13~26, 13~27, 13~28, 13~29, 13~30, 14~15, 14~16, 14~17, 14~18, 14~19, 14~20, 14~21, 14~22, 14~23, 14~24, 14~25, 14~26, 14~27, 14~28, 14~29, 14~30, 15~16, 15~ 17, 15~18, 15~19, 15~20, 15~21, 15~22, 15~23, 15~24, 15~25, 15~26, 15~27, 15~28, 15~29, 15~30, 16~17, 16~18, 16~19, 16~25, 16~21, 16~22, 16~23, 16~24, 16~25, 16~26, 16~27, 16~ 28, 16~29, 16~30, 17~18, 17~19, 17~20, 17~21, 17~22, 17~23, 17~24, 17~25, 17~26, 17~27, 17~28, 17~29, 17~30, 18~19, 18~20, 18~21, 18~22, 18~23, 18~24, 18~25, 18~26, 18~27, 18~ 28, 18~29, 18~30, 19~20, 19~21, 19~22, 19~23, 19~24, 19~25, 19~26, 19~29, 19~28, 19~29, 19~30, 20~21, 20~22, 20~23, 20~24, 20~25, 20~26, 20~27, 20~28, 20~29, 20~30, 21~22, 21~ 23, 21~24, 21~25, 21~26, 21~27, 21~28, 21~29, 21~30, 22~23, 22~24, 22~25, 22~26, 22~27, 22~28, 22~29, 22~30, 23~24, 23~25, 23~26, 23~27, 23~28, 23~29, 23~30, 24~25, 24~26, 24~ 27, 24~28, 24~29, 24~30, 25~26, 25~27, 25~28, 25~29, 25~30, 26~27, 26~28, 26~29, 26~30, An oligomeric compound comprising 27-28, 27-29, 27-30, 28-29, 28-30 or 29-30 linked nucleotides is provided.

[0251] As mentioned above, tunable REVERSIR compounds can be of any length. For example, in some embodiments, the regulatable REVERSIR compound is a modified oligonucleotide comprised of 6-30 nucleotides. For example, tunable REVERSIR compounds include 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, It may be composed of 27, 28, 29 or 30 linked nucleobases. In some embodiments, the tunable REVERSIR compound is comprised of 6-17, 7-16, or 8-15 linked nucleobases.

[0252] We have found, among others, that tunable REVERSIR compounds, ie modified oligonucleotides, composed of 15 or fewer nucleotides are particularly effective in reversing siRNA activity. Thus, in some embodiments, the regulatable REVERSIR compound is a modified oligonucleotide comprised of 8 to 15 (eg, 8, 9, 10, 11, 12, 13, 14, or 15) linked nucleotides. In some embodiments, the regulatable REVERSIR compound is a modified oligonucleotide comprised of 6-12, 7-11 or 8-10 linked nucleobases. In some embodiments, the regulatable REVERSIR compound is a modified oligonucleotide comprised of 8-9 linked nucleobases.

[0253] As described herein, a regulatable REVERSIR compound is a modified oligonucleotide that is substantially complementary to at least one strand of siRNA. Without intending to be bound by any particular theory, a regulatable REVERSIR compound that is substantially complementary to the seed region of the antisense strand of the siRNA (i.e., positions 2-8 of the 5' end of the antisense strand) Particularly effective in reducing siRNA activity. Thus, in many embodiments, the regulatory REVERSIR compound comprises nucleotides 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2 of the antisense strand of the siRNA. ~15 or substantially complementary to 2~16. Substantially complementary in this context means at least 90%, preferably at least 95% complementarity, more preferably complete complementarity.

[0254] ligand In some embodiments, oligomeric compounds are modified by covalent attachment of one or more conjugate groups. Generally, the conjugate group modifies one or more properties of the oligomeric compound to which it is attached, such as, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, These include charge and clearance. Conjugate groups are commonly used in the chemical arts and are linked to a parent compound, such as an oligomeric compound, either directly or through an optional linking moiety or group. A preferred list of conjugate groups includes, but is not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folates, lipids, phospholipids, biotin, Contains phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin and dyes.

[0255] Preferred conjugate groups suitable for the present invention include: lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid. (Manoharan et al.,Bioorg.Med.Chem.Lett.,1994,4,1053);thioethers, such as hexyl-S-tritylthiol (Manoharan et al.,Ann.N.Y.Acad.Sci.,1992,660,306;Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); aliphatic chains, e.g. dodecadiol or undecyl residues (Saison-Behmoaras et al.,EMBO J.,1991,10,111;Kabanov et al.,FEBS Lett.,1990,259,327;Svinarchuk et al.,Biochimie,1993,75,49);phospholipids, e.g. di-hexadecyl -lac-glycerol or triethylammonium-1,2-di-O-hexadecyl-lac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777); polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277,923).

[0256] In general, a wide variety of entities, such as ligands, can be attached to the oligomeric compounds described herein. Ligands can include naturally occurring molecules or recombinant or synthetic molecules. Exemplary ligands include, but are not limited to, polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glyco divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g. PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG -15K, PEG-20K, PEG-40K), MPEG, [MPEG] 2 , polyvinyl alcohol (PVA), polyurethane, poly(2-ethyl acrylic acid), N-isopropylacrylamide polymer, polyphosphazine, polyethyleneimine, cationic group, spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dentrimer Polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of polyamine, thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin, glycosylated polyamino acid, transferrin, bisphosphonate, polyglutamic acid salts, polyaspartates, aptamers, asialofetuin, hyaluronan, procollagen, immunoglobulins (e.g. antibodies), insulin, transferrin, albumin, sugar-albumin conjugates, intercalating agents (e.g. acridine), crosslinkers (e.g. , psoralen, mitomycin C), porphyrins (e.g. TPPC4, texaphyrin, sapphirin), polycyclic aromatic hydrocarbons (e.g. phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules (e.g. steroids). , bile acids, cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propane diols, heptadecyl groups, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine), peptides (e.g. alpha helical peptides, amphipathic peptides, RGD peptides, cell penetrating peptides, endosomolytic / fusion peptides), alkylating agents, phosphates, aminos, mercapto, polyaminos, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g. biotin), transport / absorption enhancers (e.g. naproxen, aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g. imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, Eu3+ complex of tetraazamacrocycle), dinitrophenyl, HRP, AP , antibodies, hormones and hormone receptors, lectins, carbohydrates, polycarbohydrates, vitamins (e.g. vitamin A, vitamin E, vitamin K, vitamin B, e.g. folic acid, B12, riboflavin, biotin and pyridoxal), vitamin cofactors, lipoproteins. Polysaccharide, p38MAP kinase activator, NF-κB activator, taxone, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanosine, myocervin, tumor necrosis factor α (TNFα), Interleukin-1β, gamma interferon, natural or recombinant low-density lipoprotein (LDL), natural or recombinant high-density lipoprotein (HDL), and cell-permeabilizing agents (eg, helical cell-permeabilizing agents).

[0257] Peptides and peptidomimetic ligands include natural or modified peptides, such as D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides with one or more amides, i.e. peptides; or a bond substituted with multiple urea, thiourea, carbamate or sulfonylurea bonds; or a cyclic peptide. Peptidomimetics (also referred to herein as oligopeptide mimetics) are molecules that can fold into well-defined three-dimensional structures similar to natural peptides. A peptide or peptidomimetic ligand can be about 5-50 amino acids long, such as 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids long.

[0258] Exemplary amphipathic peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava (S. clava) peptide, hagfish intestinal antimicrobial peptide (HFIAP), magainine, brevinin-2, dermaseptin, melittin, pleurocidin, H 2 A peptide, Xenopus peptide, esculentinis-1 and caelin.

[0259] As used herein, "endosomal ligand" refers to a molecule with endosomal properties. Endosomal ligands may be used to dissolve the composition of the present invention or its components and / or to induce the lysis of the composition of the present invention or its components in endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other cells within the cell. promotes the transport of from the endoplasmic reticulum to the cell cytoplasm. Some exemplary endosomal ligands include, but are not limited to, imidazole, poly- or oligoimidazole, linear or branched polyethyleneimine (PEI), linear and branched polyamines such as spermine, cationic linear or branched. linear or branched polymers with masked or unmasked cations or anionic charges, masked or unmasked Included are dendrimers with cationic or anionic charges, polyanionic peptides, peptidomimetics, pH-sensitive peptides, natural and synthetic fusogenic lipids, natural and synthetic cationic lipids.

[0260] Exemplary endosomal / fusogenic peptides include, but are not limited to: AALEALAEALEALAEALEALAEAAAAGGC(GALA)(SEQ ID NO: 1);AALAEALAEALAEALAEALAEALAAAAGGC(EALA)(SEQ ID NO:2);ALEALAEALEALAEA(SEQ ID NO:3);GLFEAIEGFIENGWEGMIWDYG(INF-7)(SEQ ID NO:4);GLFGAIAGFIENGWEGMIDGWYG(Inf HA-2)(SEQ ID NO:5 );GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMID GWYGC(diINF-7)(SEQ ID NO:6);GLFEAIEGFIENGWEGMIDGGCGLFEAIEGFIENGWEGMIDGGC(diINF-3)(SEQ ID NO:7);GLFGALAEALAEALAEHLAEALAEALEALAAGGSC(GLF)(SEQ ID NO:8);GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC(GALA-INF 3)(SEQ ID NO. 9);GLF EAI EGFI ENGW EGnI DG K GLF EAI EGFI ENGW EGnI DG(INF-5, n is norleucine) (SEQ ID NO: 10);LFEALLELLESLWELLLEA(JTS-1)(SEQ ID NO: 11);GLFKALLKLLKSLWKLLLKA(ppTG1)(SEQ ID NO: 12) ;GLFRALLRLLRSLWRLLLRA(ppTG20)(SEQ ID NO: 13);WEAKLAKALAKALAKHLAKALAKALKACEA(KALA)(SEQ ID NO: 14);GLFFEAIAEFIEGGWEGLIEGC(HA)(SEQ ID NO: 15);GIGAVLKVLTTGLPALISWIKRKRQQ(Melittin)(SEQ ID NO: 16);H 5 WYG (SEQ ID NO: 17); and CHK 6 HC (SEQ ID NO: 18).

[0261] Without intending to be bound by any particular theory, fusogenic lipids fuse with membranes, thereby destabilizing them. Fusogenic lipids usually have small head groups and unsaturated acyl chains. Exemplary fusogenic lipids include, but are not limited to, 1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoylphosphatidylcholine (POPC), (6Z, 9Z, 28Z, 31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di(9Z,12Z)-octadeca-9,12-dienyl)- 1,3-dioxolan-4-yl)methanamine (DLin-k-DMA) and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3 -dioxolan-4-yl)ethanamine (also referred to herein as XTC).

[0262] Synthetic polymers with endosomolytic activity suitable for the present invention are described in U.S. Patent Application Publication Nos. 2009 / 0048410; 2009 / 0023890; 2008 / 0287630; Specification; 2008 / 0281044; 2008 / 0281041; 2008 / 0269450; 2007 / 0105804; 20070036865; and 2004 / 0198687, the contents of which are incorporated herein by reference in their entirety.

[0263] Exemplary cell-penetrating peptides include, but are not limited to: RQIKIWFQNRRMKWKK (Penetratin) (SEQ ID NO: 19); GRKKRRQRRRPPQC (Tat fragment 48-60) (SEQ ID NO: 20); GALFLGWLGAAGSTMGAWSQPKKKRKV (signal sequence based peptide) (SEQ ID NO: 21); LLIILRRRIRKQAHAHSK (PVEC) (SEQ ID NO: 22); GWTLNSAGYLLKINLKALAALAKKIL (transportan) (SEQ ID NO: 23); KLALKLALKALKAALKLA (amphiphilic model peptide) (SEQ ID NO: 24); RRRRRRRRR (Arg9) ( SEQ ID NO: 25); KFFKFFKFFK (bacterial cell penetrating peptide) (SEQ ID NO: 26); LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37) (SEQ ID NO: 27); SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin P1) (SEQ ID NO: 28); ACYCRIPACIAGERRYGTCIYQGRLWAFCC (α-defensin) (SEQ ID NO: 28); No. 29); DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (β-defensin) (SEQ ID No. 30); RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (PR-39) (SEQ ID No. 31); ILPWKWPWWPWRR-NH2 (indolicidin) (SEQ ID No. 32); AAVALLPAVLLALLAP (RFGF) (SEQ ID No. 33) ); AALLPVLLAAP (RFGF analog) (SEQ ID NO: 34); and RKCRIVVIRVCR (bactenecin) (SEQ ID NO: 35).

[0264] Exemplary cationic groups include, but are not limited to, O-AMINE (AMINE=NH 2 ;alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino);aminoalkoxy, such as O(CH 2 ) n AMINE (for example, AMINE=NH 2 ;alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino);amino (e.g. NH 2 ;alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino or amino acid); and NH(CH 2 CH 2 N.H.) n CH 2 CH 2 -AMINE(AMINE=NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino).

[0265] As used herein, the term "targeting ligand" refers to a target with increased affinity for a selected target, e.g. a cell, cell type, tissue, organ, region or compartment of the body, e.g. a cell, tissue or organ compartment. Refers to any molecule that confers sex. Some exemplary targeting ligands include, but are not limited to, antibodies, antigens, folate, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA. , endothelin, GCPII, somatostatin, LDL and HDL ligands.

[0266] Carbohydrate-based targeting ligands include, but are not limited to, D-galactose, polyvalent galactose, N-acetyl-D-galactose (GalNAc), polyvalent GalNAc such as GalNAc2 and GalNAc3; D-mannose, polyvalent mannose, polyvalent Includes lactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent fucose, glycosylated polyamino acids and lectins. The term polyvalent indicates the presence of two or more monosaccharide units. Such monosaccharide subunits can be linked to each other via glycosidic bonds or to scaffold molecules.

[0267] As ligands, some folates and folate analogs suitable for the present invention are described in U.S. Pat. Nos. 2,816,110; 51410,104; No. 7,128,893, the contents of which are incorporated herein by reference in their entirety.

[0268] As used herein, the terms "PK modulating ligand" and "PK modulator" refer to molecules that can modulate the pharmacokinetics of the compositions of the invention. Some exemplary PK modulators include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogs, peptides, protein binders, vitamins, fatty acids, phenoxazine, aspirin, naproxen, ibuprofen, suprofen, Ketoprofen, (S)-(+)-pranoprofen, carprofen, PEG, biotin and transthyrethia-binding ligands (eg, tetraidotyroacetic acid, 2,4,6-triidophenol and flufenamic acid). Oligomeric compounds containing some phosphorothioate intersugar linkages are also known to bind serum proteins, and therefore short oligomeric compounds, such as about 5 to 30 nucleotides (e.g., 5 to 25 nucleotides, preferably 5 to 20 nucleotides, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides) and also contain multiple phosphorothioate linkages in the backbone. , are suitable for the present invention as ligands (eg PK modulating ligands). The PK modulating oligonucleotide comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more phosphorothioate and / or phosphorodithioate linkages. In some embodiments, all internucleotide linkages in the PK modulating oligonucleotide are phosphorothioate and / or phosphorodithioate linkages. Furthermore, aptamers that bind serum components (eg, serum proteins) are also suitable for the present invention as PK modulating ligands. Binding to serum components (eg, serum proteins) can be predicted from albumin binding assays such as those described in Oravcova, et al., Journal of Chromatography B (1996), 677:1-27.

[0269] If more than one ligand is present, the ligands may all have the same properties, all have different properties, or some ligands may have the same properties and others may have different properties. For example, a ligand can have targeting properties, endosomolytic activity, or PK modulatory properties. In a preferred embodiment, the ligands all have different properties.

[0270] In some embodiments, the ligand of one strand of the double-stranded oligomeric compound has an affinity for the ligand of the second strand. In some embodiments, the ligand is covalently attached to both strands of a double-stranded oligomeric compound. As used herein, when a ligand is linked to more than an oligomeric chain, the point of attachment of the oligomeric compound can be an atom of the ligand itself or an atom of a carrier molecule to which the ligand itself is attached.

[0271] The ligand can be attached to the oligomeric compound at various locations, such as at the 3'-terminus, at the 5'-terminus, and / or at internal positions. If more than one ligand is present, the ligands can be at opposite ends of the oligomeric compound. In preferred embodiments, the ligand is attached to the oligomeric compound via an intervening tether / linker. A ligand or tethered ligand may be present on the monomer if it is incorporated into a growing chain. In some embodiments, the ligand can also be incorporated into the "precursor" monomer by coupling after the "precursor" monomer is incorporated into the growing chain. For example, an amino-terminal tethered (i.e., no ligand attached) monomer, e.g. monomer-linker-NH 2 can be incorporated into the growing oligomeric compound chain. In the next step, i.e. after incorporation of the precursor monomer into the chain, a ligand with an electrophilic group, for example a pentafluorophenyl ester or an aldehyde group, is combined with the electrophilic group of the ligand and the terminal electrophilic group of the precursor monomer. can be attached to the tether of the precursor monomer by coupling with

[0272] In another example, a ligand with a suitable chemical group to participate in a click chemistry reaction, such as an azide or alkyne terminal tether / linker, can be incorporated. In a subsequent operation, i.e. after incorporation of the precursor monomer into the chain, a ligand with a complementary chemical group, such as an alkyne or an azide, can be attached to the precursor monomer by coupling with the alkyne and the azide. .

[0273] In the case of double-stranded oligomeric compounds, the ligand can be attached to one or both strands. In some embodiments, the siRNA includes a ligand conjugated to the sense strand. In another embodiment, the siRNA includes a ligand conjugated to the antisense strand.

[0274] In some embodiments, a ligand can be conjugated to a nucleobase, sugar moiety, or internucleoside linkage of an oligomeric compound. Conjugation to the purine nucleobase or derivative thereof may be performed at any position, including endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is attached to the conjugate moiety. Conjugation to a pyrimidine nucleobase or derivative thereof may also be performed at any position. In some embodiments, positions 2-, 5-, and 6 of the pyrimidine nucleobase can be replaced with a conjugate moiety. When conjugating a ligand to a nucleobase, preferred positions are those that do not interfere with hybridization, ie, do not interfere with the hydrogen bonding interactions necessary for base pairing.

[0275] Conjugation of the nucleoside to the sugar moiety can be performed at any carbon atom. Exemplary carbon atoms of sugar moieties that can be attached to the conjugate moiety include 2', 3' and 5' carbon atoms. The 1' position can also be attached to a conjugate moiety, such as an abasic residue. The internucleoside linkage can also carry a conjugate moiety. In the case of phosphorus-containing bonds (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidite, etc.), the conjugate moiety may be attached directly to the phosphorus atom or to an O, N or S atom attached to the phosphorus atom. Can be done. In the case of amine- or amide-containing internucleoside linkages (eg, PNA), the conjugate moiety can be attached to the nitrogen atom or adjacent carbon atom of the amine or amide.

[0276] The inventors have found, among others, that tunable REVERSIR compounds conjugated to ligands are particularly effective in reducing the activity of siRNA. Without intending to be bound by any particular theory, a ligand can increase or enhance the potency of a tunable REVERSIR compound by delivering the tunable REVERSIR compound to the desired site of action. Accordingly, in some embodiments, a tunable REVERSIR compound is conjugated to a ligand.

[0277] Ligands conjugated to regulatory REVERSIR compounds, while useful for delivery of the regulatory REVERSIR compound to the desired site of action, can negatively impact the ability of the regulatory REVERSIR compound to reduce siRNA activity. Thus, in some embodiments, the linkage between the ligand and the regulatable REVERSIR compound can be designed to undergo cleavage after the regulatable REVERSIR compound reaches the desired site of action. This can be achieved in several ways. For example, the linker that connects the regulatable REVERSIR compound to the ligand can be a cleavable linker.

[0278] The inventors further discovered that nucleotides in a regulatory REVERSIR compound linked to a ligand can affect the ability of the regulatory REVERSIR compound to reduce the activity of siRNA. The inventors have found that ligand-conjugated nucleotides containing deoxy sugars (eg, 2'-deoxyribose) are particularly effective in enhancing the ability of regulatable REVERSIR compounds to reduce siRNA activity. Thus, in some embodiments, the nucleotide conjugated to the ligand comprises a deoxy sugar, such as a 2'-deoxy sugar.

[0279] In some embodiments of the various aspects disclosed herein, the ligand is attached to a nucleotide at the 3' end of the regulatable REVERSIR compound. The inventors have found, among other things, that the internucleotide linkage between the ligand-conjugated nucleotide and the remaining regulatory REVERSIR compound can also affect the ability of the regulatory REVERSIR compound to reduce siRNA activity. Without intending to be bound by any particular theory, easily cleavable internucleotide linkages have been found to be particularly effective in enhancing the ability of regulatable REVERSIR compounds to reduce siRNA activity. Thus, in some embodiments, the ligand-conjugated nucleotide is attached to the remaining regulatable REVERSIR compound via a cleavable internucleotide bond. In some embodiments, the cleavable internucleotide bond is a phosphodiester internucleotide bond.

[0280] In some embodiments, the ligand-conjugated nucleotide comprises a deoxy sugar, which is linked to the remaining regulatable REVERSIR compound via a cleavable internucleotide bond. In some other embodiments thereof, the cleavable internucleotide bond is a phosphodiester internucleotide bond.

[0281] In some embodiments, the ligand-conjugated nucleotide comprises a deoxy sugar, which is linked to the rest of the regulatable REVERSIR compound via an internucleotide linkage that is not a phosphodiester linkage.

[0282] In some embodiments, the ligand is conjugated to a nucleotide at the 3' end of the regulatable REVERSIR compound.

[0283] In some embodiments, the ligand is conjugated to the 5' end of the tunable REVERSIR compound. In some embodiments, a first ligand is conjugated to the 5' end of the tunable REVERSIR compound and a second ligand is conjugated to the first ligand.

[0284] There are many ways to prepare conjugates of oligomeric compounds. Generally, an oligomeric compound is attached to a conjugate moiety by contacting a reactive group (eg, OH, SH, amine, carboxyl, aldehyde, etc.) on the oligomeric compound with a reactive group on the conjugate moiety. In some embodiments, one reactive group is electrophilic and the other is nucleophilic.

[0285] For example, an electrophilic group can be a carbonyl-containing functional group, and a nucleophilic group can be an amine or a thiol. Methods for conjugating nucleic acids and related oligomeric compounds with or without linking groups are described, for example, by Manoharan in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17; (incorporated herein by reference in its entirety).

[0286] Representative US patents teaching the preparation of conjugates of oligomeric compounds, such as oligonucleotides, include, but are not limited to, US Pat. No. 4,828,979; US Pat. No. 4,948,882; US Pat. Specification No. 5,525,465; Specification No. 5,541,313; Specification No. 5,545,730; Specification No. 5,552,538; Specification No. 5,578,717, Specification No. 5,580,731; Specification No. 5,580,731; Specification No. 5,591,584 No. 5,109,124; No. 5,118,802; No. 5,138,045; No. 5,414,077; No. 5,486,603; No. 5,512,439; No. 5,578,718 Specification: No. 5,608,046; No. 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,824,941 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136 5,082,830 specification; 5,112,963 specification; 5,149,782 specification; 5,214,136 specification; 5,245,022 specification; 5,254,469 specification; 5,258,506 specification; Specification No. 5,262,536; Specification No. 5,272,250; Specification No. 5,292,873; Specification No. 5,317,098; Specification No. 5,371,241, Specification No. 5,391,723; Specification No. 5,416,203, Specification Specification No. 5,451,463; Specification No. 5,510,475; Specification No. 5,512,667; Specification No. 5,514,785; Specification No. 5,565,552; Specification No. 5,567,810; Specification No. 5,574,142; Specification No. 5,585,481; Specification No. 5,587,371; Specification No. 5,595,726; Specification No. 5,597,696; Specification No. 5,599,923; Specification No. 5,599,928; Specification No. 5,672,662; Specification No. 5,688,941 No. 5,714,166; No. 6,153,737; No. 6,172,208; No. 6,300,319; No. 6,335,434; No. 6,335,437; No. 6,395,437 Specification; Specification No. 6,444,806; Specification No. 6,486,308; Specification No. 6,525,031; Specification No. 6,528,631; Specification No. 6,559,279. incorporated herein by.

[0287] In some embodiments, the oligomeric compounds described herein, including but not limited to regulatable REVERSIR compounds and siRNA, are [ka] A ligand having the structure shown in here, L G is, independently for each occurrence, a ligand, e.g. a carbohydrate, e.g. a monosaccharide, di-saccharide, trisaccharide, tetrasaccharide, polysaccharide; Z', Z'', Z''' and Z''' are each independently O or S for each occurrence.

[0288] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNA, have formulas (II), (III), (IV), or (V): [ka] A ligand having the structure shown in here, q 2A ,q 2B ,q 3A ,q 3B ,q 4A ,q 4B ,q 5A ,q 5B and q 5C is independently for each occurrence from 0 to 20, and the repeating units may be the same or different; Q and Q’ are independent for each existence, non-existence, -(p 7 -Q 7 -R 7 ) p -T 7 -or-T 7 -Q 7 -T 7’ -B-T 8’ -Q 8 -T 8 And; P 2A ,P 2B ,P 3A ,P 3B ,P 4A ,P 4B ,P 5A ,P 5B ,P 5C ,P 7 ,T 2A ,T 2B ,T 3A ,T 3B ,T 4A ,T 4B ,T 4A ,T 5B ,T 5C ,T 7 ,T 7’ ,T 8 and T 8’ are CO, NH, O, S, OC(O), NHC(O), CH 2 ,C.H. 2 NH or CH 2 is O; B is -CH 2 -N(B L )-CH 2 -is; B L -T B -Q B -T B’ -R x And; Q 2A ,Q 2B ,Q 3A ,Q 3B ,Q 4A ,Q 4B ,Q 5A ,Q 5B ,Q 5C ,Q 7 ,Q 8 and Q B is, independently for each occurrence, absent, alkylene, substituted alkylene, and one or more methylenes are O, S, S(O), SO 2 , N(R N ), C(R’)=C(R’), C≡C or C(O); T B and T B’ are independently for each presence, non-existence, CO, NH, O, S, OC(O), OC(O)O, NHC(O), NHC(O), NHC(O)NH, NHC( O) O, CH 2 ,C.H. 2 NH or CH 2 is O; R X contains lipophilic substances (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestrone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanol diol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine), vitamins (e.g. folate, vitamin A , vitamin E, biotin, pyridoxal), peptides, carbohydrates (e.g., monosaccharides, di-, trisaccharides, tetrasaccharides, oligosaccharides, polysaccharides), endosomal components, steroids (e.g., uvaol, hesigenin, diosgenin), terpenes ( for example triterpenes such as sarsa sapogenin, friederin, epifriederanol derivatized lithocholic acid) or cationic lipids; R 1 ,R 2 ,R 2A ,R 2B ,R 3A ,R 3B ,R 4A ,R 4B ,R 5A ,R 5B ,R 5C ,R 7 are, for each existence independently, non-existence, NH, O, S, CH 2 , C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-, NH-, CO, CH=N-O, [ka] or heterocyclyl; L 1 ,L 2A ,L 2B ,L 3A ,L 3B ,L 4A ,L 4B ,L 5A ,L 5B and L 5C are carbohydrates, such as monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides and polysaccharides, each independently for each occurrence; R' and R'' are each independently H, C 1 ~C 6 Alkyl, OH, SH or N(R N ) 2 And; R N are each independently H, methyl, ethyl, propyl, isopropyl, butyl or benzyl; R a is H or an amino acid side chain; Z’, Z’’, Z’’’ and Z’’’’ are each independently O or S for each occurrence; p is 0 to 20 for each entity independently.

[0289] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0290] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0291] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0292] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0293] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0294] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0295] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0296] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0297] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0298] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0299] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0300] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains monomers.

[0301] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0302] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains monomers.

[0303] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains monomers.

[0304] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains monomers.

[0305] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains monomers.

[0306] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains monomers.

[0307] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains monomers.

[0308] In some embodiments, the oligomeric compounds described herein, including but not limited to regulatable REVERSIR compounds and siRNA, have the structure: [ka] Contains the ligand of

[0309] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0310] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0311] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0312] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0313] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0314] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0315] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0316] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains the ligand of

[0317] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains monomers.

[0318] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains monomers.

[0319] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains monomers.

[0320] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains monomers.

[0321] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains monomers.

[0322] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains monomers.

[0323] In some embodiments, L 2A and L 2B are all different.

[0324] In some embodiments, L 3A and L 3B are all the same.

[0325] In some embodiments, L 3A and L 3B are all different.

[0326] In some embodiments, L 4A and L 4B are all the same.

[0327] In some embodiments, L 4A and L 4B are all different.

[0328] In some preferred embodiments, L 5A ,L 5B and L 5C are all the same.

[0329] In some embodiments, L 5A ,L 5B and L 5C The two are the same.

[0330] In some embodiments, L 5A and L 5B are the same.

[0331] In some embodiments, L 5A and L 5C are the same.

[0332] In some embodiments, L 5B and L 5C are the same.

[0333] In some embodiments, the oligomeric compounds described herein, including but not limited to regulatable REVERSIR compounds and siRNA, have the structure: [ka] Contains monomers.

[0334] In some embodiments, the oligomeric compounds described herein, including but not limited to regulatable REVERSIR compounds and siRNA, have the structure: [ka] Contains monomers.

[0335] In some embodiments, the oligomeric compounds described herein, including but not limited to regulatable REVERSIR compounds and siRNA, have the structure: [ka] Contains monomers.

[0336] In some embodiments, the oligomeric compounds described herein, including but not limited to regulatable REVERSIR compounds and siRNA, have the structure: [ka] where Y is O or S and n is 3 to 6.

[0337] In some embodiments, the oligomeric compounds described herein, including but not limited to regulatable REVERSIR compounds and siRNA, have the structure: [ka] where Y is O or S and n is 3 to 6.

[0338] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains monomers.

[0339] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] where X is O or S.

[0340] In some embodiments, the oligomeric compounds described herein, including but not limited to regulatable REVERSIR compounds and siRNA, are [ka] [ka] The monomer selected from the group consisting of:

[0341] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] monomers, where R is OH or NHCOOH.

[0342] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] monomers, where R is OH or NHCOOH.

[0343] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] monomers, where R is O or S.

[0344] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] monomers, where R is OH or NHCOOH.

[0345] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains monomers.

[0346] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] monomers, where R is OH or NHCOOH.

[0347] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] monomers, where R is OH or NHCOOH.

[0348] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] monomers, where R is OH or NHCOOH.

[0349] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] monomers, where R is OH or NHCOOH.

[0350] In some embodiments, the oligomeric compounds described herein, such as, but not limited to, regulatable REVERSIR compounds and siRNAs have the structure: [ka] Contains monomers.

[0351] In the monomers described above, X and Y each independently represent H, a protecting group, a phosphoric acid group, a phosphodiester group, an activated phosphoric acid group, an activated phosphorous acid group, a phosphoramidite, a fixed support, -P(Z')(Z'')O-nucleotide, -P(Z')(Z'')O-nucleotide, lipid, PEG, steroid, polymer, nucleotide, nucleotide or oligonucleotide; Z' and Z'' are each independently O or S for each occurrence.

[0352] In some embodiments, the tunable REVERSIR compound has the structure: [ka] is conjugated with the ligand of

[0353] In some embodiments, the conjugated siRNA has the structure: [ka] has a ligand of

[0354] In some embodiments, the tunable REVERSIR compound has the structure: [ka] where each n is independently 1-20.

[0355] In one example, a tunable REVERSIR compound has the structure: [ka] is conjugated with the ligand of

[0356] Synthesis of the aforementioned ligands and monomers is described, for example, in US Pat. No. 8,106,022, the contents of which are incorporated herein by reference in their entirety.

[0357] Linking groups or bifunctional linking moieties such as those known in the art are suitable for the compounds provided herein. Linking groups are useful, for example, for attaching chemical functional groups, conjugate groups, reporter groups, and other groups to selective sites in parent compounds such as oligomeric compounds. Generally, a difunctional binding moiety is a hydrocarbonyl moiety with two functional groups. One of the functional groups is selected to bind to the parent molecule or compound of interest, and the other is selected to essentially bind to any group selected, such as a chemical functional group or conjugate group. . In some embodiments, the linker comprises a chain structure or oligomer of repeating units, such as ethylene glycol or amino acid units. Examples of functional groups commonly used in bifunctional binding moieties include, but are not limited to, electrophilic groups for reacting with nucleophilic groups and nucleophilic groups for reacting with electrophilic groups. In some embodiments, bifunctional binding moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturation (eg, double or triple bond), and the like. Some non-limiting examples of bifunctional binding moieties include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, or substituted or unsubstituted C2-C10 alkynyl; a non-limiting list of preferred substituents includes hydroxyl, Includes amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

[0358] In some embodiments, the ligand is conjugated to the oligomeric compound via a linker.

[0359] As used herein, the term "linker" refers to an organic moiety that connects two parts of a compound. The linker is typically a direct bond or an atom such as oxygen or sulfur, NR 1 , C(O), C(O)NH, SO, SO 2 , S.O. 2 Units such as NH or substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, Heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylararylalkyl, alkynylarylalkenyl, alkynylaryl Alkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl , alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl rule, atomic chains such as alkynylhereroaryl, where one or more methylenes are O, S, S(O), SO 2 , N(R 1 ) 2 , C(O), a cleavable linking moiety, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, where R 1 is hydrogen, acyl, aliphatic or substituted aliphatic.

[0360] In one embodiment, the linker is -[(P-Q''-R) q -X-(P’-Q’’’-R’) q’ ] q’’ -T-, where P, R, T, P', R' and T are each independently present, CO, NH, O, S, OC(O), NHC( O), CH 2 ,C.H. 2 N.H., C.H. 2 O;NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CH=N-O, [ka] or heterocyclyl; Q’’ and Q’’’ are independent of each other for their existence, non-existence, -(CH 2 ) n -, -C(R 1 )(R 2 )(CH 2 ) n -,-(CH 2 ) n C(R 1 )(R 2 )-,-(CH 2 CH 2 O) m CH 2 CH 2 -or-(CH 2 CH 2 O) m CH 2 CH 2 NH-, and X is absent or a cleavable linking group; R a is H or an amino acid side chain; R 1 and R 2 are independently H, CH for each existence 3 , OH, SH or N(R N ) 2 And; R N is, for each occurrence independently, H, methyl, ethyl, propyl, isopropyl, butyl or benzyl; q, q' and q'' are each independently for each occurrence from 0 to 20, where the repeating units may be the same or different; n is 1 to 20 for each entity independently; m is 0 to 50 for each entity independently.

[0361] In some embodiments, the linker includes at least one cleavable linking group.

[0362] In some embodiments, the linker is a branched linker. The branch point of the branched linker may be at least trivalent, but may be a tetravalent, pentavalent or hexavalent atom or a group exhibiting such multiple valency. In some embodiments, the branch points are -N, -N(Q)-C, -O-C, -S-C, -SS-C, -C(O)N(Q)-C, -OC(O)N( Q)-C, -N(Q)C(O)-C or -N(Q)C(O)O-C; where Q, for each occurrence independently, is H or optionally substituted is an alkyl. In some embodiments, the branch point is glycerol or a glycerol derivative.

[0363] A cleavable linking group is one that is sufficiently stable outside the cell, but upon entering the target cell, is cleaved, releasing the two moieties that the linker holds together. In a preferred embodiment, the cleavable linking group is in the blood or serum of the subject or under a second reference condition (e.g., which can be selected to mimic or represent conditions found in blood or serum). It is cleaved at least 10 times faster, preferably at least 100 times faster, in the target cell or under the first reference conditions (which can be selected, for example, to mimic or represent conditions inside the cell).

[0364] Cleavable linking groups are sensitive to cleavage agents such as pH, redox potential or the presence of degrading molecules. Generally, cleavage agents are more prevalent or found at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include: redox agents selected for specific substrates or redox agents without substrate specificity, such as oxidizing or reductases or Reducing agents such as mercaptans present in cells that can break down binding groups that are cleavable to Enzymes capable of hydrolyzing or degrading acid-cleavable linking groups by acting as , peptidases (which may be substrate specific) and proteases as well as phosphatases.

[0365] The linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linkage group incorporated into the linker can vary depending on the cells to be targeted. For example, a liver targeting ligand can be linked to a cationic lipid via a linker containing an ester group. Hepatocytes are rich in esterases, so the linker will be cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include lung, renal cortex and testicular cells.

[0366] When targeting peptidase-rich cell types such as hepatocytes and synoviocytes, linkers containing peptide bonds can be used.

[0367] In some embodiments, the cleavable linking group is intracellular (or mimics intracellular conditions) as compared to blood or serum (or in vitro conditions selected to mimic extracellular conditions). at least 1.25, 1.5, 1.75, 2, 3, 4, 5, 10, 25, 50 or 100 times faster under in vitro conditions selected as such. In some embodiments, the cleavable linking group is in the blood (or in a manner that mimics extracellular conditions) as compared to within the cell (or under in vitro conditions selected to mimic intracellular conditions). cleaved by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less than 1% under in vitro conditions selected for

[0368] Exemplary cleavable linking groups include, but are not limited to, the following: redox-cleavable linking groups (e.g., -S-S and -C(R) 2 -S-S-, where R is H or C 1 ~C 6 alkyl and at least one R is C 1 ~C 6 Alkyl, e.g. CH 3 or CH 2 CH 3); phosphate-based cleavable linking groups (e.g., -O-P(O)(OR)-O-, -O-P(S)(OR)-O-, -O-P(S)(SR)-O- , -S-P(O)(OR)-O-, -O-P(O)(OR)-S-, -S-P(O)(OR)-S-, -O-P(S)(ORk)-S-, - S-P(S)(OR)-O-, -O-P(O)(R)-O-, -O-P(S)(R)-O-, -S-P(O)(R)-O-, -S-P( S)(R)-O-, -S-P(O)(R)-S-, -O-P(S)(R)-S-, -O-P(O)(OH)-O-, -O-P(S) (OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH )-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)- O-, -S-P(O)(H)-O-, -S-P(S)(H)-O-, -S-P(O)(H)-S- and -O-P(S)(H)-S- , where R is an optionally substituted linear or branched C 1 ~C 10 acid-cleavable linking groups (e.g. hydrazones, esters and esters of amino acids, -C=NN- and -OC(O)-); ester-based cleavable linking groups (e.g. -C (O)O-); Peptide-based cleavable linking groups (e.g., linking groups that are cleaved by intracellular enzymes such as peptidases and proteases, e.g. -NHCHR); A C(O)NHCHHR B C(O)-, where R A and R B is an R group composed of two adjacent amino acids). Peptide-based cleavable linking groups contain two or more amino acids. In some embodiments, the peptide-based cleavable linking group comprises an amino acid sequence that is a substrate for a peptidase or protease found in cells.

[0369] In some embodiments, the acid-cleavable linking group can act as a general acid or in an acidic environment having a pH of about 6.5 or less (e.g., about 6.-, 5.5, 5.0 or below). Cleaved by agents such as enzymes.

[0370] In some embodiments, the linker is an oligonucleotide linker, including but not limited to (N) n (N is independently a modified or unmodified nucleotide, and n is 1 to 23). In some embodiments, n is 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the oligonucleotide linker is GNRA, (G) 4 ,(U) 4 and (dT) 4 where N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide. Some nucleotides in the linker may participate in base pairing interactions with other nucleotides in the linker. Those skilled in the art can use any of the oligonucleotide chemical modifications or changes described herein to oligonucleotide linkers. In some embodiments, the linker is dA.

[0371] motif The present invention further includes oligomeric compounds that are chimeric oligomeric compounds. In the context of the present invention, a "chimeric" oligomeric compound or "chimera" is an oligomeric compound comprising two or more chemically distinct regions, each region comprising at least one monomer unit, i.e. in the case of an oligonucleotide; Composed of modified or unmodified nucleotides. Chimeric oligomeric compounds can be described as having specific motifs. In some embodiments, motifs include, but are not limited to, alternating motifs, gap motifs, hemimer motifs, homogeneously fully modified motifs, and positional modification motifs. As used herein, the phrase "chemically distinct region" refers to a region that differs from other regions by having a modification that is not present elsewhere in the oligomeric compound or in the absence of a modification that is present elsewhere in the oligomeric compound. Refers to the oligomer region. Oligomeric compounds may contain two or more chemically distinct regions. As used herein, regions that do not contain modifications are also considered to be chemically different.

[0372] Chemically different regions can be repeated within an oligomeric compound. Thus, a pattern of chemically different regions within an oligomeric compound can be realized such that a first chemically different region is followed by one or more second chemically different regions. Such sequences of chemically distinct regions can be repeated one or more times. Preferably, the sequence is repeated two or more times. Both strands of a double-stranded oligomeric compound can contain these sequences. Each chemically different region may actually contain only a single monomer, such as a nucleotide. In some embodiments, each of the chemically different regions is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or Contains 18 monomers, e.g. nucleotides.

[0373] In some embodiments, the alternating nucleotides have the same modification, e.g., all odd nucleotides in a strand have the same modification and / or all even nucleotides in a strand have the same modification. with similar modifications. In some embodiments, all odd nucleotides in an oligomeric compound have the same modification and all even nucleotides have a modification that is not present in the odd nucleotides, and vice versa.

[0374] When both strands of a double-stranded oligomeric compound contain alternating modification patterns, the nucleotides of one strand can be in complementary positions to similarly modified nucleotides of the second strand. In another embodiment, there is a phase shift between the patterns of modification of each first strand relative to the pattern of similar modifications of the second strand. Preferably, this shift is such that the similarly modified nucleotides of the first and second strands are not in complementary positions to each other.

[0375] In some embodiments, the first strand has an alternating modification pattern in which alternating nucleotides include 2'-modifications, such as 2'-O-methyl modifications. In some embodiments, the first strand includes alternating 2'-O-methyl modifications and the second strand includes alternating 2'-fluoro modifications. In other embodiments, both strands of the double-stranded oligonucleotide contain alternating 2'-O-methyl modifications.

[0376] When both strands of a double-stranded oligomeric compound contain alternating 2'-O-methyl modifications, such 2'-modified nucleotides may be at complementary positions in the double helix region. Instead, such 2'-modified nucleotides are not in complementary positions in the double helix region.

[0377] In some embodiments, the oligonucleotide comprises two chemically distinct regions, where each region is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides long. be.

[0378] In another embodiment, the oligomeric compound includes three chemically distinct regions. The central region is about 5 to 15 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) nucleotides in length, and each flanking or wing region independently 1 to 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides in length. All three regions may contain different modifications, or the wing regions may have similar modifications to each other. In some embodiments, the wing regions are the same length, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length.

[0379] As used herein, the term "alternating motif" refers to a series of linked monomer subunits in which the monomer subunits have two different types of sugar groups that alternate over substantially the entire sequence of the oligomeric compound. Refers to an oligomeric compound containing a sequence. Oligomeric compounds with alternating motifs can be represented by the formula: 5'-A(-L-B-L-A)n(-L-B)nn-3', where A and B are monomeric subunits with different sugar groups. each L is an internucleoside linking group, n is about 4 to about 12, and nn is 0 or 1. This allows for alternating oligomeric compounds from about 9 to about 26 monomer subunits in length. This length range is not limiting, as longer and shorter oligomeric compounds are also suitable for the present invention. In one embodiment, one of A and B is a 2'-modified nucleoside as described herein.

[0380] As used herein, the term "type of modification" with respect to a "type" of nucleoside or nucleotide refers to modification of the nucleoside and includes modified and unmodified nucleosides. Thus, unless otherwise specified, a "nucleoside having a first type of modification" may be an unmodified nucleoside.

[0381] As used herein, "type region" refers to a portion of an oligomeric compound in which the nucleosides and internucleoside linkages within said region contain the same type of modification; the nucleosides of any adjacent portions and / or the internucleoside linkage comprises at least one different type of modification. As used herein, the term "homogeneous fully modified motif" refers to an oligonucleotide that includes a contiguous array of linked monomer subunits, each having the same type of sugar group. In one embodiment, a homogeneous fully modified motif comprises a contiguous sequence of nucleosides of the invention. In one embodiment, one or both of the 3' and 5' ends of the contiguous sequence of nucleosides described herein comprises a terminal group, such as one or more unmodified nucleosides.

[0382] As used herein, the term "hemimeric motif" refers to an oligomeric compound having a short contiguous array of monomeric subunits with one type of sugar group located at the 5' or 3' end; , the remaining monomer subunits have different types of sugar groups. Generally, a hemimer is a short region (1, 2, 3, 4, or about 5 monomer subunits) that has homogeneous but different sugar groups and is located at either the 3' or 5' end of the oligomeric compound. It is an oligomeric compound of homogeneous sugar groups, further comprising: In one embodiment, the hemimeric motif comprises a contiguous sequence composed of about 10 to about 28 monomer subunits of one type and 1-5 or 2-5 of a second type located at one end. and two monomer subunits. In one embodiment, the hemimer is a contiguous chain composed of about 8 to about 20 β-D-2'-deoxyribonucleosides with 1 to 12 contiguous nucleosides of the invention located at one end. It is an array. In one embodiment, the hemimer is a contiguous sequence composed of about 8 to about 20 β-D-2'-deoxyribonucleosides with 1 to 5 contiguous nucleosides of the invention located at one end. It is. In one embodiment, the hemimer is a contiguous chain composed of about 12 to about 18 β-D-2'-deoxyribo-nucleosides with 1 to 3 contiguous nucleosides of the invention located at one end. It is an array. In one embodiment, the hemimer is a contiguous sequence composed of about 10 to about 14 β-D-2'-deoxyribonucleosides with 1 to 3 contiguous nucleosides of the invention located at one end. It is.

[0383] As used herein, the term "blockmer motif" refers to monomer subunits in which the sugar groups on each monomer subunit are the same except for intervening internal blocks of consecutive monomer subunits having different types of sugar groups. refers to an oligonucleotide containing a continuous sequence of Blockmers have some overlap in definition with gapmers, but typically, for blockmers, the only monomer subunits within the block are non-natural sugar groups; for gapmers, the monomer subunits within the external region are only non-natural sugar groups; the remaining monomer subunits within the blockmer or gapmer are β-D-2'-deoxyribonucleosides or β-D-ribonucleosides. In one embodiment, provided herein are blockmer oligonucleotides in which all of the monomer subunits contain non-natural sugar groups.

[0384] As used herein, the term "positionally modified motif" means separated by two or more regions composed of from 1 to about 5 contiguous monomer subunits bearing different types of sugar groups. It is meant to contain a continuous array of monomer subunits having one type of sugar group. Each of the two or more regions composed of 1 to about 5 consecutive monomer subunits is independently modified homogeneously with respect to the type of sugar group. In one embodiment, each of the two or more regions has the same type of sugar group. In one embodiment, each of the two or more regions has a different type of sugar group. In one embodiment, the invention comprises a sequence composed of 8 to 20 β-D-2'-deoxyribonucleosides, further comprising two or three regions composed of 2 to about 5 contiguous nucleosides. Positionally modified oligonucleotides are provided. Positionally modified oligonucleotides have gap motifs because the pattern of substitution in the region characterized by either positional motif does not match the definitions provided herein for one of these other motifs. , as distinguished from hemimer motifs, blocker motifs and alternating motifs. The term positionally modified oligomeric compound includes many different specific substitution patterns.

[0385] As used herein, the term "gapmer" or "gap oligomeric compound" refers to an oligomeric compound that has two external regions or wings and one internal region or gap. The three regions form a continuous array of monomer subunits with the sugar groups of the inner region and the sugar groups of the outer region different, where the sugar groups of each monomer subunit within a particular region are the same. When the sugar groups in the external region are the same, the gapmer is a symmetric gapmer, and when the sugar groups used in the 5'-external region are different from the sugar groups used in the 3'-external region, the gapmer is symmetrical. The mer is an asymmetric gap mer. In one embodiment, the external region is small (each independently 1, 2, 3, 4, or about 5 monomer subunits) and the monomer subunits have an internal region containing β-D-2'-deoxyribonucleosides. Contains unnatural sugar groups with In one embodiment, the external region each independently comprises from 1 to about 5 monomer subunits and the internal region comprises from 6 to 18 unmodified nucleosides. The internal region or gap generally contains β-D-2'-deoxyribo-nucleosides, but may contain non-natural sugar groups.

[0386] In one embodiment, the gap oligomeric compound comprises an internal region of β-D-2'-deoxyribonucleosides, where one of the external regions comprises a nucleoside of the invention. In one embodiment, the gap oligonucleotide comprises an internal region of β-D-2'-deoxyribonucleosides, with both external regions comprising nucleosides of the invention. In one embodiment, the gap oligonucleotide comprises an internal region of β-D-2'-deoxyribonucleosides, with both external regions comprising nucleosides of the invention. In one embodiment, provided herein is a gapped oligonucleotide in which all monomer subunits contain non-natural sugar groups. In one embodiment, it consists of one or two nucleosides of the invention at the 5' end, two or three nucleosides of the invention at the 3' end, and 10 to 16 β-D-2'-deoxyribonucleosides. Gap oligonucleotides are provided that include an internal region. In one embodiment, one nucleoside of the invention at the 5' end, two nucleosides of the invention at the 3' end, and an internal region consisting of 10-16 β-D-2'-deoxyribonucleosides. Provided are gap oligonucleotides comprising: In one embodiment, an internal region consisting of two nucleosides of the invention at the 5' end, two nucleosides of the invention at the 3' end, and 10 to 14 β-D-2'-deoxyribonucleosides. Provided are gap oligonucleotides comprising: In one embodiment, gap oligonucleotides of about 10 to about 21 monomer subunits in length are provided. In one embodiment, gap oligonucleotides of about 12 to about 16 monomer subunits in length are provided. In one embodiment, gap oligonucleotides of about 12 to about 14 monomer subunits in length are provided.

[0387] In some embodiments, the 5' terminal monomer of the oligomeric compounds of the invention includes a phosphorus-containing moiety at the 5' end. In some embodiments, the 5' terminal monomer includes a 2'-modification. In some such embodiments, the 2'-modification of the 5' terminal monomer is a cationic modification. In some embodiments, the 5' terminal monomer includes a 5'-modification. In some embodiments, the 5' terminal monomer includes a 2'-modification and a 5'-modification. In some embodiments, the 5' terminal monomer comprises a 5'-stabilizing nucleoside. In some embodiments, modification of the 5' terminal monomer stabilizes the 5'-phosphate. In some embodiments, oligomeric compounds that include modifications of the 5' terminal monomer are resistant to exonucleases. In some embodiments, oligomeric compounds that include modifications of the 5' terminal monomer have improved tunable REVERSIR properties. In some embodiments, oligomeric compounds that include modifications of the 5' terminal monomer have improved association with strands of siRNA.

[0388] In some embodiments, the 5' terminal monomer is attached to the remaining oligomeric compound by a modified linkage. In some such embodiments, the 5' terminal monomer is attached to the remaining oligomeric compound by a phosphorothioate linkage.

[0389] In some embodiments, oligomeric compounds of the invention include one or more regions of alternating modification. In some embodiments, oligomeric compounds include one or more regions of alternating nucleoside modification. In some embodiments, oligomeric compounds include one or more regions of alternating bond modification. In some embodiments, oligomeric compounds include one or more alternating nucleosides and regions of bond modification.

[0390] In some embodiments, oligomeric compounds of the invention include regions of one or more alternating 2'-F modified nucleosides and 2'-OMe modified nucleosides. In some such embodiments, such regions of alternating 2'-F modified nucleosides and 2'-OMe modified nucleosides also include alternating linkages. In some such embodiments, the linkage at the 3' end of the 2'-F modified nucleoside is a phosphorothioate linkage. In some such embodiments, the bond at the 3' end of the 2'-OMe nucleoside is a phosphodiester bond.

[0391] In some embodiments, such alternating regions: (2’-F)-(PS)-(2’-OMe)-(PO) It is.

[0392] In some embodiments, the oligomeric compound comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 such alternating regions. Such regions may be contiguous or separated by different modified nucleosides or linkages.

[0393] In some embodiments, one or more alternating regions in an alternating motif comprises more than one type of single nucleoside. For example, oligomeric compounds of the invention may include the following nucleoside motifs: ABA; ABBA; AABA; AABBAA; ABBABB; AABAAB; ABBABAABB; ABABAA; AABABAB; ABABAA; ABBAABBABABAA; BABBAABBABABAA; or ABABBAABBABABAA where A is a first type of nucleoside and B is a second type of nucleoside. In some embodiments, A and B are each selected from 2'-F, 2'-OMe, LNA, DNA, and MOE.

[0394] In some embodiments, A is DNA. In some embodiments, B is DNA. In some embodiments, A is 4’-CH 2 It is O-2’-LNA. In some embodiments, B is 4'-CH 2 It is O-2’-LNA. In some embodiments, A is DNA and B is 4'-CH 2 It is O-2’-LNA. In some embodiments, A is 4'-CH 2 In O-2'-LNA, B is DNA.

[0395] In some embodiments, A is 2'-OMe. In some embodiments, B is 2'-OMe. In some embodiments, A is 2'-OMe and B is 4'-CH 2 It is O-2’-LNA. In some embodiments, A is 4'-CH 2 In O-2'-LNA, B is 2'-OMe. In some embodiments, A is 2'-OMe and B is DNA. In some embodiments, A is DNA and B is 2'-OMe.

[0396] In some embodiments, A is (S)-cEt. In some embodiments, B is (S)-cEt. In some embodiments, A is 2'-OMe and B is (S)-cEt. In some embodiments, A is (S)-cEt and B is 2'-OMe. In some embodiments, A is DNA and B is (S)-cEt. In some embodiments, A is (S)-cEt and B is DNA.

[0397] In some embodiments, A is 2'-F. In some embodiments, B is 2'-F. In some embodiments, A is 2'-F and B is 4'-CH 2 It is O-2’-LNA. In some embodiments, A is 4'-CH 2 In O-2'-LNA, B is 2'-F. In some embodiments, A is 2'-F and B is (S)-cEt. In some embodiments, A is (S)-cEt and B is 2'-F. In some embodiments, A is 2'-F and B is DNA. In some embodiments, A is DNA and B is 2'-F. In some embodiments, A is 2'-OMe and B is 2'-F. In some embodiments, A is DNA and B is 2'-OMe. In some embodiments, A is 2'-OMe and B is DNA.

[0398] In some embodiments, oligomeric compounds having such alternating motifs also include a 5' terminal nucleoside that includes a phosphate stabilizing modification. In some embodiments, the oligomeric compounds having alternating motifs described above also include a 5' terminal nucleoside that includes a 2' cationic modification. In some embodiments, the oligomeric compounds having alternating motifs described above also include a 5' end modification.

[0399] 2-2-3 motif In some embodiments, oligomeric compounds of the invention include a region with a 2-2-3 motif. Such areas are based on the following motifs: 5’- (E) w -(A) 2 -(B) X -(A) 2 -(C) y -(A) 3 -(D) Z including; where A is a first type of modified nucleoside; B, C, D and E are nucleosides that are modified differently from A, but B, C, D and E may have the same or different modifications; w and z are 0 to 15; x and y are 1 to 15.

[0400] In some embodiments, A is a 2'-OMe modified nucleoside. In some embodiments, B, C, D and E are all 2'-F modified nucleosides. In some embodiments, A is a 2'-OMe modified nucleoside and B, C, D and E are all 2'-F modified nucleosides.

[0401] In some embodiments, all 2-2-3 motif bonds are modified bonds. In some embodiments, the bonds are all phosphorothioate bonds. In some embodiments, the 3'-end bond of each first type of modification is a phosphodiester.

[0402] In some embodiments, Z is 0. In such embodiments, the first type of three nucleoside region is at the 3' end of the oligonucleotide. In some embodiments, such a region is at the 3' end of the oligomeric compound and no other groups are attached to the 3' end of the 3-nucleoside region of the first type. In some embodiments, oligomeric compounds, including oligonucleotides where Z is 0, can include a terminal group attached to a 3' terminal nucleoside. Such terminal groups may include other nucleosides. Such other nucleosides are typically non-hybridizing nucleosides.

[0403] In some embodiments, Z is 1-3. In some embodiments, Z is 2. In some embodiments, the Z nucleoside is a 2'-MOE nucleoside. In some embodiments, Z represents a non-hybridizing nucleoside. For the avoidance of confusion, it is noted that such non-hybridizing nucleosides may also be described as 3' end groups with Z=0.

[0404] combination motif It should be understood that some of the motifs and modifications described above may be combined. Since a motif may contain only a few nucleosides, certain oligomeric compounds may contain more than one motif. As a non-limiting example, in some embodiments, oligomeric compounds can have two or more nucleotide motifs selected from LNA, phosphorothioate linkages, 2'-OMe, conjugated ligands.

[0405] Oligomeric compounds having any of the various nucleoside motifs described herein can also have any binding motif. For example, in oligomeric compounds, the first 1, 2, 3, 4, or 5 at the 5' end are modified intersugar linkages, and the first 4, 5, 6, 7, or 8 sugar linkages at the 3' end are The linkage can be a modified sugar-sugar linkage. The central region of such modified oligomeric compounds may have intersugar linkages based on any of the other motifs described herein, such as homogeneous, alternating, hemimers, gapmers, and the like. In some embodiments, the oligomeric compound has phosphorothioate linkages between the first and second monomers at the 5' end, alternating phosphorothioate / phosphodiester linkages in the central region, and 6, 7, or 8 phosphorothioate linkages at the 3' end. include.

[0406] It should be noted that the length of the region defined by the nucleoside motif and the length of the binding motif need not be the same.

[0407] In some embodiments, at least one strand of a single-stranded oligomeric compound or a double-stranded oligomeric compound includes at least one of the following motifs: (a) 5'-phosphorothioate or 5'-phosphorodithioate; (b) cationic modification of nucleotides 1 and 2 at the 5' end, where the cationic modification is located at the C5 position of the pyridimine and the C2, C6, C8 of the purine, exocyclic N2 or exocyclic N6; (c) at least one G-clamp nucleotide in the first two terminal nucleotides of the 5' end and other nucleotides with a cationic modification, where the cationic modification is at the C5 position of a pyridimine or the C2, C6, C8 of a purine. , located at the exocyclic N2 or exocyclic N6 position; (d) at least one 2'-F modified nucleotide comprising a nucleobase modification; (e) at least one gem-2'-O-methyl / 2'-F modified nucleotide comprising a nucleobase modification; preferably the methyl substituent is in an upward configuration, such as an arabinose configuration; (f) a 3'-terminal 5'-PuPu-3' dinucleotide, where either nucleotide is a 5'-PuPu-3' dinucleotide, the contents of which are incorporated herein by reference in their entirety; with a modified MOE in the 2' position as described in; (g) a 5'-terminal 5'-PuPu-3' dinucleotide, where either nucleotide includes a modified MOE at the 2' position, as described in U.S. Patent Application Publication No. 20130130378; (h) a nucleotide at the 5' end, comprising a modified MOE at the 2' position, as described in U.S. Patent Application Publication No. 20130130378; (i) 5’ terminal nucleotide with 3’-F modification: (j) 5'-terminal nucleotide containing a 4'-substituent; (k) 5’ terminal nucleotide containing O4’ modification; (l) a 3' terminal nucleotide containing a 4'-substituent; and (m) A combination of these.

[0408] In some embodiments, both strands of the double-stranded oligomeric compound independently include at least one of the motifs described above. In some other embodiments, both strands of the double-stranded oligomeric compound include at least one of the aforementioned motifs, and these motifs can be the same or different, or the same or It can be some combination of different ones.

[0409] The foregoing examples are provided to illustrate how the motifs described can be used in combination; the invention is not intended to be limited to the particular combinations or specific modifications used to illustrate the combinations. Unintentional. Furthermore, specific examples such as, but not limited to, those shown in the table above are intended to encompass more general embodiments. For example, column A of the table above illustrates regions of alternating 2'-OMe and 2'-F nucleosides. Accordingly, this same disclosure also exemplifies regions of alternating 2'-F modifications. This also exemplifies regions of alternating 2'-O-alkyl and 2'-halogen nucleosides. This also illustrates alternating regions of different modified nucleosides. All of the examples throughout this specification contemplate such an inclusive interpretation.

[0410] Note that the length of oligomeric compounds such as those exemplified in the table above can be easily manipulated by lengthening or shortening one or more of the regions described without breaking the motif.

[0411] In some embodiments, the oligomeric compound comprises two or more chemically different regions and is disclosed in International Application No. PCT / U.S. has the structure described in (incorporated herein in its entirety).

[0412] Synthesis, purification and analysis Oligomerization of modified and unmodified nucleosides and nucleotides can be performed using DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and / or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al. , Applications of Chemically synthesized RNA in RNA:Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713). can be implemented.

[0413] The oligomeric compounds provided by the present invention can be generally and routinely produced by known techniques of solid phase synthesis. Equipment for such synthesis is sold by several suppliers, including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be used. It is well known to prepare oligonucleotides such as phosphorothioate and alkylated derivatives using similar techniques. The invention is not limited by the method of antisense compound synthesis.

[0414] Methods for the purification and analysis of oligomeric compounds are well known to those skilled in the art. Analytical methods include capillary electrophoresis (CE) and electrospray mass spectrometry. Such synthetic and analytical methods can be performed in multiwell plates. The method of the present invention is not limited by the oligomer purification method.

[0415] The oligomeric compounds of the invention can be prepared using liquid phase or solid phase organic synthesis or enzymatically by methods known in the art. Organic synthesis offers the advantage that oligomeric chains containing unnatural or modified nucleotides can be easily prepared. Any other means for such synthesis known in the art may additionally or alternatively be used. It is also well known to prepare other oligomeric compounds using similar techniques, such as those containing phosphorothioates, phosphorodithioates, and alkylated derivatives of intersugar linkages. Double-stranded oligomeric compounds of the invention can be prepared using a two-step method. First, the individual strands of the double-stranded molecule are prepared separately. Next, the constituent strands are annealed.

[0416] Regardless of the method of synthesis, oligomeric compounds can be prepared in solutions suitable for formulation (eg, aqueous and / or organic solutions). For example, the oligomeric preparation can be precipitated, redissolved in pure double-distilled water, and then lyophilized. The dried oligomeric compound can then be resuspended in a solution appropriate for the intended formulation method.

[0417] Teachings regarding the synthesis of specific modified oligomeric compounds can be found in: U.S. Pat. Nos. 5,138,045 and 5,218,105 for polyamine-conjugated oligonucleotides; U.S. Pat. No. 5,212,295 for monomers; U.S. Pat. Nos. 5,378,825 and 5,541,307 for oligonucleotides with modified backbones; U.S. Patents for backbone-modified oligonucleotides and their preparation by reductive coupling No. 5,386,023; U.S. Pat. No. 5,457,191 for modified nucleobases based on the 3-deazapurine ring system and methods for their synthesis; U.S. Pat. No. 5,459,255 for modified nucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302 for a method for preparing oligonucleotides with chiral phosphorus linkages; U.S. Pat. No. 5,539,082 for peptide nucleic acids; U.S. Pat. No. 5,554,746 for oligonucleotides with a beta-lactam backbone US Patent No. 5,571,902, for methods and materials for the synthesis of oligonucleotides; nucleosides having alkylthio groups (such groups are used as linkers to other moieties attached to any of the various positions of the nucleoside); U.S. Pat. No. 5,578,718 for oligonucleotides having phosphorothioate linkages of high chiral purity; U.S. Pat. and related compounds, such as 2,6-diaminopurine compounds; U.S. Patent No. 5,587,469 for oligonucleotides with N-2 substituted purines; and 3-deazapurines. US Pat. No. 5,587,470, both relating to oligonucleotides; US Pat. No. 5,223,168 and US Pat. No. 5,608,046, both relating to conjugated 4'-desmethyl nucleoside analogs; and US Pat. No. 5,608,046, both relating to backbone-modified oligonucleotide analogs. U.S. Pat. Nos. 5,602,240 and 5,610,289; and U.S. Pat. Nos. 6,262,241 and 5,459,255, which relate to methods of synthesizing 2'-fluoro-oligonucleotides, among others.

[0418] Compositions and methods for formulating pharmaceutical compositions The oligomeric compounds can be mixed with pharmaceutically acceptable active and / or inactive substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for formulating pharmaceutical compositions will depend on a number of criteria including, but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

[0419] Oligomeric compounds such as siRNA and / or regulatable REVERSIR compounds can be used as pharmaceutical compositions by combining such oligomeric compounds with a suitable pharmaceutically acceptable diluent or carrier. Pharmaceutically acceptable diluents include phosphate buffered saline (PBS). PBS is a suitable diluent for use in compositions intended for parenteral delivery. Accordingly, in one embodiment, a pharmaceutical composition comprising an antisense compound and / or an antidote compound and a pharmaceutically acceptable diluent is used in the methods described herein. In some embodiments, the pharmaceutically acceptable diluent is PBS.

[0420] Pharmaceutical compositions containing oligomeric compounds include any pharmaceutically acceptable salts, esters or salts of such esters. In some embodiments, a pharmaceutical composition comprising an oligomeric compound is capable of imparting (directly or indirectly) a biologically active metabolite or residue thereof when administered to an animal, such as a human. contains one or more oligonucleotides that can be used. Thus, for example, the present disclosure also relates to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other biological equivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0421] Prodrugs can include the incorporation of another nucleoside at one or both ends of the oligomeric compound that is cleaved by endogenous nucleases in the body to form the active oligomeric compound.

[0422] The pharmaceutical compositions of the invention can be administered in a variety of ways depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (eg, by a transdermal patch), pulmonary, by inhalation or insufflation of a powder or aerosol, using, for example, a nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous, eg, an implantable device; or intracranial, eg, intraparenchymal, intrathecal or intraventricular administration.

[0423] Oligomeric compounds can be delivered to target specific tissues, such as the liver (eg, hepatocytes of the liver).

[0424] Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickening agents and the like may be required or desirable. Coated condoms, gloves, etc. may also be useful. Suitable topical formulations include those in which the subject iRNA of the present invention is mixed with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoyl phosphatidylcholine DMPC, disteroylphosphatidylcholine) negative (e.g. dimyristoyl phosphatidylglycerol DMPG) and cationic (e.g. dioleoylphosphatidyl choline) Methylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNA that is the subject of the invention may be encapsulated in liposomes or complexed therewith, in particular with cationic liposomes. Alternatively, iRNA can be complexed with lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicapric acid esters, tricapric acid. Acid ester, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine or C 1~20 Examples include alkyl esters (eg, isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in US Pat. No. 6,747,014, which is incorporated herein by reference.

[0425] There are many organized surfactant structures other than microemulsions that have been studied and used for drug formulation. These include monolayers, micelles, bilayers and vesicles. Vesicles such as liposomes have attracted great interest because of the specificity and duration of action they offer from a drug delivery perspective. As used herein, the term "liposome" refers to a vesicle composed of amphipathic lipids arranged within a spherical bilayer or bilayers.

[0426] Liposomes are unilamellar or multilamellar vesicles with a membrane formed from a lipophilic substance and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of being able to fuse to the cell wall. Non-cationic liposomes cannot efficiently fuse to the cell wall but are taken up by macrophages in vivo.

[0427] Further advantages of liposomes are: Liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide variety of water- and fat-soluble drugs; that drugs encapsulated within their internal compartments may be protected from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.245). Important considerations in the preparation of liposome formulations are lipid surface charge, vesicle size, and liposome water content.

[0428] Liposomes are useful for transport and delivery of active ingredients to the site of action. Liposome membranes are structurally similar to biological membranes, so when liposomes are applied to tissues, they begin to fuse with cell membranes, and as liposome-cell fusion progresses, the contents of the liposomes are absorbed into the cell. into which the active agent can act.

[0429] Liposomal formulations have been the subject of extensive research as a delivery method for many drugs. For topical administration, there is increasing evidence that liposomes offer several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target site, and the ability to administer a variety of drugs, both hydrophilic and hydrophobic, into the skin. Includes the ability to

[0430] Multiple reports have detailed the ability of liposomes to deliver drugs containing high molecular weight DNA into the skin. Compounds including painkillers, antibodies, hormones and high molecular weight DNA have been delivered to the skin. In most cases, targeting of the upper epidermis was achieved by application.

[0431] Liposomes are divided into two major classes. Cationic liposomes are positively charged liposomes that interact with negatively charged DNA molecules to form stable complexes. Positively charged DNA / liposome complexes bind to negatively charged cell surfaces and are internalized within endosomes. Due to the acidic pH inside the endosome, the liposome is destroyed and releases its contents into the cytoplasm (Wang et al., Biochem.Biophys.Res.Commun., 1987, 147, 980-985).

[0432] Liposomes that are pH sensitive or negatively charged entrap DNA rather than complexing it. Because DNA and lipids are similarly charged, repulsion occurs rather than complex formation. Still, some DNA is trapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was directed within the target cell (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0433] One major type of liposome composition contains phospholipids other than naturally occurring phosphatidylcholines. Neutral liposome compositions can be formed from, for example, dimyristoyl phosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, whereas anionic fusogenic liposomes are primarily formed from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as, for example, soy PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholines and / or cholesterol.

[0434] Several studies have evaluated topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to the skin of guinea pigs achieved relief of herpes cutaneous sores, whereas delivery of interferon by other means (e.g., as a solution or emulsion) was not effective (Weiner et al. .,Journal of Drug Targeting,1992,2,405-410). Additionally, another study tested the efficacy of interferon administered as part of a liposomal formulation versus administration of interferon using an aqueous system and concluded that liposomal formulations were superior to aqueous administration. (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[0435] Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have also been tested to determine their usefulness in drug delivery to the skin. A nonionic liposome formulation containing Novasome (trademark) I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome (trademark) II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) , was used to deliver cyclosporin-A to the dermis of mouse skin. The results showed that these nonionic liposome systems were effective in promoting the deposition of cyclosporin-A in various layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466 ).

[0436] Liposomes also include "sterically stabilized" liposomes, which term as used herein refers to liposomes that include one or more specialized lipids that, when incorporated into the liposome, Such specialized lipid-free liposomes result in increased circulation lifetime compared to liposomes. As an example of a sterically stabilized liposome, a portion of the endoplasmic reticulum-forming lipid portion of the liposome (A) is monosialoganglioside G. M1 or (B) is derivatized with one or more hydrophilic polymers such as polyethylene glycol (PEG) moieties. Without intending to be bound by any particular theory, the art has shown that for sterically stabilized liposomes containing at least gangliosides, sphingomyelins, or PEG-derivatized lipids, increased circulation life of these sterically stabilized liposomes has been demonstrated. This is thought to be due to reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765 ).

[0437] A variety of liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann.N.Y.Acad.Sci., 1987, 507, 64) reported that monosialoganglioside G, which increases the blood half-life of liposomes, M1 , reported the ability of galactocerebroside sulfate and phosphatidylinositol. These findings are explained in detail by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). US Pat. No. 4,837,028 and WO 88 / 04924 (both by Allen et al.) describe (1) sphingomyelin and (2) ganglioside G. M1 Alternatively, a liposome containing galactocerebroside sulfate is disclosed. US Pat. No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97 / 13499 (Lim et al.).

[0438] Many liposomes containing lipids derivatized with one or more hydrophilic polymers and methods for their preparation are known in the art. Sunamoto et al. (Bull.Chem.Soc.Jpn., 1980, 53, 2778) reported that a nonionic detergent containing a PEG moiety, 2C 1215G describes liposomes containing Illum et al. (FEBS Lett., 1984, 167, 79) describe that hydrophilic coating of polystyrene particles with polymeric glycols significantly increased blood half-life. Synthetic phospholipids modified by attachment of carboxylic acid groups of polyalkylene glycols (eg, PEG) have been described by Sears (US Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) conducted experiments demonstrating that liposomes containing phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have a significant increase in circulating half-life. is listed. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended these observations to other PEG-derivatized phospholipids, such as DSPE-PEG, which is formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. expanded to. Liposomes having a PEG moiety covalently attached to the outer surface are described in Fisher, EP 0 445 131 B1 and WO 90 / 04384. Liposomes containing 1 to 20 mol% of PE derivatized with PEG and methods of using the same have been described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. and European Patent No. 0 496 813B1). Liposomes containing several other lipid-polymer conjugates have been described in WO 91 / 05545 and US Pat. No. 5,225,212 (both Martin et al.) and WO 94 / 20073 (Zalipsky et al. ) is disclosed. Liposomes containing PEG-modified ceramide lipids are described in WO 96 / 10391 (Choi et al.). US Pat. No. 5,540,935 (Miyazaki et al.) and US Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surface.

[0439] Several liposomes containing nucleic acids are known in the art. WO 96 / 40062 to Thierry et al. discloses a method for encapsulating high molecular weight nucleic acids in liposomes. US Pat. No. 5,264,221 to Tagawa et al. discloses protein-bound liposomes and claims that the contents of such liposomes can include dsRNA. Rahman et al., US Pat. No. 5,665,710, describes certain methods of encapsulating oligodeoxyribonucleotides in liposomes. WO 97 / 04787 to Love et al. discloses liposomes containing dsRNA targeted to the raf gene.

[0440] Transfersomes, another type of liposome, are highly deformable lipid aggregates and are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets, which are highly deformable and can therefore easily penetrate smaller pores. Transfersomes are able to adapt to the environment in which they are used, e.g. are self-optimizing (adaptive to the shape of skin pores), self-repair, and often reach their targets without fragmentation. , often self-filling. Surface active agents, usually surfactants, can be added to standard liposome compositions to produce transfersomes. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been found to be as effective as subcutaneous injection of a solution containing serum albumin.

[0441] Liposomal compositions can be prepared by various methods known in the art. See, for example, the following documents: U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,737,323; U.S. Pat. No. 96 / 37194 pamphlet; Felgner, P.L. et al., Proc. Natl. Acad. Sci., USA (1987) 8:7413-7417, Bangham, et al. M. Mol. Biol. (1965) 23:238 , Olson, et al. Biochim. Biophys. Acta (1979) 557:9, Szoka, et al. Proc. Natl. Acad. Sci. (1978) 75:4194, Mayhew, et al. Biochim. Biophys. Acta (1984) ) 775:169, Kim, et al. Biochim. Biophys. Acta (1983) 728:339 and Fukunaga, et al. Endocrinol. (1984) 115:757.

[0442] Surfactants find widespread application in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and grade the properties of a wide variety of surfactants, both natural and synthetic, is through the use of hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means of classifying the various surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p.285).

[0443] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceuticals and cosmetics and can be used over a wide range of pH values. Generally, their HLB values ​​range from 2 to about 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Also included in this class are nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols and ethoxylated / propoxylated block polymers. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

[0444] A surfactant is classified as anionic if the surfactant molecule carries a negative charge when dissolved or dispersed in water. Examples of anionic surfactants include carboxylates such as soaps, acyl lactylates, acylamides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, Includes acyl taurate and sulfosuccinates and phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0445] A surfactant is classified as cationic if the surfactant molecule carries a positive charge when dissolved or dispersed in water. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most used members of this class.

[0446] If a surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines and phosphatides.

[0447] The use of surfactants in pharmaceutical products, formulations, and emulsions is discussed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0448] lipid particles In some embodiments, the tunable REVERSIR can be completely encapsulated within a lipid formulation, such as LNPs or other nucleic acid-lipid particles. A tunable REVERSIR encapsulated in a lipid formulation may be unconjugated or conjugated to a ligand (ie, a conjugated tunable REVERSIR).

[0449] As used herein, the term "LNP" refers to stable nucleic acid-lipid particles. LNPs include cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (eg, PEG-lipid conjugates). LNPs exhibit a long circulation life after intravenous (i.v.) injection and accumulate at distant sites (eg, physically distant from the site of administration), making them extremely useful for systemic applications. LNPs include "pSPLPs," which include encapsulated condensing agent-nucleic acid complexes as described in PCT Publication No. WO 00 / 03683. Particles of the invention typically have an average size of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm. particle size and is virtually non-toxic. Furthermore, the nucleic acids, when present in the nucleic acid-lipid particles of the invention, are resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and their preparation methods are described, for example, in U.S. Pat. No. 5,976,567; U.S. Pat. No. 5,981,501; U.S. Pat. It is disclosed in Patent Application Publication No. 2010 / 0324120 and PCT Publication No. WO 96 / 40964 pamphlet.

[0450] In some embodiments, the lipid and drug ratio (mass / mass ratio) (e.g., lipid to adjustable REVERSIR ratio) is about 1:1 to about 50:1, about 1:1 to about 25:1, about It ranges from 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also considered to be part of this invention.

[0451] Cationic lipids are, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2 ,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethyl Ammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1, 2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-( Dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3- Dimethylaminopropane (DLin-S-DMA), 1-linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin -MPZ) or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio(DOAP), 1 ,2-dilinolenyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2- Di((9Z,12Z)-ocdadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxo-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatria Conta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2) -hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanedyl)didodecan-2-ol or mixtures thereof. The cationic lipid can represent about 20 mol% to about 50 mol% or about 40 mol% of the total lipids present in the particle.

[0452] In some embodiments, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-tunable REVERSIR nanoparticles. The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in International Application PCT / US Patent Application No. 2009 / 061897, published as pamphlet of WO 2010 / 048536. , which is incorporated herein by reference.

[0453] In some embodiments, the lipid-tunable REVERSIR particles include 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% cholesterol: 10% PEG- Contains C-DOMG (mol %) with a particle size of 63.0±20 nm and a tunable REVERSIR / lipid ratio of 0.027.

[0454] Ionizable / non-cationic lipids can be anionic or neutral lipids, including but not limited to distearoyl phosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidyl Glycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleylphosphatidylethanolamine (POPC), palmitoyloleylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimide) Methyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE , 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), cholesterol or mixtures thereof. Non-cationic lipids can represent from about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% if cholesterol is included, of the total lipids present in the particles.

[0455] Conjugated lipids that inhibit particle aggregation can be, for example, polyethylene glycol (PEG)-lipids, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, Contains PEG-ceramide (Cer) or mixtures thereof. PEG-DAA conjugates are, for example, PEG-dilauryloxypropyl (C 12 ), PEG-dimyristyloxypropyl (C 14 ), PEG-dipalmityloxypropyl (C 16 ) or PEG-distearoyloxypropyl (C 18 ). Conjugated lipids that inhibit particle aggregation can account for 0 mol% to about 20 mol% or about 2 mol% of the total lipids present in the particles.

[0456] In some embodiments, the nucleic acid-lipid particles further comprise cholesterol, eg, from about 10 mol% to about 60 mol% or about 48 mol% of the total lipids present in the particle.

[0457] Other exemplary lipid-tunable REVERSIR formulations are listed in Table 1 below.

[0458] [Table 2]

[0459] [Table 3]

[0460] [Table 4]

[0461] Abbreviations in Table 1 include: DSPC: distearoyl phosphatidylcholine; DPPC: dipalmitoylphosphatidylcholine; PEG-DMG: PEG-didimyristoylglycerol (C14-PEG or PEG-C14) (PEG with average molecular weight 2000) ;PEG-DSG:PEG-distyrylglycerol (C18-PEG or PEG-C18) (PEG with an average molecular weight of 2000);PEG-cDMA:PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with an average molecular weight of 2000) ).

[0462] A formulation containing DLinDMA (1,2-dilinolenyloxy-N,N-dimethylaminopropane) is described in WO 2009 / 127060, filed on April 15, 2009, which is referred to as is incorporated herein by.

[0463] XTC-containing formulations are described, for example, in U.S. Provisional Patent Application No. 61 / 148,366, filed on January 29, 2009; in U.S. Provisional Patent Application No. 61 / 156,851, filed on March 2, 2009; U.S. Provisional Patent Application No. 61 / 228,373 filed on July 24, 2009; U.S. Provisional Patent Application No. 61 / 239,686 filed on September 3, 2009 ; and International Application No. PCT / US Patent Application Publication No. 2010 / 022614, filed January 29, 2010, which are incorporated herein by reference.

[0464] MC3-containing formulations are described, for example, in US Patent Application Publication No. 2010 / 0324120, filed June 10, 2010, the entire contents of which are incorporated herein by reference.

[0465] Biodegradable lipid-containing preparations are described, for example, in PCT Publication No. WO 2011 / 153493, filed on June 3, 2011, and PCT Publication No. WO 2013 / 086354, filed on December 7, 2012. , the entire contents of which are incorporated herein by reference.

[0466] (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine-containing preparations are described in, for example, International Publication No. 2012 / 040184 pamphlet filed on September 20, 2011. , the entire contents of which are incorporated herein by reference.

[0467] Oligomeric compounds of the invention can be prepared and formulated as micelles. As used herein, a "micelle" is a spherical structure of amphiphilic molecules such that all the hydrophobic parts on the molecule are directed inward and the hydrophilic parts remain in contact with the surrounding water layer. A specific type of molecular assembly that is located within. The opposite configuration exists if the environment is hydrophobic.

[0468] In some embodiments, the formulation comprises micelles formed from an oligonucleotide of the invention and at least one amphiphilic carrier, wherein the micelles preferably have an average diameter of less than about 100 nm. More preferred embodiments provide micelles with an average diameter of less than about 50 nm, and even more preferred embodiments provide micelles with an average diameter of less than about 30 nm or even less than about 20 nm.

[0469] Micellar formulations are aqueous solutions of oligonucleotide compositions, alkali metal C 8 ~C 22 It can be prepared by mixing an alkyl sulfate and an amphipathic carrier. The amphiphilic carrier can be added simultaneously with or after the addition of the alkali metal alkyl sulfate. Micelles are formed by mixing virtually any type of ingredients other than vigorous mixing to provide micelles of finer particle size.

[0470] The oligomeric compounds of the invention can be prepared and formulated as emulsions. As used herein, an "emulsion" is a heterogeneous system of one liquid dispersed in another liquid in the form of droplets.

[0471] Emulsions are often two-phase systems containing two immiscible liquid phases that are carefully mixed and dispersed in each other. Generally, emulsions can be either of the water-in-oil (w / o) or oil-in-water (o / w) variety. When the aqueous phase is finely divided and dispersed as microdroplets in the bulk oil phase, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, if the oil phase is finely divided and dispersed as microdroplets in the bulk water phase, the resulting composition is called an oil-in-water (o / w) emulsion. Emulsions may contain additional ingredients in addition to the dispersed phase and the active drug, which may be present as a solution in an aqueous phase, an oil phase, or as a separate phase itself. Formulation excipients such as emulsifiers, stabilizers, dyes and antioxidants may also be present in the emulsion if desired. Pharmaceutical emulsions can be multiple emulsions composed of three or more phases, such as in the case of oil-in-water-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. . Such complex formulations often offer several advantages that simple two-component emulsions do not. A multiple emulsion in which individual oil droplets of an o / w emulsion surround tiny water droplets constitutes a w / o / w emulsion. Similarly, a system of oil droplets surrounded by droplets of water stabilized in an oily continuous phase constitutes an o / w / o emulsion.

[0472] Emulsions are characterized by little or no thermodynamic stability. Often the dispersed or discontinuous phase of an emulsion is well dispersed in the outer or continuous phase and is maintained in this form using emulsifiers or by the viscosity of the formulation. Any of the emulsion phases can be semi-solid or solid, as are emulsion-type ointment bases and creams. Other means of stabilizing emulsions include the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can be broadly classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorbent bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.) , 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0473] Synthetic surfactants, also known as surfactants, have wide applicability in emulsion formulation and are reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988 ,Marcel Dekker,Inc.,New York,N.Y.,volume 1,p.285;Idson,in Pharmaceutical Dosage Forms,Lieberman,Rieger and Banker(Eds.),Marcel Dekker,Inc.,New York,N.Y.,1988,volume 1, p.199). Surfactants are typically amphipathic and include hydrophilic and hydrophobic portions. The hydrophilic to hydrophobic ratio of a surfactant is referred to as the hydrophilic / lipophilic balance (HLB) and is a valuable tool in classifying and selecting surfactants in formulation preparation. Surfactants can be classified into various classes based on their hydrophilic properties: nonionic, anionic, cationic, and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.285).

[0474] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorbent bases have hydrophilic properties such as anhydrous lanolin and hydrophilic petrolatum to absorb water and form a w / o emulsion while still maintaining its semi-solid consistency. Finely divided solids are also used as excellent emulsifiers, especially in combination with surfactants and in viscous preparations. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and non-swelling clays such as carbon. Mention may be made of polar solids or glyceryl tristearate.

[0475] A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, wetting agents, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.). ), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0476] Hydrophilic colloids or hydrocolloids include polysaccharides (e.g. acacia, agar, alginic acid, carrageenan, guar gum, karaya gum and tragacanth), cellulose derivatives (e.g. carboxymethyl cellulose and carboxypropyl cellulose) and synthetic polymers (e.g. carbomers, cellulose ethers). and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize the emulsion by forming a strong interfacial film around the dispersed phase droplets and by increasing the viscosity of the external phase. .

[0477] These formulations often contain preservatives, as emulsions often contain several ingredients such as carbohydrates, proteins, sterols and phosphatides that can easily support bacterial growth. . Commonly used preservatives included in emulsion formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent degradation of the formulation. The antioxidants used are free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene or reducing agents such as ascorbic acid and sodium metabisulfite and antioxidants such as citric acid, tartaric acid and lecithin. It can be an oxidant synergist.

[0478] In some embodiments, the composition is formulated as a microemulsion. As used herein, "microemulsion" refers to a system of water, oil, and amphiphile that is a single optically isotropic and thermodynamically stable solution. Microemulsions also include thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surface-active molecules.

[0479] A microemulsion can be defined as a system of water, oil, and amphiphiles that is a single optically isotropic and thermodynamically stable solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically, microemulsions are made by first dispersing the oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component (generally an intermediate chain length alcohol) to form a clear A system prepared by forming a system. Microemulsions are therefore described as thermodynamically stable, isotropically transparent dispersions composed of two immiscible liquids stabilized by an interfacial film of surface-active molecules ( Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions are generally prepared using a combination of three to five ingredients including oil, water, surfactants, cosurfactants, and electrolytes. Whether a microemulsion is of water-in-oil (w / o) or oil-in-water (o / w) type depends on the properties of the oil and surfactant used and the polar head and It depends on the structure and geometric packing of the hydrocarbon tail (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0480] Phenomenological methods using phase diagrams have been extensively studied, and those skilled in the art have gained a comprehensive knowledge of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in the formulation of thermodynamically stable droplets that form spontaneously.

[0481] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij96, polyethylene oleyl ether, polyglycerol fatty acids, alone or in combination with co-surfactants. Esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), deca monooleate Examples include glycerol (MO750), decaglycerol sequioleate (SO750), and decaglycerol sequioleate (DAO750). Co-surfactants, which are usually short-chain alcohols such as ethanol, 1-propanol and 1-butanol, penetrate into the surfactant thin film, resulting in irregularities due to the void spaces created between the surfactant molecules. By forming a thin film, it plays a role in increasing interfacial fluidity. However, microemulsions can be prepared without the use of co-surfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, aqueous solutions of drugs, glycerol, PEG300, PEG400, polyglycerols, propylene glycol and ethylene glycol derivatives. The oil phase includes, but is not limited to, Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono-, di- and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolization. It may include materials such as glycerides, saturated polyglycolated C8-C10 glycerides, vegetable oils and silicone oils.

[0482] Microemulsions are of particular interest from the standpoint of drug solubilization and enhanced drug absorption. Lipid-based microemulsions (both o / w and w / o) have been proposed to improve the oral bioavailability of drugs containing peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385- 1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions offer improved drug solubilization, protection of drugs from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced modification of membrane fluidity and permeability, ease of preparation, and solid dosage forms. (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J.Pharm.Sci., 1996,85,138-143). In many cases, microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating heat-labile drugs, peptides or dsRNA. Microemulsions are also useful for transdermal delivery of active ingredients in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will promote increased systemic absorption of dsRNA and nucleic acids from the gastrointestinal tract and improved local cellular uptake of dsRNA and nucleic acids.

[0483] Furthermore, the microemulsions of the present invention may contain additional agents such as sorbitan monostearate (Grill3), Labrasol and permeation enhancers to improve the properties of the formulation and enhance the absorption of the dsRNA and nucleic acids of the present invention. may contain ingredients and additives. The permeation enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes is discussed above.

[0484] Dermatology, the application of emulsion formulations by oral and parenteral routes and methods of their preparation are described in the literature, for example Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York; N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; and Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335, the contents of which are incorporated herein by reference in their entirety. incorporated into the book.

[0485] The oligomeric compound of the present invention is a lipid particle, such as (a) an oligonucleotide of the present invention (the oligonucleotide is conjugated to a lipophilic substance) and (b) the conjugated oligonucleotide is aggregated, mixed, or bonded. can be prepared and formulated as formulated lipid particles (FLiPs) comprising at least one lipid component, such as an emulsion, liposome, isolated lipoprotein, reconstituted lipoprotein or phospholipid. The stoichiometry of the oligonucleotide and lipid components can be 1:1. Alternatively, the stoichiometry can be 1:poly, poly:1 or poly:poly, where poly is 2 or more.

[0486] FLiPs can include triacylglycerols, phospholipids, glycerol, and one or more lipid binding proteins aggregated, mixed, or linked with oligonucleotides via lipophilic linker molecules. Surprisingly, due to said one or more lipid binding proteins in combination with said lipids, FLiP exhibits an affinity for liver, intestine, kidney, steroid producing organs, heart, lung and / or muscle tissue. It is clear that These FLiPs can therefore serve as carriers of oligonucleotides to these tissues. For example, lipid-conjugated oligonucleotides, such as cholesterol-conjugated oligonucleotides, bind to HDL and LDL lipoprotein particles and, upon binding to their respective receptors, mediate cellular uptake, thereby leading to liver, intestine, kidney and steroidogenic organs. Direct oligonucleotide delivery to. See Wolfrum et al. Nature Biotech. (2007), 25:1145-1157.

[0487] FLiPs can be lipid particles containing 15-25% triacylglycerols, about 0.5-2% phospholipids and 1-3% glycerol and one or more lipid binding proteins. FLiPs can be lipid particles comprising about 15-25% triacylglycerols, about 1-2% phospholipids, about 2-3% glycerol, and one or more lipid binding proteins. In some embodiments, the lipid particles include about 20% triacylglycerols, about 1.2% phospholipids, and about 2.25% glycerol and one or more lipid binding proteins.

[0488] Another lipid component suitable for FLiP is a lipoprotein, such as an isolated lipoprotein or more preferably a reconstituted lipoprotein. Exemplary lipoproteins include chylomicrons, VLDL (very low density lipoprotein), IDL (intermediate density lipoprotein), LDL (low density lipoprotein) and HDL (high density lipoprotein). Methods for producing reconstituted lipoproteins are known in the art and are described, for example, in A. Jones, Experimental Lung Res. 6, 255-270 (1984), U.S. Pat. See Publication No. 87 / 02062, Canadian Patent No. 2,138,925. Other methods of producing reconstituted lipoproteins, particularly apolipoproteins A-I, A-II, A-IV, apoC and apoE, are described by A. Jonas, Methods in Enzymology 128, 553-582 (1986) and G. Franceschini et al.J. Biol. Chem., 260(30), 16321-25 (1985).

[0489] One preferred lipid component for FLiP is Intralipid. Intralipid® is the trademark for the first safe fat emulsion for human use. Intralipid® 20% (20% intravenous fat emulsion) is composed of 20% soybean oil, 1.2% egg yolk phospholipids, 2.25% glycerin, and water for injection. Furthermore, it is within the present invention that other suitable oils, such as safflower oil, can be used to produce the lipid component of the FLiP.

[0490] The FLiPs may have a particle size in the range of about 20-50 nm or about 30-50 nm, such as about 35-40 nm. In some embodiments, the FLiP has a particle size of at least about 100 nm. Alternatively, the FLiP, whether featuring liposome or emulsion based, may be about 100-150 nm, such as about 110 nm, about 120 nm, about 130 nm or about 140 nm. It is also possible to aggregate multiple FLiPs and deliver them together, so the particle size can exceed 100 nm.

[0491] A method of producing lipid particles includes (a) mixing a lipid component with one or more lipophilic (e.g., cholesterol) conjugated oligonucleotides that can be chemically modified; and (b) combining the mixture with a lipid component. It includes a step of sorting. In some embodiments, the method includes an additional step of selecting a fraction having a particle size of 30-50 nm, preferably about 40 nm.

[0492] Some exemplary lipid particle formulations suitable for the present invention are described in U.S. Patent Application No. 12 / 412,206, filed March 26, 2009, the contents of which are incorporated by reference in their entirety. Incorporated herein.

[0493] In some embodiments, oligomeric compounds can be formulated into yeast cell wall particles (“YCWP”). Yeast cell wall particles include an extracted yeast cell wall exterior and a core, where the core includes a payload (eg, an oligonucleotide). The exterior of the particle includes yeast glucan (eg, β-glucan, β-1,3-glucan, β-1,6-glucan), yeast mannan, or a combination thereof. Yeast cell wall particles are typically spherical particles about 1-4 μm in diameter.

[0494] Preparation of yeast cell wall particles is known in the art and is described, for example, in U.S. Pat. No. 4,992,540; U.S. Pat. No. 5,082,936; U.S. Pat. Specification No. 5,401,727; Specification No. 5,504,079; Specification No. 5,607,677; Specification No. 5,741,495; Specification No. 5,830,463; Specification No. 5,968,811; Specification No. 6,444,448 ; and US Pat. is incorporated herein by reference. Applications of yeast cell-like particles for drug delivery are described, for example, in U.S. Pat. No. 5,032,401; U.S. Pat. No. 5,607,677; U.S. Pat. No. 2005 / 0281781 and No. 2008 / 0044438, the contents of which are incorporated herein by reference in their entirety. U.S. Patent Application Publication No. 2009 / 0226528, the contents of which are incorporated herein by reference, describes the formulation of nucleic acids using yeast cell wall particles for the delivery of oligonucleotides to cells. are doing.

[0495] Exemplary formulations of oligomeric compounds are described in U.S. Patent Nos. 4,897,355; 4,394,448; 4,235,871; 4,231,877; Specification: No. 4,673,567; No. 4,247,411; No. 4,814,270; No. 5,567,434; No. 5,552,157; ​​No. 5,565,213; No. 5,738,868 5,795,587 specification; 5,922,859 specification; 6,077,663 specification; 7,906,484 specification; and 8,642,076; PCT Publication International Publication No. 2009 / 132131 specification pamphlet and US patent Publication No. 2006 / 0240093, Publication No. 2007 / 0135372, Publication No. 2011 / 0117125, Publication No. 2009 / 0291131, Publication No. 2012 / 0316220, Publication No. 2009 / No. 0163705 and No. 2013 / 0129785, the contents of which are incorporated herein by reference in their entirety. Behr (1994) Bioconjugate Chem. 5:382-389 and Lewis et al. (1996) PNAS 93:3176-3181) also describe the formulation of oligonucleotides suitable for the present invention; Incorporated herein by reference.

[0496] siRNA As used herein, the term "siRNA" refers to an agent that mediates targeted cleavage of RNA transcripts. These substances bind to a cytoplasmic multiprotein complex known as the RNAi-induced silencing complex (RISC). Substances effective in inducing RNA interference are also referred to herein as siRNA, RNAi agents or iRNA agents. As used herein, the term siRNA includes microRNA and pre-microRNA.

[0497] As used herein, the term "siRNA" refers to an agent that mediates targeted cleavage of RNA transcripts. These substances bind to a cytoplasmic multiprotein complex known as the RNAi-induced silencing complex (RISC). Substances effective in inducing RNA interference are also referred to herein as siRNA, dsRNA, RNAi agents or iRNA agents.

[0498] As used herein, the terms "siRNA activity" and "RNAi activity" refer to gene silencing by siRNA.

[0499] As used herein, "gene silencing" by an RNA interference molecule means, for a target gene, at least about 5%, at least about 10%, at least at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, up to 100% Refers to a decrease in the level of mRNA in a cell by about 99% and any integer percentage therebetween. In one preferred embodiment, the mRNA level is at least about 99% and any integer % from 5% to 100%, including at least about 70%, at least about 80%, at least about 90%, at least about 95%, up to 100%. shows a decrease in

[0500] As used herein, the term "regulate gene expression" means to up-regulate or down-regulate the expression of a gene encoding one or more proteins or protein subunits or the level of an RNA molecule or equivalent RNA molecule. By controlling is meant such that expression, level or activity is higher or lower than that observed in the absence of the modulator. For example, the term "modulate" can mean "inhibit," although use of the term "modulate" is not limited to this definition.

[0501] As used herein, gene expression regulation means that the expression of genes encoding one or more proteins or protein subunits or the level of RNA molecules or equivalent RNA molecules encoding them is controlled by siRNA, e.g. RNAi at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2x, 3x that observed in the absence of the agent , occurs when they differ by a factor of 4 or 5 or more. The % and / or fold difference can be calculated, for example, relative to a control or non-control as follows.

number

[0502] As used herein, the term "inhibit," "downregulate," or "reduce," with respect to gene expression, refers to the expression of a gene encoding one or more proteins or protein subunits; It means that the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits, is reduced below that observed in the absence of the modulator. The expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits compared to a corresponding non-regulatory control. , at least 10% lower, preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or most preferably 100% Gene expression is down-regulated when it is reduced as low as (ie, no gene expression).

[0503] As used herein, the terms "increase", "upregulate", with respect to gene expression, refer to the expression of a gene encoding one or more proteins or protein subunits, or an RNA molecule or equivalent. It means that the level of an RNA molecule or the activity of one or more proteins or protein subunits is increased above that observed in the absence of the modulator. The expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits compared to a corresponding non-regulatory control. , at least 10%, preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 100%, 1.1 times, 1.25 times, Gene expression is up-regulated when increased by 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more.

[0504] As used herein, the term "increased" or "increases" generally means an increase in a statistically significant amount; for the avoidance of doubt, "increased" means an increase in an increase of at least 10% compared to a reference level, such as 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 less than 100%, or any increase from 10 to 100%, or at least about 2 times, at least about 3 times, at least about 4 times, at least It means an increase of about 5 times or at least 10 times, or any increase from 2 times to 10 times or more.

[0505] As used herein, the term "reduced" or "reducing" generally means a decrease in a statistically significant amount. However, for the avoidance of doubt, "reduced" means a reduction of at least 10% compared to a reference level, such as at least about 20%, or at least about 30%, or at least about 40% compared to a reference level; 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 100% (i.e., zero level compared to a reference sample) or less or less than the reference level. Means any reduction of 10~100% in comparison.

[0506] Those skilled in the art will be well aware that double-stranded oligonucleotides containing double helical structures of 20-23, especially 21 base pairs are recognized as being particularly effective in inducing RNA interference. (Elbashir et al., EMBO 2001, 20:6877-6888). However, it has been discovered that other, shorter or longer double-stranded oligonucleotides may also be useful.

[0507] Double-stranded oligonucleotides include two oligonucleotide strands that are sufficiently complementary to hybridize to form a double helical structure. Generally, the double helix structure is 15-30, more typically 18-25, even more typically 19-24, most commonly 19-21 base pairs in length. In some embodiments, longer double-stranded oligonucleotides of 25-30 base pairs in length are preferred. In some embodiments, shorter double-stranded oligonucleotides, 10-15 base pairs in length, are preferred. In another embodiment, the double-stranded oligonucleotide is at least 21 nucleotides long.

[0508] In some embodiments, the double-stranded oligonucleotide includes a sense strand and an antisense strand, where the antisense RNA strand has a region of complementarity that is complementary to at least a portion of the target sequence. However, the double helix region is 14-30 nucleotides long. Similarly, the region of complementarity to the target sequence is 14-30, more typically 18-25, even more typically 19-24, most commonly 19-21 nucleotides long.

[0509] As used herein, the term "antisense strand" refers to an oligomeric compound that is substantially complementary or 100% complementary to the intended target sequence. The phrase "antisense strand" includes antisense regions of both oligomeric compounds that are formed from two separate strands as well as monomolecular compounds that can form hairpin or dumbbell-shaped structures. The terms "antisense strand" and "guide strand" are used interchangeably herein.

[0510] The term "sense strand" refers to an oligomeric compound having a nucleoside sequence that is identical, in whole or in part, to a target sequence, such as a sequence of messenger RNA or DNA. The terms "sense strand" and "passenger strand" are used interchangeably herein.

[0511] "Specifically hybridizable" and "complementary" mean that a nucleic acid is capable of forming hydrogen bonds with another nucleic acid sequence, either by traditional Watson-Crick or other non-traditional types. means. In the context of the nucleic acid molecules of the invention, the free energy of binding between the nucleic acid molecule and its complementary sequence is sufficient to drive the relevant function of the nucleic acid, such as RNAi activity. Determination of the binding free energy of nucleic acid molecules is well known in the art (e.g., Turner et al, 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; see Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). Percent complementarity refers to the percentage of contiguous residues (e.g., 5 out of 10, 6 out of 10) in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence. , 7, 8, 9, 10 are 50%, 60%, 70%, 80%, 90% and 100% complementary). "Fully complementary" or 100% complementarity means that all consecutive residues of a nucleic acid sequence hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. Less than perfect complementarity refers to a situation in which some (but not all) nucleotide units of two strands are able to hydrogen bond with each other. "Substantial complementarity" refers to polynucleotide strands that exhibit 90% or more complementarity, excluding regions of the polynucleotide strands that are selected to be non-complementary (eg, overhangs). Specific binding involves binding to a non-target under the conditions in which specific binding is desired, i.e., in the case of in vivo assays or therapeutic treatments, under physiological conditions, or, in the case of in vitro assays, under the conditions under which the assay is performed. A sufficient degree of complementarity is required to avoid non-specific binding of the sequences and oligomeric compounds. Non-target sequences typically differ by at least 5 nucleotides.

[0512] The term "off-target" and the phrase "off-target effect" refer to the effects of siRNA directed against a given target due to its interaction, either directly or indirectly, with another mRNA sequence, DNA sequence, or cellular protein or other part. , refers to any case that causes an unintended effect. For example, "off-target effects" can occur when partial homology or complementarity between other transcripts and the sense and / or antisense strand of the siRNA results in simultaneous degradation of other transcripts. .

[0513] In some embodiments, the double-stranded regions of the double-stranded oligomeric compound are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24 , 25, 26, 27, 28, 29 or 30 nucleotide pairs.

[0514] In some embodiments, the antisense strand of the double-stranded oligomeric compound is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, It is equal to or at least as long as 29 or 30 nucleotide pairs.

[0515] In some embodiments, the sense strand of the double-stranded oligomeric compound is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25 , 26, 27, 28, 29 or 30 nucleotide pairs.

[0516] In some embodiments, one strand has at least one stretch of 1-5 single-stranded nucleotides within the double-stranded region. "A section of single-stranded nucleotides within a double-stranded region" means that there is at least one nucleotide base pair at both ends of the single-stranded section. In some embodiments, both strands have at least one stretch comprised of 1 to 5 (eg, 1, 2, 3, 4, or 5) single-stranded nucleotides within the double-stranded region. If both strands have a stretch of 1 to 5 (e.g. 1, 2, 3, 4 or 5) single-stranded nucleotides within the double-stranded region, such single-stranded nucleotides are , they can be opposite to each other (e.g., mismatched intervals), or they can be arranged such that the second strand does not contain single-stranded nucleotides opposite to the single-stranded oligonucleotides of the first strand. , and vice versa (eg, single-stranded loops) are also possible. In some embodiments, the single-stranded nucleotides are within 8 nucleotides from either end, e.g., 8, 7, 6, Present in 5, 4, 3 or 2 nucleotides.

[0517] In some embodiments, each strand of the double-stranded oligonucleotide has a ZXY structure, such as that described in PCT Publication No. WO 2004080406, the contents of which are incorporated herein by reference in their entirety. have

[0518] In some embodiments, two strands of a double-stranded oligomeric compound can be linked together. The two chains can be linked to each other at both ends or only at one end. Linking at one end means linking the 5' end of the first strand to the 3' end of the second strand, or linking the 3' end of the first strand to the 5' end of the second strand. means. When two strands are linked together at both ends, the 5' end of the first strand is linked to the 3' end of the second strand, and the 3' end of the first strand is linked to the 5' end of the second strand. Connect to. The two strands can be linked to each other by an oligonucleotide linker, including but not limited to (N) n (N is independently a modified or unmodified nucleotide, and n is 3 to 23). In some embodiments, n is 3-10, such as 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the oligonucleotide linker is GNRA, (G) 4 ,(U) 4 and (dT) 4 where N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide. Some of the nucleotides in the linker may participate in base pairing interactions with other nucleotides in the linker. The two chains can also be linked to each other by a non-nucleoside linker, such as a linker described herein. Those skilled in the art will appreciate that any of the oligonucleotide chemical modifications or alterations described herein can be used in oligonucleotide linkers.

[0519] Hairpin and dumbbell type oligomeric compounds can have a double helix region of at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24 or 25 nucleotide pairs or more. The double helix region can be up to 200, 100 or 50 in length. In some embodiments, the double helix region ranges from 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length.

[0520] Hairpin oligomeric compounds can have a single-stranded overhang or terminal unpaired region on the 3' in some embodiments and on the antisense side of the hairpin in some embodiments. In some embodiments, the overhang was 1-4, more typically 2-3 nucleotides in length. Hairpin oligomeric compounds capable of inducing RNA interference are also referred to herein as "shRNA."

[0521] In some embodiments, the two oligomeric chains are present under conditions in which specific binding is desired, i.e., under physiological conditions for in vivo assays or therapeutic treatments, and under physiological conditions for in vitro assays. The antisense compound hybridizes specifically if there is a sufficient degree of complementarity to avoid nonspecific binding of the non-target nucleic acid sequence and the antisense compound under the conditions described above.

[0522] As used herein, "stringent hybridization conditions" or "stringent conditions" refer to conditions under which an antisense compound hybridizes to its target sequence, but to a minimal number of other sequences. Stringency conditions are sequence-dependent and different in various situations, and the "stringency conditions" under which antisense compounds hybridize to a target sequence are determined by the nature and composition of the antisense compounds and the assay in which they are tested. .

[0523] It is understood in the art that with the incorporation of nucleotide affinity modifications, higher numbers of mismatches can be achieved compared to unmodified activation. Similarly, certain oligonucleotide sequences may be more tolerant to mismatches than other oligonucleotide sequences. One skilled in the art can determine the appropriate number of mismatches between oligonucleotides or between oligonucleotides and target nucleic acids, for example, by determining melting temperatures (Tm). Tm or ΔTm can be calculated by techniques well known to those skilled in the art. For example, techniques described in Freier et al. (Nucleic Acids Research, 1997, 25, 22:4429-4443) allow one skilled in the art to evaluate nucleotide modifications for their ability to increase the melting temperature of RNA:DNA double helices. can do.

[0524] Regulation of target expression In some embodiments, the target nucleic acid is mRNA. In some such embodiments, the siRNA is designed to modulate its target mRNA or its expression. In some embodiments, designing antisense compounds to a target nucleic acid molecule can be a multi-step process. Typically, this process begins with the identification of the target protein, its activity to be modulated, followed by identification of the nucleic acid whose expression produces the target protein. In some embodiments, antisense compound design results in an antisense compound that hybridizes to a target nucleic acid molecule. In some embodiments, the antisense compound is an antisense oligonucleotide or antisense oligonucleoside. In some embodiments, the antisense compound and target nucleic acid are complementary to each other. In some such embodiments, the antisense compound is fully complementary to the target nucleic acid. In some embodiments, the antisense compound contains one mismatch. In some embodiments, the antisense compound contains two mismatches. In some embodiments, antisense compounds contain three or more mismatches.

[0525] Modulation of a target nucleic acid can be achieved by altering any number of nucleic acid functions. In some embodiments, the functions of the RNA to be modulated include, but are not limited to, translocation functions (including, but not limited to, translocation of the RNA to sites of protein translation, sites within the cell distal to the site of RNA synthesis). translocation of RNA into other proteins) as well as translation of proteins from RNA. RNA processing functions that can be modulated include, but are not limited to, splicing of RNA to obtain one or more RNA species, capping of RNA, 3' maturation and catalytic activity of RNA or those involved in RNA or RNA complex formation involving RNA, which can be promoted by Modulation of expression can result in increased levels of one or more nucleic acid species or decreased levels of one or more nucleic acid species, either transiently or by net steady state levels. Thus, in one embodiment, modulating expression may mean increasing or decreasing target RNA or protein levels. In another embodiment, modulating expression may mean increasing or decreasing one or more RNA splice products or changing the ratio of two or more splice products.

[0526] In some embodiments, the siRNA is a conjugated siRNA. As used herein, the term "conjugated siRNA" refers to an RNAi agent conjugated with a ligand. For example, RNAi agents conjugated to the ligands described herein.

[0527] In some other embodiments, the siRNA is unconjugated siRNA. As used herein, the term "unconjugated siRNA" refers to an RNAi agent that is not conjugated to a ligand, such as a ligand described herein.

[0528] In one aspect, the invention relates to double-stranded RNA (dsRNA) agents, or siRNAs, for inhibiting target gene expression. dsRNA agents include a sense strand and an antisense strand, each having 14-40 nucleotides. The dsRNA agent has the formula (I): [ka] Represented by

[0529] In formula (I), B1, B2, B3, B1', B2', B3' and B4' each independently represent 2'-O-alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2' - A nucleotide containing a modification selected from the group consisting of halo, ENA and BNA / LNA. In one embodiment, B1, B2, B3, B1', B2', B3' and B4' each include a 2'-OMe modification.

[0530] C1 is a heat-destabilized nucleotide located at a position opposite the seed region of the antisense strand (ie, positions 2-8 of the 5' end of the antisense strand). For example, C1 is at one position on the sense strand that pairs with nucleotides 2-8 of the 5' end of the antisense strand. C1 nucleotides carry thermodestabilizing modifications, which include abasic modifications; mismatches with the opposite nucleotide in the double helix; as well as 2'-deoxy modifications or non-cyclic nucleotides, such as unlocked nucleic acids (UNA) or May include sugar modifications such as glycerol nucleic acids (GNA). In one embodiment, C1 is i) a mismatch with the opposite nucleotide of the antisense strand; ii) [ka] an abasic modification selected from the group consisting of; and iii) [ka] wherein B is a modified or unmodified nucleobase; 1 and R 2 are independently H, halogen, OR 3 or alkyl ;R 3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar. In one embodiment, the thermal destabilizing modification at C1 is G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, a mismatch selected from the group consisting of U:U, T:T and U:T; and optionally, at least one nucleobase in the mismatch pair is a 2'-deoxynucleobase. In one example, a thermally destabilizing modification at C1 [ka] It is.

[0531] T1, T1', T2' and T3' each independently represent a nucleotide containing a modification that imparts to the nucleotide a steric bulkiness that is less than or equal to the steric bulkiness of the 2'-OMe modification. The modification may be at the 2' position of the ribose sugar of the nucleotide or may be a modification to a non-ribose nucleotide, a non-cyclic nucleotide or the backbone of the nucleotide, the modification being similar or equivalent to the 2' position of the ribose sugar; In addition, it imparts steric bulk to the nucleotide that is lower than the steric bulk of 2'-OMe modification. For example, T1, T1', T2' and T3' are each independently selected from DNA, RNA, LNA, 2'-F and 2'-F-5'-methyl. In one embodiment, T1 is DNA. In one embodiment, T1' is DNA, RNA or LNA. In one embodiment, T2' is DNA or RNA. In one embodiment, T3' is DNA or RNA.

[0532] n 1 ,n 3 and q 1 are independently 4 to 15 nucleotides long.

[0533] n 5 ,q 3 and q 7 are independently 1 to 6 nucleotides long.

[0534] n 4 ,q 2 and q 6 are independently 1 to 3 nucleotides long.

[0535] q 5 are independently 0 to 10 nucleotides long.

[0536] n 2 and q 4 are independently 0 to 3 nucleotides long.

[0537] Instead, n 4 is 0 to 3 nucleotides long.

[0538] In one embodiment, n 4 can be 0. In one example, n 4 is 0 and q 2 and q 6 is 1. In another example, n 4 is 0 and q 2 and q 6 is 1, where the sense strand has two phosphorothioate internucleotide bond modifications within positions 1-5 (counting from the 5' end of the sense strand) and 2 at positions 1 and 2 of the antisense strand. It also has two phosphorothioate internucleotide bond modifications within positions 18-23 (counting from the 5' end of the antisense strand).

[0539] In one embodiment, n 4 ,q 2 and q 6 are each 1.

[0540] In one embodiment, n 2 ,n 4 ,q 2 ,q 4 and q 6 are each 1.

[0541] In one embodiment, the sense strand is 19-22 nucleotides long and n 4 is 1, C1 is located at positions 14-17 of the 5' end of the sense strand.

[0542] In one embodiment, T3' begins at position 2 from the 5' end of the antisense strand. In one example, T3' is at position 2 from the 5' end of the antisense strand and q 6 is equal to 1.

[0543] In one embodiment, T1' begins at position 14 from the 5' end of the antisense strand. In one example, T1' is at position 14 from the 5' end of the antisense strand and q 2 is equal to 1.

[0544] In one embodiment, T1' and T3' are separated by a length of 11 nucleotides (ie, T1' and T3' nucleotides are not counted).

[0545] In one embodiment, T1' is at position 14 from the 5' end of the antisense strand. In one example, T1' is at position 14 from the 5' end of the antisense strand and q 2 is equal to 1, and non-ribose, non-cyclic or backbone modifications at one or more 2' positions confer less steric bulk than the 2'-OMe ribose modification.

[0546] In one embodiment, T3' is at position 2 from the 5' end of the antisense strand. In one example, T3' is at position 2 from the 5' end of the antisense strand and q 6 is equal to 1, and non-ribose, non-cyclic or backbone modifications at one or more 2' positions confer less steric bulk than the 2'-OMe ribose modification.

[0547] In one embodiment, T1 is at the cleavage site of the sense strand. In one example, the sense strand is 19-22 nucleotides long and n 2 is 1, T1 is at position 11 from the 5' end of the sense strand.

[0548] In one embodiment, T2' begins at position 6 from the 5' end of the antisense strand. In one example, T2' is located at positions 6-10 from the 5' end of the antisense strand, and q 4 is 1.

[0549] In one embodiment, B1 is 2'-OMe or 2'-F and n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, and n 3 is 7 and n 4 is 0, B3 is 2’-OMe, and n 5 is 3, B1’ is 2’OMe or 2’-F, and q 1 is 9, T1’ is 2’-F, and q 2 is 1, B2’ is 2’-OMe or 2’-F, and q 3 is 4, T2’ is 2’-F, and q 4 is 2, B3’ is 2’-OMe or 2’-F, and q 5 is 5, T3’ is 2’-F, and q 6 is 1, B4’ is 2’-OMe, and q 7 is 1.

[0550] In one embodiment, B1 is 2'-OMe or 2'-F and n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, and n 3 is 7 and n 4 is 0, B3 is 2’-OMe, and n 5 is 3, B1’ is 2’-OMe or 2’-F, and q 1 is 9, T1’ is 2’-F, and q 2 is 1, B2’ is 2’-OMe or 2’-F, and q 3 is 4, T2’ is 2’-F, and q 4 is 2, B3’ is 2’-OMe or 2’-F, and q 5 is 5, T3’ is 2’-F, and q 6 is 1, B4’ is 2’-OMe, and q 7 is 1; has two phosphorothioate internucleotide bond modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate nucleotides at positions 1 and 2 of the antisense strand. It also has two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand).

[0551] In one embodiment, B1 is 2'-OMe or 2'-F and n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, and n 3 is 7 and n 4 is 0, B3 is 2’-OMe, and n 5 is 3, B1’ is 2’-OMe or 2’-F, and q 1 is 9, T1’ is 2’-F, and q 2 is 1, B2’ is 2’-OMe or 2’-F, and q 3 is 4 and q 4 is 0, B3’ is 2’-OMe or 2’-F, and q 5 is 7, T3’ is 2’-F, and q 6 is 1, B4’ is 2’-OMe, and q 7 is 1.

[0552] In one embodiment, B1 is 2'-OMe or 2'-F and n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, and n 3 is 7 and n 4 is 0, B3 is 2’-OMe, and n 5 is 3, B1’ is 2’-OMe or 2’-F, and q 1 is 9, T1’ is 2’-F, and q 2 is 1, B2’ is 2’-OMe or 2’-F, and q 3 is 4 and q 4 is 0, B3’ is 2’-OMe or 2’-F, and q 5 is 7, T3’ is 2’-F, and q 6 is 1, B4’ is 2’-OMe, and q 7 is 1; has two phosphorothioate internucleotide bond modifications in positions 1-5 of the sense strand (counting from the 5' end) and two phosphorothioate internucleotide bond modifications in positions 1 and 2 of the antisense strand. and also has two phosphorothioate internucleotide bond modifications within positions 18-23 (counting from the 5' end).

[0553] In one embodiment, B1 is 2'-OMe or 2'-F and n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, and n 3 is 7 and n 4 is 0, B3 is 2’-OMe, and n 5 is 3, B1’ is 2’-OMe or 2’-F, and q 1 is 9, T1’ is 2’-F, and q 2 is 1, B2’ is 2’-OMe or 2’-F, and q 3 is 4, T2’ is 2’-F, and q 4 is 2, B3’ is 2’-OMe or 2’-F, and q 5 is 5, T3’ is 2’-F, and q 6 is 1, B4’ is 2’-F, and q 7 is 1.

[0554] In one embodiment, B1 is 2'-OMe or 2'-F and n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, and n 3 is 7 and n 4 is 0, B3 is 2’-OMe, and n 5 is 3, B1’ is 2’-OMe or 2’-F, and q 1 is 9, T1’ is 2’-F, and q 2 is 1, B2’ is 2’-OMe or 2’-F, and q 3 is 4, T2’ is 2’-F, and q 4 is 2, B3’ is 2’-OMe or 2’-F, and q 5 is 5, T3’ is 2’-F, and q 6 is 1, B4’ is 2’-F, and q 7 has two phosphorothioate internucleotide bond modifications (counting from the 5' end of the sense strand) in positions 1-5 of the sense strand and 2 in positions 1 and 2 of the antisense strand. It also has two phosphorothioate internucleotide bond modifications within positions 18-23 (counting from the 5' end of the antisense strand).

[0555] In one embodiment, B1 is 2'-OMe or 2'-F and n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, and n 3 is 7 and n 4 is 0, B3 is 2’-OMe, and n 5 is 3, B1’ is 2’-OMe or 2’-F, and q 1 is 9, T1’ is 2’-F, and q 2 is 1, B2’ is 2’-OMe or 2’-F, and q 3 is 4 and q 4 is 0, B3’ is 2’-OMe or 2’-F, and q 5 is 7, T3’ is 2’-F, and q 6 is 1, B4’ is 2’-F, and q 7 is 1.

[0556] In one embodiment, B1 is 2'-OMe or 2'-F and n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, and n 3 is 7 and n 4 is 0, B3 is 2’-OMe, and n 5 is 3, B1’ is 2’-OMe or 2’-F, and q 1 is 9, T1’ is 2’-F, and q 2 is 1, B2’ is 2’-OMe or 2’-F, and q 3 is 4 and q 4 is 0, B3’ is 2’-OMe or 2’-F, and q 5 is 7, T3’ is 2’-F, and q 6 is 1, B4’ is 2’-F, and q 7 has two phosphorothioate internucleotide bond modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and also has two phosphorothioate internucleotide bond modifications within positions 1-5 of the sense strand and It has two phosphorothioate internucleotide linkage modifications at the top and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand).

[0557] In one embodiment, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% are modified.

[0558] In one embodiment, each of the sense and antisense strands of the dsRNA agent independently comprises a non-cyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-O-N-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) , 2'-O-aminopropyl (2'-O-AP) or 2'-ara-F.

[0559] In one embodiment, the sense and antisense strands of the dsRNA agent each contain at least two different modifications.

[0560] In one embodiment, the dsRNA agent of formula (I) further comprises a 3' and / or 5' overhang of 1 to 10 nucleotides in length. In one example, the dsRNA agent of formula (I) includes a 3' overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand. In another example, the dsRNA agent has a 5' overhang at the 5' end of the sense strand.

[0561] In one embodiment, a dsRNA agent of the invention does not contain any 2'-F modifications.

[0562] In one embodiment, the sense and / or antisense strand of the dsRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand includes one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand includes two blocks of two phosphorothioate or methylphosphonate internucleotide linkages. For example, two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16-18 phosphate internucleotide linkages.

[0563] In one embodiment, the sense and antisense strands of the dsRNA agent each have 15-30 nucleotides. In one example, the sense strand has 19-22 nucleotides and the antisense strand has 19-25 nucleotides. In another example, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

[0564] In one embodiment, the first nucleotide at the 5' end of the antisense strand in the double helix is ​​selected from the group consisting of A, dA, dU and dT. In one embodiment, at least one of the first, second and third base pairs from the 5' end of the antisense strand is an AU base pair.

[0565] In one embodiment, the antisense strand of a dsRNA agent of the invention is 100% complementary to and hybridizes to the target RNA to inhibit its expression by RNA interference. In another embodiment, the antisense strand of a dsRNA agent of the invention is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60% %, at least 55% or at least 50% complementary.

[0566] In one aspect, the invention relates to dsRNA agents capable of inhibiting target gene expression. dsRNA agents include a sense strand and an antisense strand, each strand having 14-40 nucleotides. The sense strand contains at least one heat-destabilized nucleotide, wherein the at least one heat-destabilized nucleotide is located opposite the seed region of the antisense strand (i.e., at the 5' end of the antisense strand). 2 to 8) or around it. For example, when the sense strand is 21 nucleotides long, the heat-destabilizing nucleotides are present at positions 14-17 at the 5' end of the sense strand. The antisense strand contains at least two modified nucleic acids that are smaller than the sterically demanding 2'-OMe modification. Preferably, the two modified nucleic acids, which are less than the sterically demanding 2'-OMe modification, are separated by a length of 11 nucleotides. For example, the two modified nucleic acids are at positions 2 and 14 of the 5' end of the antisense strand.

[0567] In one embodiment, the sense strand sequence of the dsRNA agent has the formula (Is): [ka] is expressed by During the ceremony, B1, B2 and B3 are each independently a nucleotide containing a modification selected from the group consisting of 2'-O alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA and BNA / LNA; indicates; C1 is a heat-destabilized nucleotide (e.g., UNA or GNA, mismatched, abasic or DNA non-cyclic nucleotides such as; T1 indicates a nucleotide that is non-ribose, acyclic or contains a chemical modification at the 2' or equivalent position of the backbone, which confers less steric bulk to the nucleotide than the 2'-OMe modification; e.g. T1 is selected from the group consisting of DNA, RNA, LNA, 2'-F and 2'-F-5'-methyl; n 1 or n 3 are independently 4 to 15 nucleotides long; n 5 is 1 to 6 nucleotides long; n 4 is 1-3 nucleotides long; n 2 is 0 to 3 nucleotides long.

[0568] In one embodiment, the 19, 20, 21 or 22 nucleotide long sense strand sequence of the dsRNA agent has the formula (Is): [ka] is expressed by During the ceremony, B1, B2 and B3 are each independently a nucleotide containing a modification selected from the group consisting of 2'-O alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA and BNA / LNA; indicates; C1 is a heat-destabilized nucleotide (e.g., UNA or GNA, mismatched, abasic or DNA non-cyclic nucleotides such as; T1 indicates a nucleotide containing a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F and 2'-F-5'-methyl; n 1 or n 3 are independently 4 to 15 nucleotides long; n 5 is 1 to 6 nucleotides long; n 4 is 1-3 nucleotides long; n2 is 0 to 3 nucleotides long.

[0569] In one embodiment, the dsRNA agent of formula (Is) further comprises a 3' and / or 5' overhang of 1 to 10 nucleotides in length. In one example, the dsRNA agent of formula (Is) includes a 5' overhang.

[0570] In one embodiment, C1 includes one heat-destabilizing nucleotide at position 14, 15, 16 or 17 from the 5' end of the sense strand. For example, C1 is a non-cyclic nucleotide (eg UNA or GNA), a mismatch, an abasic or DNA. In one specific example, C1 is GNA.

[0571] In one embodiment, T1 comprises DNA, RNA, LNA, 2'-F or 2'-F-5'-methyl at position 11 from the 5' end of the sense strand.

[0572] In one embodiment, the dsRNA agent of the invention comprises a sense strand (Is), where C1 is a non-cyclic nucleotide (e.g., UNA or GNA), mismatched, abasic or DNA; T1 is a sense strand (Is); Contains DNA, RNA, LNA, 2'-F or 2'-F-5'-methyl at position 11 from the 5' end of the chain.

[0573] In one embodiment, the antisense strand of the dsRNA agent has the formula (Ia): [ka] is expressed by During the ceremony, B1', B2', B3' and B4' are each independently selected from the group consisting of 2'-O alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA and BNA / LNA indicates a nucleotide containing a modification; T1', T2' and T3' each independently represent a nucleotide that is non-ribose, non-cyclic, or contains a chemical modification at the 2' position of the backbone or an equivalent position, and this modification is less steric than the 2'-OMe modification. imparting bulk to the nucleotide; for example, T1', T2' and T3' are each independently selected from the group consisting of DNA, RNA, LNA, 2'-F and 2'-F-5'-methyl. is; q 1 are independently 4 to 15 nucleotides long; q 3 or q 7 are independently 1 to 6 nucleotides long; q 2 or q 6 are independently 1 to 3 nucleotides long; q 4 are independently 0 to 3 nucleotides long; q 5 are independently 0 to 10 nucleotides long.

[0574] In one embodiment, the 19, 20, 21, 22, 23, 24 or 25 nucleotide long antisense strand sequence of the dsRNA agent has the formula (Ia): [ka] is expressed by During the ceremony, B1', B2', B3', and B4' are each independently from the group consisting of 2'-O alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and BNA / LNA; Indicates the nucleotide containing the selected modification; T1', T2' and T3' each independently represent a nucleotide containing a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F and 2'-F-5'-methyl; q 1 are independently 4 to 15 nucleotides long; q 3 or q 7 are independently 1 to 6 nucleotides long; q 2 or q 6 are independently 1 to 3 nucleotides long; q 4 are independently 0 to 3 nucleotides long; q 5 are independently 0 to 10 nucleotides long.

[0575] In one embodiment, the dsRNA agent of formula (Ia) further comprises a 3' and / or 5' overhang of 1 to 10 nucleotides in length. In one example, the dsRNA agent of Formula (Ia) has a 3' overhang.

[0576] In one embodiment, the invention relates to double-stranded RNA (dsRNA) agents for inhibiting target gene expression. dsRNA agents have sense and antisense strands, each strand having 14-40 nucleotides: [ka] including; During the ceremony, B1, B2, B3, B1', B2', B3' and B4' are each independently 2'-O alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA and BNA / Indicates a nucleotide containing a modification selected from the group consisting of LNA; C1 is an acyclic nucleotide (e.g. UNA or GNA); T1, T1', T2' and T3' each independently represent a nucleotide containing a chemical modification selected from t...

Claims

1. A modified oligonucleotide consisting of 8 to 15 linked nucleotides, wherein one, two, or three of the linked nucleotides are high-affinity monomers, and the high-affinity monomers are LNAs; the modified oligonucleotide comprises a nucleotide sequence that is at least 90% complementary to positions 2 to 9 of an antisense strand of an siRNA; and one of the high-affinity monomers in the modified oligonucleotide base pairs with the sixth nucleotide from the 5'-end of the antisense strand of the siRNA.

2. The modified oligonucleotide of claim 1 , comprising three LNA nucleotides.

3. The modified oligonucleotide of claim 1 , comprising a nucleotide sequence that is at least 95% complementary to the antisense strand.

4. The modified oligonucleotide of claim 1 , which is perfectly complementary to the antisense strand.

5. The modified oligonucleotide of claim 1 , comprising at least one modified internucleotide linkage.

6. The modified oligonucleotide of claim 5 , wherein the internucleotide linkage is phosphorothioate.

7. 7. The modified oligonucleotide of claim 6, comprising no more than three or four phosphorothioate modifications.

8. The modified oligonucleotide of claim 1 , conjugated to a ligand.

9. The ligand is 【Chemical 1】 9. The modified oligonucleotide of claim 8, wherein:

10. The modified oligonucleotide of claim 8, wherein the ligand is conjugated to the 3'-end of the modified oligonucleotide.

11. The modified oligonucleotide of claim 1 , wherein the modified oligonucleotide is conjugated to a ligand, and the ligand is conjugated to a nucleotide having a deoxy sugar in the modified oligonucleotide.

12. 12. The modified oligonucleotide of claim 11, wherein the deoxy sugar is 2'-deoxyribose.

13. The modified oligonucleotide of claim 1 , wherein the siRNA is targeted to mRNA, pre-mRNA, microRNA, or pre-microRNA.

14. The modified oligonucleotide of claim 1 , wherein the siRNA is conjugated to a ligand.

15. The modified oligonucleotide of claim 1 , wherein the modified oligonucleotide consists of 9, 10, or 11 linked nucleotides.

16. 2. The modified oligonucleotide of claim 1, wherein only two of the nucleotides are high-affinity monomers.

17. A kit comprising the modified oligonucleotide of claim 1.

18. A kit comprising an siRNA and the modified oligonucleotide of claim 1.

19. 17. A composition comprising a modified oligonucleotide according to any one of claims 1 to 16 for use in a method for evaluating the efficacy and safety of a pharmaceutical composition for use in the treatment or prevention of a disease, said method comprising: (1) deriving mRNA levels and / or other biomarkers for subjects treated with the pharmaceutical composition over a first treatment period; (2) separating responding members from non-responding members of said treated subjects; (3) randomizing and stratifying said responder members into at least two further subgroups; (4) selecting members of one subgroup in (3) for subsequent treatment with said pharmaceutical composition and selecting members of the other subgroup for treatment with said modified oligonucleotide over a second treatment period; (5) deriving mRNA levels and / or other biomarkers for the subpopulation after undergoing the treatment in (4); (6) comparing the mRNA levels and / or other biomarkers in (5) with the mRNA levels and / or other biomarkers in (1); (7) using the comparison in (6) to derive efficacy and safety measures for the pharmaceutical composition. Including, the disease is caused by abnormal expression of a target gene, and the pharmaceutical composition comprises an oligonucleotide; composition.

20. 17. A composition comprising a modified oligonucleotide according to any one of claims 1 to 16 for use in a method for evaluating the efficacy and safety of a pharmaceutical composition for use in the treatment or prevention of a disease, said method comprising: (1) stratifying a subject population into at least two subgroups, with members of a first subgroup treated with a pharmaceutical composition for a first treatment period and members of a second subgroup treated with a blinded placebo; (2) deriving mRNA levels and / or biomarkers for said subpopulations; (3) selecting members of the first subgroup for treatment with the modified oligonucleotide and selecting members of the second subgroup for treatment with the pharmaceutical composition for a second treatment period; (4) deriving mRNA levels and / or other biomarkers for the subpopulation after undergoing the treatment in (3); (5) comparing the mRNA levels and / or other biomarkers in (4) with the mRNA levels and / or other biomarkers in (2); (6) using the comparison in (5) to derive efficacy and safety measures for the pharmaceutical composition. Including, the disease is caused by abnormal expression of a target gene, and the pharmaceutical composition comprises an oligonucleotide; composition.

21. The composition described in claim 19 or 20, wherein the oligonucleotide in the pharmaceutical composition is an antisense or siRNA.

22. 20. The composition of claim 19, wherein the modified oligonucleotide of claim 1 is used to standardize and enable randomized treatment discontinuation of the treated members.

23. 20. The composition of claim 19, wherein administration of the modified oligonucleotide of claim 1 is blinded along with placebo administration of the pharmaceutical composition.

24. 21. The composition of claim 19 or 20, wherein the modified oligonucleotide of claim 1 induces washout in the treated subpopulation.

25. 23. The composition of claim 22, wherein the modified oligonucleotide of claim 1 resets the baseline of the RNAi activity of the oligonucleotide in the pharmaceutical composition.

26. 21. The composition of claim 20, wherein the pharmaceutical composition can be re-administered at least 1, 2, 3, or 4 weeks after treatment with the modified oligonucleotide.

27. The target genes include TTR, AGT, ALAS-1, GO1, AT3, factor XI, factor XII, CC3, CC5, AAT, Eg5, PCSK9, TPX2, apoB, SAA, RSV, PDGFβ gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1 / 2 gene, PCNA (p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, cyclin D gene, VEGF gene, and EGFR.

21. The composition of claim 19 or 20, wherein the mutation is selected from the group consisting of a mutation in a CYP3A1 gene, a cyclin A gene, a cyclin E gene, a WNT-I gene, a β-catenin gene, a c-MET gene, a PKC gene, a NFKB gene, a STAT3 gene, a survivin gene, a Her2 / Neu gene, a topoisomerase I gene, a topoisomerase IIα gene, a mutation in the p73 gene, a mutation in the p21 (WAF1 / CIP1) gene, a mutation in the p27 (KIP1) gene, a mutation in the PPM1D gene, a mutation in the RAS gene, a mutation in the caveolin I gene, a mutation in the MIB I gene, a mutation in the MTAI gene, a mutation in the M68 gene, a mutation in a tumor suppressor gene, and a mutation in the p53 tumor suppressor gene.

28. 20. The composition of claim 19, wherein the first treatment period is until the completion of an open-label or single-blind study.

29. 20. The composition of claim 19, wherein the second treatment period is from 1 week to 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months.

30. 20. The composition of claim 19, wherein the second treatment period is until the completion of a clinical trial.

31. The composition of any one of claims 19 to 30, wherein treatment with the pharmaceutical composition constitutes a clinical trial or a system for conducting a clinical trial to validate said pharmaceutical composition.

32. A composition described in any one of claims 19 to 30, wherein the method is for evaluating the effectiveness of a treatment regimen using the pharmaceutical composition.

Images