Regulatory reversir (TM) compound

Abstract

To provide a medicine that regulates pharmacodynamic activity of siRNA.SOLUTION: Provided is a regulatory REVERSIR compound including eight or nine modified nucleotides, where at least three of the modified nucleotides are high affinity monomers, and one of the high affinity monomers base-pairs 6-th nucleotide from 5'-terminal of a target chain of siRNA.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62 / 546,779, filed August 17, 2017, the contents of which are incorporated herein by reference in their entirety.

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

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

[0004] The present inventors have surprisingly found that adjusting the appropriate level of the regulating REVERSIR agent is necessary to effectively restore the RNAi activity of an siRNA compound after re-administration of the same siRNA compound. The regulating duration of REVERSIR action is achieved by optimizing design parameters that allow for effective restoration of RNAi pharmacological efficacy after re-administration of the same siRNA, thereby providing dissociation and binding rate control for adjusting the therapeutic efficacy of siRNA therapeutics in vivo. The regulating duration of the regulating REVERSIR agent can be extended to control siRNA effects when designing clinical trials using siRNA therapeutics in humans.

[0005] In some embodiments, the invention provides tunable REVERSIR compounds comprising a modified oligonucleotide comprised of 8-10 (e.g., 8, 9, or 10) linked nucleotides and having a nucleobase sequence substantially complementary to at least one strand of an siRNA compound (e.g., conjugated or unconjugated siRNA). In some embodiments, the tunable REVERSIR compound comprises a modified oligonucleotide comprised of 8-10 (e.g., 8, 9, or 10) linked nucleotides and has a nucleobase sequence substantially complementary to the antisense strand of an siRNA compound. In some embodiments, the tunable REVERSIR compound comprises a modified oligonucleotide comprised of 8-10 (e.g., 8, 9, or 10) linked nucleotides and has a nucleobase sequence substantially complementary to the sense strand of an siRNA compound.

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

[0008] In some embodiments, the modulating REVERSIR compound comprises at least one modified internucleotide or intersugar linkage. In some such embodiments, at least one (e.g., 1, 2, 3, 4, 5, 6, and all) internucleotide linkage is a phosphorothioate internucleotide linkage.

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

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

[0011] In some embodiments, each nucleotide in the tuning REVERSIR compound is a 2'-modified nucleotide, and the tuning REVERSIR compound comprises at least one (eg, 1, 2, or 3) G-clamp nucleobase.

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

[0013] In some embodiments, the regulating REVERSIR compound comprises at least one modification. In some such embodiments, the regulating REVERSIR compound comprises one or more nucleotide modifications and / or one or more linkage modifications. In some embodiments, the regulating REVERSIR compound comprises one or more modifications selected from a sugar modification, a linkage modification, a nucleobase modification, a conjugate (e.g., a ligand), and any combination thereof.

[0014] In some embodiments, the present invention provides a modulating REVERSIR compound that includes 8 or 9 modified nucleotides.

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

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

[0017] In some embodiments, the tunable REVERSIRs of the present invention further comprise up to three or four phosphorothioate (PS) backbone modifications.

[0018] In some embodiments, the modulatory REVERSIR compound modulates the RISC pathway.In some embodiments, the modulatory REVERSIR compound inhibits the RISC pathway.

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

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

[0021] In some embodiments, the action of the regulatory REVERSIR compound allows for dosing and withdrawal control of the therapeutic action of the siRNA agent, where sequential dosing and withdrawal of the siRNA can be achieved by sequential administration of the regulatory REVERSIR compound after and / or before administration of the therapeutic siRNA.

[0022] In some embodiments, the present invention provides methods for inhibiting RNAi activity of conjugated or unconjugated siRNA in a cell. The methods generally include the step of inhibiting RNAi activity in a cell by contacting the cell with a regulatory REVERSIR compound of the present invention. In some such embodiments, the cell is in vivo. In some embodiments, the cell is in vitro. In some embodiments, the cell is ex vivo. In some embodiments, the cell is within a subject. In still other embodiments, the cell is from an animal. In some embodiments, the animal is a human.

[0023] In some embodiments, the invention provides methods comprising contacting a cell with conjugated or unconjugated siRNA; detecting RNAi activity; and contacting the cell with a regulatory REVERSIR compound. In some embodiments, the method of detecting RNAi activity comprises 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 comprise detecting regulatory REVERSIR activity by contacting the cell with a regulatory REVERSIR compound and then measuring RNAi activity. In some such embodiments, the cell is 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 for ameliorating side effects of siRNA therapeutics, comprising the steps of contacting a cell with conjugated or unconjugated siRNA; detecting side effects; and contacting the cell with a regulatory REVERSIR compound, thereby ameliorating side effects of the siRNA, endogenous, exogenous microRNA, or a combination thereof.

[0025] In some embodiments, the present invention provides methods of treating a patient, comprising administering a conjugated or unconjugated siRNA to the patient; monitoring the patient for siRNA activity; and administering a regulatory REVERSIR compound if siRNA activity becomes higher than desired. 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 comprise detecting regulatory REVERSIR activity by measuring siRNA activity after administration of the regulatory REVERSIR compound. In some embodiments, the patient is a mammal. In some embodiments, the patient is human.

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

[0027] In some embodiments, the structure and properties of the modulatory REVERSIR compound are designed such that the modulatory REVERSIR compound achieves maximal inhibition or reversal of siRNA activity in vivo, for example, maximal inhibition or reversal of siRNA activity occurs when mRNA levels are reduced by at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, up to 100%, and any integer between 5% and 100%.

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

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

[0030] In some embodiments, the structure and properties of the regulatory REVERSIR compounds are designed such that they allow for effective re-administration of siRNA and restoration of RNAi in vivo.

[0031] The present invention also generally relates to methods for demonstrating the efficacy and safety of pharmaceutical compositions for use in treating or preventing diseases, such as diseases caused by the abnormal expression of target genes. In particular, the present invention relates to improved methods for demonstrating efficacy and safety in situations where pharmaceutical compositions, such as siRNAs, have sustained pharmacodynamics (PD) and long-term safety and efficacy in open-label, single-arm clinical trials are desirable but not feasible. The present invention also relates to situations, such as rare disease situations, where patient recruitment is difficult and few subjects receive placebo or low-activity therapeutics, thereby hindering detection of therapeutic efficacy and safety.

[0032] Thus, 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, wherein the randomized treatment withdrawal method comprises: (1) treating all subjects with a pharmaceutical composition for a first treatment period; (2) deriving mRNA levels and / or physiological outcome measures for all subjects; (3) separating responding members from non-responding members of the treated subjects; (4) randomizing and stratifying the responder members into at least two further subgroups; (5) continuing to treat one subgroup member in (4) with the pharmaceutical composition and the other subgroup member with a REVERSIR compound of claim 1 for a second treatment period; (6) deriving mRNA levels and / or physiological outcome measures for these subpopulations; (7) comparing the outcome in (6) with the outcome in (2); and (8) Using the comparison in (7), deriving efficacy and safety measures for the pharmaceutical composition. Includes.

[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 a disease, the crossover method comprising: (1) stratifying the subject group into at least two subgroups; (2) treating members of one subgroup with the pharmaceutical composition and 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 subpopulations; (4) treating members of the treated subgroup with a modulating REVERSIR compound and 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 subpopulations; (6) comparing the outcomes in (5) with the outcomes in (3); and (7) Using the comparison in (6), deriving efficacy and safety measures for the pharmaceutical composition. Includes. [Brief explanation of the drawings]

[0034] [Figure 1] We show the in vivo activity of exemplary 15-mer REVERSIR compounds that target transthyretin-directed (TTR) siRNA and have varying abilities to reverse RNAi activity based on different base pairing with the g6 position (the sixth nucleotide from the 5'-end of the target strand of the siRNA) of the guide (antisense) siRNA strand. [Figure 2] 1 shows the in vivo activity of exemplary 9-mer tunable REVERSIR compounds that target TTR siRNA and have varying abilities to reverse RNAi activity based on base pairing with the g6 position of the guide siRNA strand. [Figure 3]1 shows the in vivo activity of exemplary 9-mer regulative REVERSIR compounds targeting TTR siRNA and having different contents of locked nucleic acid (LNA) nucleotides and phosphorothioate (PS) backbone modifications. [Figure 4] 1 shows the in vivo activity of exemplary REVERSIR compounds with long-lasting RNAi activity reversal upon multiple rechallenge with TTR siRNA agents. [Figure 5] 1 shows the in vivo activity at various doses of an exemplary REVERSIR compound that has long-lasting RNAi activity reversal upon a single rechallenge with a TTR siRNA agent. [Figure 6] 1 shows the in vivo activity of exemplary REVERSIR compounds that have long-lasting RNAi activity reversal upon a single rechallenge with a TTR siRNA agent. [Figure 7] 1 shows the in vivo activity of exemplary tunable REVERSIR compounds with tunable and long-lasting RNAi activity reversal upon a single rechallenge with a TTR siRNA agent. [Figure 8] 1 shows the in vivo activity of an exemplary tunable REVERSIR compound with tunable and persistent RNAi activity reversal upon a single rechallenge with a TTR siRNA agent, as well as subsequent complete restoration of RNAi activity. [Figure 9]

[0023] Figure 1 shows the Phase 1 / 2a Coordinated REVERSIR Crossover Clinical Trial. [Figure 10] The Phase 2 / 3 Coordinated REVERSIR Randomized Treatment Withdrawal Clinical Trial is presented. [Figure 11] 1 shows exemplary data expected from a coordinated REVERSIR randomized treatment withdrawal clinical trial design. [Figure 12] 1 shows miR-122 target mRNA liver qPCR data from an in vivo study in male rats with miR-122 modulating REVERSIR compounds. [Figure 13] 1 shows the miR-122 biomarker time course from an in vivo study in male rats with miR-122 modulating REVERSIR compounds. [Figure 14] 1 shows the miR-122 biomarker time course from an in vivo study in male rats with miR-122 modulating REVERSIR compounds. [Figure 15] 1 shows the miR-122 biomarker time course from an in vivo study in male rats with miR-122 modulating REVERSIR compounds. [Figure 16] 1 shows the fold change in miR-122 biomarker compared to saline group from an in vivo study in male rats with miR-122 modulating REVERSIR compounds. [Figure 17] 1 shows LFT assessments from in vivo studies in male rats with miR-122 modulating REVERSIR compounds. [Figure 18] 1 shows histopathology data from in vivo studies in male rats with miR-122 modulating REVERSIR compounds. [Figure 19] 1 shows histopathology data from in vivo studies in male rats with miR-122 modulating REVERSIR compounds. DETAILED DESCRIPTION OF THE INVENTION

[0035] It is to be understood that both the foregoing general description and the following detailed description are intended to be exemplary and explanatory, but are not limiting of the invention, as defined in the claims. As used herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "comprising" and other forms, such as "comprises" and "includes," is not limiting. Also, terms such as "element" or "component" encompass both elements and components that include one unit and elements and components that include two or more subunits, unless specifically stated otherwise.

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

[0037] In some embodiments, the present invention provides a tunable REVERSIR compound comprising eight or nine 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 sixth 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 modulating REVERSIR compound comprises 3 or 4 LNA nucleotides.

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

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

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

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

[0044] In some embodiments, the modulating REVERSIR compound comprises no more than three or four phosphorothioate modifications.

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

[0046] In some embodiments, the modulating REVERSIR compound is [ka] The ligand includes a ligand in which

[0047] In some embodiments, the modulating REVERSIR compound comprises a ligand conjugated to the 3'-end of the compound.

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

[0049] In some embodiments, the present invention provides a kit comprising a modulating REVERSIR compound comprising eight or nine 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 sixth nucleotide from the 5'-end of the target strand of the siRNA.

[0050] In some embodiments, the invention provides a kit comprising an siRNA and a modulating REVERSIR compound comprising eight or nine 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 sixth nucleotide from the 5'-end of the target strand of the siRNA.

[0051] In some embodiments, the present 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 a pharmaceutical composition for a first treatment period; (2) deriving mRNA levels and / or physiological outcome measures for all subjects; (3) separating responding members from non-responding members of the treated subjects; (4) randomizing and stratifying the responder members into at least two further subgroups; (5) continuing to treat one subgroup member in (4) with the pharmaceutical composition and the other subgroup member with a REVERSIR compound of claim 1 for a second treatment period; (6) deriving mRNA levels and / or physiological outcome measures for these subpopulations; (7) comparing the outcome in (6) with the outcome in (2); and (8) Using the comparison in (7), deriving efficacy and safety measures for the pharmaceutical composition. Includes.

[0052] In some embodiments, the present 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 a pharmaceutical composition for a first treatment period; (2) deriving mRNA levels and / or physiological outcome measures for all subjects; (3) separating responding members from non-responding members of the treated subjects; (4) randomizing and stratifying the responder members into at least two further subgroups; (5) continuing to treat one subgroup member in (4) with the pharmaceutical composition and the other subgroup member with a REVERSIR compound of claim 1 for a second treatment period; (6) deriving mRNA levels and / or physiological outcome measures for these subpopulations; (7) comparing the outcome in (6) with the outcome in (2); and (8) Using the comparison in (7), deriving efficacy and safety measures for the pharmaceutical composition. In this study, subjects were divided into four subgroups and followed the Williams design and randomization in a crossover clinical trial (Journal of Statistical Software, V29, February 2009).

[0053] In one example, the number of treatments in the study is four, and the following series of treatments are administered:

[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, ActCtrl includes the initial dose of the pharmaceutical composition, an active control drug or other standard of care (i.e., other treatments available for the disease), and Placebo includes the group containing the REVERSIR treatment.

[0056] In some embodiments, the present invention provides methods or systems for evaluating the efficacy and safety of a pharmaceutical composition for use in treating or preventing a disease, wherein the disease is caused by aberrant expression of a target gene.

[0057] In some embodiments, the present invention provides methods or systems 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.

[0058] In some embodiments, the present invention provides methods or systems 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.

[0059] In some embodiments, the present invention provides methods or systems for evaluating the efficacy and safety of pharmaceutical compositions for use in the treatment or prevention of disease, wherein the oligonucleotides provided sustainable pharmacodynamics.

[0060] In some embodiments, the present invention provides methods or systems for evaluating the efficacy and safety of pharmaceutical compositions for use in the treatment or prevention of disease, wherein a modulating REVERSIR compound is used to standardize and enable randomized treatment discontinuation of treated members.

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

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

[0063] In some embodiments, the siRNA may be re-administered at least 1, 2, 3, or 4 weeks after the regulatory 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, PDGF beta 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 , a mutation in the EGFR 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.

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

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

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

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

[0069] definition Unless specifically defined, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis and chemical analysis. Some such techniques and procedures can be found, for example, in "Carbohydrate Modifications in Antisense Research," Edited by Sangvi and Cook, American Chemical Society, Washington, DC, 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 materials referred to in this disclosure are incorporated herein by reference in their entirety.

[0070] Unless otherwise indicated, the following terms have the following meanings:

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

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

[0073] As used herein, the term "nucleoside" refers to a glycosylamine comprising a nucleobase and a sugar. Nucleosides include, but are not limited to, naturally occurring nucleosides, abasic nucleosides, modified nucleosides, and nucleosides with mimetic base and / or sugar groups.

[0074] As used herein, the term "nucleotide" refers to a glycosomine comprising 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 is capable of hydrogen bonding to a base of another nucleic acid.

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

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

[0078] As used herein, "oligonucleoside" refers to an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.

[0079] As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising multiple linked nucleotides. In some embodiments, one or more nucleotides of an oligonucleotide are modified. In some embodiments, an oligonucleotide comprises ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, an oligonucleotide is composed of natural and / or non-natural nucleobases, sugars, and covalent internucleotide linkages, and may further comprise 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 linkage" refers to the covalent phosphorus bond between adjacent nucleotides.

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

[0083] As used herein, the term "detecting siRNA activity" or "measuring siRNA activity" means that a test for detecting or measuring siRNA activity is carried out on a specific sample and compared with a test on a control sample. Such detection and / or measurement may include a value of zero. Thus, even if the test for detecting siRNA activity shows that there is no siRNA activity (zero siRNA activity), the "step of detecting siRNA activity" is still carried out.

[0084] As used herein, the term "control sample" refers to a sample that has not been contacted with a reporter oligomeric 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 can hybridize with at least one strand of a conjugated or unconjugated siRNA. Without limitation, a regulatory REVERSIR compound can not only block unintended target PD effects, but also block any potential off-target activity that may occur with conjugated or unconjugated siRNA.

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

[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 is different from at least one other internucleotide linkage of the oligomeric compound.

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

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

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

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

[0093] As used herein, the terms "targeting" or "targeted" refer to the association of the antisense strand of an siRNA with a specific target nucleic acid molecule or a specific region of nucleotides within a target nucleic acid molecule.

[0094] As used herein, the term "nucleobase complementarity" refers to a nucleobase that can base pair 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, complementary nucleobase refers to the nucleobase of an antisense compound that can base pair with the nucleobase of its target nucleic acid.For example, if the nucleobase at a specific position of an antisense compound can hydrogen bond with the nucleobase at a specific position of target nucleic acid, the hydrogen bond position between oligonucleotide and target nucleic acid is considered to be complementary at this nucleobase pair.

[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 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, an oligomeric compound and its target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases that can bind to each other, allowing stable association between the antisense compound and the target. Those skilled in the art will recognize that mismatches are possible without eliminating the ability of the oligomeric compound to maintain association. Thus, described herein are oligomeric compounds (e.g., tunable REVERSIR compounds, siRNAs, etc.) that can contain up to about 20% mismatched nucleotides (i.e., nucleobases that are not complementary to the corresponding nucleotides of the target). Preferably, oligomeric compounds such as tunable REVERSIR compounds and siRNAs contain no more than about 15%, more preferably no more than about 10%, and most preferably no more than 5% mismatches. The remaining nucleotides are complementary nucleobases or do not otherwise disrupt hybridization (e.g., universal bases). Those skilled in the art will recognize that the compounds provided herein may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target nucleic acid.

[0097] As used herein, "hybridization" refers to the pairing of complementary oligomeric compounds (for example, the antisense strand of siRNA and its target nucleic acid or target siRNA regulatory REVERSIR). Without being limited to a specific mechanism, the most common pairing mechanism involves hydrogen bonding between complementary nucleotides or nucleotide bases (nucleobases), which can be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds. For example, the natural base adenine is a nucleobase that pairs with the natural nucleobases thymidine and uracil through the formation of hydrogen bonds. The natural base guanine is a nucleobase that pairs with the natural bases cytosine and 5-methylcytosine. Hybridization can occur under various circumstances.

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

[0099] As used herein, "design" or "designed" refers to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.

[0100] As used herein, the term "modulation" refers to a perturbation of function or activity 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 suppression). As another example, modulation of expression can include a perturbation of splice site selection in pre-mRNA processing.

[0101] As used herein, the term "expression" refers to all the functions and steps by which a gene's coded information is converted into structures present and operating in a cell, including, but not limited to, the products of transcription and translation.

[0102] As used herein, "variant" refers to alternative RNA transcripts 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 in either their start or end positions, including both intron and exon sequences. Further 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" refers to a monomer that has at least one modified nucleobase, internucleotide linkage, or sugar moiety, compared to a naturally occurring monomer, such that this modification increases the affinity of the antisense compound comprising the high-affinity modified monomer for its target nucleic acid. High-affinity modifications include, but are not limited to, monomers (e.g., nucleosides and nucleotides) that contain 2'-modified sugars.

[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-C1-C2 10 Included are BNAs and monomers (e.g., nucleosides and nucleotides) having alkyl, -OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-ON(Rm)(Rn), or O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is independently H or a substituted or unsubstituted C1-C 10 In some embodiments, the oligomeric compound has the formula 2'-O(CH2) nIn some embodiments, the oligomeric compounds comprise 2' modified monomers that lack H (where n is 1 to 6). In some embodiments, the oligomeric compounds comprise 2' modified monomers that lack 2'-OCH3. In some embodiments, the oligomeric compounds comprise 2' modified monomers that lack the above formula or, in another embodiment, 2'-O(CH2)2OCH3.

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

[0106] As used herein, unless otherwise indicated, the term "methyleneoxyLNA" refers solely to β-D-methyleneoxyLNA.

[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 comprising a central region (the "gap") and regions on either side of the central region (the "wings"), wherein the gap contains at least one modification that differs from each wing. Such modifications include nucleobase, monomer linkage, and sugar modifications, as well as the absence of a modification (unmodified). Thus, in some embodiments, the nucleotide linkage in each of the wings differs from the nucleotide linkage in the gap. In some embodiments, each wing contains nucleotides with a high-affinity modification, and the gap contains nucleotides without that modification. In some embodiments, the nucleotides in the gap and the nucleotides in the wings all contain a high-affinity modification, but the high-affinity modification in the gap differs from the high-affinity modification in the wings. In some embodiments, the modifications in the wings are the same as each other. In some embodiments, the modifications in the wings differ from each other. In some embodiments, the nucleotides in the gap are unmodified and the nucleotides in the wings are modified. In some embodiments, the modifications in each wing are the same. In some embodiments, the modification in one wing differs from the modification in the other wing. In some embodiments, the oligomeric compounds are gapmers having 2'-deoxynucleotides in the gap and nucleotides containing high affinity modifications in the wings.

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

[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 undesired toxicological effects.

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

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

[0113] As used herein, the term "amelioration" refers to a reduction in at least one activity or severity indicator 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 present invention to effect an alteration or improvement of a disease or condition. Prevention, improvement, and / or treatment may require the administration of multiple doses at regular intervals or prior to the onset of the condition or disease to alter the course of the disease or condition. Furthermore, a single agent may be used sequentially or simultaneously in one individual for the prevention, improvement, and treatment of a condition or disease, respectively.

[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 an amount of a pharmaceutical agent that confers a therapeutic benefit on 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 medical 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, the two or more pharmaceutical agents are administered together. In some embodiments, the two or more pharmaceutical agents are administered separately. In some embodiments, the two or more pharmaceutical agents are administered simultaneously. In some embodiments, the two or more pharmaceutical agents are administered at different times. In some embodiments, the two or more pharmaceutical agents are administered by the same route of administration. In some embodiments, the two or more pharmaceutical agents are administered by different routes of administration. In some embodiments, the two or more pharmaceutical agents are contained in the same pharmaceutical formulation. In some embodiments, the 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 may comprise an antisense oligonucleotide and a sterile aqueous solution. In some embodiments, a pharmaceutical composition comprises 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 (e.g., an animal or plant), but in an artificial environment, such as a test tube or reaction vessel, in cell culture. As used herein, the term "ex vivo" refers to cells removed from a living organism and cultured outside the organism (e.g., in a test tube). As used herein, the term "in vivo" refers to events that occur within an organism (e.g., an animal, plant, and / or microorganism).

[0121] As used herein, the term "subject" or "patient" refers to any living organism to which a composition disclosed herein can be administered, for example, for experimental, diagnostic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. Typically, animals are vertebrates such as primates, rodents, livestock, or sport animals. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys (Rhesus). Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and sport animals include cattle, horses, pigs, deer, bison, water buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, and ostriches, and fish, such as trout, catfish, and salmon. A patient or subject includes any subset of those listed above, e.g., all of the foregoing, but excluding 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, e.g., a primate, e.g., a human. The terms "subject" and "patient" are used interchangeably herein. A subject can be either male or female.

[0122] Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can be advantageously used as subjects to serve as animal models of human diseases and disorders. Furthermore, the compounds, compositions, and methods described herein can also be used with 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 for a disease model. The term does not denote a particular age or sex. Thus, it is intended to encompass adult and newborn subjects, as well as fetuses, whether male or female. 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 will be limited to prescribing a controlled substance to the human subject for self-administration by any method (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation consistent with the statute or regulation defining patentable subject matter is intended. In jurisdictions that do not prohibit the patenting of methods performed on the human body, "administration" of a composition will encompass both the method performed on the human body and the aforementioned activities.

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

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

[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 easily delivered by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In some embodiments, a dose may be administered in two or more injections to minimize injection site reactions in individuals.

[0128] As used herein, the term "unit dosage form" refers to the form in which a 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 the substance in a pharmaceutical composition that imparts a desired effect.

[0130] As used herein, the term "side effect" refers to a physiological reaction attributed to 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, liver toxicity, central nervous system abnormalities, and muscle disorders. For example, elevated serum aminotransferase levels may indicate liver toxicity or liver dysfunction. For example, elevated bilirubin may indicate liver toxicity or liver dysfunction.

[0131] As used herein, the term "alkyl," as used herein, refers to a saturated straight-chain 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), with 1 to about 6 carbon atoms being more preferred. As used herein, the term "lower alkyl" contains 1 to about 6 carbon atoms. As used herein, alkyl groups can optionally include one or more additional substituents.

[0132] As used herein, the term "alkenyl" refers to a straight-chain or branched hydrocarbon radical containing up to 24 carbon atoms and having at least one carbon-carbon double bond. 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 from 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-chain or branched hydrocarbon radical containing up to 24 carbon atoms and having at least one carbon-carbon triple bond. 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 from 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. This term is meant to include C1-C12 alkyl groups with an amino substituent at any position, where the alkyl group attaches the aminoalkyl group to 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" refers to a straight-chain or branched hydrocarbon radical containing up to 24 carbon atoms, with the saturation between any two carbons being a single, double, or triple bond. Aliphatic groups preferably contain from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms, with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of the aliphatic group may be interrupted by one or more heteroatoms, including nitrogen, oxygen, sulfur, and phosphorus. Examples of such heteroatom-interrupted aliphatic groups include, but are not limited to, polyalkoxy groups, such as polyalkylene glycols, polyamines, and polyimines. The aliphatic groups used herein may optionally contain other substituents.

[0136] As used herein, the term "alicyclic" or "alicyclyl" refers to a ring system in which the ring is aliphatic. The ring system can 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, alicyclics can 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 is used to attach the alkoxy group to the parent molecule. 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. As used herein, alkoxy groups can optionally include other substituents. As used herein, the terms "halo" and "halogen," as used herein, refer to an atom selected from fluorine, bromine, and iodine.

[0137] As used herein, the terms "aryl" and "aromatic," as used herein, refer to a monocyclic or polycyclic carbocyclic ring system radical 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 from about 5 to about 20 carbon atoms in the ring or rings. As used herein, aryl groups can 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 the aralkyl group to the parent molecule. Examples include, but are not limited to, benzyl, phenethyl, and the like. Aralkyl groups as used herein can optionally include additional substituents attached to the alkyl, aryl, or both groups that form the radical group.

[0139] As used herein, the term "heterocyclic radical" refers to a radical monocyclic or polycyclic ring system that contains at least one heteroatom and is unsaturated, partially saturated, or fully saturated, and thus includes heteroaryl groups. Heterocyclic is also meant to include fused ring systems, where one or more of the fused rings contain at least one heteroatom, and the other rings may contain one or more heteroatoms, or optionally no 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, tetrahydrofuryl, and the like. Heterocyclic groups as used herein may optionally contain other substituents. As used herein, the terms "heteroaryl" and "heteroaromatic," as used herein, refer to a radical containing a monocyclic or polycyclic aromatic ring, ring system, or fused ring system in which at least one of the rings is aromatic and contains one or more heteroatoms. Heteroaryl is also meant to include fused ring systems, including systems in which one or more fused rings do not contain heteroatoms. Heteroaryl groups typically contain 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, quinoxalinyl, and the like. 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 can optionally include other substituents.

[0140] As used herein, the term "heteroarylalkyl" refers to a heteroaryl group, as previously defined, having an alkyl radical that can attach the heteroarylalkyl group to a parent molecule. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl, naphthyridinylpropyl, and the like. Heteroarylalkyl groups as used herein can optionally include other substituents on either or both of the heteroaryl or alkyl portions.

[0141] As used herein, the term "monocyclic or polycyclic structure" encompasses all ring systems, whether monocyclic or polycyclic with fused or linked rings, including monocyclic or mixed ring systems selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic, and heteroarylalkyl. Such monocyclic and polycyclic structures can be homogeneous or contain rings with varying degrees of saturation, such as fully saturated, partially saturated, or fully unsaturated. Each ring can contain ring atoms selected from C, N, O, and S, thereby forming heterocyclic rings and mixed motifs containing only C ring atoms, such as benzimidazole, where one ring contains only carbon atoms and the fused ring has two nitrogen atoms. The monocyclic or polycyclic structure can be further substituted with substituents, such as phthalimide, which has two =O groups attached to one of the rings. In another embodiment, the monocyclic or polycyclic structure can be attached to a parent molecule directly through a ring atom, via a substituent, or via a bifunctional linking molecule.

[0142] As used herein, the term "acyl," as used herein, refers to a radical formed by removal of a hydroxyl group from an organic acid, which has the general formula -C(O)-X, where X is typically aliphatic, alicyclic, or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates, and the like. As used herein, acyl groups can optionally include other substituents.

[0143] As used herein, the term "hydrocarbyl" includes groups containing C, O, and H. Included are straight-chain, branched, and cyclic groups of any degree of saturation. 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 As used herein, the term "group" includes groups that are typically added to other groups or parent compounds to enhance a desired property or impart a desired effect. Substituents can be either protected or unprotected and can be added to one available site or to multiple 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)Ra), carboxyl (-C(O)O-Ra), aliphatic groups, alicyclic groups, alkoxy, substituted oxo (-O-Ra), aryl, aralkyl, heterocyclic, heteroaryl, heteroarylalkyl, amino (-NRbbRcc), imino (=NRbb), amido (-C(O)N-RbbRcc or -N(Rbb)C(O)Ra), azido (-N3), nitro (-NO2), and the like. ), 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 Ra, Rbb, and Rcc is independently H, an optionally linked chemical functionality, or another substituent, with a preferred list including, but not limited to, H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic, and heteroarylalkyl.

[0145] The regulating REVERSIR compounds disclosed herein are particularly effective in reducing the activity of siRNA. For example, the regulating REVERSIR compounds disclosed herein can reduce 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., to a level that is nonexistent compared to a reference sample), or can result in any reduction in the range of 50-100% compared to the reference level. The reference level can be the siRNA activity in the absence of the regulating REVERSIR compound.

[0146] In some embodiments, the modulatory REVERSIR compounds described herein can reduce the activity of an siRNA by at least about 75%, e.g., 80%, 85%, 90%, 95%, or more, within less than 7 days (e.g., within 6, 5, 4, 3, 2, or 1 day) of administration or use of the modulatory REVERSIR compound, including more complete reduction or inhibition of siRNA activity.

[0147] In some embodiments, the modulatory REVERSIR compound can completely reduce the activity of the siRNA within four days of administration or use of the modulatory REVERSIR compound, where complete reduction of the activity of the siRNA means a reduction of the activity of the siRNA by at least 80% relative to baseline levels.

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

[0149] monomer In some embodiments, oligomeric compounds comprise one or more modified monomers. In some such embodiments, oligomeric compounds comprise one or more high-affinity monomers. In some embodiments, such high-affinity monomers are selected from monomers (e.g., nucleosides and nucleotides) containing 2'-modified sugars, including, but not limited to, BNAs and monomers (e.g., nucleosides and nucleotides) having 2'-substituents, such as allyl, amino, azido, thio, O-allyl, O-C1-C2 10 alkyl, -OCF3-, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-ON(Rm)(Rn), or O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is independently H or a substituted or unsubstituted C1-C 10 It is alkyl.

[0150] In some embodiments, oligomeric compounds, including but not limited to, the regulatory REVERSIR compounds and siRNAs of the present invention comprise one or more high affinity monomers.

[0151] In some embodiments, oligomeric compounds, including but not limited to, regulatory REVERSIR compounds and siRNAs of the present invention comprise one or more β-D-methyleneoxy (4'-CH2-O-2') LNA monomers.

[0152] In some embodiments, oligomeric compounds, including but not limited to, regulative REVERSIR compounds and siRNAs of the present invention comprise one or more α-D-methyleneoxy (4′-CH2-O-2′) LNA monomers.

[0153] In some embodiments, oligomeric compounds, including but not limited to, regulatory REVERSIR compounds and siRNAs of the present invention comprise one or more (S)-cEt monomers.

[0154] In some embodiments, oligomeric compounds, including but not limited to, the regulatory REVERSIR compounds and siRNAs of the present invention, comprise one or more high affinity monomers, provided that the oligomeric compounds do not contain a 2'-O(CH2) n It does not contain nucleotides containing H (n is 1 to 6).

[0155] In some embodiments, oligomeric compounds, including but not limited to, regulatory REVERSIR compounds and siRNAs, comprise one or more high affinity monomers, with the proviso that the oligomeric compounds do not comprise nucleotides containing 2'-OCH3 or 2'-O(CH2)2OCH3.

[0156] In some embodiments, oligomeric compounds, including but not limited to, regulatory REVERSIR compounds and siRNAs, comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) high affinity monomers, with the proviso that the oligomeric compounds do not comprise α-L-methyleneoxy(4'-CH2-O-2') LNA.

[0157] In some embodiments, oligomeric compounds, including but not limited to, regulatory REVERSIR compounds and siRNAs, comprise one or more high affinity monomers, with the proviso that the oligomeric compounds do not comprise β-D-methyleneoxy(4'-CH2-O-2')LNA.

[0158] In some embodiments, oligomeric compounds, including but not limited to, regulatory REVERSIR compounds and siRNAs, comprise one or more high affinity monomers, with the proviso that the oligomeric compounds do not comprise α-L-methyleneoxy(4'-CH2-O-2')LNA or β-D-methyleneoxy(4'-CH2-O-2')LNA.

[0159] Certain nucleobases The naturally occurring base moiety of a nucleoside is typically a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. In nucleosides containing a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. When forming oligonucleotides, these phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. Within oligonucleotides, the phosphate groups are generally referred to as forming the internucleoside or internucleotide backbone of the oligonucleotide. The naturally occurring linkage or backbone of RNA and DNA is the 3'-5' phosphodiester bond.

[0160] In addition to "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), many modified nucleobases or nucleobase mimetics known to those skilled in the art can be used in the compounds described herein. Unmodified or natural nucleobases can be modified or substituted to provide oligonucleotides with improved properties. For example, nuclease-resistant oligonucleotides can be prepared using these bases or synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidin) and any one of the oligomeric modifications described herein. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be used. When a natural base is replaced with a non-natural and / or universal base, the nucleotide is said herein to comprise a modified nucleobase and / or nucleobase modification. Modified nucleobases and / or nucleobase modifications also include natural, non-natural, and universal bases containing conjugate moieties, such as the ligands described herein. Preferred conjugate moieties for conjugation to nucleobases include cationic amino groups, which can be conjugated to the nucleobases via linkers having suitable alkyl, alkenyl or amide bonds.

[0161] The oligomeric compounds described herein can also include nucleobase (often simply referred to in the art 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). 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 cytosine, 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-(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-(allylamino)uracil, 5-(aminoallyl)uracil 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-(methoxycarbonylmethyl)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 Douracil, 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 pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-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)-phenoxazin-1, ,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-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)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl (hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidin, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deazaino Indolyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl allyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stivenyl, 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 pyrimidines, N, 2 -substituted purines, N6 -substituted purines, 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- Other synthetic or natural nucleic acid bases include 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- or N-alkylated derivative thereof. Alternatively, substituted or modified analogs of any of the above-listed bases and "universal bases" can also be used.

[0162] As used herein, a universal nucleobase is capable of base pairing with all four naturally occurring nucleobases without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of the oligonucleoside duplex. Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazur, 4-methylbenzimidazur, 3-methylisocarbostyrilyl, 5-methylisocarbostyrilyl, 3-methyl-7-propynylisocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, Examples include 9-methyl-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-propynylisocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-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 include those disclosed in U.S. Pat. No. 3,687,808; those disclosed in International Application PCT / US Patent Application No. 09 / 038425, filed March 26, 2009; those disclosed in Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990; those disclosed by English et al., Angewandte Chemie, International Edition, 1991, 30,613; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, p. Ed. Wiley-VCH, 2008; and those disclosed in Sanghvi, YS, Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, the contents of all of which are incorporated herein by reference.

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

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

[0166] Certain sugars The oligomeric compounds provided herein may contain one or more monomers, such as nucleosides or nucleotides, having modified sugar moieties. For example, the furanosyl sugar ring of a nucleoside can be modified in several ways, including, but not limited to, the addition of a substituent or bridging two non-geminal ring atoms to form a locked nucleic acid or bicyclic nucleic acid. In some embodiments, the oligomeric compounds contain one or more monomers that are LNAs.

[0167] In some embodiments of the locked nucleic acid, the 2' position of the 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 -ON(R1)-, -C(R1)=C(R2)-O-, -C(R1)=N-, -C(R1)=NO-, -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 - 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, a 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 cycloaliphatic radical, substituted C5-C7 cycloaliphatic 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 is independently 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, a heterocyclic radical, a substituted heterocyclic radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.

[0168] In one embodiment, each of the linkers of the LNA compound is independently -[C(R1)(R2)] n -, -[C(R1)(R2)] n In another embodiment, each of the linkers is independently 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', 4'-CH2-ON(R1)-2', and 4'-CH2-N(R1)-O-2'-, where each R1 is independently H, a protecting group, or a C1-C12 alkyl.

[0169] Several LNAs have been prepared and described 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. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO 94 / 14226; WO 2005 / 021570; Singh ... al., J. Org. Chem., 1998, 63, 10035-10039; examples of issued U.S. patents and published applications disclosing LNAs include, for example, U.S. Pat. Nos. 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Patent Application Publication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004-0143114; and 20030082807.

[0170] Additionally, LNAs have been described in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy (4'-CH2-O-2') bond to form a bicyclic sugar moiety (discussed in 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; see also U.S. Pat. Nos. 6,268,490 and 6,670,461). The linkage can be a methylene (-CH2-) group bridging the 2' oxygen atom and the 4' carbon atom, in which case the term methyleneoxy(4'-CH2-O-2')LNA is used for the bicyclic moiety; if there is an ethylene group at this position, the term ethyleneoxy(4'-CH2CH2-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'-CH2-O-2')LNA and other bicyclic sugar analogs exhibit very high duplex thermal stability with complementary DNA and RNA (Tm = +3 to +10°C), stability against 3'-exonuclease degradation, and good solubility. Potent and non-toxic antisense oligonucleotides, including BNAs, have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0171] An isomer of methyleneoxy(4'-CH2-O-2')LNA that has also been discussed is α-L-methyleneoxy(4'-CH2-O-2')LNA, which has been shown to have excellent stability against 3'-exonucleases. α-L-methyleneoxy(4'-CH2-O-2')LNA has been incorporated into antisense gapmers and chimeras, which have shown potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0172] The synthesis and preparation of methyleneoxy (4'-CH2-O-2') 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). BNAs and their preparation are also described in WO 98 / 39352 and WO 99 / 14226.

[0173] Furthermore, analogs of methyleneoxy(4'-CH2-O-2')LNA, phosphorothioate-methyleneoxy(4'-CH2-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 duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99 / 14226). Furthermore, the synthesis of a novel conformationally restricted, 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 thermal stability of their duplexes with complementary RNA and DNA strands has been reported.

[0174] Modified sugar moieties are well known and can be used to alter, 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, bicyclic modified sugars such as methyleneoxy (4'-CH2-O-2') LNA and ethyleneoxy (4'-(CH2)2-O-2'-bridged) ENA; substituted sugars, particularly 2'-substituted sugars having a 2'-F, 2'-OCH3, or 2'-O(CH2)2-OCH3 substituent; and 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 of skill in the art. Some representative patents and publications that teach the preparation of such modified sugars include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; Nos. 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; 6,531,584; and 6,600,032; and WO 2005 / 121371.

[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(CH2CHO) nCH2CH2OR, n=1-50; "locked" nucleic acids (LNA) in which the furanose portion of the nucleoside contains a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system; O-AMINE or O-(CH2) n AMINE (n=1 to 10, AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine or polyamino); and O-CH2CH2(NCH2CH2NMe2)2.

[0176] "Deoxy" modifications include hydrogen (i.e., the deoxyribose sugar particularly associated with overhanging single strands); halo (e.g., fluoro); amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CHCHNH) n Included are CH2CH2-AMINE (AMINE = NH2; 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] The modification at the 2' position can be in the arabinose configuration. The term "arabinose configuration" refers to the placement of the substituent on C2' of the ribose in the same configuration as the 2'-OH in arabinose.

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

[0180] Furthermore, oligomeric compounds may also contain abasic sugars, i.e., sugars lacking a nucleobase at C-1' or having another chemical group in place of the nucleobase at C1'. See, for example, U.S. Pat. No. 5,998,203, the contents of which are incorporated herein by reference in their entirety. These abasic sugars may also contain modifications to one or more of the constituent sugar atoms. Oligomeric compounds may also contain one or more sugars that are L-isomers, e.g., L-nucleosides. Furthermore, modifications to the sugar group may include substitution of the 4'-O with sulfur, an optionally substituted nitrogen, or a CH2 group. In some embodiments, the linkage between C1' and the nucleobase is in the α-configuration.

[0181] Sugar modifications can also include acyclic nucleotides in which a C-C bond between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4') is absent and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is absent from the nucleotide. In some embodiments, an acyclic nucleotide is [ka] wherein B is a modified or unmodified nucleobase, R and R are independently H, halogen, OR, or alkyl; and R is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar.

[0182] In some embodiments, the sugar modification is selected from the group consisting of 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-CH2-(4'-C) (LNA), 2'-O-CH2CH2-(4'-C) (ENA), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and gem 2'-OMe / 2'F with 2'-O-Me in the arabinose configuration.

[0183] It should be understood that when a particular nucleotide is linked to the next nucleotide through its 2' position, the sugar modifications described herein can be located at the 3' position of the sugar for that particular nucleotide, e.g., a nucleotide linked to the next nucleotide through its 2' position. The 3' modification can be in the xylose configuration. The term "xylose configuration" refers to the placement of the substituent on the C3' of the ribose in the same configuration as the 3'-OH of the xylose sugar.

[0184] The hydrogen attached to C4' and / or C1' can be replaced by a straight or branched, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, and the backbone of the alkyl, alkenyl, and alkynyl can contain one or more of O, S, S(O), SO2, N(R'), C(O), N(R')C(O)O, OC(O)N(R'), CH(Z'), a 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, and Z' is OR 11 , C.O.R. 11 , CO2R 11 , [ka] , N.R. 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 R51 , SO2R 11 , SOR 11 , S.R. 11 and substituted or unsubstituted heterocyclic; R 21 and R 31 is, for each occurrence, independently hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R. 11 , CO2R 11 or NR 11 R 11 ' or R 21 and R 31 together with the atoms to which they are attached form a heterocyclic ring; R 41 and R 51 is, for each occurrence, independently hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R. 11 or CO2R 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 heterocyclic ring, which contains 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, for each occurrence, is independently an alkylmetal or transition metal with a total charge of +1; Y is O, S, or NR'; where R' is hydrogen or an optionally substituted aliphatic. This modification is preferably at the 5' end of the oligonucleotide.

[0186] In some embodiments, the LNA has the formula: [ka] and a bicyclic nucleotide having the formula: 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 to C6 alkyl, C2 to C6 alkenyl, C2 to C6 alkynyl, substituted C1 to C6 alkyl, substituted C2 to C6 alkenyl, substituted C2 to C6 alkynyl, acyl, substituted acyl, or substituted amido.

[0187] In one embodiment, each of the substituted groups is independently mono- or polysubstituted with optionally protected substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN (each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1).

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

[0189] In some embodiments, the Z group is C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN (each J, J, and J is independently H or C-C alkyl and X is O, S, or NJ). In other embodiments, the Z group is C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH—), substituted alkoxy, or azido.

[0190] In some embodiments, the Z group is -CH2Xx, where Xx is selected from 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 other embodiments, the Z group is -CH2Xx, where Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3-), or azido.

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

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

[0193] In some embodiments, each of T1 and T2 is a hydroxyl protecting group. A preferred list of hydroxyl protecting groups includes benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In some embodiments, T1 is a hydroxyl protecting group selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldimethylsilyl, and dimethoxytrityl, and a more preferred hydroxyl protecting group is T1 which is 4,4'-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 the expression: [ka] or the expression: [ka] and having 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, monomeric 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, monomeric subunit, or oligomeric compound; wherein at least one of T3 and T4 is an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; Z is C1 to C6 alkyl, C2 to C6 alkenyl, C2 to C6 alkynyl, substituted C1 to C6 alkyl, substituted C2 to C6 alkenyl, substituted C2 to C6 alkynyl, acyl, substituted acyl, or substituted amido.

[0196] In one embodiment, each of the substituted groups is independently mono- or polysubstituted with optionally protected substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN (each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1).

[0197] In one embodiment, each of the substituted groups is mono- or polysubstituted with substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, and NJ3C(=X)NJ1J2 (each J1, J2, and J3 is 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 (e.g., at least one Z is C1-C6 alkyl substituted with one or more C1-C6 alkoxy). In other embodiments, 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 CHO- (e.g., at least one Z is CHOCH-). In other embodiments, each C1-C6 alkoxy substituent is CHO- (e.g., each Z is CHOCH-).

[0201] In some embodiments, at least one substituent is halogen (e.g., at least one Z is C1-C6 alkyl substituted with one or more halogens). In some embodiments, each substituent is independently halogen (e.g., each Z is independently 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 CH2FCH2-, CHF2CH2-, or CF3CH2-). In some embodiments, each halo substituent is fluoro (e.g., each Z is CH2FCH2-, CHF2CH2-, or CF3CH2-).

[0202] In some embodiments, at least one substituent is hydroxyl (e.g., at least one Z is C1-C6 alkyl substituted with one or more hydroxyl). In some embodiments, each substituent is independently hydroxyl (e.g., each Z is independently C1-C6 alkyl substituted with one or more hydroxyl). In some embodiments, at least one Z is HOCH2-. In other embodiments, each Z is HOCH2-.

[0203] In some embodiments, at least one Z is CH3-, CH3CH2-, CH2OCH3-, CH2F-, or HOCH2-. In some embodiments, each Z is CH3-, CH3CH2-, CH2OCH3-, CH2F-, or HOCH2-.

[0204] In some embodiments, at least one Z group is C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN; each J, J, and J is independently H or C-C alkyl, and X is O, S, or NJ. In other embodiments, at least one Z group is C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO—), or azido.

[0205] In some embodiments, each Z group is independently C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN; each J, J, and J is independently H or C-C alkyl, and X is O, S, or NJ. In other embodiments, each Z group is independently C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO—), or azido.

[0206] In some embodiments, at least one Z group is -CH2Xx, where each 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 some embodiments, at least one Z group is -CH2Xx, where each Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), or azido.

[0207] In some embodiments, each Z group is independently -CH2Xx, 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, each Z group is independently -CH2Xx, where each Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), or azido.

[0208] In some embodiments, at least one Z group is CH3-. In other embodiments, each Z group is CH3-.

[0209] In some embodiments, the Z group of at least one monomer has the formula: [ka] or the expression: [ka] or the expression: [ka] It has an (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 the expression: [ka] or the expression: [ka] The (S)-configuration is 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 other embodiments, T3 is an internucleoside linking group attached to a nucleoside, nucleotide, or monomer subunit. In some embodiments, T4 is an internucleoside linking group attached to a nucleoside, nucleotide, or monomer subunit. In some embodiments, T3 is an internucleoside linking group attached to an oligonucleoside or oligonucleotide. In some embodiments, T4 is an internucleoside linking group attached to an oligonucleoside or oligonucleotide. In some embodiments, T3 is an internucleoside linking group attached to an oligomeric compound. In some embodiments, T4 is an internucleoside linking group attached to an oligomeric compound. In some embodiments, at least one of T3 and T4 is an internucleoside linking group selected from phosphodiester or phosphorothioate.

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

[0215] In some such embodiments, the LNA may include, but is not limited to: [ka] As shown in the table, examples include (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-ON(R)-2') LNA, and (E) oxyamino (4'-CH2-N(R)-O-2') LNA.

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

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

[0218] In some embodiments, the oligomeric compound, e.g., the tuning 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)). In some embodiments, the nucleotide sequence comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) nucleotide selected from:

[0219] In some embodiments, the monomer comprises a sugar mimetic. In some such embodiments, the mimetic is used in place of the sugar or sugar-internucleoside linkage combination, while maintaining the nucleobase for hybridization with a selected target. 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 bonds. In some cases, the mimetic is used in place of the nucleobase. 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 synthesizing sugar, nucleoside, nucleotide, and nucleobase mimetics are well known to those skilled in the art.

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

[0222] In some embodiments, the tunable REVERSIR compound comprises at least one PNA and at least one G-clamp nucleobase, e.g., the tunable REVERSIR compound comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more PNAs and 1, 2, 3, 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, the tunable REVERSIR compound comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more LNA monomers; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more PNAs 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 referred to as intersugar linkages. 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, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative phosphorus-containing linkages include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thiocarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Oligomeric compounds with non-phosphorus linking groups are called oligonucleosides. Compared to natural phosphodiester linkages, modified linkages can be used to alter, typically increase, the nuclease resistance of oligomeric compounds. In some embodiments, linkages with chiral atoms can be prepared as individual enantiomers or 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 can be increased resistance of the oligonucleotide to nucleolytic degradation. Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphates, phosphoramidates, alkyl or aryl phosphates, and phosphate triesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the linkage can be replaced with S, Se, BR3 (R is hydrogen, alkyl, or aryl), C (i.e., alkyl, aryl, etc.), H, NR2 (R is hydrogen, optionally substituted alkyl, or aryl), or OR (R is optionally substituted alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygen atoms with one of the above atoms or atomic groups makes the phosphorus atom chiral. In other words, the phosphorus atom in such a modified phosphate group is a stereocenter. The stereogenic phosphorus atom 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 phosphorothioates is achiral, which eliminates the formation of oligonucleotide diastereomers. Therefore, without intending to be bound by a particular theory, modifications to both non-bridging oxygen atoms may be desirable, as they eliminate the formation of chiral centers, such as phosphorothioates, and cannot produce diastereomeric mixtures. Thus, the non-bridging oxygen atoms can independently be O, S, Se, B, C, H, N, or OR (R is alkyl or aryl).

[0227] Phosphate linkers can also be modified by substitution of the bridging oxygen (i.e., the oxygen connecting the phosphate to the sugar of the monomer) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates). This substitution can be made at either or both linking oxygens. When the bridging oxygen is the 3'-oxygen of the nucleoside, substitution with carbon is preferred. When the bridging oxygen is the 5'-oxygen of the nucleoside, substitution with nitrogen is preferred.

[0228] Modified phosphate linkages in which at least one of the oxygens linked to the phosphate is replaced or the phosphate group is replaced by a non-phosphorus group are also referred to as "non-phosphodiester intersugar linkages" or "non-phosphodiester linkers."

[0229] In some embodiments, the phosphate group can be replaced by a non-phosphorus-containing connector, such as a dephospho linker. Dephospho linkers 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 for nucleic acid degradation, its replacement with 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 a modification that replaces the charged phosphate group with a neutral moiety.

[0230] Examples of moieties that can replace the phosphate group include, but are not limited to, amide (e.g., amide-3(3'-CH2-C(=O)-N(H)-5') and amide-(3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylic acid ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3'-O-CH2-O-5'), oxime, methyl Nonionic bonds containing mixed N, O, S, and CH moieties, such as phenylimino, methylenecarbonylamino, methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ether (C3'-O-C5'), thioether (C3'-S-C5'), thioacetamide (C3'-N(H)-C(=O)-CH2-S-5', C3'-OP(O)-O-SS-C5', C3'-CH2-NH-NH-C5', 3'-NHP(O)(OCH3)-O-5', and 3'-NHP(O)(OCH3)-O-5') and mixed N, O, S, and CH2 moieties, e.g., Carbohydrate See Modifications in Antisense Research; YS Sanghvi and PDCook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65). Preferred embodiments include methylenemethylimino (MMI), methylenecarbonylamino, amide, carbamate, and ethylene oxide linkers.

[0231] Those skilled in the art will appreciate that, in some cases, substitution of a non-bridging oxygen atom may result in enhanced cleavage of the intersugar bond by the adjacent 2'-OH, and therefore in many cases, modification of the non-bridging oxygen atom may require modification of the 2'-OH, e.g., a modification that does not participate in cleavage of the adjacent intersugar bond, e.g., arabinose sugars, 2'-O-alkyl, 2'-F, LNA, and ENA.

[0232] Preferred non-phosphodiester intersugar linkages include phosphorothioates, phosphorothioates containing at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more enantiomeric excess of the Sp isomer, phosphorothioates containing at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more enantiomeric excess of the Rp isomer, phosphorodithioates, phosphotriesters, aminoalkylphosphotrioesters, alkyl-phosphonates (e.g., methyl-phosphonates), selenophosphates, phosphoramidates (e.g., N-alkylphosphoramidates), and boranophosphonates.

[0233] In some embodiments, the oligomeric compound, e.g., the regulating REVERSIR compound or siRNA, comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more and inclusive) modified or non-phosphodiester bond. In one embodiment, the oligomeric compound, e.g., the regulating REVERSIR compound or siRNA, comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more and inclusive) phosphorothioate bond.

[0234] In some embodiments, all internucleotide linkages in a reverser compound are phosphorothioate (PS) internucleotide linkages. In some embodiments, a regulating REVERSIR compound includes at least one phosphorothioate (PS) internucleotide linkage, but not all internucleotide linkages in the regulating 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, the modulating REVERSIR compound comprises at least one phosphorothioate internucleotide linkage and at least one internucleoside or internucleotide linkage that is not phosphorothioate. For example, the modulating REVERSIR compound comprises at least one phosphorothioate internucleotide linkage and at least one phosphodiester internucleotide linkage. In some embodiments, the non-phosphorothioate internucleotide linkage is between the terminal and penultimate nucleotide.

[0236] In some embodiments, the internucleotide linkage between the 3'-terminal nucleobase of the coordinating REVERSIR compound and the remaining coordinating REVERSIR compounds is a phosphodiester linkage, hi some embodiments, all internucleotide linkages in the reverser compound are phosphorothioate, except for the internucleotide linkage between the 3'-terminal nucleoside of the coordinating REVERSIR compound and the remaining coordinating REVERSIR compounds.

[0237] Oligomeric compounds can also be constructed in which the phosphate linker and sugar are replaced by nuclease-resistant nucleosides, nucleotides, or nucleotide surrogates. Without intending to be bound by any particular theory, it is believed that a repeatedly charged backbone weakens binding to proteins that recognize polyanions (e.g., nucleases). Again, without intending to be bound by any particular theory, in some embodiments, it may be desirable to introduce modifications in which the bases are 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 and thus give rise to enantiomers, diastereomers, and other configurations, which may be defined with respect to absolute stereochemistry as (R) or (S), such as in the case of sugar anomers, or (D) or (L), such as in the case of amino acids. The antisense compounds described herein include all such possible isomers as well as their racemic and optionally pure forms.

[0239] terminal modification The termini of the oligomeric compound can be modified. Such modifications are possible at one or both termini. For example, the 3' and / or 5' termini of the oligonucleotide can be conjugated to a labeling moiety, such as a fluorophore (e.g., pyrene, TAMRA, fluorescein, Cy3, or Cy5 dye) or other functional molecular entity, such as a protecting group (e.g., sulfur, silicon, boron, or ester-based). The functional molecular entity can be attached to the sugar via a phosphate group and / or a linker. The terminal atom of the linker can be linked to or replace the linking 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 linked to or replace the terminal atom of a nucleotide surrogate (e.g., PNA).

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

[0241] Terminal modifications useful for modulating activity include modification of the 5' end of an oligomeric compound with phosphate or a phosphate analog. In some embodiments, the 5' end of an oligomeric compound is phosphorylated or comprises a phosphoryl analog. Exemplary 5' phosphate modifications include those that are compatible with RISC-mediated gene silencing. 5' end modifications may also be useful for stimulating or inhibiting a subject's immune system. In some embodiments, the 5' end of an oligomeric compound is modified [ka] wherein W, X, and Y are each independently selected from O, OR (wherein R is hydrogen, alkyl, or aryl), S, Se, BR3 (wherein R is hydrogen, alkyl, or aryl), BH3 - , C (i.e., alkyl, aryl, etc.), H, NR (R is hydrogen, alkyl, aryl), or OR (R is hydrogen, alkyl, or aryl); A and Z, for each occurrence, are each independently absent, O, S, CH, NR (R is hydrogen, alkyl, aryl), or optionally substituted alkylene, where the alkylene backbone can contain internal and / or terminal O, S, SS, and NR (R is hydrogen, alkyl, aryl); and n is 0-2. 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 5-8 membered heterocyclic compound, where W and Y are each independently O, S, NR', or alkylene. The heterocyclic ring is preferably substituted with aryl or heteroaryl. In some embodiments, one or both hydrogens on the 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)pO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)PO-5'), 5'-phosphorothiolate ((HO)2(O)PS-5'); 5'-alpha-thiotriphosphate; 5'-beta-thiotriphosphate; 5'-gamma-thiotriphosphate; 5'-phosphoramidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'). Other 5'-modifications include 5'-alkyl phosphates (R(OH)(O)PO-5', R = alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-alkyl ether phosphonates (R(OH)(O)PO-5', R = alkyl ether, e.g., methoxymethyl (CHOMe), ethoxymethyl, etc.). Other exemplary 5'-modifications include those in which Z is an alkyl optionally substituted at least once, e.g., ((HO)(X)PO[-(CH) a -OP(X)(OH)-O] b -5', ((HO)2(X)PO[-(CH2) a -P(X)(OH)-O] b -5', ((HO)2(X)P-[-(CH2) a -OP(X)(OH)-O] b -5'; Dialkyl-terminated phosphates and phosphate mimetics; HO[-(CH2) a -OP(X)(OH)-O] b -5', H2N[-(CH2) a -OP(X)(OH)-O] b -5', H[-(CH2) a -OP(X)(OH)-O] b -5', Me2N[-(CH2) a -OP(X)(OH)-O] b -5', HO[-(CH2) a -P(X)(OH)-O] b-5', H2N[-(CH2) a -P(X)(OH)-O] b -5', H[-(CH2) a -P(X)(OH)-O] b -5', Me2N[-(CH2) a -P(X)(OH)-O] b -5', where a and b are each independently 1 to 10. Other embodiments include BH3, BH3 - and / or containing substitution of oxygen and / or sulfur by Se.

[0243] Terminal modifications can also be useful for monitoring distribution; in such cases, preferred groups to add include fluorophores, such as fluorescein, or Alexa dyes, such as Alexa 488. Terminal modifications can also be useful for enhancing uptake; useful modifications for this purpose include targeting ligands. Terminal modifications can also be useful for crosslinking the oligonucleotide to another moiety; useful modifications for this purpose include mitomycin C, psoralens, and their derivatives.

[0244] oligomeric compounds In some embodiments, provided herein are oligomeric compounds having reactive phosphorus groups useful for forming linkages such as, for example, phosphodiester and phosphorothioate internucleotide linkages. Methods for preparing and / or purifying precursors or oligomeric compounds are not intended to limit the compositions or methods described herein. Methods for synthesizing and purifying oligomeric compounds, such as DNA, RNA, oligonucleotides, oligonucleosides, and antisense compounds, are well known to those skilled in the art.

[0245] Generally, oligomeric compounds comprise multiple monomer 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 siRNAs. Thus, these compounds can be introduced in the form of single-stranded, double-stranded, circular, branched, or hairpin structures, and they can contain structural elements such as internal or terminal bulges or loops. Oligomeric double-stranded compounds can be either double-stranded, capable of hybridizing to form a double-stranded compound, or single-stranded, with sufficient self-complementarity to allow hybridization and the formation of a complete or partial double-stranded compound.

[0246] In some embodiments, the present invention provides chimeric oligomeric compounds. In some such embodiments, the chimeric oligomeric compounds are chimeric oligonucleotides. In some such embodiments, the chimeric oligonucleotides comprise various modified nucleotides. In some embodiments, the chimeric oligonucleotides are mixed backbone antisense oligonucleotides.

[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 mimicking groups can comprise a chimeric oligomeric compound described herein.

[0248] In some embodiments, chimeric oligomeric compounds typically contain at least one region that has been modified to confer increased resistance to nuclease degradation, enhanced cellular uptake, and / or increased binding affinity for target nucleic acids, hi some embodiments, another region of the oligomeric compound may serve as a substrate for enzymes 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 in the gap. In some such embodiments, the mixed backbone oligonucleotide has phosphodiester linkages in the wings and phosphorothioate linkages in the gap. In some embodiments where the internucleotide linkages in the wings are different from the internucleotide linkages in the gap, the internucleotide linkages bridging the wing and the gap are the same as the internucleotide linkages in the wing. In some embodiments where the internucleotide linkages in the wing are different from the internucleotide linkages in the gap, the internucleotide linkages bridging the wing and the gap are the same as the internucleotide linkages in the gap.

[0250] In some embodiments, the present invention provides an oligomeric compound comprising siRNAs of any length within various length ranges and a regulatory REVERSIR compound. In some embodiments, the present invention provides an oligomeric compound composed of X-Y linked oligonucleotides, where X and Y are each independently selected from 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; provided that X < Y. For example, in some embodiments, the present application relates to 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 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 5, 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, Oligomeric compounds are provided, each comprising 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, 27-28, 27-29, 27-30, 28-29, 28-30, or 29-30 linked nucleotides.

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

[0252] The inventors have found that, among others, regulating REVERSIR compounds, i.e., modified oligonucleotides, composed of 15 or fewer nucleotides are particularly effective in reversing siRNA activity. Thus, in some embodiments, the regulating REVERSIR compound is a modified oligonucleotide composed of 8-15 (e.g., 8, 9, 10, 11, 12, 13, 14, or 15) linked nucleotides. In some embodiments, the regulating REVERSIR compound is a modified oligonucleotide composed of 6-12, 7-11, or 8-10 linked nucleobases. In some embodiments, the regulating REVERSIR compound is a modified oligonucleotide composed of 8-9 linked nucleobases.

[0253] As described herein, a regulating REVERSIR compound is a modified oligonucleotide that is substantially complementary to at least one strand of an siRNA. While not intending to be bound by theory, regulating REVERSIR compounds that are substantially complementary to the seed region of the antisense strand of an siRNA (i.e., positions 2-8 of the 5' end of the antisense strand) are particularly effective in reducing siRNA activity. Thus, in many embodiments, a regulating REVERSIR compound is substantially complementary to nucleotides 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, or 2-16 of the antisense strand of an siRNA. In this context, substantially complementary means at least 90%, preferably at least 95%, and more preferably complete complementarity.

[0254] Ligand In some embodiments, oligomeric compounds are modified by the covalent attachment of one or more conjugate groups. Generally, the conjugate group modifies one or more properties of the attached oligomeric compound, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and clearance. Conjugate groups are commonly used in the chemical arts and are linked to parent compounds, such as oligomeric compounds, directly or via an optional linking moiety or linking 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, folate, lipids, phospholipids, biotin, 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. NY 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, such as 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, such as di-hexadecyl-lacto-glycerol or triethylammonium-1,2-di-O-hexadecyl-lacto-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantaneacetic 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] Generally, a wide variety of entities, e.g., 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-glycolide) copolymer, 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-ethylacrylic acid), N-isopropylacrylamide polymer, polyphosphazine, polyethyleneimine, cationic groups, spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, and the like of polyamines. Quaternary salts, thyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucins, glycosylated polyamino acids, transferrin, bisphosphonates, polyglutamates, polyaspartates, aptamers, asialofetuin, hyaluronan, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, sugar-albumin conjugates, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralen, mitogens), mycin C), porphyrins (e.g., TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., steroids, bile acids, cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine), peptides (e.g., α-helical peptides, amphipathic peptides, RGD peptides, cell-penetrating peptides, endosomolytic / fusogenic peptides), alkylating agents, phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., naproxen, aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitro Phenylephrine, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, polyvalent carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, such as folic acid, B12, riboflavin, biotin, and pyridoxal), vitamin cofactors, lipopolysaccharides, activators of p38 MAP kinase, activators of NF-κB, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, 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-penetrating agents (e.g., spiral cell-penetrating agents).

[0257] Peptide and peptidomimetic ligands include natural or modified peptides, such as D- or L-peptides; α, β, or γ peptides; N-methylpeptides; azapeptides; peptides with one or more amide, i.e., peptides, one or more urea, thiourea, carbamate, or sulfonylurea bonds substituted; or cyclic peptides. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. Peptide or peptidomimetic ligands can be about 5 to 50 amino acids in length, e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0258] Exemplary amphipathic peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, hagfish intestinal antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaseptin, melittin, pleurocidin, H2A peptide, Xenopus peptide, esculentinis-1, and caerin.

[0259] As used herein, "endosomotropic ligand" refers to a molecule having endosomal properties. Endosomotropic ligands promote the dissolution of the compositions of the present invention or their components and / or the transport of the compositions of the present invention or their components from endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other intracellular endoplasmic reticulum to the cytoplasm of the cell. Some exemplary endosomotropic ligands include, but are not limited to, imidazoles, poly- or oligoimidazoles, linear or branched polyethyleneimines (PEI), linear and branched polyamines, such as spermine, cationic linear or branched polyamines, polycarboxylates, polycations, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, linear or branched polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges, polyanionic peptides, peptidomimetics, pH-sensitive peptides, natural and synthetic fusogenic lipids, and natural and synthetic cationic lipids.

[0260] Exemplary endosomal / fusogenic peptides include, but are not limited to, the following: AALEALAEALEALAEALEALAEAAAAGGC (GALA) (SEQ ID NO: 1); AALEALAEALAEALAEALAEALAEALAAAAGGC (EALA) (SEQ ID NO: 2); ALEALEALEALAEA (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-INF3) (SEQ ID NO: 9); GLF EAI EGFI ENGW EGnI DG K GLF EAI EGFI ENGW EGnI DG (INF-5, where 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); H5WYG (SEQ ID NO: 17); and CHK6HC (SEQ ID NO: 18).

[0261] Without intending to be bound by any particular theory, fusogenic lipids fuse with membranes, thereby destabilizing them. Fusogenic lipids typically 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; 2008 / 0287628; 2008 / 0281044; 2008 / 0281041; 2008 / 0269450; 2007 / 0105804; 20070036865; and 2004 / 0198687, the contents of which are incorporated herein by reference in their entireties.

[0263] Exemplary cell-penetrating peptides include, but are not limited to, the following: 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 (amphipathic model peptide) (SEQ ID NO: 24); RRRRRRRRR (Arg9) (SEQ ID NO: 25); KFFKFFKFFK (bacterial cell-penetrating peptide) (SEQ ID NO: 26); LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNL VPRTES(LL-37) (SEQ ID NO: 27); SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin P1) (SEQ ID NO: 28); ACYCRIPACIAGERRYGTCIYQGRLWAFCC (α-defensin) (SEQ ID 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; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino); aminoalkoxy, e.g., O(CH) nAMINE (e.g., AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino); amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); and NH(CH2CH2NH) n Protonated amino groups derived from CH2CH2-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino) are included.

[0265] As used herein, the term "targeting ligand" refers to any molecule that confers increased affinity to a selected target, such as a cell, cell type, tissue, organ, body region, or compartment, e.g., a cell, tissue, or organ compartment. 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, multivalent galactose, N-acetyl-D-galactose (GalNAc), multivalent GalNAc, such as GalNAc2 and GalNAc3; D-mannose, multivalent mannose, multivalent lactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent fucose, glycosylated polyamino acids and lectins.The term multivalent indicates the presence of two or more monosaccharide units.These monosaccharide subunits can be linked to each other or to a scaffold molecule via glycosidic bonds.

[0267] As ligands, several folates and folate analogs suitable for the present invention are described in U.S. Patent Nos. 2,816,110; 5,1410,104; 5,552,545; 6,335,434 and 7,128,893, the contents of which are incorporated herein by reference in their entireties.

[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 present 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 transthyretin-binding ligands (e.g., tetrahydrothyroacetic acid, 2,4,6-trimethylolpropane, and flufenamic acid). Oligomeric compounds containing several phosphorothioate intersugar linkages are also known to bind to serum proteins. Therefore, short oligomeric compounds, e.g., oligonucleotides containing about 5-30 nucleotides (e.g., 5-25 nucleotides, preferably 5-20 nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides) and containing multiple phosphorothioate linkages in the backbone, are also suitable as ligands (e.g., PK-modulating ligands) for the present invention. PK-modulating oligonucleotides contain 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 to serum components (e.g., serum proteins) are also suitable as PK-modulating ligands for the present invention. 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] When there are two or more ligands, the ligands can all have the same properties, or all have different properties, or some ligands can have the same properties and the rest have different properties.For example, the ligands can have targeting properties, endosomolytic activity, or PK regulation properties.In a preferred embodiment, the ligands all have different properties.

[0270] In some embodiments, the ligand of one strand of a double-stranded oligomeric compound has 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 one oligomeric chain, the point of attachment of the oligomeric compound can be an atom of the ligand itself or an atom of the carrier molecule to which the ligand itself is attached.

[0271] Ligands can be attached to oligomeric compounds at various locations, such as the 3'-terminus, 5'-terminus, and / or internal positions. When two or more ligands are present, the ligands can be at both termini of the oligomeric compound. In preferred embodiments, the ligand is attached to the oligomeric compound via an intervening tether / linker. The ligand or tethered ligand can be present on a monomer as it is incorporated into a growing chain. In some embodiments, a ligand can also be incorporated into a "precursor" monomer by coupling after the monomer is incorporated into the growing chain. For example, an amino-terminated tethered (i.e., unliganded) monomer, such as monomer-linker-NH, can be incorporated into a growing oligomeric compound chain. Subsequent to incorporation of the precursor monomer into the chain, a ligand bearing an electrophilic group, such as a pentafluorophenyl ester or a ligand bearing an aldehyde group, can be attached to the tether of the precursor monomer by coupling the electrophilic group of the ligand with the terminal electrophilic group of the precursor monomer.

[0272] In another example, a ligand bearing a chemical group suitable for participating 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 bearing a complementary chemical group, such as an alkyne or azide, can be attached to the precursor monomer by coupling with the alkyne and 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 comprises a ligand conjugated to the sense strand. In other embodiments, the siRNA comprises a ligand conjugated to the antisense strand.

[0274] In some embodiments, the ligand can be conjugated to the nucleobase, sugar moiety, or internucleoside bond of the oligomeric compound. Conjugation to the purine nucleobase or its derivative can be performed at any position, such as the endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is linked to the conjugate moiety. Conjugation to the pyrimidine nucleobase or its derivative can also be performed at any position. In some embodiments, the 2-, 5-, and 6-position of the pyrimidine nucleobase can be replaced with the conjugate moiety. When conjugating the ligand to the nucleobase, the preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bond interaction required for base pairing.

[0275] Conjugation of a nucleoside to a sugar moiety can occur at any carbon atom. Exemplary carbon atoms of the sugar moiety that can be attached to a conjugate moiety include the 2', 3', and 5' carbon atoms. The 1' position can also be attached to a conjugate moiety, such as an abasic residue. An internucleoside linkage can also carry a conjugate moiety. In the case of phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidite, etc.), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. In the case of amine- or amide-containing internucleoside linkages (e.g., 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 that, among other things, modulating REVERSIR compounds conjugated to a ligand are particularly effective in reducing the activity of siRNA. Without intending to be bound by any particular theory, the ligand can increase or enhance the potency of the modulating REVERSIR compound by delivering the modulating REVERSIR compound to the desired site of action. Thus, in some embodiments, the modulating REVERSIR compound is conjugated to a ligand.

[0277] Ligands conjugated to a regulating REVERSIR compound, while useful for delivering the regulating REVERSIR compound to a desired site of action, can negatively affect the ability of the regulating REVERSIR compound to reduce siRNA activity. Thus, in some embodiments, the link between the ligand and the regulating REVERSIR compound can be designed to undergo cleavage after the regulating REVERSIR compound reaches a desired site of action. This can be achieved in several ways. For example, the linker connecting the regulating REVERSIR compound to the ligand can be a cleavable linker.

[0278] The inventors have further found that the nucleotide in the ligand-linked modulating REVERSIR compound can affect the ability of the modulating REVERSIR compound to reduce the activity of siRNA. The inventors have found that ligand-conjugated nucleotides containing deoxy sugars (e.g., 2'-deoxyribose) are particularly effective in enhancing the ability of the modulating REVERSIR compound to reduce siRNA activity. Thus, in some embodiments, the nucleotide conjugated to the ligand contains a deoxy sugar, for example, a 2'-deoxy sugar.

[0279] In some embodiments of the various aspects disclosed herein, the ligand is attached to the 3'-terminal nucleotide of the coordinating REVERSIR compound. The inventors have found that, among other things, the internucleotide bond between the ligand-conjugated nucleotide and the remaining coordinating REVERSIR compound can also affect the ability of the coordinating REVERSIR compound to reduce siRNA activity. Without intending to be bound by any particular theory, it has been found that a readily cleavable internucleotide bond is particularly effective in enhancing the ability of the coordinating REVERSIR compound to reduce siRNA activity. Thus, in some embodiments, the ligand-conjugated nucleotide is attached to the remaining coordinating 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 that is linked to the remainder of the coordinating REVERSIR compound via a cleavable internucleotide bond, which in some other embodiments is a phosphodiester internucleotide bond.

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

[0282] In some embodiments, the ligand is conjugated to the 3' terminal nucleotide of the modulating REVERSIR compound.

[0283] In some embodiments, the ligand is conjugated to the 5' end of the coordinating REVERSIR compound. In some embodiments, a first ligand is conjugated to the 5' end of the coordinating 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 (e.g., OH, SH, amine, carboxyl, aldehyde, etc.) of 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, the electrophilic group can be a carbonyl-containing functional group, and the nucleophilic group can be an amine or thiol. Methods for conjugating nucleic acids and related oligomeric compounds, with or without linking groups, are well described in the literature, for example, Manoharan in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, which is incorporated herein by reference in its entirety.

[0286] Representative United States patents that teach the preparation of oligomeric compounds, e.g., conjugates of oligonucleotides, include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; and 5,109,124. specification; Specification No. 7,044; Specification No. 4,605,735; Specification No. 4,667,025; Specification No. 4,762,779; Specification No. 4, Specification No. 789,737; Specification No. 4,824,941; Specification No. 4,835,263; Specification No. 4,876,335; Specification No. 4,876,335; Specification No. 4,904,582; Specification No. 4,958,013; Specification No. 5,082,830; Specification No. 5,112,963 ; Specification No. 5,214,136; Specification No. 5,082,830; Specification No. 5,112,963; Specification No. 5,149,782 Specifications; Specification No. 5,214,136; Specification No. 5,245,022; Specification No. 5,254,469; Specification No. 5,258,506 No. 5,262,536; No. 5,272,250; No. 5,292,873; No. 5,317,0 Specification No. 98; Specification No. 5,371,241, Specification No. 5,391,723; Specification No. 5,416,203, Specification No. 5,451,463; Specification No. 5,510,475; Specification No. 5,512,667; Specification No. 5,514,785; Specification No. 5, Specification No. 565,552; Specification No. 5,567,810; Specification No. 5,574,142; Specification No. 5,585,481; 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;Nos. 5,599,928; 5,672,662; 5,688,941; 5,714,166; 6,153,737; 6,172,208; 6,300,319; 6,335,434; 6,335,437; 6,395,437; 6,444,806; 6,486,308; 6,525,031; 6,528,631; and 6,559,279, the contents of which are incorporated herein by reference in their entirety.

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

[0288] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the formula (II), (III), (IV), or (V): [ka] and a ligand having the structure shown in where: q 2A , q 2B , q 3A , q 3B , q 4A , q 4B , q 5A , q 5B and q 5Cis, independently for each occurrence, 0 to 20, and the repeat units can be the same or different; Q and Q' are, for each occurrence, independently: non-existence, -(p 7 -Q 7 -R 7 ) p -T 7 -or-T 7 -Q 7 -T 7’ -BT 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’ is, independently for each occurrence, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; B is -CH2-N(B L )-CH2-; 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 QB is, independently for each occurrence, absent, alkylene, substituted alkylene, and one or more methylenes are O, S, S(O), SO, N(R N ), C(R')=C(R'), C≡C, or C(O); T B and T B’ is, independently for each occurrence, absent, CO, NH, O, S, OC(O), OC(O)O, NHC(O), NHC(O), NHC(O)NH, NHC(O)O, CH, CHNH, or CHO; R X may contain lipophilic substances (e.g., cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanoldiol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl or is phenoxazine), vitamins (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal), peptides, carbohydrates (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, polysaccharides), endosomal components, steroids (e.g., uvaol, hesigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friedelin, epifriedelanol-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 is, independently for each occurrence, absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-, NH-, CO, CH=NO, [ka] or heterocyclyl; L 1 , L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C is, each independently for each occurrence, a carbohydrate, e.g., a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, and a polysaccharide; R' and R'' are each independently H, C 1 ~C6 alkyl, OH, SH or N(R N )2; 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 for each occurrence O or S; p is, independently for each occurrence, 0 to 20.

[0289] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0290] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0291] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0292] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0293] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0294] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0295] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0296] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0297] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0298] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0299] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0300] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

[0301] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0302] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

[0303] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

[0304] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

[0305] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

[0306] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

[0307] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

[0308] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0309] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0310] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0311] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0312] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0313] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0314] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0315] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0316] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The ligands include:

[0317] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

[0318] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

[0319] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

[0320] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

[0321] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

[0322] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

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

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

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

[0326] In some embodiments, L 4A and L 4B are both 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, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

[0334] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

[0335] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

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

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

[0338] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

[0339] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative 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, regulating REVERSIR compounds and siRNAs, [ka] [ka] The monomer comprises a monomer selected from the group consisting of:

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

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

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

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

[0345] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

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

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

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

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

[0350] In some embodiments, oligomeric compounds described herein, including but not limited to, regulative REVERSIR compounds and siRNAs, have the structure: [ka] The monomers include:

[0351] In the foregoing monomers, X and Y, for each occurrence, are each independently H, a protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, -P(Z')(Z")O-nucleotide, -P(Z')(Z")O-nucleotide, a lipid, PEG, a steroid, a polymer, a nucleotide, a nucleotide, or an oligonucleotide; Z' and Z" are each independently O or S for each occurrence.

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

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

[0354] In some embodiments, the modulating REVERSIR compound has the structure: [ka] wherein each n is independently 1 to 20.

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

[0356] The 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, bifunctional linking moieties are hydrocarbon moieties having two functional groups. One functional group is selected to attach to a parent molecule or compound of interest, and the other is selected to essentially attach to any selected group, 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 linking 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 linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturation (e.g., double or triple bonds), and the like. Some non-limiting examples of bifunctional linking 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, with a non-limiting list of preferred substituents including hydroxyl, 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" means an organic moiety that connects two parts of a compound. A linker is typically a direct bond or an atom such as oxygen or sulfur, NR 1, C(O), C(O)NH, SO, SO2, SO2NH, or a unit such as 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, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkyl and alkylaryl, alkenylaryl, alkynylaryl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, and the like, wherein one or more methylenes are selected from O, S, S(O), SO, N(R 1 )2, C(O), a cleavable bond, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, wherein 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-, wherein P, R, T, P', R', and T are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, CHO; NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CH=NO, [ka] or heterocyclyl; Q" and Q"' are each independently for each occurrence absent, -(CH2) n -, -C(R 1 )(R 2 )(CH2) n -, -(CH2) n C(R 1 )(R 2 )-, -(CH2CH2O) m CH2CH2- or -(CH2CH2O) m CH2CH2NH-, and X is absent or a cleavable linking group; R a is H or an amino acid side chain; R 1 and R 2 is, independently for each occurrence, H, CH, OH, SH, or N(R N )2; R N is independently for each occurrence H, methyl, ethyl, propyl, isopropyl, butyl, or benzyl; q, q', and q'' are each independently for each occurrence 0 to 20, wherein the repeating units may be the same or different; n is, independently for each occurrence, 1 to 20; m is, independently for each occurrence, 0 to 50.

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

[0362] In some embodiments, the linker is a branched linker. The branch point of a branched linker can be at least trivalent, but can also be a tetravalent, pentavalent, or hexavalent atom or group exhibiting such multiple valencies. In certain embodiments, the branch point is -N, -N(Q)-C, -OC, -SC, -SS-C, -C(O)N(Q)-C, -OC(O)N(Q)-C, -N(Q)C(O)-C, or -N(Q)C(O)OC; where Q is, for each occurrence, independently H or an optionally substituted 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 a cell, but upon entering a target cell, is cleaved to release the two moieties held together by the linker. In preferred embodiments, the cleavable linking group is cleaved at least 10 times faster, and preferably at least 100 times faster, inside the target cell or under first reference conditions (which can, e.g., be selected to mimic or represent conditions inside the cell) than in the subject's blood or serum or under second reference conditions (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

[0364] Cleavable linking groups are sensitive to cleaving agents, such as pH, redox potential, or the presence of degradative molecules. Generally, cleaving agents are more prevalent or found at higher levels or activity inside cells than in serum or blood. Examples of such degradative agents include: redox agents that are selective for specific substrates or that lack substrate specificity, such as oxidizing or reducing enzymes or reducing agents present in cells, such as mercaptans, that can degrade redox-cleavable linking groups by reduction; esterases; amidases; agents that can create endosomes or acidic environments, such as those that result in a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which may be substrate-specific), and proteases, as well as phosphatases.

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

[0366] When targeting cell types that are rich in peptidases, such as hepatocytes and synoviocytes, linkers containing peptide bonds can be used.

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

[0368] Exemplary cleavable linking groups include, but are not limited to, redox cleavable linking groups (e.g., -SS and -C(R)2-SS-, where R is H or C1-C6 alkyl and at least one R is C1-C6 alkyl, e.g., CH3 or CH2CH3); phosphate-based cleavable linking groups (e.g., -OP(O)(OR)-O-, -OP(S)(OR)-O-, -OP(S)(SR)-O-, -SP(O)(OR)-O-, -OP(O)(OR)-S-, -SP(O)(OR)-S-, -OP(S)(ORk)-S-, -SP(S)(OR)-O-, -OP(O)(R)-O-, -OP(S)(R)-O-, -SP(O)(R)-O-, -SP(S)(R)-O-, -SP(O)(R)-S-, -OP(S)(R)-S-, -OP(O) (OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(O -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S- and -OP(S)(H)-S-, where R is an optionally substituted straight or branched C1-C 10 alkyl); 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 enzymes such as intracellular peptidases and proteases, e.g., -NHCHR A C(O)NHCHR B C(O)-, where R A and R B is an R group composed of two adjacent amino acids. Peptide-based cleavable linking groups comprise 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 is cleaved in an acidic environment having a pH of about 6.5 or less (e.g., about 6.-, 5.5, 5.0, or less) or by an agent such as an enzyme that can act as a general acid.

[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 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of 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. One of skill in the art can use any of the oligonucleotide chemical modifications or alterations described herein in the oligonucleotide linker. In some embodiments, the linker is dA.

[0371] motif The present invention also 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 that contains two or more chemically distinct regions, each region being composed of at least one monomeric unit, i.e., in the case of oligonucleotides, modified or unmodified nucleotides. Chimeric oligomeric compounds can be described as having a particular motif. In some embodiments, motifs include, but are not limited to, alternating motifs, gap motifs, hemimeric motifs, uniformly fully modified motifs, and positionally modified motifs. As used herein, the phrase "chemically distinct region" refers to an oligomeric region that differs from other regions in that it contains a modification not present elsewhere in the oligomeric compound or in that it lacks a modification present elsewhere in the oligomeric compound. An oligomeric compound may contain two or more chemically distinct regions. As used herein, a region that does not contain a modification is also considered chemically distinct.

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

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

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

[0375] In some embodiments, the first strand has an alternating modification pattern in which alternating nucleotides contain 2'-modifications, such as 2'-O-methyl modifications. In some embodiments, the first strand contains alternating 2'-O-methyl modifications, and the second strand contains 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 can be at complementary positions in the double-helical region. Alternatively, such 2'-modified nucleotides are not at complementary positions in the double-helical 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 in length.

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

[0379] As used herein, the term "alternating motif" refers to an oligomeric compound comprising a continuous sequence of linked monomeric subunits having two different types of sugar groups that alternate throughout substantially the entire sequence of the oligomeric compound. Oligomeric compounds having an alternating motif can be represented by the formula: 5'-A(-LBLA)n(-LB)nn-3', where A and B are monomeric subunits having different sugar groups, each L is an internucleoside linking group, n is from about 4 to about 12, and nn is 0 or 1. This allows for alternating oligomeric compounds having lengths of from about 9 to about 26 monomeric subunits. This length range is not intended to be 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 the modification of the nucleoside and includes modified and unmodified nucleosides. Thus, unless otherwise specified, a "nucleoside having a first type of modification" can be an unmodified nucleoside.

[0381] As used herein, a "type region" refers to a portion of an oligomeric compound, where the nucleosides and internucleoside linkages within said region contain the same type of modification; and the nucleosides and / or internucleoside linkages of any adjacent portion contain at least one different type of modification. As used herein, the term "homogeneously fully modified motif" refers to an oligonucleotide comprising a contiguous sequence of linked monomer subunits, each having the same type of sugar group. In one embodiment, a homogeneously 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 sequence of monomeric subunits with one type of sugar group located at the 5' or 3' end, where the remaining monomeric subunits have a different type of sugar group. Generally, a hemimer is an oligomeric compound of homogeneous sugar groups, but further comprising a short region (1, 2, 3, 4, or about 5 monomeric subunits) with different sugar groups located at either the 3' or 5' end of the oligomeric compound. In one embodiment, a hemimeric motif comprises a contiguous sequence of about 10 to about 28 monomeric subunits of one type and 1 to 5 or 2 to 5 monomeric subunits of a second type located at one end. In one embodiment, a hemimer is a contiguous sequence of about 8 to about 20 β-D-2'-deoxyribonucleosides with 1 to 12 contiguous nucleosides of the invention located at one end. In one embodiment, a hemimer is a contiguous sequence of about 8 to about 20 β-D-2'-deoxyribonucleosides, with one to five consecutive nucleosides of the invention at one terminus. In one embodiment, a hemimer is a contiguous sequence of about 12 to about 18 β-D-2'-deoxyribonucleosides, with one to three consecutive nucleosides of the invention at one terminus. In one embodiment, a hemimer is a contiguous sequence of about 10 to about 14 β-D-2'-deoxyribonucleosides, with one to three consecutive nucleosides of the invention at one terminus.

[0383] As used herein, the term "blockmir motif" refers to an oligonucleotide comprising a contiguous sequence of monomer subunits in which the sugar groups of each monomer subunit are the same except for intervening internal blocks of consecutive monomer subunits having different types of sugar groups. Blockmirs overlap somewhat in definition with gapmers, but typically, in blockmirs, only the monomer subunits within the blocks have unnatural sugar groups, whereas in gapmers, only the monomer subunits within the exterior regions have unnatural sugar groups, with the remaining monomer subunits within the blockmer or gapmer being β-D-2'-deoxyribonucleosides or β-D-ribonucleosides. In one embodiment, provided herein are blockmer oligonucleotides in which all of the monomer subunits contain unnatural sugar groups.

[0384] As used herein, the term "positionally modified motif" refers to a contiguous sequence of monomer subunits having one type of sugar group interrupted by two or more regions consisting of one to about five consecutive monomer subunits having another type of sugar group. Each of the two or more regions consisting of one to about five consecutive monomer subunits is independently homogeneously modified 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, a positionally modified oligonucleotide of the present invention is provided, comprising a sequence of 8 to 20 β-D-2'-deoxyribonucleosides, further comprising two or three regions consisting of two to about five consecutive nucleosides. Positionally modified oligonucleotides are distinguished from gap motifs, hemimer motifs, blockmir motifs, and alternating motifs, in that the substitution pattern in the region characterized by any one of the positional motifs does not conform to the definitions set forth herein for one of these other 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 having two exterior regions or wings and one interior region or gap. The three regions form a continuous array of monomer subunits with sugar groups in the exterior regions that are different from the sugar groups in the interior regions, where the sugar groups of each monomer subunit within a particular region are the same. When the sugar groups in the exterior regions are the same, the gapmer is a symmetric gapmer; when the sugar groups used in the 5'-exterior region are different from the sugar groups used in the 3'-exterior region, the gapmer is an asymmetric gapmer. In one embodiment, the exterior regions are small (each independently 1, 2, 3, 4, or about 5 monomer subunits), and the monomer subunits comprise unnatural sugar groups with the interior region comprising β-D-2'-deoxyribonucleosides. In one embodiment, the exterior regions each independently comprise 1 to about 5 monomer subunits, and the interior region comprises 6 to 18 unmodified nucleosides. The internal region or gap typically comprises β-D-2′-deoxyribo-nucleosides but may contain unnatural sugar groups.

[0386] In one embodiment, a gap oligomer compound comprises an internal region of β-D-2'-deoxyribonucleosides, wherein one of the external regions comprises a nucleoside of the present invention. In one embodiment, a gap oligonucleotide comprises an internal region of β-D-2'-deoxyribonucleosides, wherein both external regions comprise a nucleoside of the present invention. In one embodiment, a gap oligonucleotide comprises an internal region of β-D-2'-deoxyribonucleosides, wherein both external regions comprise a nucleoside of the present invention. In one embodiment, a gap oligonucleotide is provided herein, wherein all of the monomer subunits comprise a non-natural sugar group. In one embodiment, a gap oligonucleotide is provided, comprising one or two nucleosides of the present invention at the 5'-terminus, two or three nucleosides of the present invention at the 3'-terminus, and an internal region consisting of 10 to 16 β-D-2'-deoxyribonucleosides. In one embodiment, a gap oligonucleotide is provided that comprises one nucleoside of the present invention at the 5'-terminus, two nucleosides of the present invention at the 3'-terminus, and an internal region composed of 10 to 16 β-D-2'-deoxyribonucleosides. In one embodiment, a gap oligonucleotide is provided that comprises two nucleosides of the present invention at the 5'-terminus, two nucleosides of the present invention at the 3'-terminus, and an internal region composed of 10 to 14 β-D-2'-deoxyribonucleosides. In one embodiment, a gap oligonucleotide is provided that is about 10 to about 21 monomer subunits in length. In one embodiment, a gap oligonucleotide is provided that is about 12 to about 16 monomer subunits in length. In one embodiment, a gap oligonucleotide is provided that is about 12 to about 14 monomer subunits in length.

[0387] In some embodiments, the 5'-terminal monomer of an oligomeric compound of the present invention comprises a phosphorus-containing moiety at the 5'-terminus. In some embodiments, the 5'-terminal monomer comprises 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 comprises a 5'-modification. In some embodiments, the 5'-terminal monomer comprises a 2'-modification and a 5'-modification. In some embodiments, the 5'-terminal monomer comprises a 5'-stabilizing nucleoside. In some embodiments, the modification of the 5'-terminal monomer stabilizes the 5'-phosphate. In some embodiments, oligomeric compounds comprising modified 5'-terminal monomers are exonuclease resistant. In some embodiments, oligomeric compounds comprising modified 5'-terminal monomers have improved tunability / reversal properties. In some embodiments, oligomeric compounds comprising modified 5'-terminal monomers have improved association with siRNA strands.

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

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

[0390] In some embodiments, oligomeric compounds of the present invention comprise a region of one or more alternating 2'-F modified nucleosides and 2'-OMe modified nucleosides. In some such embodiments, such a region of alternating 2'-F modified nucleosides and 2'-OMe modified nucleosides also comprises 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 linkage at the 3' end of the 2'-OMe nucleoside is a phosphodiester linkage.

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

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

[0393] In some embodiments, one or more alternating regions in an alternating motif comprises a plurality of a single nucleoside of a certain type. For example, an oligomeric compound of the invention may comprise the following nucleoside motif: 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'-CHO-2'-LNA. In some embodiments, B is 4'-CHO-2'-LNA. In some embodiments, A is DNA and B is 4'-CHO-2'-LNA. In some embodiments, A is 4'-CHO-2'-LNA and 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'-CHO-2'-LNA. In some embodiments, A is 4'-CHO-2'-LNA and 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'-CHO-2'-LNA. In some embodiments, A is 4'-CHO-2'-LNA and 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, oligomeric compounds having the above alternating motifs also include a 5'-terminal nucleoside that includes a 2'-cationic modification. In some embodiments, oligomeric compounds having the above alternating motifs also include a 5'-terminal modification.

[0399] 2-2-3 motif In some embodiments, oligomeric compounds of the invention comprise a region having a 2-2-3 motif. Such a region may have the following motif: 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 with different modifications than A, but B, C, D and E may have the same or different modifications from each other; 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, the linkages in the 2-2-3 motif are all modified linkages. In some embodiments, the linkages are all phosphorothioate linkages. In some embodiments, the linkage at the 3' end of each first type of modification is a phosphodiester.

[0402] In some embodiments, Z is 0. In these embodiments, the region of the first type of three nucleosides 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 group is attached to the 3' end of the region of the first type of three nucleosides. In some embodiments, an oligomeric compound comprising an oligonucleotide in which Z is 0 can include a terminal group attached to the 3'-terminal nucleoside. Such a terminal group can include another nucleoside. Such another nucleoside is typically a non-hybridizing nucleoside.

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

[0404] Combination motifs It should be understood that some of the motifs and modifications described above may be combined. A motif may contain only a small number of nucleosides, and therefore a particular oligomeric compound may contain more than one motif. As a non-limiting example, in some embodiments, an oligomeric compound may have two or more nucleotide motifs selected from LNA, phosphorothioate linkages, 2'-OMe, and conjugated ligands.

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

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

[0407] In some embodiments, at least one strand of a single stranded oligomeric compound or a double stranded oligomeric compound comprises at least one of the following motifs: (a) 5'-phosphorothioate or 5'-phosphorodithioate; (b) cationic modifications of 5′-terminal nucleotides 1 and 2, wherein the cationic modifications are located at the C5 position of the pyrimidine and at C2, C6, C8, exocyclic N2, or exocyclic N6 of the purine; (c) at least one G-clamp nucleotide and another nucleotide having a cationic modification in the first two terminal nucleotides of the 5' end, where the cationic modification is located at the C5 position of a pyrimidine or the C2, C6, C8, exocyclic N2, or exocyclic N6 position of a purine; (d) at least one 2'-F modified nucleotide containing 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 the up configuration, e.g., the arabinose configuration; (f) a 3'-terminal 5'-PuPu-3' dinucleotide, wherein both nucleotides contain a modified MOE at the 2' position as described in U.S. Patent Application Publication No. 20130130378, the contents of which are incorporated herein by reference in their entirety; (g) a 5'-terminal 5'-PuPu-3' dinucleotide, where both nucleotides contain a modified MOE at the 2' position, as described in U.S. Patent Application Publication No. 20130130378; (h) a 5'-terminal nucleotide comprising a modified MOE at the 2' position, as described in U.S. Patent Application Publication No. 20130130378; (i) a 5'-terminal nucleotide having a 3'-F modification: (j) a 5'-terminal nucleotide containing a 4'-substituent; (k) a 5′-terminal nucleotide containing an O4′ modification; (l) a 3'-terminal nucleotide containing a 4'-substituent; and (m) Combinations of these.

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

[0409] The foregoing examples are provided to illustrate how the described motifs can be used in combination and are not intended to limit the invention to specific combinations or the specific modifications used to exemplify the combinations. Furthermore, without limitation, specific examples, such as those shown in the table above, are intended to encompass more general embodiments. For example, column A of the table above exemplifies a region of alternating 2'-OMe and 2'-F nucleosides. Accordingly, this same disclosure also exemplifies regions of alternating 2'-F modifications. It also exemplifies regions of alternating 2'-O-alkyl and 2'-halogen nucleosides. It also exemplifies regions of alternating different modified nucleosides. All examples throughout this specification contemplate this inclusive interpretation.

[0410] It is noted that the length of oligomeric compounds such as those exemplified in the table above can be easily manipulated by extending or shortening one or more of the regions described without disrupting the motif.

[0411] In some embodiments, the oligomeric compounds contain two or more chemically distinct regions and have the structure described in International Application PCT / US Ser. No. 09 / 038433, filed Mar. 26, 2009, the contents of which are incorporated herein in their entirety.

[0412] Synthesis, purification and analysis Oligomerization of modified and unmodified nucleosides and nucleotides can be routinely carried out according to literature procedures for 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).

[0413] The oligomeric compounds provided herein can be generally and routinely prepared by the known technique 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 use similar techniques to prepare oligonucleotides such as phosphorothioates and alkylated derivatives. The present invention is not limited by the method of antisense compound synthesis.

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

[0415] The oligomeric compounds of the present 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 of easily preparing oligomeric strands containing unnatural or modified nucleotides. Any other means for such synthesis known in the art can additionally or alternatively be used. It is also well known to use similar techniques to prepare other oligomeric compounds, such as those containing phosphorothioate, phosphorodithioate, and alkylated derivatives of the intersugar linkage. The double-stranded oligomeric compounds of the present invention can be prepared using a two-step method. First, the individual strands of the double-stranded molecule are prepared separately. Then, the constituent strands are annealed.

[0416] Regardless of the method of synthesis, the oligomeric compounds can be prepared in a solution (e.g., water and / or organic solution) appropriate for formulation. 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 certain 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 for the preparation of oligonucleotides with chiral phosphorus linkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, for oligonucleotides with modified backbones; U.S. Pat. Nos. 5,378,825 and 5,541,307, for backbone-modified oligonucleotides and their preparation by reductive coupling; No. 5,386,023; U.S. Pat. No. 5,457,191, relating to modified nucleobases based on 3-deazapurine ring systems and methods for their synthesis; U.S. Pat. No. 5,459,255, relating to modified nucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302, relating to methods for preparing oligonucleotides with chiral phosphorus linkages; U.S. Pat. No. 5,539,082, relating to peptide nucleic acids; U.S. Pat. No. 5,554,746, relating to oligonucleotides with β-lactam backbones; U.S. Pat. No. 5,571,902, which relates to methods and materials for the synthesis of nucleotides; U.S. Pat. No. 5,578,718, which relates to nucleosides having alkylthio groups (such groups can be used as linkers to other moieties attached to any of the various positions of the nucleoside); U.S. Pat. Nos. 5,587,361 and 5,599,797, which relate to oligonucleotides having phosphorothioate linkages of high chiral purity; 2'-O-alkylguanosine and related compounds (2,6-diaminopurine compounds); U.S. Pat. No. 5,506,351, relating to methods for preparing oligonucleotides containing N-2 substituted purines; U.S. Pat. No. 5,587,469, relating to oligonucleotides containing 3-deazapurines; U.S. Pat. Nos. 5,223,168 and 5,608,046, both relating to conjugated 4'-desmethylnucleoside analogs; U.S. Pat. Nos. 5,602,240 and 5,610,289, relating to backbone-modified oligonucleotide analogs;and U.S. Patent Nos. 6,262,241 and 5,459,255, among others, relating to methods for synthesizing 2'-fluoro-oligonucleotides;

[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. The composition and method for the formulation of pharmaceutical compositions depend on several 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 regulatory 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. Thus, 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 comprising oligomeric compounds include any pharmaceutically acceptable salts, esters, or salts of such esters. In some embodiments, pharmaceutical compositions comprising oligomeric compounds include one or more oligonucleotides that can provide (directly or indirectly) biologically active metabolites or residues thereof upon administration to an animal, such as a human. 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 involve the incorporation of additional nucleosides at one or both termini of the oligomeric compound that are cleaved by endogenous nucleases in the body to form the active oligomeric compound.

[0422] The pharmaceutical compositions of the present 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 (e.g., via a transdermal patch), pulmonary, e.g., using a nebulizer, by inhalation or insufflation of powders or aerosols; intratracheal, intranasal, epidermal, and transdermal; oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous, e.g., via an implantable device; or intracranial, e.g., intraparenchymal, intrathecal, or intraventricular, administration.

[0423] The oligomeric compounds can be delivered in a manner that targets 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, thickeners, and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful. Suitable topical formulations include those in which the iRNA of the present invention is mixed with a topical delivery agent, 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, dimyristoylphosphatidylcholine DMPC, disteroylphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA) lipids. The iRNA of the present invention can be encapsulated in or complexed with liposomes, particularly cationic liposomes. Alternatively, the 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, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1~20 Examples of suitable topical formulations include alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0425] There are many organized surface-active 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 from the perspective of drug delivery due to the specificity and prolonged action they offer. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayer.

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

[0427] Further advantages of liposomes are as follows: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a variety of water- and lipid-soluble drugs; liposomes can protect the drugs encapsulated in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, 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 transporting and delivering active ingredients to the site of action. Because liposome membranes are structurally similar to biological membranes, when liposomes are applied to tissues, they begin to fuse with cell membranes, and as the fusion of liposomes with cells progresses, the contents of the liposomes are transferred into the cells, where 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 growing 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 wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

[0430] Several reports have detailed the ability of liposomes to deliver drugs, including high molecular weight DNA, into the skin. Painkillers, antibodies, hormones, and compounds containing 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. The positively charged DNA / liposome complexes bind to the negatively charged cell surface and are internalized in endosomes. Due to the acidic pH inside the endosomes, the liposomes are ruptured, releasing their 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 both DNA and lipids are similarly charged, repulsion occurs rather than complexation. Nevertheless, some DNA is entrapped 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 cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

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

[0434] Several studies have evaluated the topical delivery of liposomal drug formulations to the skin. When liposomes containing interferon were applied to the skin of guinea pigs, relief of cutaneous herpes was achieved, whereas delivery of interferon by other means (e.g., as a solution or emulsion) was ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Furthermore, another study tested the efficacy of interferon administered as part of a liposomal formulation versus administration using an aqueous system, concluding that the liposomal formulation was 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 delivering drugs to the skin. Nonionic liposomal formulations containing Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver cyclosporine A to the dermis of mouse skin. Results indicated that these nonionic liposomal systems were effective in promoting the deposition of cyclosporine A into various layers of the skin (Hu et al., STP Pharma. Sci., 1994, 4, 6, 466).

[0436] Liposomes also include "sterically stabilized" liposomes, which term, as used herein, refers to liposomes that contain one or more specialized lipids that, when incorporated into the liposome, result in increased circulation life compared to liposomes without such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the vesicle-forming lipid portion of 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, it is believed in the art that for sterically stabilized liposomes containing at least gangliosides, sphingomyelin, or PEG-derivatized lipids, the increased circulation lifetime of these sterically stabilized liposomes is 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] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) report the use of monosialoganglioside G, which improves the blood half-life of liposomes. M1 (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88 / 04924 (both by Allen et al.) report the ability of (1) sphingomyelin and (2) ganglioside G M1 or galactocerebroside sulfate esters. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. WO 97 / 13499 (Lim et al.) discloses liposomes containing 1,2-sn-dimyristoylphosphatidylcholine.

[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) describe a method for preparing liposomes containing a nonionic detergent, 2C, containing a PEG moiety. 1215G(FEBS Lett., 1984, 167, 79) described liposomes containing PEG-derivatized phosphatidylethanolamine (PE) or PEG stearate. Illum et al. (FEBS Lett., 1984, 167, 79) described that hydrophilic coating of polystyrene particles with polymeric glycols significantly increased their blood half-life. Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899) described synthetic phospholipids modified by the attachment of carboxylic acid groups of polyalkylene glycols (e.g., PEG). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes containing PEG-derivatized phosphatidylethanolamine (PE) or PEG stearate have significantly increased blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended these observations to other PEG-derivatized phospholipids, such as DSPE-PEG, formed from a combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes with covalently bound PEG moieties on their outer surface are described by Fisher in European Patent No. 0 445 131 B1 and International Publication No. WO 90 / 04384. Liposomes containing 1-20 mole % PEG-derivatized PE and methods for their use are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. 0 496 813 B1). Liposomes containing several other lipid-polymer conjugates are disclosed in WO 91 / 05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and WO 94 / 20073 (Zalipsky et al.). Liposomes containing PEG-modified ceramide lipids are described in WO 96 / 10391 (Choi et al.). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

[0439] Some liposomes containing nucleic acids are known in the art. Thierry et al., International Publication No. 96 / 40062, discloses a method for encapsulating high molecular weight nucleic acids into liposomes. Tagawa et al., U.S. Patent No. 5,264,221, discloses protein-bound liposomes, and claims that the contents of these liposomes can contain dsRNA. Rahman et al., U.S. Patent No. 5,665,710, describes a specific method for encapsulating oligodeoxyribonucleotides into liposomes. Love et al., International Publication No. 97 / 04787, discloses liposomes containing dsRNA targeted to raf gene.

[0440] Transfersomes, another type of liposome, are highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets, which are highly deformable and can easily penetrate pores smaller than these lipid droplets. Transfersomes can adapt to the environment in which they are used, for example, self-optimizing (adapting to the shape of skin pores), self-repairing, often reaching their target without fragmentation, and often self-filling. To prepare transfersomes, a surface-activating agent, usually a surfactant, can be added to a standard liposome composition. 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] Liposome compositions can be prepared by various methods known in the art.See, for example, the following documents: U.S. Patent No. 4,235,871; U.S. Patent No. 4,737,323; U.S. Patent No. 4,897,355 and U.S. Patent No. 5,171,678; WO 96 / 14057 and WO 96 / 37194; Felgner, PL 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. 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 a wide range of applications in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the properties of a wide variety of surfactants, both natural and synthetic, is by using hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as "head") provides the most useful means of classifying the various surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0443] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find widespread application in pharmaceuticals and cosmetics and are usable over a wide range of pH values. Their HLB values ​​generally range from 2 to approximately 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. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers are also included in this class. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

[0444] When dissolved or dispersed in water, if the surfactant molecule carries a negative charge, this surfactant is classified as anionic.Anionic surfactants include carboxylates such as soap, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzenesulfonates, acyl isethionates, acyl taurates, sulfosuccinates and phosphates.The most important members of anionic surfactant class are alkyl sulfates and soaps.

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

[0446] If the 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-alkylbetaines, 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, NY, 1988, p. 285).

[0448] lipid particles In some embodiments, the tunable REVERSIR can be fully encapsulated in a lipid formulation, such as an LNP or other nucleic acid-lipid particle. The tunable REVERSIR encapsulated in the lipid formulation can be unconjugated or conjugated to a ligand (i.e., a conjugated tunable REVERSIR).

[0449] As used herein, the term "LNP" refers to stable nucleic acid-lipid particles. LNPs contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). LNPs exhibit long circulating life after intravenous (iv) injection and accumulate at distant sites (e.g., sites physically distant from the administration site), making them extremely useful for systemic applications. LNPs include "pSPLPs," which contain encapsulated condensing agent-nucleic acid complexes as described in PCT Publication WO 00 / 03683. The particles of the present invention typically have an average particle 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, and most typically about 70 nm to about 90 nm, and are substantially nontoxic. Furthermore, when present in the nucleic acid-lipid particles of the present invention, the nucleic acid is resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Patent Application Publication No. 2010 / 0324120; and PCT Publication No. WO 96 / 40964.

[0450] In some embodiments, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to modulating REVERSIR ratio) ranges from about 1:1 to about 50:1, about 1:1 to about 25:1, about 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-listed ranges are also considered part of this invention.

[0451] Examples of cationic lipids include 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-trimethylammonium 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-dilinoleyl Carbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleyl-3-dimethylaminopropane (DLin-DAP), 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-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3 The cationic lipid may be (aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxo-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-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)ethylazanediyl)didodecan-2-ol, or a mixture thereof. The cationic lipid may comprise about 20 mol% to about 50 mol% or about 40 mol% of the total lipid present in the particle.

[0452] In some embodiments, the lipid-regulated REVERSIR nanoparticles can be prepared using the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane. The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in International Application PCT / US2009 / 061897, published as WO2010 / 048536, which is incorporated herein by reference.

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

[0454] The ionized / non-cationic lipids may be anionic or neutral lipids, including but not limited to distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleylphosphatidylcholine (POPC), palmitoyloleylphosphatidylethanolamine (POPC), and the like. Examples of suitable lipids include dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), cholesterol, or mixtures thereof. The non-cationic lipid can comprise about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% if cholesterol is included, of the total lipid present in the particle.

[0455] The conjugated lipid that inhibits particle aggregation can be, for example, a polyethylene glycol (PEG)-lipid, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. PEG-DAA conjugates include, for example, PEG-dilauryloxypropyl (C 12), PEG-dimyristyloxypropyl (C 14 ), PEG-dipalmityloxypropyl (C 16 ) or PEG-distearoyloxypropyl (C 18 The conjugated lipid that inhibits particle aggregation can comprise from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

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

[0457] Other exemplary lipid-modulated REVERSIR formulations are set forth in Table 1 below.

[0458] [Table 2]

[0459] [Table 3]

[0460] [Table 4]

[0461] Abbreviations in Table 1 include the following: DSPC: distearoylphosphatidylcholine; DPPC: dipalmitoylphosphatidylcholine; PEG-DMG: PEG-dimyristoylglycerol (C14-PEG or PEG-C14) (PEG with an average molecular weight of 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] Formulations containing DLinDMA (1,2-dilinolenyloxy-N,N-dimethylaminopropane) are described in WO 2009 / 127060, filed April 15, 2009, which is incorporated herein by reference.

[0463] Formulations containing XTC are described, for example, in U.S. Provisional Patent Application No. 61 / 148,366, filed January 29, 2009; U.S. Provisional Patent Application No. 61 / 156,851, filed March 2, 2009; U.S. Provisional Patent Application No. 61 / 228,373, filed July 24, 2009; U.S. Provisional Patent Application No. 61 / 239,686, filed September 3, 2009; and International Application No. PCT / US2010 / 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 formulations are described, for example, in PCT Publication No. WO 2011 / 153493, filed June 3, 2011, and PCT Publication No. WO 2013 / 086354, filed December 7, 2012, the entire contents of which are incorporated herein by reference.

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

[0467] The oligomeric compounds of the present invention can be prepared and formulated as micelles. As used herein, a "micelle" is a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all of the hydrophobic portions of the molecules are oriented inward, while the hydrophilic portions remain in contact with the surrounding aqueous layer. If the environment is hydrophobic, the opposite arrangement exists.

[0468] In some embodiments, the formulation comprises micelles formed from the oligonucleotides 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 having an average diameter of less than about 50 nm, and even more preferred embodiments provide micelles having an average diameter of less than about 30 nm or even less than about 20 nm.

[0469] The micelle formulation contains an aqueous solution of the oligonucleotide composition, an alkali metal C8-C 22 They can be prepared by mixing an alkyl sulfate and an amphiphilic carrier. The amphiphilic carrier can be added simultaneously with or after the addition of the alkali metal alkyl sulfate. Micelles can be formed by virtually any type of mixing of the components, except with vigorous mixing, to provide micelles of smaller particle size.

[0470] The oligomeric compounds of the present 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 biphasic systems containing two immiscible liquid phases intimately mixed and dispersed with each other. Generally, emulsions can be either water-in-oil (w / o) or oil-in-water (o / w) types. If the aqueous phase is finely divided and dispersed as minute droplets 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 minute droplets in the bulk aqueous phase, the resulting composition is called an oil-in-water (o / w) emulsion. Emulsions may contain additional components in addition to the dispersed phase and active drug, which may be present in the aqueous phase, as a solution in the oil phase, or as a separate phase of their own. Optional formulation excipients, such as emulsifiers, stabilizers, dyes, and antioxidants, may also be present in emulsions. Pharmaceutical emulsions can be multiple emulsions composed of three or more phases, such as, for example, 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 binary emulsions do not. Multiple emulsions in which individual oil droplets of an o / w emulsion surround tiny water droplets constitute w / o / w emulsions. Similarly, systems of oil droplets surrounded by small droplets of water stabilized in an oily continuous phase constitute o / w / o emulsions.

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

[0473] Synthetic surfactants, also known as surface active agents, 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, NY, volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p. 199). Surfactants are typically amphiphilic, containing hydrophilic and hydrophobic moieties. The ratio of hydrophilic to hydrophobic properties of a surfactant, referred to as the hydrophile / lipophile balance (HLB), is a valuable tool for classifying and selecting surfactants for formulation preparation. Surfactants can be divided into various classes based on the nature of the hydrophilic group: nonionic, anionic, cationic, and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285).

[0474] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin, and acacia. Absorbent bases, such as anhydrous lanolin and hydrophilic petrolatum, have hydrophilic properties that allow them to absorb water to form water-in-oil emulsions while still maintaining their semisolid consistency. Finely divided solids have also been 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-polar solids such as carbon or glyceryl tristearate.

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

[0476] Hydrophilic colloids, or hydrocolloids, include natural gums and synthetic polymers such as 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., carbomer, cellulose ethers, and carboxyvinyl polymers), which disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed phase droplets and by increasing the viscosity of the external phase.

[0477] Emulsions often contain several components, such as carbohydrates, proteins, sterols, and phosphatides, which can easily support bacterial growth, so these preparations often contain preservatives.Common preservatives contained in emulsion preparations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.Antioxidants are also commonly added to emulsion preparations to prevent the preparation from decomposing.The antioxidants used can be free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, and butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0478] In some embodiments, the composition is formulated as a microemulsion. As used herein, "microemulsion" refers to a system of water, oil, and amphiphiles 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] Microemulsions 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, NY, volume 1, p. 245). Typically, microemulsions are prepared by first dispersing oil in an aqueous surfactant solution, and then adding a sufficient amount of a fourth component (generally a medium-chain alcohol) to form a transparent system. Thus, microemulsions are 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 components, including oil, water, surfactant, cosurfactant, and electrolyte. Whether a microemulsion is water-in-oil (w / o) or oil-in-water (o / w) depends on the properties of the oil and surfactant used and the structure and geometric packing of the polar head and hydrocarbon tail of the surfactant molecule (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

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

[0481] Surfactants used in preparing microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyethylene oleyl ether, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), and decaglycerol decaoleate (DAO750), either alone or in combination with cosurfactants. The cosurfactants, which are typically short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, serve to increase interfacial fluidity by penetrating the surfactant film, resulting in the formation of an irregular film due to the void spaces created between the surfactant molecules. However, microemulsions can be prepared without the use of cosurfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG 300, PEG 400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase can include, but is not limited to, materials such as 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, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

[0482] Microemulsions are particularly interesting 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, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions offer the following advantages: improved drug solubilization, protection of the drug from enzymatic hydrolysis, potential enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration compared to solid dosage forms, improved clinical efficacy, and reduced toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). In many cases, microemulsions can spontaneously form when their components are combined at ambient temperature. This can be particularly advantageous when formulating heat-labile drugs, peptides, or dsRNA. Microemulsions are also effective 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 ingredients and additives, such as sorbitan monostearate (Grill 3), Labrasol, and permeation enhancers, to improve formulation properties and enhance the absorption of the dsRNA and nucleic acids of the present invention.The permeation enhancers used in the microemulsions of the present invention can be classified into one of five major 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] The application of emulsion formulations by dermatological, oral and parenteral routes, as well as methods for their manufacture, are discussed in the literature, e.g., Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; and Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335, the contents of which are incorporated herein by reference in their entireties.

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

[0486] FLiPs may contain triacylglycerol, phospholipids, glycerol, and one or more lipid-binding proteins aggregated, mixed, or bound to oligonucleotides via lipophilic linker molecules. Surprisingly, it has been found that the one or more lipid-binding proteins combined with the aforementioned lipids allow FLiPs to exhibit affinity for the liver, intestine, kidney, steroid-producing organs, heart, lung, and / or muscle tissues. Therefore, these FLiPs can serve as carriers for oligonucleotides in these tissues. For example, lipid-conjugated oligonucleotides, such as cholesterol-conjugated oligonucleotides, bind to HDL and LDL lipoprotein particles and mediate cellular uptake upon binding to their respective receptors, thereby directing oligonucleotide delivery to the liver, intestine, kidney, and steroid-producing organs. See Wolfrum et al. Nature Biotech. (2007), 25:1145-1157.

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

[0488] Another lipid component suitable for FLiP is lipoprotein, such as isolated lipoprotein or more preferably 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, see, for example, A. Jones, Experimental Lung Res. 6, 255-270 (1984), U.S. Patent Nos. 4,643,988 and 5,128,318, WO 87 / 02062, and Canadian Patent No. 2,138,925. Other methods for producing reconstituted lipoproteins, particularly apolipoproteins AI, A-II, A-IV, apoC, and apoE, are described in 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 of 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 scope of the present invention that other suitable oils, such as safflower oil, can be used to prepare the lipid component of FLiP.

[0490] FLiPs can have particle sizes ranging from about 20-50 nm or about 30-50 nm, e.g., about 35-40 nm. In some embodiments, FLiPs have a particle size of at least about 100 nm. Alternatively, FLiPs, whether characterized as liposome- or emulsion-based, can be about 100-150 nm, e.g., about 110 nm, about 120 nm, about 130 nm, or about 140 nm. Multiple FLiPs can also be aggregated and delivered together, resulting in particle sizes greater than 100 nm.

[0491] The method for producing lipid particles includes (a) mixing one or more lipophilic substance (e.g., cholesterol)-conjugated oligonucleotides, which may be chemically modified, with lipid components; and (b) fractionating the mixture. In some embodiments, the method includes the 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 herein by reference in their entirety.

[0493] In some embodiments, oligomeric compounds can be formulated into yeast cell wall particles ("YCWP"). Yeast cell wall particles comprise an extracted yeast cell wall exterior and a core, the core comprising a payload (e.g., an oligonucleotide). The particle exterior comprises yeast glucan (e.g., β-glucan, β-1,3-glucan, β-1,6-glucan), yeast mannan, or a combination thereof. Yeast cell wall particles are typically spherical particles with a diameter of about 1-4 μm.

[0494] Preparation of yeast cell wall particles is known in the art, see, e.g., U.S. Pat. Nos. 4,992,540; 5,082,936; 5,028,703; 5,032,401; 5,322,841; 5,401,727; 5,504,079; 5,607,677; 5,741,495; The application of yeast cell-like particles for drug delivery is described in, for example, U.S. Patent Nos. 5,830,463; 5,968,811; 6,444,448; and 6,476,003, U.S. Patent Application Publication Nos. 2003 / 0216346 and 2004 / 0014715 and International Publication No. WO 2002 / 12348, the contents of which are incorporated herein by reference in their entirety.The application of yeast cell-like particles for drug delivery is described in, for example, U.S. Patent Nos. 5,032,401; 5,607,677; 5,741,495; and 5,830,463 and U.S. Patent Application Publication Nos. 2005 / 0281781 and 2008 / 0044438, the contents of which are incorporated herein by reference in their entirety. US 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.

[0495] Exemplary formulations of oligomeric compounds are described in U.S. Pat. Nos. 4,897,355; 4,394,448; 4,235,871; 4,231,877; 4,224,179; 4,753,788; 4,673,567; 4,247,411; 4,814,270; 5,567,434; 5,552,157; ​​5,565,213; 5,738,868; 5,795,587; and 5,922,859. Nos. 6,077,663; 7,906,484; and 8,642,076; PCT Publication WO 2009 / 132131 and U.S. Patent Application Publication Nos. 2006 / 0240093, 2007 / 0135372, 2011 / 0117125, 2009 / 0291131, 2012 / 0316220, 2009 / 0163705, and 2013 / 0129785, the contents of which are incorporated herein by reference in their entireties. Behr (1994) Bioconjugate Chem. 5:382-389 and Lewis et al. (1996) PNAS 93:3176-3181) also describe formulations of oligonucleotides suitable for the present invention, the contents of which are incorporated herein by reference in their entireties.

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

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

[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 refers to a reduction in mRNA levels in a cell of a target gene by at least about 5%, at least about 10%, 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%, at least about 99%, including up to 100%, and any integer percentage between these, of the mRNA levels found in a cell in the absence of the miRNA or RNA interference molecule. In one preferred embodiment, the mRNA levels are reduced by at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, including up to 100%, and any integer percentage between 5% and 100%.

[0500] As used herein, the term "modulate gene expression" means up-regulating or down-regulating the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, such that the 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 modulation occurs when the expression of a gene encoding one or more proteins or protein subunits, or the level of the RNA molecules or equivalent RNA molecules that encode them, differs by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, or more from that observed in the absence of an siRNA, e.g., an RNAi agent. The percentage and / or fold difference can be calculated, for example, relative to a control or non-control, as follows:

number

[0502] As used herein, the terms "inhibit," "down-regulate," or "reduce," with respect to gene expression, mean that 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, is reduced below that observed in the absence of a modulator. Gene expression is down-regulated when 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, is reduced by at least 10%, preferably by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or most preferably 100% (i.e., no gene expression), compared to a corresponding unmodulated control.

[0503] As used herein, the terms "increase" and "up-regulate," with respect to gene expression, mean that 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, is increased above that observed in the absence of a modulator. Gene expression is up-regulated when 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, is increased by at least 10%, preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 100%, 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more, compared to a corresponding unmodulated control.

[0504] As used herein, the term "increased" or "increase" generally refers to an increase by a statistically significant amount; for the avoidance of doubt, "increased" means an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% compared to a reference level, or any increase between 10 and 100%, or at least about a 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, or at least 10-fold increase, or any increase between 2-fold and 10-fold or more compared to a reference level.

[0505] As used herein, the term "reduced" or "reducing" generally refers to a statistically significant decrease. However, for the avoidance of doubt, "reduced" refers to a decrease of at least 10% compared to a reference level, such as a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or a decrease of less than 100% (i.e., zero level compared to a reference sample), or any decrease between 10 and 100% compared to a reference level.

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

[0507] A double-stranded oligonucleotide comprises two oligonucleotide strands that are sufficiently complementary to hybridize and form a double-helical structure. Typically, the double-helical structure is 15-30, more commonly 18-25, even more commonly 19-24, and most commonly 19-21 base pairs in length. In some embodiments, longer double-stranded oligonucleotides, such as 25-30 base pairs in length, are preferred. In some embodiments, shorter double-stranded oligonucleotides, such as 10-15 base pairs in length, are preferred. In another embodiment, the double-stranded oligonucleotide is at least 21 nucleotides in length.

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

[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 term "antisense strand" includes the antisense region of both oligomeric compounds formed from two separate strands, as well as unimolecular compounds that can form hairpin or dumbbell structures. The terms "antisense strand" and "guide strand" are used interchangeably herein.

[0510] The phrase "sense strand" refers to an oligomeric compound that has all or part of the same nucleoside sequence as 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 can form hydrogen bonds with another nucleic acid sequence, either through traditional Watson-Crick or other non-traditional types. In the context of the nucleic acid molecules of the present invention, the binding free energy between a nucleic acid molecule and its complementary sequence is sufficient to allow the relevant function of the nucleic acid, such as RNAi activity, to proceed. Determination of the binding free energy of nucleic acid molecules is well known in the art (see, for example, Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83: 9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109: 3783-3785). Percent complementarity refers to the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" or 100% complementarity means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous 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 can hydrogen bond with each other. "Substantial complementarity" refers to polynucleotide strands that exhibit 90% or greater complementarity, except for regions of the polynucleotide strands selected to be non-complementary (e.g., overhangs). Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under the conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, or under the conditions in which the assay is performed in the case of in vitro assays. Non-target sequences typically differ by at least 5 nucleotides.

[0512] The term "off-target" and the phrase "off-target effect" refer to any case in which an siRNA for a given target causes an unintended effect by directly or indirectly interacting with another mRNA sequence, DNA sequence, or cellular protein or other moiety. For example, an "off-target effect" can occur when partial homology or complementarity between the sense and / or antisense strands of an siRNA and another transcript leads to simultaneous degradation of the other transcript.

[0513] In some embodiments, the double-stranded region of a double-stranded oligomeric compound is equal to or at least 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 in length.

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

[0515] In some embodiments, the sense strand of a double-stranded oligomeric compound is at least equal to or has a length of 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 to 5 single-stranded nucleotides within the double-stranded region. A "single-stranded nucleotide stretch within a double-stranded region" means that there is at least one nucleotide base pair on both ends of the single-stranded stretch. In some embodiments, both strands have at least one stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides within the double-stranded region. When 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, these single-stranded nucleotides can be opposite each other (e.g., a mismatched stretch), or they can be positioned such that the second strand does not contain the opposite single-stranded nucleotides of the single-stranded oligonucleotides of the first strand, or vice versa (e.g., a single-stranded loop). In some embodiments, the single-stranded nucleotides are present within 8 nucleotides from either end, for example, 8, 7, 6, 5, 4, 3, or 2 nucleotides from either the 5' or 3' end of the region of complementarity between the two strands.

[0517] In some embodiments, each strand of the double-stranded oligonucleotide has a ZXY structure, such as that described in PCT Publication WO 2004080406, the contents of which are incorporated herein by reference in their entirety.

[0518] In some embodiments, the two strands of a double-stranded oligomeric compound can be linked to each other. The two strands can be linked to each other at both ends or only one end. Linking at one end means that the 5' end of the first strand is linked to the 3' end of the second strand, or the 3' end of the first strand is linked to the 5' end of the second strand. When the two strands are linked to each other 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. The two strands can be linked to each other by an oligonucleotide linker, such as, 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 to 10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of 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 strands may also be linked to each other by a non-nucleoside linker, such as a linker described herein. One of skill in the art will recognize that any of the oligonucleotide chemical modifications or alterations described herein may be used in the oligonucleotide linker.

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

[0520] Hairpin oligomeric compounds may have single-stranded overhangs or terminal unpaired regions, in some embodiments at the 3', and in some embodiments, at the antisense side of the hairpin. In some embodiments, the overhangs are 1-4, more typically 2-3 nucleotides in length. Hairpin oligomeric compounds capable of inducing RNA interference are also referred to herein as "shRNAs."

[0521] In some embodiments, two oligomer strands specifically hybridize when there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense compound to non-target nucleic acid sequences under the conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, and under the conditions in which the assay is performed in the case of in vitro assays.

[0522] As used herein, "stringent hybridization conditions" or "stringent conditions" refers to conditions under which an antisense compound will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will vary in various circumstances; the "stringent conditions" under which an antisense compound will hybridize to its target sequence are determined by the nature and composition of the antisense compound and the assay in which it is tested.

[0523] It is understood in the art that the incorporation of nucleotide affinity modifications can achieve a greater number of mismatches compared to unmodified activation.Similarly, certain oligonucleotide sequences may be more tolerant of mismatches than other oligonucleotide sequences.Those 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 temperature (Tm).Tm or ΔTm can be calculated by techniques well known to those skilled in the art.For example, those skilled in the art can evaluate nucleotide modifications for their ability to increase the melting temperature of RNA:DNA double helix by the technique described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443).

[0524] Regulation of target expression In some embodiments, the target nucleic acid is mRNA. In some such embodiments, the siRNA is designed to regulate the target mRNA or its expression. In some embodiments, designing an antisense compound for a target nucleic acid molecule can be a multi-step process. Typically, this process begins with identifying the target protein, the activity of which is to be regulated, followed by identifying the nucleic acid whose expression produces the target protein. In some embodiments, the antisense compound is designed to obtain an antisense compound that hybridizes with the target nucleic acid molecule. In some embodiments, the antisense compound is an antisense oligonucleotide or antisense oligonucleoside. In some embodiments, the antisense compound and the 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, the antisense compound contains three or more mismatches.

[0525] Modulation of target nucleic acids can be achieved by altering any number of nucleic acid functions. In some embodiments, the RNA function to be modulated includes, but is not limited to, translocation functions (such as, but not limited to, translocation of RNA to a protein translation site, translocation of RNA to a site within the cell distal to the site of RNA synthesis, etc.) and translation of protein 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 complex formation involving RNA that may be associated with or facilitated by RNA. 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, modulation of expression can refer to an increase or decrease in target RNA or protein levels. In another embodiment, modulation of expression can refer to an increase or decrease in one or more RNA splice products or a change in the ratio of two or more splice products.

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

[0527] In some other embodiments, the siRNA is an 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 present invention relates to a double-stranded RNA (dsRNA) agent, i.e., an siRNA, for inhibiting expression of a target gene. The dsRNA agent includes a sense strand and an antisense strand, each having 14 to 40 nucleotides. The dsRNA agent has a structure represented by formula (I): [ka] It is expressed by:

[0529] In Formula (I), B1, B2, B3, B1', B2', B3', and B4' 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. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification.

[0530] C1 is a thermolabile nucleotide located at the site opposite the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand). For example, C1 is located at a position on the sense strand that pairs with nucleotides 2-8 of the 5' end of the antisense strand. The C1 nucleotide carries a thermolabile modification, which may include an abasic modification; a mismatch with the opposite nucleotide in the duplex; and a sugar modification, such as a 2'-deoxy modification or an acyclic nucleotide, e.g., an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA). In one embodiment, C1: i) a mismatch with the opposite nucleotide of the antisense strand; ii) [ka] an abasic modification selected from the group consisting of: [ka] wherein B is a modified or unmodified nucleobase; and R 1 and R 2are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. In one embodiment, the thermolabilizing modification in C1 is a mismatch selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T; and optionally, at least one nucleobase in the mismatch pair is a 2'-deoxynucleobase. In one example, the thermolabilizing modification in C1 is GNA or [ka] is.

[0531] T1, T1', T2', and T3' each independently represent a nucleotide containing a modification that confers steric bulk to the nucleotide equal to or less than that of a 2'-OMe modification. The modification can be at the 2' position of the ribose sugar of the nucleotide, or it can be a non-ribose nucleotide, acyclic nucleotide, or modification to the backbone of the nucleotide, which is similar or equivalent to the 2' position of the ribose sugar and confers steric bulk to the nucleotide equal to or less than that of a 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 in length.

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

[0534] n 4 , q 2 and q6 are independently 1 to 3 nucleotides in length.

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

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

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

[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, which has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand, and two phosphorothioate internucleotide linkage 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 to 22 nucleotides in length, and 4 When C1 is 1, C1 is at positions 14 to 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 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 2 is equal to 1.

[0544] In one embodiment, T1' and T3' are separated by a length of 11 nucleotides (i.e., not counting the T1' and T3' nucleotides.

[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 the non-ribose, acyclic, or backbone 2'-position modification(s) impart 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, acyclic, or backbone 2'-position modifications impart less steric bulk than 2'-OMe ribose modifications.

[0547] In one embodiment, T1 is at the cleavage site of the sense strand. In one example, the sense strand is 19-22 nucleotides in length and 2 When T1 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 at positions 6-10 from the 5' end of the antisense strand, and 4 is 1.

[0549] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and 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, and 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 7is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand, and 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, and 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, and 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 q5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (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, and 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, and 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 2is 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; wherein the sense strand has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand), and the antisense strand has two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage 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, and 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n5 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, wherein the sense strand has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) and the antisense strand has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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% of the dsRNA agents of the invention are modified.

[0558] In one embodiment, each of the sense and antisense strands of a dsRNA agent is independently modified with an acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-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) comprises 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 include any 2'-F modifications.

[0562] In one embodiment, the sense strand and / or antisense strand of a dsRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16 to 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 nucleotide at position 1 of the 5' end of the antisense strand in the duplex 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 the target RNA, hybridizes therewith, and inhibits 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 to the target RNA.

[0566] In one aspect, the present invention relates to a dsRNA agent capable of inhibiting expression of a target gene. The dsRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand contains at least one thermolabile nucleotide, where the at least one thermolabile nucleotide is located at or near the site opposite the seed region of the antisense strand (i.e., positions 2 to 8 of the 5' end of the antisense strand). For example, if the sense strand is 21 nucleotides long, the thermolabile nucleotide is located at positions 14 to 17 of the 5' end of the sense strand. The antisense strand contains at least two modified nucleic acids with less than sterically demanding 2'-OMe modifications. Preferably, the two modified nucleic acids with less than sterically demanding 2'-OMe modifications are separated by a length of 11 nucleotides. For example, the two modified nucleic acids are located 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 represented by During the ceremony, B1, B2, and B3 each independently represent 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; C1 is a heat-labile nucleotide (e.g., a non-cyclic nucleotide such as UNA or GNA, a mismatch, an abasic nucleotide, or DNA) located at a site opposite the seed region of the antisense strand (i.e., positions 2 to 8 of the 5' end of the antisense strand); T1 represents a non-ribose, acyclic or nucleotide containing a chemical modification at the 2'-position or equivalent position of the backbone, which modification confers less steric bulk to the nucleotide than a 2'-OMe modification; for example, 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 in length; n 5 is 1 to 6 nucleotides in length; n 4 is 1 to 3 nucleotides in length; n 2 is 0 to 3 nucleotides in length.

[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 represented by During the ceremony, B1, B2, and B3 each independently represent 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; C1 is a heat-labile nucleotide (e.g., a non-cyclic nucleotide such as UNA or GNA, a mismatch, an abasic nucleotide, or DNA) located at a site opposite the seed region of the antisense strand (i.e., positions 2 to 8 of the 5' end of the antisense strand); T1 represents 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 in length; n 5 is 1 to 6 nucleotides in length; n 4 is 1 to 3 nucleotides in length; n 2 is 0 to 3 nucleotides in length.

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

[0570] In one embodiment, C1 comprises one heat-destabilizing nucleotide at position 14, 15, 16, or 17 from the 5' end of the sense strand. For example, C1 is an acyclic nucleotide (e.g., UNA or GNA), a mismatch, an abasic nucleotide, 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, a dsRNA agent of the invention comprises a sense strand (Is), wherein C1 is an acyclic nucleotide (e.g., UNA or GNA), a mismatch, an abasic, or DNA; and T1 comprises DNA, RNA, LNA, 2'-F, or 2'-F-5'-methyl at position 11 from the 5' end of the sense strand.

[0573] In one embodiment, the antisense strand of the dsRNA agent has the formula (Ia): [ka] is represented by During the ceremony, B1', B2', B3', and B4' each independently represent 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; T1', T2', and T3' each independently represent a non-ribose, acyclic, or nucleotide containing a chemical modification at the 2'-position of the backbone or an equivalent position, which modification confers less steric bulk to the nucleotide than a 2'-OMe modification; for example, T1', T2', and T3' each independently are selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; q 1 are independently 4 to 15 nucleotides in length; q 3 or q 7 are independently 1 to 6 nucleotides in length; q 2 or q 6 are independently 1 to 3 nucleotides in length; q 4 are independently 0 to 3 nucleotides in length; q 5 are independently 0 to 10 nucleotides in length.

[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 represented by During the ceremony, B1', B2', B3', and B4' each independently represent 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; 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 in length; q 3 or q 7 are independently 1 to 6 nucleotides in length; q 2 or q 6 are independently 1 to 3 nucleotides in length; q 4 are independently 0 to 3 nucleotides in length; q 5 are independently 0 to 10 nucleotides in length.

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

[0576] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides: [ka] Including, During the ceremony, B1, B2, B3, B1', B2', B3', and B4' each independently represent 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; 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 the group consisting of DNA, RNA, LNA, 2'-F and 2'-F-5'-methyl; n 1 , n 3 or q 1 are independently 4 to 15 nucleotides in length; n 5 , q 3 or q 7 are independently 1 to 6 nucleotides in length; n 4 , q 2 or q 6 are independently 1 to 3 nucleotides in length; n 2 or q 4 are indepen...

Claims

1. A tunable REVERSIR compound comprising eight or nine modified nucleotides, at least three of which are high affinity monomers, and one of which base pairs with the sixth nucleotide from the 5'-end of the target strand of an siRNA.

2. 10. The tunable REVERSIR compound of claim 1, wherein the high affinity monomer is an LNA.

3. 3. The modulatory REVERSIR compound of claim 2, comprising three or four LNA nucleotides.

4. 10. The tunable REVERSIR compound of claim 1, which is a single-stranded oligonucleotide that is at least 90% complementary to the antisense strand.

5. 10. The modulatory REVERSIR compound of claim 1, which is fully complementary to the antisense strand.

6. 10. The tunable REVERSIR compound of claim 1, comprising at least one modified internucleotide linkage.

7. 7. The regulatable REVERSIR compound of claim 6, wherein the internucleotide linkage is phosphorothioate.

8. 8. The tunable REVERSIR compound of claim 7, comprising no more than three or four phosphorothioate modifications.

9. 10. The tunable REVERSIR compound of claim 1 conjugated to a ligand.

10. The ligand is 【Chemistry 1】 10. The tunable REVERSIR compound of claim 9, wherein:

11. 10. The tunable REVERSIR compound of claim 9, wherein the ligand is conjugated to the 3'-end of the compound.

12. 10. The tunable REVERSIR compound 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 tunable REVERSIR compound.

13. 13. The modulatory REVERSIR compound of claim 12, wherein said deoxy sugar is 2'-deoxyribose.

14. 10. The regulatory REVERSIR compound of claim 1, wherein the siRNA is targeted to an mRNA, a pre-mRNA, a microRNA, or a pre-microRNA.

15. 10. The modulatory REVERSIR compound of claim 1, wherein the siRNA is conjugated to a ligand.

16. A kit comprising the tunable REVERSIR compound of claim 1.

17. A kit comprising an siRNA and a regulatory REVERSIR compound of claim 1.

18. 1. A method or system for evaluating the efficacy and safety of a pharmaceutical composition for use in treating or preventing a disease, said 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 of said subjects; (3) separating responding members from non-responding members of said treated subjects; (4) randomizing and stratifying said responder members into at least two further subgroups; (5) continuing to treat one subgroup member in (4) with said pharmaceutical composition and the other subgroup member with a REVERSIR compound of claim 1 for a second treatment period; (6) deriving mRNA levels and / or physiological outcome measures for said subpopulations; (7) comparing the outcome in (6) with the outcome in (2); (8) using the comparison in (7) to derive efficacy and safety measures for the pharmaceutical composition. A method or system comprising:

19. 1. A method or system for evaluating the efficacy and safety of a pharmaceutical composition for use in treating or preventing a disease, the system comprising: (1) stratifying a subject group into at least two subgroups; (2) treating members of one subgroup with said pharmaceutical composition and 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 said subpopulations; (4) treating members of the treated subgroup with the modulating REVERSIR of claim 1 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 said subpopulations; (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. A method or system comprising:

20. 20. The method or system of claim 18 or 19, wherein the disease is caused by abnormal expression of a target gene.

21. 21. The method or system of claim 20, wherein the pharmaceutical composition is an oligonucleotide.

22. 22. The method or system of claim 21, wherein the oligonucleotide is an antisense or siRNA.

23. 22. The method or system of claim 21, wherein the oligonucleotide provided sustained pharmacodynamics.

24. 20. The method or system of claim 18, wherein the modulatory REVERSIR compound of claim 1 is used to standardize and enable randomized treatment discontinuation of the treated members.

25. 20. The method or system of claim 18, wherein administration of the modulatory REVERSIR compound of claim 1 is blinded along with placebo administration of the pharmaceutical composition.

26. 20. The method or system of claim 18 or 19, wherein the modulatory REVERSIR compound of claim 1 induces washout in the treated subpopulation.

27. 20. The method or system of claim 18, wherein the modulatory REVERSIR compound of claim 1 resets the baseline of RNAi acidity.

28. 20. The method or system of claim 19, wherein the siRNA can be re-administered at least 1, 2, 3, or 4 weeks after the regulatory REVERSIR treatment.

29. 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 method or system of claim 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.

30. 20. The method or system of claim 18, wherein the first treatment period is until the completion of an open-label or single-blind study.

31. 20. The method or system of claim 18, wherein the second treatment period is from about 1 week to 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months.

32. 20. The method or system of claim 18, wherein the second treatment period is until completion of a clinical trial.

33. 33. The method or system of any one of claims 18 to 32, comprising a clinical trial or a system for conducting a clinical trial to validate said pharmaceutical agent.

34. 34. The method or system of any one of claims 18 to 33, for evaluating the effectiveness of a treatment regimen using said pharmaceutical agent.

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