WO2022094615A1 - Compounds and methods for increased antisense activity - Google Patents

Compounds and methods for increased antisense activity Download PDF

Info

Publication number
WO2022094615A1
WO2022094615A1 PCT/US2021/072140 US2021072140W WO2022094615A1 WO 2022094615 A1 WO2022094615 A1 WO 2022094615A1 US 2021072140 W US2021072140 W US 2021072140W WO 2022094615 A1 WO2022094615 A1 WO 2022094615A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
nucleic acid
alkyl
antisense
certain embodiments
Prior art date
Application number
PCT/US2021/072140
Other languages
French (fr)
Inventor
Aimee EDINGER
Stephen Hanessian
Michael TANOWITZ
Punit P. Seth
Brendan T. FINICLE
Original Assignee
The Regents Of The University Of California
Ionis Pharmaceuticals, Inc.
Université de Montréal
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Regents Of The University Of California, Ionis Pharmaceuticals, Inc., Université de Montréal filed Critical The Regents Of The University Of California
Publication of WO2022094615A1 publication Critical patent/WO2022094615A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/401—Proline; Derivatives thereof, e.g. captopril
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy

Definitions

  • the disclosure is generally directed to compounds and methods for increasing activity of an antisense compound, including compounds that agonize PP2A activity.
  • RNAi refers to antisense-mediated gene silencing through a mechanism that utilizes the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • RNA target function is by an occupancy-based mechanism such as is employed naturally by microRNA.
  • MicroRNA molecules are small non-coding RNAs that regulate the expression of protein-coding RNAs. The binding of an antisense compound to a microRNA prevents that microRNA from binding to its messenger RNA targets, and thus interferes with the function of the microRNA. MicroRNA mimics can enhance native microRNA function. Certain antisense compounds alter splicing of pre- mRNA. Regardless of the specific mechanism, sequence-specificity makes antisense compounds attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in the pathogenesis of diseases.
  • Antisense technology is an effective means for modulating the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications.
  • Chemically modified nucleosides may be incorporated into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target nucleic acid.
  • New chemical modifications have improved the potency and efficacy of antisense compounds, uncovering the potential for oral delivery as well as enhancing subcutaneous administration, decreasing potential for side effects, and leading to improvements in patient convenience.
  • New compounds or methods that increase the potency of antisense compounds would allow administration of lower doses, which reduces the potential for toxicity, as well as decreasing overall cost of therapy.
  • new compounds or methods that increase the potency of antisense compounds or that improve cellular uptake of antisense compounds would result in slower clearance from the body, allowing for less frequent dosing.
  • various tissues and cell types, such as cancer cells are highly resistant to antisense compound uptake.
  • New compounds and methods that improve cellular uptake of antisense compounds would permit antisense compound methodologies to be performed on these traditionally resistant tissues and cell types.
  • the present disclosure provides compounds and methods of increasing the efficacy and potency of antisense compounds, and in certain instances, promotes antisense compound uptake in cells and tissues that are traditionally resistant.
  • small molecule compounds and methods improve cellular uptake and/or potency of antisense compounds.
  • the resulting antisense activity is greater at a concentration of antisense compound than the antisense activity at the same concentration of the antisense compound in the absence of a small molecule compound.
  • FIG. 1 illustrates a mechanism of protein phosphatase 2 (PP2A) agonists increasing antisense activity through alteration of cellular trafficking, utilized in accordance with various embodiments.
  • P2A protein phosphatase 2
  • FIG. 2 illustrates reaction pathways for the production of therapeutic small molecule analogs, utilized in accordance with various embodiments.
  • FIG. 3 illustrates reaction pathways for the production of therapeutic small molecule analogs, utilized in accordance with various embodiments.
  • FIGs. 4A-4F provide data graphs showing sphingolipid-like molecules increase antisense targeting in accordance with various embodiments.
  • FIGs. 5A and 5B provide data graphs and tables showing sphingolipid-like molecules increase antisense compound activity in multiple cell lines in accordance with various embodiments.
  • FIGs. 6A-6D provide microscopy images and data graphs showing sphingolipid-like molecules decrease antisense compound accumulation in lysosomes in accordance with various embodiments.
  • FIGs. 7A and 7B provide microscopy images and data graphs showing antisense compound accumulate intracellularly and fail to reach the lysosome in cells treated with sphingolipid-like molecules in accordance with various embodiments.
  • FIGs. 8A-8F provide data graphs and microscopy images showing sphingolipid-like molecules increase antisense compound activity better than established antisense compound agonists in accordance with various embodiments.
  • FIGs. 9A-9D provide microscopy images and data graphs showing sphingolipid-like molecules decrease lysosomal accumulation of antisense compounds similar to established PlKfyve inhibitors but are more effective than PlKfyve inhibitors at increasing antisense compound activity in accordance with various embodiments.
  • FIGs. 10A-10D provide microscopy images and data graphs showing sphingolipid-like molecules increase intracellular accumulation of antisense compounds similar to established ARF6 inhibitors in accordance with various embodiments.
  • FIGs. 11A-11 F provide microscopy images and data graphs showing sphingolipid-like molecules decrease lysosomal accumulation and increase intracellular accumulation of antisense compounds similar to established PP2A agonists in accordance with various embodiments.
  • FIGs. 12A-12C provide data graphs showing Dihydro-C2-ceramide does not activate PP2A or increase antisense compound activity in accordance with various embodiments.
  • FIGs.13A-13C provide data graphs showing sphingolipid-like molecules increase antisense targeting in vivo within the liver and lungs in accordance with various embodiments.
  • an agonist of antisense activity is utilized in conjunction with an antisense compound to increase antisense activity of the antisense compound.
  • a biological cell is contacted with an agonist of antisense activity and an antisense compound to induce antisense activity in the biological cell.
  • an animal is administered an agonist of antisense activity and an antisense compound to induce antisense activity in the animal.
  • an agonist of antisense activity and an antisense compound is used in a course of treatment.
  • an antisense compound in accord with various embodiments, can be any compound inclusive of a nucleic acid having a sequence for hybridizing with a target to provide an antisense effect on that target.
  • nucleic acids within antisense compounds include (but are not limited to) antisense oligomers (ASOs), double stranded RNA molecules (dsRNA), small interfering RNA (siRNA), short-hairpin RNA molecules (shRNA), RNA/DNA hybrids, microRNA, and microRNA mimics.
  • ASOs antisense oligomers
  • dsRNA double stranded RNA molecules
  • siRNA small interfering RNA
  • shRNA short-hairpin RNA molecules
  • RNA/DNA hybrids microRNA, and microRNA mimics.
  • antisense compounds are taken up into cells through endocytosis. Once an antisense compound comes into contact with a cell, the antisense compound can interact with the cell surface, where multiple pathways can lead to internalization of the antisense compound (Fig. 1 ). Antisense compounds are most active when within the cell and available to contact RNA compounds therein. There are several factors, however, that can prevent antisense compounds from performing their activity. For instance, the cytosolic enzyme ADP Ribosylation Factor 6 (ARF6) induces some antisense compoundcontaining endocytic vesicles to be recycled, fusing with the plasma membrane and eliminating the antisense compound from the cell.
  • ADP Ribosylation Factor 6 ADP Ribosylation Factor 6
  • endosomes that are not recycled are eventually trafficked to the lysosome via multivesicular bodies (MVBs), resulting in antisense compound inactivation.
  • Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing (PlKfyve) is one enzyme that promotes lysosome fusion.
  • the present disclosure provides compounds and methods of increasing antisense activity through blocking or slowing endosome recycling and lysosomal fusion.
  • endosome recycling is reduced by utilizing ARF6 antagonists.
  • lysosomal fusion is reduced by utilizing PlKfyve antagonists.
  • protein phosphatase 2 (PP2A) agonists inhibit endosome recycling and lysosomal fusion. Accordingly, in certain embodiments, endosome recycling and lysosomal fusion is reduced utilizing PP2A agonists.
  • PP2A protein phosphatase 2
  • Certain cell types demonstrate excellent uptake and activity of antisense compounds. Other cell types, however, demonstrate reduced uptake and/or activity of antisense compounds.
  • the present disclosure provides compounds that increase the uptake and/or activity of antisense compounds in different cell types.
  • the present disclosure provides methods of increasing the amount or activity of an antisense compound in a cancer cell.
  • the present disclosure provides methods of increasing the amount or activity of an antisense compound in a pancreatic or breast cancer cell.
  • the present disclosure provides methods of increasing the amount or activity of an antisense compound in a tumor cell.
  • the present disclosure provides a method of increasing the amount or activity of an antisense compound in non-neoplastic cells.
  • 2’-deoxynucleoside means a nucleoside comprising 2’-H(H) furanosyl sugar moiety, as found in naturally occurring deoxyribonucleic acids (DNA).
  • a 2’-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).
  • 2’-substituted nucleoside or “2-modified nucleoside” means a nucleoside comprising a 2’-substituted or 2’-modified sugar moiety.
  • “2’-substituted” or “2-modified” in reference to a sugar moiety means a sugar moiety comprising at least one 2'-substituent group other than H or OH.
  • Antisense activity means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid.
  • antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.
  • antisense activity is a change in splicing of a pre-mRNA nucleic acid target.
  • antisense activity is an increase in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.
  • Antisense compound means a compound comprising an antisense nucleic acid and optionally one or more additional features, such as a conjugated group or a terminal group.
  • Nucleic acids within antisense compounds include (but are not limited to) antisense oligomers (ASOs), double stranded RNA molecules (dsRNA), small interfering RNA (siRNA), short-hairpin RNA molecules (shRNA), RNA/DNA hybrids, microRNA, microRNA mimics, and derivatives thereof.
  • ASOs antisense oligomers
  • dsRNA double stranded RNA molecules
  • siRNA small interfering RNA
  • shRNA short-hairpin RNA molecules
  • RNA/DNA hybrids microRNA, microRNA mimics, and derivatives thereof.
  • Antisense oligonucleotide means an oligonucleotide that (1 ) has a nucleobase sequence that is at least partially complementary to a target nucleic acid and that (2) is capable of producing an antisense activity in a cell or animal.
  • “Ameliorate” in reference to a treatment means improvement in at least one symptom relative to the same symptom in the absence of the treatment.
  • amelioration is the reduction in the severity or frequency of a symptom or the delayed onset or slowing of progression in the seventy or frequency of a symptom.
  • Bicyclic nucleoside or “BNA” means a nucleoside comprising a bicyclic sugar moiety.
  • bicyclic sugar or “bicyclic sugar moiety” means a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure.
  • the first ring of the bicyclic sugar moiety is a furanosyl moiety.
  • the bicyclic sugar moiety does not comprise a furanosyl moiety.
  • Branching group means a group of atoms having at least 3 positions that are capable of forming covalent linkages to at least 3 groups.
  • a branching group provides a plurality of reactive sites for connecting tethered ligands to an oligonucleotide via a conjugate linker and/or a cleavable moiety.
  • Cell-targeting moiety means a conjugate group or portion of a conjugate group that is capable of binding to a particular cell type or particular cell types.
  • “Cleavable moiety” means a bond or group of atoms that is cleaved under physiological conditions, for example, inside a cell, an animal, or a human.
  • “Complementary” in reference to an oligonucleotide means that at least 70% of the nucleobases of such oligonucleotide or one or more regions thereof and the nucleobases of another nucleic acid or one or more regions thereof are capable of hydrogen bonding with one another when the nucleobase sequence of the oligonucleotide and the other nucleic acid are aligned in opposing directions.
  • Complementary nucleobases means nucleobases that are capable of forming hydrogen bonds with one another.
  • Complementary nucleobase pairs include, but unless otherwise specified are not limited to, adenine (A) and thymine (T), adenine (A) and uracil (II), cytosine (C) and guanine (G), 5-methyl cytosine ( m C) and guanine (G).
  • Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated.
  • oligonucleotides are complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide.
  • Conjugate group means a group of atoms that is directly or indirectly attached to an oligonucleotide.
  • Conjugate groups include a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.
  • Conjugate linker means a group of atoms comprising at least one bond that connects a conjugate moiety to an oligonucleotide.
  • Conjugate moiety means a group of atoms that is attached to an oligonucleotide via a conjugate linker.
  • Contiguous in the context of an oligonucleotide refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to each other.
  • contiguous nucleobases means nucleobases that are immediately adjacent to each other in a sequence.
  • Duplex means two oligomeric compounds that are paired. In certain embodiments, the two oligomeric compounds are paired via hybridization of complementary nucleobases.
  • Extra-hepatic cell type means a cell type that is not a hepatocyte.
  • Extra-hepatic nucleic acid target means a target nucleic acid that is expressed in tissues other than liver.
  • extra-hepatic nucleic acid targets are not expressed in the liver or not expressed in the liver at a significant level.
  • extra-hepatic nucleic acid targets are expressed outside the liver and also in the liver.
  • Extra-hepatic disease means a disease or condition where one or more symptoms or causes of the disease or condition occur in tissues other than liver.
  • Extra-hepatic tissue means a tissue other than liver.
  • “Fully modified” in reference to a modified oligonucleotide means a modified oligonucleotide in which each sugar moiety is modified.
  • “Uniformly modified” in reference to a modified oligonucleotide means a fully modified oligonucleotide in which each sugar moiety is the same.
  • the nucleosides of a uniformly modified oligonucleotide can each have a 2’-MOE modification but different nucleobase modifications, and the internucleoside linkages may be different.
  • “Gapmer” means an antisense compound comprising an internal region having a plurality of nucleosides that support RNase H cleavage positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions.
  • the internal region may be referred to as the “gap” and the external regions may be referred to as the “wings.”
  • “Hybridization” means the pairing or annealing of complementary oligonucleotides and/or nucleic acids. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
  • Inhibiting the expression or activity refers to a reduction or blockade of the expression or activity relative to the expression of activity in an untreated or control sample and does not necessarily indicate a total elimination of expression or activity.
  • Internucleoside linkage means a group or bond that forms a covalent linkage between adjacent nucleosides in an oligonucleotide.
  • modified internucleoside linkage means any internucleoside linkage other than a naturally occurring, phosphodiester internucleoside linkage. Non-phosphate linkages are referred to herein as modified internucleoside linkages.
  • Phosphorothioate linkage means a modified phosphodiester linkage in which one of the non-bridging oxygen atoms is replaced with a sulfur atom.
  • a phosphorothioate internucleoside linkage is a modified internucleoside linkage.
  • Linker-nucleoside means a nucleoside that links, either directly or indirectly, an oligonucleotide to a conjugate moiety. Linker-nucleosides are located within the conjugate linker of an oligomeric compound. Linker-nucleosides are not considered part of the oligonucleotide portion of an oligomeric compound even if they are contiguous with the oligonucleotide.
  • Lipophilic group or “lipophilic” in reference to a chemical group means a group of atoms that is more soluble in lipids or organic solvents than in water and/or has a higher affinity for lipids than for water.
  • lipophilic groups comprise a lipid.
  • lipid means a molecule that is not soluble in water or is less soluble in water than in organic solvents.
  • compounds of the present invention comprise lipids selected from saturated or unsaturated fatty acids, steroids, fat soluble vitamins, phospholipids, sphingolipids, hydrocarbons, mono-, di-, and triglycerides, and synthetic derivatives thereof.
  • Non-bicyclic modified sugar or “non-bicyclic modified sugar moiety” means a modified sugar moiety that comprises a modification, such as a substituent, that does not form a bridge between two atoms of the sugar to form a second ring.
  • Linked nucleosides are nucleosides that are connected in a continuous sequence (/.e. no additional nucleosides are present between those that are linked).
  • mismatch or “non-complementary” means a nucleobase of a first oligonucleotide that is not complementary with the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligomeric compound are aligned.
  • MOE means methoxyethyl.
  • 2’-MOE means a -OCH2CH2OCH3 group at the 2’ position of a furanosyl ring.
  • Motif means the pattern of unmodified and/or modified sugar moieties, nucleobases, and/or internucleoside linkages, in an oligonucleotide.
  • Multi-tissue disease or condition means a disease or condition affects or is affected by more than one tissue. In treating a multi-tissue disease or condition, it is desirable to affect more than one tissue type. In certain embodiments, treatment of disease or condition may be enhanced by treating the disease or condition in multiple tissues. For example, in certain embodiments, a disease or condition may manifest itself in the liver tissue and the muscle tissue. In certain embodiments, treating the disease or condition in the liver tissue and the muscle tissue will be more effective than treating the disease in either the liver tissue or the muscle tissue.
  • Nucleobase means an unmodified nucleobase or a modified nucleobase.
  • an “unmodified nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (II), and guanine (G).
  • a “modified nucleobase” is a group of atoms other than unmodified A, T, C, II, or G capable of pairing with at least one unmodified nucleobase.
  • a universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases.
  • nucleobase sequence means the order of contiguous nucleobases in a nucleic acid or oligonucleotide independent of any sugar or internucleoside linkage modification.
  • Nucleoside means a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each, independently, unmodified or modified.
  • modified nucleoside means a nucleoside comprising a modified nucleobase and/or a modified sugar moiety. Modified nucleosides include abasic nucleosides, which lack a nucleobase.
  • Olemeric compound means a compound consisting of an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group.
  • Oligonucleotide means a strand of linked nucleosides connected via internucleoside linkages, wherein each nucleoside and internucleoside linkage may be modified or unmodified. Unless otherwise indicated, oligonucleotides consist of 8-50 linked nucleosides. As used herein, “modified oligonucleotide” means an oligonucleotide, wherein at least one nucleoside or internucleoside linkage is modified. As used herein, “unmodified oligonucleotide” means an oligonucleotide that does not comprise any nucleoside modifications or internucleoside modifications.
  • “Pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal. Certain such carriers enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspension and lozenges for the oral ingestion by a subject.
  • a pharmaceutically acceptable carrier or diluent is sterile water; sterile saline; or sterile buffer solution.
  • “Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of compounds, such as oligomeric compounds, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • “Pharmaceutical composition” means a mixture of substances suitable for administering to a subject.
  • a pharmaceutical composition may comprise an antisense compound and a sterile aqueous solution.
  • a pharmaceutical composition shows activity in free uptake assay in certain cell lines.
  • “Phosphorus moiety” means a group of atoms comprising a phosphorus atom.
  • a phosphorus moiety comprises a mono-, di-, or tri-phosphate, or phosphorothioate.
  • Prodrug means a therapeutic agent in a form outside the body that is converted to a different form within the body or cells thereof. Typically, conversion of a prodrug within the body is facilitated by the action of an enzymes (e.g., endogenous or viral enzyme) or chemicals present in cells or tissues and/or by physiologic conditions.
  • an enzymes e.g., endogenous or viral enzyme
  • chemicals present in cells or tissues and/or by physiologic conditions.
  • RNAi compound means an antisense compound that acts, at least in part, through RISC or Ago2 to modulate a target nucleic acid and/or protein encoded by a target nucleic acid.
  • RNAi compounds include, but are not limited to double-stranded siRNA, single-stranded RNA (ssRNA), and microRNA, including microRNA mimics.
  • an RNAi compound modulates the amount, activity, and/or splicing of a target nucleic acid.
  • the term RNAi compound excludes antisense compounds that exclusively act through RNase H.
  • Single-stranded in reference to an oligomeric compound means such a compound that is not paired with a second oligomeric compound to form a duplex.
  • Self- complementary in reference to an oligonucleotide means an oligonucleotide that at least partially hybridizes to itself.
  • a single-stranded antisense or oligomeric compound may be capable of binding to a complementary oligomeric compound to form a duplex, in which case it would no longer be single-stranded.
  • “Sugar moiety” means an unmodified sugar moiety or a modified sugar moiety.
  • “unmodified sugar moiety” means a 2’-OH(H) furanosyl moiety, as found in RNA (an “unmodified RNA sugar moiety”), or a 2’-H(H) moiety, as found in DNA (an “unmodified DNA sugar moiety”).
  • Unmodified sugar moieties have one hydrogen at each of the T, 3’, and 4’ positions, an oxygen at the 3’ position, and two hydrogens at the 5’ position.
  • “modified sugar moiety” or “modified sugar” means a modified furanosyl sugar moiety or a sugar surrogate.
  • modified furanosyl sugar moiety means a furanosyl sugar comprising a non-hydrogen substituent in place of at least one hydrogen of an unmodified sugar moiety.
  • a modified furanosyl sugar moiety is a 2’-substituted sugar moiety.
  • modified furanosyl sugar moieties include bicyclic sugars and non-bicyclic sugars.
  • sugar surrogate means a modified sugar moiety having other than a furanosyl moiety that can link a nucleobase to another group, such as an internucleoside linkage, conjugate group, or terminal group in an oligonucleotide.
  • Modified nucleosides comprising sugar surrogates can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to complementary oligomeric compounds or nucleic acids.
  • Target nucleic acid means an identified nucleic acid to which an antisense compound is designed to hybridize to at least a portion of the nucleic acid.
  • target nucleic acids are endogenous cellular nucleic acids, including, but not limited to RNA transcripts, pre-mRNA, mRNA, microRNA.
  • target nucleic acids are viral nucleic acids.
  • target nucleic acids are nucleic acids that an antisense compound is designed to affect.
  • Target region means a portion of a target nucleic acid to which an antisense compound is designed to hybridize.
  • TCA motif means three nucleosides having the nucleobase sequence TCA (5’-3’). Such nucleosides may have modified sugar moieties and/or modified internucleosides linkages. Unless otherwise indicated, the nucleosides of TCA motifs comprise unmodified 2’-deoxy sugar moieties and unmodified phosphodiester internucleoside linkages.
  • Terminal group means a chemical group or group of atoms that is covalently linked to a terminus of an oligonucleotide.
  • Alcohol means a compound with an -OH group bonded to a saturated, alkane-like compound, (ROH).
  • Alkyl refers to the partial structure that remains when a hydrogen atom is removed from an alkane.
  • Alkane means a compound of carbon and hydrogen that contains only single bonds.
  • Alkyne refers to a hydrocarbon structure that contains a carbon-carbon triple bond.
  • Alkoxy refers to a portion of a molecular structure featuring an alkyl group bonded to an oxygen atom.
  • Aryl refers to any functional group or substituent derived from an aromatic ring.
  • “Amine” molecules are compounds containing one or more organic substituents bonded to a nitrogen atom, RNH 2 , R 2 NH, or R3N.
  • amino acid refers to a difunctional compound with an amino group on the carbon atom next to the carboxyl group, RCH(NH 2 )CO 2 H.
  • Cyanide refers to CN.
  • Ester is a compound containing the -CO 2 R functional group.
  • Ether refers to a compound that has two organic substituents bonded to the same oxygen atom, i.e. , R-O-R’.
  • Halogen or “halo” means fluoro (F), chloro (Cl), bromo (Br), or iodo (I).
  • Hydrocarbon means an organic chemical compound that consists entirely of the elements carbon (C) and hydrogen (H).
  • Phosphate means a compound containing the elements phosphorous (P) and oxygen (O).
  • R in the molecular formula above and throughout are meant to indicate any suitable organic molecule.
  • the disclosure provides oligonucleotides, which consist of linked nucleosides.
  • Oligonucleotides may be unmodified oligonucleotides (RNA or DNA) or may be modified oligonucleotides.
  • Modified oligonucleotides comprise at least one modification relative to unmodified RNA or DNA (i.e. , comprise at least one modified nucleoside (comprising a modified sugar moiety and/or a modified nucleobase) and/or at least one modified internucleoside linkage).
  • Modified nucleosides comprise a modified sugar moiety or a modified nucleobase or both a modified sugar moiety and a modified nucleobase.
  • modified sugar moieties are non-bicyclic modified sugar moieties.
  • modified sugar moieties are bicyclic or tricyclic sugar moieties.
  • modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of other types of modified sugar moieties.
  • modified sugar moieties are non-bicyclic modified sugar moieties comprising a furanosyl ring with one or more acyclic substituent, including but not limited to substituents at the 2’, 4’, and/or 5’ positions.
  • one or more acyclic substituent of non-bicyclic modified sugar moieties is branched.
  • 2’-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 2’-F, 2'-OCH3 (“OMe” or “O-methyl”), and 2'-O(CH2)2OCH3 (“MOE”).
  • these 2'-substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl.
  • Examples of 4’-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al., WO 2015/106128.
  • Examples of 5’ -substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 5’-methyl (R or S), 5' -vinyl, and 5’-methoxy.
  • non-bicyclic modified sugars comprise more than one non-bridging sugar substituent, for example, 2'-F-5'-methyl sugar moieties and the modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008/101157 and Rajeev et al., US2013/0203836.).
  • a non-bridging 2’-substituent group selected from: F, NH 2
  • a 2’-substituted nucleoside or 2’- non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2’-substituent group selected from: F, OCH3, and OCH 2 CH 2 OCH3.
  • Nucleosides comprising modified sugar moieties may be referred to by the position(s) of the substitution(s) on the sugar moiety of the nucleoside.
  • nucleosides comprising 2’-substituted or 2- modified sugar moieties are referred to as 2’-substituted nucleosides or 2-modified nucleosides.
  • modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety.
  • the bicyclic sugar moiety comprises a bridge between the 4' and the 2' furanose ring atoms.
  • Examples of such 4’ to 2’ bridging sugar substituents include but are not limited to: 4'- CH 2 -2', 4'-(CH 2 ) 2 -2', 4'-(CH 2 ) 3 -2', 4'-CH 2 -O-2' (“LNA”), 4'-CH 2 -S-2', 4'-(CH 2 ) 2 -O-2' (“ENA”), 4'-CH(CH3)-O-2' (referred to as “constrained ethyl” or “cEt” when in the S configuration), 4’-CH 2 -O-CH 2 -2’, 4’-CH 2 -N(R)-2’, 4'-CH(CH 2 OCH 3 )-O-2' (“constrained MOE” or “cMOE”) and analogs thereof (see, e.g., Seth et al., U.S.
  • each R, R a , and R b is, independently, H, a protecting group, or Ci-Ci 2 alkyl (see, e.g. Imanishi et al., U.S. 7,427,672).
  • bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration.
  • an LNA nucleoside (described herein) may be in the a-L configuration or in the
  • modified sugar moieties comprise one or more nonbridging sugar substituent and one or more bridging sugar substituent (e.g., 5’ -substituted and 4’-2’ bridged sugars).
  • modified sugar moieties are sugar surrogates.
  • the oxygen atom of the sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen atom.
  • such modified sugar moieties also comprise bridging and/or non-bridging substituents as described herein.
  • certain sugar surrogates comprise a 4’-sulfur atom and a substitution at the 2'- position (see, e.g., Bhat et al., U.S. 7,875,733 and Bhat et al., U.S. 7,939,677) and/or the 5’ position.
  • sugar surrogates comprise rings having other than 5 atoms.
  • a sugar surrogate comprises a sixmembered tetrahydropyran (“THP”).
  • TTP tetrahydropyrans
  • Such tetrahydropyrans may be further modified or substituted.
  • Nucleosides comprising such modified tetrahydropyrans include but are not limited to hexitol nucleic acid (“HNA”), anitol nucleic acid (“ANA”), manitol nucleic acid (“MNA”) (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841 -854), fluoro HNA:
  • F-HNA see e.g.Swayze et al., U.S. 8,088,904; Swayze et al., U.S.
  • F-HNA can also be referred to as a F-THP or 3' -fluoro tetrahydropyran), and nucleosides comprising additional modified THP compounds having the formula: wherein, independently, for each of said modified THP nucleoside: Bx is a nucleobase moiety;
  • T3 and T4 are each, independently, an internucleoside linking group linking the modified THP nucleoside to the remainder of an oligonucleotide or one of T3 and T4 is an internucleoside linking group linking the modified THP nucleoside to the remainder of an oligonucleotide and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or 3'-terminal group; qi, q2, qs, q4, qs, qe and q?
  • modified THP nucleosides are provided wherein qi, q2, qs, q4, qs, qe and q? are each H. In certain embodiments, at least one of qi, q2, qs, q4, qs, qe and q? is other than H. In certain embodiments, at least one of qi, q2, qs, q4, qs, qe and q? is methyl. In certain embodiments, modified THP nucleosides are provided wherein one of R1 and R2 is F. In certain embodiments, R1 is F and R2 is H, in certain embodiments, R1 is methoxy and R2 is H, and in certain embodiments, R1 is methoxyethoxy and R2 is H.
  • sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom.
  • nucleosides comprising morpholino sugar moieties and their use in oligonucleotides have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. 5,698,685; Summerton et al., U.S. 5,166,315; Summerton et al., U.S. 5,185,444; and Summerton et al., U.S. 5,034,506).
  • morpholino means a sugar surrogate having the following structure: [0115]
  • morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure.
  • sugar surrogates are referred to herein as “modified morpholinos.”
  • sugar surrogates comprise acyclic moieites.
  • nucleosides and oligonucleotides comprising such acyclic sugar surrogates include but are not limited to: peptide nucleic acid (“PNA”), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and nucleosides and oligonucleotides described in Manoharan et al., WO2011/133876.
  • modified oligonucleotides comprise one or more nucleoside comprising an unmodified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside that does not comprise a nucleobase, referred to as an abasic nucleoside.
  • modified nucleobases are selected from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6 substituted purines.
  • modified nucleobases are selected from: 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2- propyladenine , 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-C ⁇ C-CHs) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyla
  • nucleobases include tricyclic pyrimidines, such as 1 ,3-diazaphenoxazine-2-one, 1 ,3- diazaphenothiazine-2-one and 9-(2-aminoethoxy)-1 ,3-diazaphenoxazine-2-one (G- clamp).
  • Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine and 2-pyridone.
  • Further nucleobases include those disclosed in Merigan et al., U.S.
  • nucleosides of modified oligonucleotides may be linked together using any internucleoside linkage.
  • the two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom.
  • Modified internucleoside linkages compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide.
  • internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers.
  • Representative chiral internucleoside linkages include but are not limited to alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous- containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.
  • Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research', Y.S. Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2 component parts.
  • modified oligonucleotides comprise one or more modified nucleoside comprising a modified sugar.
  • modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase.
  • modified oligonucleotides comprise one or more modified internucleoside linkage.
  • the modified, unmodified, and differently modified sugar moieties, nucleobases, and/or internucleoside linkages of a modified oligonucleotide define a pattern or motif.
  • the patterns of sugar moieties, nucleobases, and internucleoside linkages are each independent of one another.
  • a modified oligonucleotide may be described by its sugar motif, nucleobase motif and/or internucleoside linkage motif (as used herein, nucleobase motif describes the modifications to the nucleobases independent of the sequence of nucleobases).
  • oligonucleotides comprise one or more type of modified sugar and/or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or sugar motif.
  • sugar motifs include but are not limited to any of the sugar modifications discussed herein.
  • modified oligonucleotides comprise or consist of a region having a gapmer motif, which comprises two external regions or “wings” and a central or internal region or “gap.”
  • the three regions of a gapmer motif (the 5’-wing, the gap, and the 3’ -wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap.
  • the sugar moieties of the nucleosides of each wing that are closest to the gap differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap (i.e., the wing/gap junction).
  • the sugar moieties within the gap are the same as one another.
  • the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap.
  • the sugar motifs of the two wings are the same as one another (symmetric gapmer).
  • the sugar motif of the 5'-wing differs from the sugar motif of the 3' -wing (asymmetric gapmer).
  • the wings of a gapmer comprise 1 -5 nucleosides. In certain embodiments, the wings of a gapmer comprise 2-5 nucleosides. In certain embodiments, the wings of a gapmer comprise 3-5 nucleosides. In certain embodiments, the nucleosides of a gapmer are all modified nucleosides. [0127] In certain embodiments, the gap of a gapmer comprises 7-12 nucleosides. In certain embodiments, the gap of a gapmer comprises 7-10 nucleosides. In certain embodiments, the gap of a gapmer comprises 8-10 nucleosides. In certain embodiments, the gap of a gapmer comprises 10 nucleosides. In certain embodiment, each nucleoside of the gap of a gapmer is an unmodified 2’-deoxy nucleoside.
  • the gapmer is a deoxy gapmer.
  • the nucleosides on the gap side of each wing/gap junction are unmodified 2’-deoxy nucleosides and the nucleosides on the wing sides of each wing/gap junction are modified nucleosides.
  • each nucleoside of the gap is an unmodified 2’-deoxy nucleoside.
  • each nucleoside of each wing is a modified nucleoside.
  • modified oligonucleotides comprise or consist of a region having a fully modified sugar motif.
  • each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety.
  • each nucleoside to the entire modified oligonucleotide comprises a modified sugar moiety.
  • modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif.
  • a fully modified oligonucleotide is a uniformly modified oligonucleotide.
  • each nucleoside of a uniformly modified comprises the same 2’-modification.
  • oligonucleotides comprise modified and/or unmodified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or motif.
  • each internucleoside linking group of a modified oligonucleotide is independently selected from a phosphorothioate and phosphate internucleoside linkage.
  • the sugar motif of a modified oligonucleotide is a gapmer and the internucleoside linkages within the gap are all modified.
  • some or all of the internucleoside linkages in the wings are unmodified phosphate linkages.
  • the terminal internucleoside linkages are modified.
  • oligonucleotides can have any of a variety of ranges of lengths.
  • oligonucleotides consist of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number nucleosides in the range.
  • 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.
  • oligonucleotides consist of 12 to 13, 12 to 14, 12 to 15,
  • the above modifications are incorporated into a modified oligonucleotide.
  • modified oligonucleotides are characterized by their modification motifs and overall lengths. In certain embodiments, such parameters are each independent of one another. Thus, unless otherwise indicated, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications.
  • the internucleoside linkages within the wing regions of a sugar gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region of the sugar motif.
  • sugar gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications.
  • an oligonucleotide is described by an overall length or range and by lengths or length ranges of two or more regions (e.g., a region of nucleosides having specified sugar modifications), in such circumstances it may be possible to select numbers for each range that result in an oligonucleotide having an overall length falling outside the specified range.
  • a modified oligonucleotide consists if of 15-20 linked nucleosides and has a sugar motif consisting of three regions, A, B, and C, wherein region A consists of 2-6 linked nucleosides having a specified sugar motif, region B consists of 6-10 linked nucleosides having a specified sugar motif, and region C consists of 2-6 linked nucleosides having a specified sugar motif.
  • Such embodiments do not include modified oligonucleotides where A and C each consist of 6 linked nucleosides and B consists of 10 linked nucleosides (even though those numbers of nucleosides are permitted within the requirements for A, B, and C) because the overall length of such oligonucleotide is 22, which exceeds the upper limit of the overall length of the modified oligonucleotide (20).
  • a and C each consist of 6 linked nucleosides and B consists of 10 linked nucleosides (even though those numbers of nucleosides are permitted within the requirements for A, B, and C) because the overall length of such oligonucleotide is 22, which exceeds the upper limit of the overall length of the modified oligonucleotide (20).
  • a description of an oligonucleotide is silent with respect to one or more parameter, such parameter is not limited.
  • a modified oligonucleotide described only as having a gapmer sugar motif without further description may have any
  • oligonucleotides are further described by their nucleobase sequence.
  • oligonucleotides have a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid.
  • a region of an oligonucleotide has a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid.
  • the nucleobase sequence of a region or entire length of an oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% complementary to the second oligonucleotide or nucleic acid, such as a target nucleic acid.
  • the disclosure provides oligomeric compounds, which consist of an oligonucleotide (modified or unmodified) and optionally one or more conjugate groups and/or terminal groups.
  • Conjugate groups consist of one or more conjugate moiety and a conjugate linker which links the conjugate moiety to the oligonucleotide.
  • Conjugate groups may be attached to either or both ends of an oligonucleotide and/or at any internal position.
  • conjugate groups are attached to the 2'-position of a nucleoside of a modified oligonucleotide.
  • conjugate groups that are attached to either or both ends of an oligonucleotide are terminal groups.
  • conjugate groups or terminal groups are attached at the 3’ and/or 5’-end of oligonucleotides. In certain such embodiments, conjugate groups (or terminal groups) are attached at the 3’-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 3’-end of oligonucleotides. In certain embodiments, conjugate groups (or terminal groups) are attached at the 5’-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 5’-end of oligonucleotides.
  • terminal groups include but are not limited to conjugate groups, capping groups, phosphate moieties, protecting groups, modified or unmodified nucleosides, and two or more nucleosides that are independently modified or unmodified.
  • oligonucleotides are covalently attached to one or more conjugate groups.
  • conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance.
  • conjugate groups impart a new property on the attached oligonucleotide, e.g., fluorophores or reporter groups that enable detection of the oligonucleotide.
  • conjugate groups and conjugate moieties have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci.
  • oligomeric compounds are metabolized in the liver or kidneys, which can reduce the half-life of the oligomeric compound in a subject.
  • an oligomeric compound administered to a subject may distribute to the kidneys and then be excreted out in the subject’s urine.
  • conjugating an oligomeric compound may lead to metabolism in the liver.
  • an oligomeric compound administered to a subject is both metabolized by the liver and excreted out through the kidneys.
  • the present disclosure provides oligomeric compounds comprising a modified oligonucleotide and a conjugate group, wherein the conjugate group enhances delivery of the modified oligonucleotide.
  • the conjugate group enhances delivery of the modified oligonucleotide to a tissue selected from among: skeletal muscle, cardiac muscle, smooth muscle, adipose, white adipose, spleen, bone, intestine, adrenal, testes, ovary, pancreas, pituitary, prostate, skin, uterus, bladder, brain, glomerulus, distal tubular epithelium, breast, lung, heart, kidney, ganglion, frontal cortex, spinal cord, trigeminal ganglia, sciatic nerve, dorsal root ganglion, epididymal fat, diaphragm, and colon.
  • a tissue selected from among: skeletal muscle, cardiac muscle, smooth muscle, adipose, white adipose, spleen, bone, intestine, adrenal, testes, ovary, pancreas, pituitary, prostate, skin, uterus, bladder, brain, glomerulus, distal tubular epithelium
  • Oligomeric compounds typically show good uptake in hepatocytes.
  • the present disclosure provides oligomeric compounds comprising a modified oligonucleotide and a conjugate group, wherein the conjugate group enhances uptake in a particular cell type.
  • the conjugate group enhances uptake in macrophages.
  • the conjugate group enhances uptake in cardiomyocytes.
  • the conjugate group enhances uptake in fibroblasts.
  • the conjugate group enhances uptake in endothelial cells.
  • the conjugate group enhances uptake in heart cells.
  • Conjugate moieties include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.
  • a conjugate moiety comprises a compound found endogenously in a subject.
  • the conjugate may be a steroid, such as cholesterol.
  • cholesterol is endogenously produced in a subject and has certain physiological activities, cholesterol may be used as a conjugate to alter or improve one or more properties of a modified oligonucleotide.
  • cholesterol conjugated to a modified oligonucleotide may increase the modified oligonucleotide’s binding affinity for a given protein, such as HDL.
  • a conjugate moiety comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5- triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • an active drug substance for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, car
  • conjugate moieties impart a new property on the attached oligonucleotide, which may alter the oligonucleotides distribution or pharmacokinetic profile.
  • certain conjugate moieties selected from among lipids, vitamins, steroids, C5-C30 saturated alkyl groups, C5-C30 unsaturated alkyl groups, fatty acids, or lipophilic groups may increase the distribution of an oligonucleotide to various tissues or organs within a subject.
  • certain conjugate moieties selected from among lipids, vitamins, steroids, C5-C30 saturated alkyl groups, C5-C30 unsaturated alkyl groups, fatty acids, or lipophilic groups increase affinity for an oligonucleotide with one or more serum proteins, such as albumin.
  • certain conjugate moieties selected from among lipids, vitamins, steroids, C5-C30 saturated alkyl groups, C5-C30 unsaturated alkyl groups, fatty acids, or lipophilic groups increase affinity for an oligonucleotide to an extra-hepatic tissue.
  • conjugated oligonucleotides are selected from among lipids, vitamins, steroids, C5-C30 saturated alkyl groups, C5-C30 unsaturated alkyl groups, fatty acids, or lipophilic groups increase affinity for an extra-hepatic tissue selected from among: skeletal muscle, cardiac muscle, smooth muscle, adipose, white adipose, spleen, bone, intestine, adrenal, testes, ovary, pancreas, pituitary, prostate, skin, uterus, bladder, brain, glomerulus, distal tubular epithelium, breast, lung, heart, kidney, ganglion, frontal cortex, spinal cord, trigeminal ganglia, sciatic nerve, dorsal root ganglion, epididymal fat, diaphragm
  • Conjugate moieties are attached to oligonucleotides through conjugate linkers.
  • the conjugate linker is a single chemical bond (i.e. , the conjugate moiety is attached directly to an oligonucleotide through a single bond).
  • the conjugate linker comprises a chain structure, such as a hydrocarbyl chain, or an oligomer of repeating units such as ethylene glycol, nucleosides, or amino acid units.
  • a conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises groups selected from alkyl, amino, oxo, amide and ether groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker includes at least one neutral linking group.
  • conjugate linkers are bifunctional linking moieties, e.g., those known in the art to be useful for attaching conjugate groups to parent compounds, such as the oligonucleotides provided herein.
  • a bifunctional linking moiety comprises at least two functional groups. One of the functional groups is selected to bind to a particular site on a parent compound and the other is selected to bind to a conjugate group. Examples of functional groups used in a bifunctional linking moiety include but are not limited to electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups.
  • bifunctional linking moieties comprise one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.
  • conjugate linkers include but are not limited to pyrrolidine, 8- amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA).
  • ADO 8- amino-3,6-dioxaoctanoic acid
  • SMCC succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate
  • AHEX or AHA 6-aminohexanoic acid
  • conjugate linkers include but are not limited to substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
  • conjugate linkers comprise 1 -10 linker-nucleosides. In certain embodiments, conjugate linkers comprise 2-5 linker-nucleosides. In certain embodiments, conjugate linkers comprise exactly 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise the TCA motif. In certain embodiments, such linker-nucleosides are modified nucleosides. In certain embodiments such linker- nucleosides comprise a modified sugar moiety. In certain embodiments, linker- nucleosides are unmodified.
  • linker-nucleosides comprise an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine.
  • a cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5- methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is typically desirable for linker-nucleosides to be cleaved from the oligomeric compound after it reaches a target tissue. Accordingly, linker- nucleosides are typically linked to one another and to the remainder of the oligomeric compound through cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.
  • linker-nucleosides are not considered to be part of the oligonucleotide. Accordingly, in embodiments in which an oligomeric compound comprises an oligonucleotide consisting of a specified number or range of linked nucleosides and/or a specified percent complementarity to a reference nucleic acid and the oligomeric compound also comprises a conjugate group comprising a conjugate linker comprising linker-nucleosides, those linker-nucleosides are not counted toward the length of the oligonucleotide and are not used in determining the percent complementarity of the oligonucleotide for the reference nucleic acid.
  • an oligomeric compound may comprise (1 ) a modified oligonucleotide consisting of 8-30 nucleosides and (2) a conjugate group comprising 1-10 linker-nucleosides that are contiguous with the nucleosides of the modified oligonucleotide.
  • the total number of contiguous linked nucleosides in such an oligomeric compound is more than 30.
  • an oligomeric compound may comprise a modified oligonucleotide consisting of 8-30 nucleosides and no conjugate group. The total number of contiguous linked nucleosides in such an oligomeric compound is no more than 30.
  • conjugate linkers comprise no more than 10 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 5 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 2 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 1 linker-nucleoside.
  • a conjugate group it is desirable for a conjugate group to be cleaved from the oligonucleotide.
  • oligomeric compounds comprising a particular conjugate moiety are better taken up by a particular cell type, but once the oligomeric compound has been taken up, it is desirable that the conjugate group be cleaved to release the unconjugated or parent oligonucleotide.
  • certain conjugate linkers may comprise one or more cleavable moieties.
  • a cleavable moiety is a cleavable bond.
  • a cleavable moiety is a group of atoms comprising at least one cleavable bond.
  • a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds.
  • a cleavable moiety is selectively cleaved inside a cell or subcellular compartment, such as a lysosome.
  • a cleavable moiety is selectively cleaved by endogenous enzymes, such as nucleases.
  • a cleavable bond is selected from among: an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide.
  • a cleavable bond is one or both of the esters of a phosphodiester.
  • a cleavable moiety comprises a phosphate or phosphodiester.
  • the cleavable moiety is a phosphate linkage between an oligonucleotide and a conjugate moiety or conjugate group.
  • a cleavable moiety comprises or consists of one or more linker-nucleosides.
  • the one or more linker- nucleosides are linked to one another and/or to the remainder of the oligomeric compound through cleavable bonds.
  • such cleavable bonds are unmodified phosphodiester bonds.
  • a cleavable moiety is 2'-deoxy nucleoside that is attached to either the 3' or 5'-terminal nucleoside of an oligonucleotide by a phosphate internucleoside linkage and covalently attached to the remainder of the conjugate linker or conjugate moiety by a phosphate or phosphorothioate linkage.
  • the cleavable moiety is 2'-deoxyadenosine.
  • antisense compounds which comprise or consist of an oligomeric compound comprising an antisense nucleic acid, having a nucleobase sequence complementary to that of a target nucleic acid.
  • antisense compounds are single-stranded.
  • Such single-stranded antisense compounds typically comprise or consist of an oligomeric compound that comprises or consists of a modified oligonucleotide and optionally a conjugate group.
  • antisense compounds are double-stranded.
  • Such double-stranded antisense compounds comprise a first oligomeric compound having a region complementary to a target nucleic acid and a second oligomeric compound having a region complementary to the first oligomeric compound.
  • the first oligomeric compound of such double stranded antisense compounds typically comprises or consists of a modified oligonucleotide and optionally a conjugate group.
  • the oligonucleotide of the second oligomeric compound of such double-stranded antisense compound may be modified or unmodified.
  • Either or both oligomeric compounds of a double-stranded antisense compound may comprise a conjugate group.
  • the oligomeric compounds of double-stranded antisense compounds may include non-complementary overhanging nucleosides.
  • oligomeric compounds of antisense compounds are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity.
  • antisense compounds selectively affect one or more target nucleic acid.
  • Such selective antisense compounds comprise a nucleobase sequence that hybridizes to one or more target nucleic acid, resulting in one or more desired antisense activity and does not hybridize to one or more non-target nucleic acid or does not hybridize to one or more non-target nucleic acid in such a way that results in significant undesired antisense activity.
  • hybridization of an antisense compound to a target nucleic acid results in recruitment of a protein that cleaves the target nucleic acid.
  • certain antisense compounds result in RNase H mediated cleavage of the target nucleic acid.
  • RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex.
  • the DNA in such an RNA:DNA duplex need not be unmodified DNA.
  • the invention provides antisense compounds that are sufficiently “DNA-like” to elicit RNase H activity. Further, in certain embodiments, one or more non-DNA-like nucleoside in the gap of a gapmer is tolerated.
  • an antisense compound or a portion of an antisense compound is loaded into an RNA-induced silencing complex (RISC), ultimately resulting in cleavage of the target nucleic acid.
  • RISC RNA-induced silencing complex
  • certain antisense compounds result in cleavage of the target nucleic acid by Argonaute.
  • Antisense compounds that are loaded into RISC are RNAi compounds. RNAi compounds may be double-stranded (siRNA) or single-stranded (ssRNA).
  • hybridization of an antisense compound to a target nucleic acid does not result in recruitment of a protein that cleaves that target nucleic acid. In certain such embodiments, hybridization of the antisense compound to the target nucleic acid results in alteration of splicing of the target nucleic acid. In certain embodiments, hybridization of an antisense compound to a target nucleic acid results in inhibition of a binding interaction between the target nucleic acid and a protein or other nucleic acid. In certain such embodiments, hybridization of an antisense compound to a target nucleic acid results in alteration of translation of the target nucleic acid.
  • Antisense activities may be observed directly or indirectly.
  • observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid, a change in the ratio of splice variants of a nucleic acid or a protein, and/or a phenotypic change in a cell or an animal.
  • antisense compounds comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid.
  • the target nucleic acid is an endogenous RNA molecule.
  • the target nucleic acid encodes a protein.
  • the target nucleic acid is selected from: an mRNA and a pre-mRNA, including intronic, exonic and untranslated regions.
  • the target RNA is an mRNA.
  • the target nucleic acid is a pre-mRNA.
  • the target region is entirely within an intron.
  • the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron.
  • the target nucleic acid is a non-coding RNA.
  • the target non-coding RNA is selected from: a long-non-coding RNA, a short non-coding RNA, an intronic RNA molecule, a snoRNA, a scaRNA, a microRNA (including pre-microRNA and mature microRNA), a ribosomal RNA, and promoter directed RNA.
  • the target nucleic acid is a nucleic acid other than a mature mRNA. In certain embodiments, the target nucleic acid is a nucleic acid other than a mature mRNA or a microRNA.
  • the target nucleic acid is a non-coding RNA other than a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA or an intronic region of a pre-mRNA. In certain embodiments, the target nucleic acid is a long non-coding RNA. In certain embodiments, the target nucleic acid is a non-coding RNA associated with splicing of other pre-mRNAs. In certain embodiments, the target nucleic acid is a nuclear-retained non-coding RNA.
  • antisense compounds described herein are complementary to a target nucleic acid comprising a single-nucleotide polymorphism (SNP).
  • the antisense compound is capable of modulating expression of one allele of the SNP-containing target nucleic acid to a greater or lesser extent than it modulates another allele.
  • an antisense compound hybridizes to a (SNP)-containing target nucleic acid at the single-nucleotide polymorphism site.
  • antisense compounds are at least partially complementary to more than one target nucleic acid.
  • antisense compounds of the present invention may mimic microRNAs, which typically bind to multiple targets.
  • antisense compounds comprise an antisense nucleic acid that are complementary to the target nucleic acid over the entire length of the oligonucleotide.
  • such oligonucleotides are 99% complementary to the target nucleic acid.
  • such oligonucleotides are 95% complementary to the target nucleic acid.
  • such oligonucleotides are 90% complementary to the target nucleic acid.
  • such oligonucleotides are 85% complementary to the target nucleic acid.
  • such oligonucleotides are 80% complementary to the target nucleic acid.
  • an antisense nucleic acid is at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide and comprise a region that is 100% or fully complementary to a target nucleic acid.
  • the region of full complementarity is from 6 to 20 nucleobases in length.
  • the region of full complementarity is from 10 to 18 nucleobases in length.
  • the region of full complementarity is from 18 to 20 nucleobases in length.
  • the oligomeric compounds of antisense compounds comprise one or more mismatched nucleobases relative to the target nucleic acid.
  • the mismatch is specifically positioned within an oligonucleotide having a gapmer motif. In certain such embodiments, the mismatch is at position 1 , 2, 3, 4, 5, 6, 7, or 8 from the 5’-end of the gap region. In certain such embodiments, the mismatch is at position 9, 8, 7, 6, 5, 4, 3, 2, 1 from the 3’-end of the gap region. In certain such embodiments, the mismatch is at position 1 , 2, 3, or 4 from the 5’-end of the wing region. In certain such embodiments, the mismatch is at position 4, 3, 2, or 1 from the 3’-end of the wing region.
  • protein phosphatase 2 (PP2A) agonists are agonists of antisense activity.
  • one or more antisense compounds are combined with one or more PP2A agonists to increase the activity of the oligomeric compounds.
  • PP2A agonists increase intracellular accumulation of oligomeric compounds within a biological cell.
  • PP2A agonists include (but are not limited to) sphingolipids, sphingolipid-like compounds, sphingolipid-like compound 893, sphingolipid-like compound 1090, sphingolipid-like compound 325, FTY720, derivatives of FTY720, ceramide, sphingosine, sphinganine, phytosphingosine, dimethylsphingosine, safingol, perphenazine, perphenazine derivatives (e.g., small molecule activator of PP2A (SMAP), iHAP (2-chloro-10-(4-methoxybenzoyl)-10H-phenothiazine)), SET inhibitors, CIP2a inhibitors, Withaferin A, OSLI-2S, and derivatives thereof.
  • SMAP small molecule activator of PP2A
  • iHAP iHAP (2-chloro-10-(4-methoxybenzoyl)-10H-phen
  • combining PP2A agonists with antisense compounds reduces the amount of oligomeric compound needed to produce the requisite antisense activity.
  • PP2A agonists increase antisense activity between 2- fold and 100-fold.
  • PP2A agonists increase antisense activity 2- fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55- fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, or greater than 100-fold.
  • ARF6 antagonists are agonists of antisense activity.
  • antisense compounds are combined with ARF6 antagonists. As discussed herein, ARF6 antagonists increase the ability of oligomeric compounds to accumulate within biological cells.
  • ARF6 antagonists include (but are not limited to) sphingolipids, sphingolipid-like compounds, sphingolipid-like compound 893, sphingolipid-like compound 1090, sphingolipid-like compound 325, FTY720, derivatives of FTY720, ceramide, NAV2729, SecinH3, perphenazine, and derivatives thereof. Numerous ARF6 antagonists are described in the literature and can be utilized in certain embodiments as described herein (see B. T. Finicle, et al., J Cell Sci. 2018;131 (12):jcs213314; J. H. Yoo, et al., Cancer Cell. 2016;29(6):889-904; and M.
  • combining ARF6 antagonists with PlKfyve antagonists and with antisense compounds reduces the amount of antisense compound needed to produce the requisite antisense activity.
  • ARF6 antagonists in combination with PlKfyve antagonists increase antisense activity between 2-fold and 100- fold.
  • ARF6 agonists increase antisense activity 2-fold, 5-fold, 10- fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, or greater than 100- fold.
  • inhibitors of multivesicular body fusion with lysosomes such as Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing (PlKfyve) antagonists are agonists of antisense activity.
  • antisense compounds are combined with PlKfyve antagonists to increase the activity of the oligomeric compounds. As discussed herein, PlKfyve antagonists increase the ability of oligomeric compounds to accumulate within biological cells.
  • PlKfyve antagonists include (but are not limited to) sphingolipids, sphingolipid-like compounds, sphingolipid-like compound 893, sphingolipid-like compound 1090, sphingolipid-like compound 325, FTY720, derivatives of FTY720, YM201636, APY0201 , Apilimod, Late Endosome Trafficking Inhibitor EGA, and derivatives thereof.
  • Numerous PlKfyve antagonists are described in the literature and can be utilized in certain embodiments as described herein (see S. M. Kim, et al., J Clin Invest. 2016;126(11 ):4088-4102; H. B. Jefferies, et al., EMBO Rep. 2008;9(2): 164-170; and X. Cai, et al., Chem Biol. 2013;20(7):912-921 ; the disclosures of which are incorporated by reference).
  • PlKfyve antagonists reduces the amount of oligomeric compound needed to produce the requisite antisense activity.
  • PlKfyve antagonists increase antisense activity between 2-fold and 100-fold.
  • PlKfyve agonists increase antisense activity 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, or greater than 100-fold.
  • an agonist of antisense activity is applied concurrently with an antisense compound. Accordingly, in certain embodiments, a biological cell is simultaneously contacted with an agonist of antisense activity and an antisense compound. In certain embodiments, an animal is simultaneously administered an agonist of antisense activity and antisense compound.
  • an agonist of antisense activity is applied as a pretreatment prior to an application of an antisense compound. Accordingly, in certain embodiments, a biological cell is pretreated with an agonist of antisense activity prior to contact with an antisense compound. In certain embodiments, a biological cell is pretreated by contacting the cell with an agonist of antisense activity for a time period of between 1 minute and 300 minutes prior to contact with an antisense compound.
  • a biological cell is pretreated by contacting the cell with an agonist of antisense activity for a time period of 1 minute, 2 minutes, 3, minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, 240 minutes, 270 minutes, 300 minutes, or more than 300 minutes prior to contact with an antisense compound.
  • an animal is pretreated by administration of an agonist of antisense activity for a time period of between 1 minute and 720 minutes prior to administration of an antisense compound.
  • an animal is pretreated by administration of an agonist antisense activity for a time period of 1 minute, 2 minutes, 3, minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, 240 minutes, 270 minutes, 300 minutes, 330 minutes, 360 minutes, 390 minutes, 420 minutes, 450 minutes, 480 minutes, 510 minutes, 540 minutes, 570 minutes, 600 minutes, 630 minutes, 660 minutes, 690 minutes, 720 minutes, or more than 720 minutes prior to administration of an antisense compound.
  • an agonist of antisense activity is utilized at concentration between 1 nM to 100 pM. In various embodiments, an agonist of antisense activity is utilized at a concentration less than 1 nM, approximately 1 nM, approximately 10 nM, approximately 100 nM, approximately 1 pM, approximately 10 pM, approximately 100 pM, or greater than 100 pM. When referring to concentration of agonists, approximately is to be interpreted as within an order of magnitude (e.g., approximately 1 nM is 1 nM to less than 10 nM).
  • an agonist of antisense activity is based on O-benzyl pyrrolidines. In certain embodiments, an agonist of antisense activity is of formula:
  • Ri is an optional functional group selected from an alkyl chain, (CH2)nOH, CHOH- alkyl, CHOH-alkyne, (CH2)nO-alkyl, (CH2)nO-alkene, (CH2)nO-alkyne, wherein an akyl, alkyne, or alkene is an aliphatic chain up to ten carbons;
  • R2 is an aliphatic chain (Ce - C10)
  • R3 is a mono-, di-, tri- or quad- aromatic substituent comprising H, halogen, alkyl, alkoxy, azide (N3), ether, NO2, or cyanide (CN);
  • R1 and R4 is an alcohol (CH2OH) or H;
  • L is O-CH2; and n is an independently selected integer selected from 1 , 2, or 3; wherein the phenyl can be moved about the five-carbon ring, e.g., extending from ring positions 3, 4, or 5.
  • the O-benzyl group can be moved to position 4 (shown above) or 3 as shown below: [0176] In certain embodiments, alkyl, CH2OH, or (CH2)nOH groups can be added to position 5.
  • one of R1 or R4 is an alkyl having 1 to 6 carbons.
  • an agonist of antisense activity is based on diastereomeric 3- and 4-C-aryl pyrrolidines.
  • an agonist of antisense activity is of formula:
  • R1 is an optional functional group selected from an alkyl chain, (CH2)nOH, CHOH- alkyl, CHOH-alkyne, (CH2)nO-alkyl, (CH2)nO-alkene, (CH2)nO-alkyne, wherein the akyl, the alkyne, or the alkene is an aliphatic chain up to ten carbons;
  • R2 is an aliphatic chain (Ce - C14);
  • R3 is a mono-, di-, tri- or tetra- aromatic substituent comprising hydrogen, halogen, alkyl, alkoxy, azide (N3), ether, NO2, or cyanide (CN); n is an independently selected integer selected from 1 , 2, or 3; and wherein the phenyl can be moved about the five-carbon ring, e.g., extending from ring positions 3 or 4, while maintaining the 3- and 4-C-aryl chemistries. [0180] In certain embodiments of diastereomeric 3- and 4-C-aryl 2-hydroxymethyl pyrrolidines, the C-aryl group can be moved to position 3 (shown above) or 4 as shown below:
  • alkyl, CH2OH, or (CH2)nOH groups can be added to position 5.
  • R2 is an unsaturated hydrocarbon chain.
  • the R1 is an alkyl having 1 to 6 carbons.
  • an agonist of antisense activity is compound 893, having the formula:
  • an agonist of antisense activity is compound 1090, having the formula: C. Sphingolipid-like Compounds Based on Azacycles with Heteroaromatic Appendage
  • an agonist of antisense activity is based on azacycles with an attached heteroaromatic appendage.
  • an agonist of antisense activity is of formula: or a pharmaceutically acceptable salt thereof;
  • R is an optionally substituted heteroaromatic moiety such as an optionally substituted pyridazine, optionally substituted pyridine, optionally substituted pyrimidine, phenoxazine, or optionally substituted phenothiazine.
  • Ri is H, alkyl such as C1-6 alkyl or C1-4 alkyl including methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, etc, Ac, Boc, guanidine moiety.
  • R2 is an aliphatic chain comprising 6 to 14 carbons.
  • R3 is a 1 , 2, 3, or 4 substituents, wherein each substituent, independently, is H, halogen, alkyl, alkoxy, N3, NO2, and CN.
  • n is independently 1 , 2, 3, or 4.
  • m is independently 1 or 2.
  • R2 is an unsaturated hydrocarbon chain.
  • R2 is C6-14 alkyl, C6-10 alkyl, C7-9 alkyl, CeH , C7H15, CsHn, C9H19, C10H21, C11H23, C12H25, C13H27, Or Cl4H29.
  • R3 is H.
  • n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4.
  • n is 1. In some embodiments, m is 2.
  • the R2 and R3 substituents can have different combinations around the phenyl ring with regard to their position.
  • the Ri is an alkyl having 1 to 6 carbons.
  • R is a 1 ,2-pyridazine having the formula:
  • R4 and Rs are functional groups independently selected from: alkyl including methyl, optionally substituted aryl (i.e., unsubstituted aryl or substituted aryl) including optionally substituted phenyl, and optionally substituted heteroaryl including optionally substituted pyridine and optionally substituted pyrimidine.
  • the pyridazine moiety is connected to the azacycle at the position 4 or 5 of the pyridazine.
  • any substituents of R4 and Rs, if present, are independently a halogen (including F), an alkyl, a terminal alkyne, or an azide.
  • R4 is C1-6 alkyl, such as CH3, C2 alkyl, C3 alkyl, C4 alkyl, Cs alkyl, or Ce alkyl; unsubstituted aryl or substituted aryl, including unsubstituted phenyl, or phenyl having 1 , 2, 3, 4, or 5 substituents; unsubstituted heteroaryl or substituted heteroaryl, including unsubstituted pyridine or pyridine having 1 , 2, 3, or 4 substituents, or unsubstituted pyrimidine or pyrimidine having 1 , 2, or 3 substituents.
  • any substituent may be used in the substituted aryl (e.g., substituted phenyl) or substituted heteroaryl (e.g., substituted pyridine or substituted pyrimidine).
  • Rs is C1-6 alkyl, such as CHs, C 2 alkyl, Cs alkyl, C4 alkyl, Cs alkyl, or Ce alkyl; unsubstituted aryl or substituted aryl, including unsubstituted phenyl, or phenyl having 1 , 2, 3, 4, or 5 substituents; unsubstituted heteroaryl or substituted heteroaryl, including unsubstituted pyridine or pyridine having 1 , 2, 3, or 4 substituents, or unsubstituted pyrimidine or pyrimidine having 1 , 2, or 3 substituents.
  • any substituent may be used in the substituted aryl (e.g., substituted phenyl) or substituted heteroaryl (e.g., substituted pyridine or substituted pyrimidine).
  • R4 and Rs are the same functional group. [0201] In some embodiments, R4 and Rs are different functional groups.
  • R4 is C1-6 alkyl, such as methyl, and Rs is optionally substituted phenyl.
  • R4 is C1-6 alkyl, such as methyl, and Rs is optionally substituted pyridine.
  • R4 is C1-6 alkyl, such as methyl, and Rs is optionally substituted pyrimidine.
  • R4 is optionally substituted pyridine and Rs is optionally substituted pyridine.
  • R4 is optionally substituted phenyl and Rs is optionally substituted phenyl.
  • R4 is optionally substituted phenyl and Rs is optionally substituted pyrimidine.
  • R is an optionally substituted phenoxazine or an optionally substituted phenothiazine, such as phenoxazine or phenthiazine having the formula: which may additionally have substituents on any available ring position.
  • X is selected from: O and S.
  • R is attached to the azacycle via R’s nitrogen.
  • Substituents of R may independently include halogen, alkyl (e.g., C1-6 alkyl, such as CH3, C2 alkyl, C3 alkyl, C4 alkyl, Cs alkyl, or Ce alkyl), alkoxy (e.g., C1-6 alkoxy, such as -OCH3, C2 alkoxy, C3 alkoxy, C4 alkoxy, Cs alkoxy, or Ce alkoxy), N3, NO2, and CN.
  • alkyl e.g., C1-6 alkyl, such as CH3, C2 alkyl, C3 alkyl, C4 alkyl, Cs alkyl, or Ce alkyl
  • alkoxy e.g., C1-6 alkoxy, such as -OCH3, C2 alkoxy, C3 alkoxy, C4 alkoxy, Cs alkoxy, or Ce alkoxy
  • an agonist of antisense activity is compound 325, having the formula:
  • an agonist of antisense activity is based on diastereomeric 2-C-aryl azacycles. In certain embodiments, an agonist of antisense activity is of formula:
  • R 2 is an aliphatic chain (Ce - C14).
  • R3 is a mono-, di-, tri- or tetra- aromatic substituent that includes hydrogen, halogen, alkyl, alkoxy, azide (N3), ether, NO 2 , cyanide (CN), or a combination thereof.
  • R4 is a functional group selected from H, alkyl including methyl (Me), ester, or acyl.
  • X’ is an anion of the suitable acid.
  • n is an independently selected integer selected from 1 , 2, or 3.
  • m is an independently selected integer selected from 0, 1 or 2.
  • R1 is H, OH, or CH 2 OH. In some embodiments, R1 is H. In some embodiments, R1 is OH. In some embodiments, R1 is CH 2 OH.
  • R 2 is C6-14 alkyl, C6-10 alkyl, C7-9 alkyl, CeH , C7H15, CsHn, C9H19, CIOH 2 I , CH H 2 3, CI 2 H 2 5, CISH 2 7, or Ci4H 2 9. In some embodiments, R 2 is CsHn.
  • R3 is H.
  • n is 1 .
  • m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3.
  • the linking group connecting the phenyl ring to the azacycle includes a cyclic carbon chain extending from the alpha, beta or gamma positions with regard to the azacycle back to the N of the azacycle, so that the azaycle with the linking group form an optionally substituted bicyclic ring of the formula:
  • R4 is H.
  • R4 is C1-6 alkyl, such as CH3, C 2 HS, C3H7, C4H9, C5H11, CeH , C1-3 alkyl, etc., C1-6 acyl, or C1-6 ester.
  • R4 is methyl.
  • the R 2 and R3 substituents can have different combinations around the phenyl ring with regard to their position.
  • R 2 is an unsaturated hydrocarbon chain.
  • the R1 is an alkyl having 1 to 6 carbons.
  • Certain sphingolipid-like compounds can also be related to pharmaceutically acceptable salts.
  • a “pharmaceutically acceptable salt” retains the desirable biological activity of the compound without undesired toxicological effects.
  • Salts can be salts with a suitable acid, including, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, benzoic acid, pamoic acid, alginic acid, methanesulfonic acid, naphthalenesulphonic acid, and the like.
  • incorporated cations can include ammonium, sodium, potassium, lithium, zinc, copper, barium, bismuth, calcium, and the like; or organic cations such as tetraalkylammonium and trialkylammonium cations.
  • organic cations such as tetraalkylammonium and trialkylammonium cations.
  • acidic and cationic salts include salts of other acids and/or cations, such as salts with trifluoroacetic acid, chloroacetic acid, and trichloroacetic acid.
  • the present disclosure provides pharmaceutical compositions comprising one or more antisense compounds or a salt thereof.
  • the pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier.
  • a pharmaceutical composition comprises a sterile saline solution, one or more antisense compounds, and one or more agonists of antisense activity.
  • such pharmaceutical composition consists of a sterile saline solution, one or more antisense compounds, and one or more agonists of antisense activity.
  • the sterile saline is pharmaceutical grade saline.
  • a pharmaceutical composition comprises sterile water, one or more antisense compounds, and one or more agonists of antisense activity.
  • the water is pharmaceutical grade water.
  • a pharmaceutical composition comprises phosphate-buffered saline (PBS), one or more antisense compounds, and one or more agonists of antisense activity.
  • the PBS is pharmaceutical grade PBS.
  • pharmaceutical compositions comprise one or more antisense compound, one or more agonists of antisense activity, and one or more excipients.
  • excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.
  • antisense compounds and agonists of antisense activity may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations.
  • Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
  • compositions comprising an antisense compound encompass any pharmaceutically acceptable salts of the antisense compound, esters of the antisense compound, or salts of such esters.
  • pharmaceutical compositions comprising an agonist of antisense activity encompass any pharmaceutically acceptable salts of the agonist of antisense activity.
  • pharmaceutical compositions comprising one or more antisense compounds and one or more agonists of antisense activity, upon administration to an animal, including a human, are capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
  • the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
  • Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
  • prodrugs comprise one or more conjugate group attached to an oligonucleotide, wherein the conjugate group is cleaved by endogenous nucleases within the body.
  • Lipid moieties have been used in nucleic acid therapies in a variety of methods.
  • the nucleic acid such as an antisense compound, is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids.
  • DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.
  • compositions comprise a delivery system.
  • delivery systems include, but are not limited to, liposomes and emulsions.
  • Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds.
  • certain organic solvents such as dimethyl sulfoxide (DMSO) are used.
  • compositions comprise one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types.
  • pharmaceutical compositions include liposomes coated with a tissuespecific antibody.
  • compositions comprise a co-solvent system.
  • co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • co-solvent systems are used for hydrophobic compounds.
  • a nonlimiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80TM and 65% w/v polyethylene glycol 300.
  • the proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics.
  • co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80TM; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
  • dimethyl sulfoxide (DMSO) is utilized as a co-solvent.
  • cremophor or cremophor EL
  • pharmaceutical compositions comprise one or more compounds that increase bioavailability.
  • 2-hydroxypropyl-beta-cyclodextrin can be utilized in pharmaceutical compositions and may increase bioavailability.
  • DMSO, cremophor and 2-hydroxypropyl-beta-cyclodextrin is utilized to increase bioavailability of various agonists, especially sphingolipid-like compounds.
  • compositions are prepared for oral administration.
  • pharmaceutical compositions are prepared for buccal administration.
  • a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.).
  • a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives).
  • injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like.
  • compositions for injection are presented in unit dosage form, e.g., in ampoules or in multidose containers.
  • Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.
  • a pharmaceutical composition is administered in a therapeutically effective amount as part of a course of treatment.
  • to "treat” means to ameliorate or prevent at least one symptom of the disorder to be treated or to provide a beneficial physiological effect.
  • a therapeutically effective amount can be an amount sufficient to prevent reduce, ameliorate or eliminate the symptoms of diseases or pathological conditions susceptible to such treatment.
  • a therapeutically effective amount is an amount sufficient to increase antisense activity of an antisense compound.
  • Dosage, toxicity and therapeutic efficacy of a pharmaceutical composition can be determined, e.g., by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds that exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to non-neoplastic cells and, thereby, reduce side effects.
  • a pharmaceutical composition for use in humans. If a pharmaceutical composition is provided systemically, the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration or within the local environment to be treated in a range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of neoplastic growth) as determined in cell culture.
  • IC50 concentration of the test compound that achieves a half-maximal inhibition of neoplastic growth
  • levels in plasma may be measured, for example, by liquid chromatography coupled to mass spectrometry.
  • an "effective amount” is an amount sufficient to effect beneficial or desired results.
  • a therapeutic amount is one that achieves the desired therapeutic effect.
  • This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • a therapeutically effective amount of a composition depends on the composition selected.
  • the compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
  • treatment of a subject with a therapeutically effective amount of a pharmaceutical composition described herein can include a single treatment or a series of treatments. For example, several divided doses may be administered daily, one dose, or cyclic administration of the compounds to achieve the desired therapeutic result. A single small molecule compound may be administered, or combinations of various small molecule compounds may also be administered.
  • a pharmaceutical composition can be formulated with one or more adjuvants and/or pharmaceutically acceptable carriers according to the selected route of administration.
  • adjuvants and/or pharmaceutically acceptable carriers for oral applications, gelatin, flavoring agents, or coating material can be added.
  • carriers may include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles can include sodium chloride and potassium chloride, among others.
  • intravenous vehicles can include fluid and nutrient replenishers, electrolyte replenishers and the like.
  • the coating agent is one which acts as a coating agent in conventional delayed release oral formulations, including polymers for enteric coating.
  • examples include hypromellose phthalate (hydroxy propyl methyl cellulose phthalate; HPMCP); hydroxypropylcellulose (HPC; such as KLLICEL®); ethylcellulose (such as ETHOCEL®); and methacrylic acid and methyl methacrylate (MAA/MMA; such as EU DRAG IT®).
  • a pharmaceutical composition also includes at least one disintegrating agent, as well as diluent.
  • a disintegrating agent is a super disintegrant agent.
  • a diluent is a bulking agent such as a polyalcohol.
  • bulking agents and disintegrants are combined, such as, for example, PEARLITOL FLASH®, which is a ready to use mixture of mannitol and maize starch (mannitol/maize starch).
  • any polyalcohol bulking agent can be used when coupled with a disintegrant or a super disintegrant agent.
  • Additional disintegrating agents include, but are not limited to, agar, calcium carbonate, maize starch, potato starch, tapioca starch, alginic acid, alginates, certain silicates, and sodium carbonate.
  • Suitable super disintegrating agents include, but are not limited to crospovidone, croscarmellose sodium, AMBERLITE (Rohm and Haas, Philadelphia, Pa.), and sodium starch glycolate.
  • diluents are selected from the group consisting of mannitol powder, spray dried mannitol, microcrystalline cellulose, lactose, dicalcium phosphate, tricalcium phosphate, starch, pregelatinized starch, compressible sugars, silicified microcrystalline cellulose, and calcium carbonate.
  • a pharmaceutical composition further utilizes other components and excipients.
  • sweeteners include, but are not limited to, fructose, sucrose, glucose, maltose, mannose, galactose, lactose, sucralose, saccharin, aspartame, acesulfame K, and neotame.
  • flavoring agents and flavor enhancers that may be included in the formulations described herein include, but are not limited to, maltol, vanillin, ethyl vanillin, menthol, citric acid, fumaric acid, ethyl maltol and tartaric acid.
  • a pharmaceutical composition also includes a surfactant.
  • surfactants are selected from the group consisting of Tween 80, sodium lauryl sulfate, and docusate sodium.
  • a pharmaceutical composition further utilizes a binder.
  • binders are selected from the group consisting of povidone (PVP) K29/32, hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC), ethylcellulose (EC), com starch, pregelatinized starch, gelatin, and sugar.
  • a pharmaceutical composition also includes a lubricant.
  • lubricants are selected from the group consisting of magnesium stearate, stearic acid, sodium stearyl fumarate, calcium stearate, hydrogenated vegetable oil, mineral oil, polyethylene glycol, polyethylene glycol 4000- 6000, talc, and glyceryl behenate.
  • Preservatives and other additives like antimicrobial, antioxidant, chelating agents, and inert gases, can also be present. (See generally, Remington's Pharmaceutical Sciences, 16th Edition, Mack, (1980), the disclosure of which is incorporated herein by reference.)
  • sphingolipid-like compound 893 (begins with compound 4c.
  • the first intermediate, 5a ((2S,3R)-te/Y-Butyl 2-((fe/t- butyldiphenylsilyloxy)methyl)-3-(4-octylphenyl)pyrrolidine-1 -carboxylate) is synthesized in the following manner: A solution of compound 4c (120 mg, 0.187 mmol) in anhydrous THF (2.4 mL) is cooled to 0 °C.
  • HCI 4M in 1 ,4-dioxane, 0.98 mL, 3.9 mmol
  • compound 5b 15 mg, 0.039 mmol
  • TLC 24 to 48 hours
  • the solvent is removed under reduced pressure and 1 ,4-dioxane (2 mL) is added to the flask and evaporated to remove the residual HCI.
  • Sphingolipid-like compound 1090 was obtained according to the procedure for synthesizing Compound 12, which is described below (Fig. 3).
  • (2S,3R)-2-((3- (6-methyl-3-(pyrimidin-2-yl)pyridazin-4-yl)propoxy)methyl)-3-(4-octylphenyl)pyrrolidin-1 - ium chloride is eluted for first as a colorless gel;
  • (2S,3R)-2-((3-(3-methyl-6-(pyrimidin-2- yl)pyridazin-4-yl)propoxy)methyl)-3-(4-octylphenyl)pyrrolidin-1 -ium chloride is eluted for last as a colorless gel (20 mg, 14% over 2 steps).
  • Example 2 Effect of sphingolipid-like compound pre-treatment on antisense compound activity in vitro
  • Isis No. 353382 targets the mouse Scavenger Receptor Class B, Number 1 (SRB-1 ) transcript (GENBANK accession number NM_016741.1 , SEQ ID NO: 1 ).
  • the sequence of Isis No. 353382 is 5’- GCTTCAGTCATGACTTCCTT-3’ (SEQ ID NO: 2).
  • 353382 activity in vitro mouse hepatocellular SV40 large T-antigen (MHT) carcinoma cells were treated with either no sphingolipid-like compound (control), 3 pM FTY-720, 1 pM COMPOUND 1090, 3 pM COMPOUND 1090, 1 pM COMPOUND 893, or 3 pM COMPOUND 893.
  • control no sphingolipid-like compound
  • 3 pM FTY-720, 1 pM COMPOUND 1090, 3 pM COMPOUND 1090, 1 pM COMPOUND 893, or 3 pM COMPOUND 893 was added 2 hours before Isis No. 353382 at the concentrations shown in the tables below.
  • RNA samples were lysed 24 hours following oligonucleotide addition, and total RNA was purified.
  • SRB-1 mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results are shown in the tables below as the average SRB-1 mRNA levels in cells treated with Isis No. 353382 relative to control cells that did not receive oligonucleotide treatment. The values were plotted on a dose-response curve and ICso values were then calculated. The results show that pre-treatment with either COMPOUND 893 or COMPOUND 1090 potentiated the effect of Isis No. 353382 in vitro relative to the control (no pretreatment).
  • Isis No. 395251 targets metastasis associated lung adenocarcinoma transcript 1 (MALAT-1 ) (GENBANK Accession No. NR_002847.2; SEQ ID NO: 3).
  • ISIS 395251 has the nucleobase sequence CCAGGCTGGTTATGACTCAG; SEQ ID NO: 4.
  • ISIS 395251 is a 5-10-5 MOE gapmer, wherein the central gap segment comprises ten 2’-deoxynucleosides and is flanked on both sides (in the 5’ and 3’ directions) by wings comprising 5 nucleosides each.
  • Each nucleoside in the 5’ wing segment and each nucleoside in the 3’ wing segment has a 2’-MOE modification.
  • control LSL murine embryonic fibroblast cells LSL MEFs
  • murine embryonic fibroblast cells expressing an activated mutant of KRAS KRAS MEFs
  • MDA-MB-231 and MDA-MB-468 cells were treated with either no sphingolipid-like compound (control), 2.5 pM COMPOUND 1090, 5 pM COMPOUND 1090, or 5 pM COMPOUND 893. Each of 2.5 pM COMPOUND 1090, 5 pM COMPOUND 1090, or 5 pM COMPOUND 893 was added 2 hours before Isis No.
  • Panc-1 and BxPC3 cells were treated with either no sphingolipid-like compound (control), 2.5 pM COMPOUND 1090, 5 pM COMPOUND 1090, or 5 pM COMPOUND 893. Each of 2.5 pM COMPOUND 1090, 5 pM COMPOUND 1090, or 5 pM COMPOUND 893 was added 2 hours before Isis No.
  • Example 8 Synthetic Sphingolipid-like Compounds Increase Antisense Compound Accumulation in Non-lysosomal Compartments
  • COMPOUND 893 increases accumulation of intracellular ASO in non- lysosomal compartments.
  • HeLa cells were co-treated with untagged 5-10-5 2’MOE ASO in the presence or absence of 5 pM 893 for 6 h and then imaged by confocal microscopy (Fig. 6A).
  • Total intracellular accumulation and colocalization with the lysosome marker LAMP2 was determined using Imaged (Fig. 6B).
  • the experiment was repeated with untagged 3-10-3 cET ASO (Figs. 6C and 6D).
  • COMPOUND 893 reduces ASO co-localization with acidified lysosomes (Figs. 7A and 7B).
  • HeLa cells co-treated with 2 pM 5’FAM-conjugated ASO in the presence or absence of 6 pM 893 for 0, 1 , 3, or 6 hours, stained with Lysotracker Red to visualize lysosomes, and then imaged by confocal microscopy.
  • Colocalization of ASO and Lysotracker Red was measured using Imaged. Preventing ASO from reaching the lysosome may increase the time available for ASO escape into the cytosol.
  • COMPOUND 893 improves the effectiveness of ASOs better than the established small molecule ASO potentiators. 893 was compared with the small molecules 6-bromo-indirubin-3'-oxime (6BIO) and AZD8055 for their ability to reduce MALAT 1 levels in HeLa cells treated with the 3-10-3 cET gapmer, as determined by qPCR (Figs. 8A and 8B). As can be seen in the figures, 893 provides a significant improvement of ASO function in HeLa cells as compared to 6BIO and AZD8055.
  • COMPOUND 893 was also compared with the ASO enhancer UNC10217938A.
  • the 3-10-3 cET gapmer ASO targeting MALAT1 was added to HeLa cells and then 16 hours later treated with 893 (5 pM) or UNC10217938A (10 pM) for two hours (Fig. 8C).
  • RNA was collected 48 hours after ASO addition and quantified (Fig. 8D).
  • the ASO enhancer UNC10217938A dramatically improved the ASOs ability to reduce MALAT1 levels, however UNC10217938A also had a dramatic effect on the healthy appearance of cells.
  • Example 11 PlKfyve inhibition blocks delivery of ASOs to lysosomes but fails to increase ASO uptake or activity to the same extent as Synthetic Sphingolipid-like Compound 893
  • 893 has at least a dual effect to improve ASO function by increasing total amount of ASOs within the cell in addition to reducing the amount of ASO sequestered in the lysosome.
  • Structurally distinct COMPOUND 893, small molecule that activates PP2A (SMAP), and perphenazine (PPZ) are each agonists of protein phosphatase 2 (PP2A). Activation of PP2A reduces both endosomal recycling and lysosomal fusion (Fig. 11 A).
  • SMAP protein phosphatase 2
  • PPZ perphenazine
  • Treatment with 893 or a PP2A agonist reduced ASO colocalization with lysosomal marker LAMP1 and increased total intracellular ASOs (Fig. 11 B).
  • Treatment with 893 or a PP2A agonist increased 3-10-3 cEt ASO activity on MALAT 1 in HeLa cells (Fig. 11 C) as well as mouse embryonic fibroblasts (Fig. 11 D).
  • Treatment with 893 or a PP2A agonist also increased the activity of a 5-10-5 2’MOE gapmer targeting MALAT 1 (Fig. 11 E) and ACTN1 (Fig. 11 F) in HeLa cells.
  • Dihydro-C2-ceramide has a similar structure to C2-ceramide but lacks the ability to agonize PP2A.
  • Dihydro-C2-ceramide fails to increase ASO activity (Figs. 12A - 12C).
  • An in vitro phosphatase assay was performed using immunoprecipitated PP2A. The results show that ceramide, and COMPOUND 893 increase PP2A activity but dihydroceramide does not (Fig. 12A).
  • HeLa cells were co-treated with 40 pM C2-ceramide or 40 pM dihydro-C2-ceramide with varying concentrations of MALAT1 cEt ASO for 24 hours. RNA was then collected and qRT-PCR for MALAT1 RNA levels performed (Figs 12B and 12C). The data suggest that PP2A activation is necessary for ASO potentiation.
  • COMPOUND 893 The ability of COMPOUND 893 to increase ASO activity in mice was assessed. Mice were treated with 120 mg/kg of compound 893 by oral gavage and 2 hours later were injected subcutaneously with various concentrations of 3-10-3 cEt gapmer ASO targeting MALAT1. Within the liver, 893 lowers the dose required to achieve efficient knockdown approximately 10-fold (Fig. 13A). As exemplified in the data, 5 mg/kg of cEt gapmer ASO with 893 treatment achieved approximately the same MALAT1 reduction as 50 mg/kg of the cEt gapmer ASO without any treatment.
  • cEt gapmer ASO with 893 treatment achieved approximately the same MALAT1 reduction as 5 mg/kg of the cEt gapmer ASO without any treatment. Improvement of ASO activity was also noticed within the lungs with systemic administration of 50 mg/kg of cEt gapmer ASO with 893 treatment (Fig. 13B) and with 5 mg/kg of cEt gapmer ASO with 893 treatment at 24 and 72 hours (Fig. 13C).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Agonists of antisense activity are described. Agonists of antisense activity can be utilized in conjunction with antisense compounds to increase the antisense activity of the antisense compound. In some instances, a biological cell is contacted with an agonist of antisense activity and an antisense compound to induce antisense activity in the biological cell. In some instances, an animal is administered an agonist of antisense activity and an antisense compound to induce antisense activity in the animal. Therapeutics and treatments involving agonists of antisense activity and antisense compounds are also described.

Description

COMPOUNDS AND METHODS FOR INCREASED ANTISENSE ACTIVITY
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application Ser. No. 63/107,371 entitled “Compounds and Methods for Increased Antisense Activity,” filed October 29, 2020, which is herein incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled CORE0142USLSEQ_ST25.txt, created on May 28, 2019 which is 13 KB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0003] The disclosure is generally directed to compounds and methods for increasing activity of an antisense compound, including compounds that agonize PP2A activity.
BACKGROUND
[0004] The principle behind antisense technology is that an antisense compound hybridizes to a target nucleic acid and modulates the amount, activity, and/or function of the target nucleic acid. For example, in certain instances, antisense compounds result in altered transcription or translation of a target. Such modulation of expression can be achieved by, for example, target mRNA degradation or occupancy-based inhibition. An example of modulation of RNA target function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound. Another example of modulation of gene expression by target degradation is RNA interference (RNAi). RNAi refers to antisense-mediated gene silencing through a mechanism that utilizes the RNA-induced silencing complex (RISC). An additional example of modulation of RNA target function is by an occupancy-based mechanism such as is employed naturally by microRNA. MicroRNA molecules are small non-coding RNAs that regulate the expression of protein-coding RNAs. The binding of an antisense compound to a microRNA prevents that microRNA from binding to its messenger RNA targets, and thus interferes with the function of the microRNA. MicroRNA mimics can enhance native microRNA function. Certain antisense compounds alter splicing of pre- mRNA. Regardless of the specific mechanism, sequence-specificity makes antisense compounds attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in the pathogenesis of diseases.
[0005] Antisense technology is an effective means for modulating the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications. Chemically modified nucleosides may be incorporated into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target nucleic acid. New chemical modifications have improved the potency and efficacy of antisense compounds, uncovering the potential for oral delivery as well as enhancing subcutaneous administration, decreasing potential for side effects, and leading to improvements in patient convenience. New compounds or methods that increase the potency of antisense compounds would allow administration of lower doses, which reduces the potential for toxicity, as well as decreasing overall cost of therapy. Additionally, new compounds or methods that increase the potency of antisense compounds or that improve cellular uptake of antisense compounds would result in slower clearance from the body, allowing for less frequent dosing. Furthermore, various tissues and cell types, such as cancer cells, are highly resistant to antisense compound uptake. New compounds and methods that improve cellular uptake of antisense compounds would permit antisense compound methodologies to be performed on these traditionally resistant tissues and cell types.
SUMMARY
[0006] The present disclosure provides compounds and methods of increasing the efficacy and potency of antisense compounds, and in certain instances, promotes antisense compound uptake in cells and tissues that are traditionally resistant. In certain embodiments, small molecule compounds and methods improve cellular uptake and/or potency of antisense compounds. In certain embodiments, the resulting antisense activity is greater at a concentration of antisense compound than the antisense activity at the same concentration of the antisense compound in the absence of a small molecule compound.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] The description and claims will be more fully understood with reference to the following figures and data graphs, which are presented as exemplary embodiments and should not be construed as a complete recitation of the scope of the disclosure.
[0008] FIG. 1 illustrates a mechanism of protein phosphatase 2 (PP2A) agonists increasing antisense activity through alteration of cellular trafficking, utilized in accordance with various embodiments.
[0009] FIG. 2 illustrates reaction pathways for the production of therapeutic small molecule analogs, utilized in accordance with various embodiments.
[0010] FIG. 3 illustrates reaction pathways for the production of therapeutic small molecule analogs, utilized in accordance with various embodiments.
[0011] FIGs. 4A-4F provide data graphs showing sphingolipid-like molecules increase antisense targeting in accordance with various embodiments.
[0012] FIGs. 5A and 5B provide data graphs and tables showing sphingolipid-like molecules increase antisense compound activity in multiple cell lines in accordance with various embodiments.
[0013] FIGs. 6A-6D provide microscopy images and data graphs showing sphingolipid-like molecules decrease antisense compound accumulation in lysosomes in accordance with various embodiments.
[0014] FIGs. 7A and 7B provide microscopy images and data graphs showing antisense compound accumulate intracellularly and fail to reach the lysosome in cells treated with sphingolipid-like molecules in accordance with various embodiments.
[0015] FIGs. 8A-8F provide data graphs and microscopy images showing sphingolipid-like molecules increase antisense compound activity better than established antisense compound agonists in accordance with various embodiments. [0016] FIGs. 9A-9D provide microscopy images and data graphs showing sphingolipid-like molecules decrease lysosomal accumulation of antisense compounds similar to established PlKfyve inhibitors but are more effective than PlKfyve inhibitors at increasing antisense compound activity in accordance with various embodiments.
[0017] FIGs. 10A-10D provide microscopy images and data graphs showing sphingolipid-like molecules increase intracellular accumulation of antisense compounds similar to established ARF6 inhibitors in accordance with various embodiments.
[0018] FIGs. 11A-11 F provide microscopy images and data graphs showing sphingolipid-like molecules decrease lysosomal accumulation and increase intracellular accumulation of antisense compounds similar to established PP2A agonists in accordance with various embodiments.
[0019] FIGs. 12A-12C provide data graphs showing Dihydro-C2-ceramide does not activate PP2A or increase antisense compound activity in accordance with various embodiments.
[0020] FIGs.13A-13C provide data graphs showing sphingolipid-like molecules increase antisense targeting in vivo within the liver and lungs in accordance with various embodiments.
DETAILED DESCRIPTION
[0021] Turning now to the drawings and data, agonists of antisense activity and antisense compounds, and methods of their use are described in accordance with the various embodiments. In some embodiments, an agonist of antisense activity is utilized in conjunction with an antisense compound to increase antisense activity of the antisense compound. In some embodiments, a biological cell is contacted with an agonist of antisense activity and an antisense compound to induce antisense activity in the biological cell. In some embodiments, an animal is administered an agonist of antisense activity and an antisense compound to induce antisense activity in the animal. In some embodiments, an agonist of antisense activity and an antisense compound is used in a course of treatment. An antisense compound, in according with various embodiments, can be any compound inclusive of a nucleic acid having a sequence for hybridizing with a target to provide an antisense effect on that target. Thus, in various embodiments, nucleic acids within antisense compounds include (but are not limited to) antisense oligomers (ASOs), double stranded RNA molecules (dsRNA), small interfering RNA (siRNA), short-hairpin RNA molecules (shRNA), RNA/DNA hybrids, microRNA, and microRNA mimics.
[0022] Typically, antisense compounds are taken up into cells through endocytosis. Once an antisense compound comes into contact with a cell, the antisense compound can interact with the cell surface, where multiple pathways can lead to internalization of the antisense compound (Fig. 1 ). Antisense compounds are most active when within the cell and available to contact RNA compounds therein. There are several factors, however, that can prevent antisense compounds from performing their activity. For instance, the cytosolic enzyme ADP Ribosylation Factor 6 (ARF6) induces some antisense compoundcontaining endocytic vesicles to be recycled, fusing with the plasma membrane and eliminating the antisense compound from the cell. In addition, endosomes that are not recycled are eventually trafficked to the lysosome via multivesicular bodies (MVBs), resulting in antisense compound inactivation. Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing (PlKfyve) is one enzyme that promotes lysosome fusion. Here, the present disclosure provides compounds and methods of increasing antisense activity through blocking or slowing endosome recycling and lysosomal fusion. In certain embodiments, endosome recycling is reduced by utilizing ARF6 antagonists. In certain embodiments, lysosomal fusion is reduced by utilizing PlKfyve antagonists. Furthermore, it is now known that protein phosphatase 2 (PP2A) agonists inhibit endosome recycling and lysosomal fusion. Accordingly, in certain embodiments, endosome recycling and lysosomal fusion is reduced utilizing PP2A agonists.
[0023] Certain cell types, such as hepatocytes, demonstrate excellent uptake and activity of antisense compounds. Other cell types, however, demonstrate reduced uptake and/or activity of antisense compounds. The present disclosure provides compounds that increase the uptake and/or activity of antisense compounds in different cell types. In certain embodiments, the present disclosure provides methods of increasing the amount or activity of an antisense compound in a cancer cell. In certain embodiments, the present disclosure provides methods of increasing the amount or activity of an antisense compound in a pancreatic or breast cancer cell. In certain embodiments, the present disclosure provides methods of increasing the amount or activity of an antisense compound in a tumor cell. In certain embodiments, the present disclosure provides a method of increasing the amount or activity of an antisense compound in non-neoplastic cells.
[0024] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.
[0025] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated-by- reference for the portions of the document discussed herein, as well as in their entirety.
Definitions
[0026] Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety.
[0027] Unless otherwise indicated, the following terms have the following meanings: [0028] “2’-deoxynucleoside” means a nucleoside comprising 2’-H(H) furanosyl sugar moiety, as found in naturally occurring deoxyribonucleic acids (DNA). In certain embodiments, a 2’-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil). [0029] “2’-substituted nucleoside” or “2-modified nucleoside” means a nucleoside comprising a 2’-substituted or 2’-modified sugar moiety. As used herein, “2’-substituted” or “2-modified” in reference to a sugar moiety means a sugar moiety comprising at least one 2'-substituent group other than H or OH.
[0030] “Antisense activity” means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound. In certain embodiments, antisense activity is a change in splicing of a pre-mRNA nucleic acid target. In certain embodiments, antisense activity is an increase in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.
[0031] “Antisense compound” means a compound comprising an antisense nucleic acid and optionally one or more additional features, such as a conjugated group or a terminal group. Nucleic acids within antisense compounds include (but are not limited to) antisense oligomers (ASOs), double stranded RNA molecules (dsRNA), small interfering RNA (siRNA), short-hairpin RNA molecules (shRNA), RNA/DNA hybrids, microRNA, microRNA mimics, and derivatives thereof.
[0032] “Antisense oligonucleotide” means an oligonucleotide that (1 ) has a nucleobase sequence that is at least partially complementary to a target nucleic acid and that (2) is capable of producing an antisense activity in a cell or animal.
[0033] “Ameliorate” in reference to a treatment means improvement in at least one symptom relative to the same symptom in the absence of the treatment. In certain embodiments, amelioration is the reduction in the severity or frequency of a symptom or the delayed onset or slowing of progression in the seventy or frequency of a symptom.
[0034] “Bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclic sugar moiety. As used herein, “bicyclic sugar” or “bicyclic sugar moiety” means a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In certain embodiments, the bicyclic sugar moiety does not comprise a furanosyl moiety.
[0035] “Branching group” means a group of atoms having at least 3 positions that are capable of forming covalent linkages to at least 3 groups. In certain embodiments, a branching group provides a plurality of reactive sites for connecting tethered ligands to an oligonucleotide via a conjugate linker and/or a cleavable moiety.
[0036] “Cell-targeting moiety” means a conjugate group or portion of a conjugate group that is capable of binding to a particular cell type or particular cell types.
[0037] “Cleavable moiety” means a bond or group of atoms that is cleaved under physiological conditions, for example, inside a cell, an animal, or a human.
[0038] “Complementary” in reference to an oligonucleotide means that at least 70% of the nucleobases of such oligonucleotide or one or more regions thereof and the nucleobases of another nucleic acid or one or more regions thereof are capable of hydrogen bonding with one another when the nucleobase sequence of the oligonucleotide and the other nucleic acid are aligned in opposing directions. Complementary nucleobases means nucleobases that are capable of forming hydrogen bonds with one another. Complementary nucleobase pairs include, but unless otherwise specified are not limited to, adenine (A) and thymine (T), adenine (A) and uracil (II), cytosine (C) and guanine (G), 5-methyl cytosine (mC) and guanine (G). Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. As used herein, “fully complementary” or “100% complementary” in reference to oligonucleotides means that such oligonucleotides are complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide.
[0039] “Conjugate group” means a group of atoms that is directly or indirectly attached to an oligonucleotide. Conjugate groups include a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.
[0040] “Conjugate linker” means a group of atoms comprising at least one bond that connects a conjugate moiety to an oligonucleotide. [0041] “Conjugate moiety” means a group of atoms that is attached to an oligonucleotide via a conjugate linker.
[0042] "Contiguous" in the context of an oligonucleotide refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to each other. For example, “contiguous nucleobases” means nucleobases that are immediately adjacent to each other in a sequence.
[0043] “Duplex” means two oligomeric compounds that are paired. In certain embodiments, the two oligomeric compounds are paired via hybridization of complementary nucleobases.
[0044] “Extra-hepatic cell type” means a cell type that is not a hepatocyte.
[0045] “Extra-hepatic nucleic acid target” means a target nucleic acid that is expressed in tissues other than liver. In certain embodiments, extra-hepatic nucleic acid targets are not expressed in the liver or not expressed in the liver at a significant level. In certain embodiments, extra-hepatic nucleic acid targets are expressed outside the liver and also in the liver.
[0046] “Extra-hepatic disease” means a disease or condition where one or more symptoms or causes of the disease or condition occur in tissues other than liver.
[0047] “Extra-hepatic tissue” means a tissue other than liver.
[0048] “Fully modified” in reference to a modified oligonucleotide means a modified oligonucleotide in which each sugar moiety is modified. “Uniformly modified” in reference to a modified oligonucleotide means a fully modified oligonucleotide in which each sugar moiety is the same. For example, the nucleosides of a uniformly modified oligonucleotide can each have a 2’-MOE modification but different nucleobase modifications, and the internucleoside linkages may be different.
[0049] “Gapmer” means an antisense compound comprising an internal region having a plurality of nucleosides that support RNase H cleavage positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the “gap” and the external regions may be referred to as the “wings.” [0050] "Hybridization" means the pairing or annealing of complementary oligonucleotides and/or nucleic acids. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
[0051] "Inhibiting the expression or activity" refers to a reduction or blockade of the expression or activity relative to the expression of activity in an untreated or control sample and does not necessarily indicate a total elimination of expression or activity.
[0052] “Internucleoside linkage” means a group or bond that forms a covalent linkage between adjacent nucleosides in an oligonucleotide. As used herein “modified internucleoside linkage” means any internucleoside linkage other than a naturally occurring, phosphodiester internucleoside linkage. Non-phosphate linkages are referred to herein as modified internucleoside linkages. “Phosphorothioate linkage” means a modified phosphodiester linkage in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. A phosphorothioate internucleoside linkage is a modified internucleoside linkage.
[0053] “Linker-nucleoside” means a nucleoside that links, either directly or indirectly, an oligonucleotide to a conjugate moiety. Linker-nucleosides are located within the conjugate linker of an oligomeric compound. Linker-nucleosides are not considered part of the oligonucleotide portion of an oligomeric compound even if they are contiguous with the oligonucleotide.
[0054] “Lipophilic group” or “lipophilic” in reference to a chemical group means a group of atoms that is more soluble in lipids or organic solvents than in water and/or has a higher affinity for lipids than for water. In certain embodiments, lipophilic groups comprise a lipid. As used herein “lipid” means a molecule that is not soluble in water or is less soluble in water than in organic solvents. In certain embodiments, compounds of the present invention comprise lipids selected from saturated or unsaturated fatty acids, steroids, fat soluble vitamins, phospholipids, sphingolipids, hydrocarbons, mono-, di-, and triglycerides, and synthetic derivatives thereof. [0055] “Non-bicyclic modified sugar” or “non-bicyclic modified sugar moiety” means a modified sugar moiety that comprises a modification, such as a substituent, that does not form a bridge between two atoms of the sugar to form a second ring.
[0056] “Linked nucleosides” are nucleosides that are connected in a continuous sequence (/.e. no additional nucleosides are present between those that are linked).
[0057] “Mismatch” or “non-complementary” means a nucleobase of a first oligonucleotide that is not complementary with the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligomeric compound are aligned.
[0058] “MOE” means methoxyethyl. ”2’-MOE” means a -OCH2CH2OCH3 group at the 2’ position of a furanosyl ring.
[0059] “Motif” means the pattern of unmodified and/or modified sugar moieties, nucleobases, and/or internucleoside linkages, in an oligonucleotide.
[0060] “Multi-tissue disease or condition” means a disease or condition affects or is affected by more than one tissue. In treating a multi-tissue disease or condition, it is desirable to affect more than one tissue type. In certain embodiments, treatment of disease or condition may be enhanced by treating the disease or condition in multiple tissues. For example, in certain embodiments, a disease or condition may manifest itself in the liver tissue and the muscle tissue. In certain embodiments, treating the disease or condition in the liver tissue and the muscle tissue will be more effective than treating the disease in either the liver tissue or the muscle tissue.
[0061] “Naturally occurring” means found in nature.
[0062] "Nucleobase" means an unmodified nucleobase or a modified nucleobase. As used herein an “unmodified nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (II), and guanine (G). As used herein, a “modified nucleobase” is a group of atoms other than unmodified A, T, C, II, or G capable of pairing with at least one unmodified nucleobase. A universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases. As used herein, “nucleobase sequence” means the order of contiguous nucleobases in a nucleic acid or oligonucleotide independent of any sugar or internucleoside linkage modification. [0063] “Nucleoside” means a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each, independently, unmodified or modified. As used herein, “modified nucleoside” means a nucleoside comprising a modified nucleobase and/or a modified sugar moiety. Modified nucleosides include abasic nucleosides, which lack a nucleobase.
[0064] "Oligomeric compound" means a compound consisting of an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group.
[0065] "Oligonucleotide" means a strand of linked nucleosides connected via internucleoside linkages, wherein each nucleoside and internucleoside linkage may be modified or unmodified. Unless otherwise indicated, oligonucleotides consist of 8-50 linked nucleosides. As used herein, “modified oligonucleotide” means an oligonucleotide, wherein at least one nucleoside or internucleoside linkage is modified. As used herein, “unmodified oligonucleotide” means an oligonucleotide that does not comprise any nucleoside modifications or internucleoside modifications.
[0066] “Pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal. Certain such carriers enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspension and lozenges for the oral ingestion by a subject. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile water; sterile saline; or sterile buffer solution.
[0067] “Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of compounds, such as oligomeric compounds, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
[0068] “Pharmaceutical composition” means a mixture of substances suitable for administering to a subject. For example, a pharmaceutical composition may comprise an antisense compound and a sterile aqueous solution. In certain embodiments, a pharmaceutical composition shows activity in free uptake assay in certain cell lines. [0069] “Phosphorus moiety” means a group of atoms comprising a phosphorus atom. In certain embodiments, a phosphorus moiety comprises a mono-, di-, or tri-phosphate, or phosphorothioate.
[0070] “Prodrug” means a therapeutic agent in a form outside the body that is converted to a different form within the body or cells thereof. Typically, conversion of a prodrug within the body is facilitated by the action of an enzymes (e.g., endogenous or viral enzyme) or chemicals present in cells or tissues and/or by physiologic conditions.
[0071] “RNAi compound” means an antisense compound that acts, at least in part, through RISC or Ago2 to modulate a target nucleic acid and/or protein encoded by a target nucleic acid. RNAi compounds include, but are not limited to double-stranded siRNA, single-stranded RNA (ssRNA), and microRNA, including microRNA mimics. In certain embodiments, an RNAi compound modulates the amount, activity, and/or splicing of a target nucleic acid. The term RNAi compound excludes antisense compounds that exclusively act through RNase H.
[0072] “Single-stranded” in reference to an oligomeric compound means such a compound that is not paired with a second oligomeric compound to form a duplex. “Self- complementary” in reference to an oligonucleotide means an oligonucleotide that at least partially hybridizes to itself. A compound consisting of one oligomeric compound, wherein the oligonucleotide of the oligomeric compound is self-complementary, is a singlestranded compound. A single-stranded antisense or oligomeric compound may be capable of binding to a complementary oligomeric compound to form a duplex, in which case it would no longer be single-stranded.
[0073] “Sugar moiety” means an unmodified sugar moiety or a modified sugar moiety. As used herein, “unmodified sugar moiety” means a 2’-OH(H) furanosyl moiety, as found in RNA (an “unmodified RNA sugar moiety”), or a 2’-H(H) moiety, as found in DNA (an “unmodified DNA sugar moiety”). Unmodified sugar moieties have one hydrogen at each of the T, 3’, and 4’ positions, an oxygen at the 3’ position, and two hydrogens at the 5’ position. As used herein, “modified sugar moiety” or “modified sugar” means a modified furanosyl sugar moiety or a sugar surrogate. As used herein, modified furanosyl sugar moiety means a furanosyl sugar comprising a non-hydrogen substituent in place of at least one hydrogen of an unmodified sugar moiety. In certain embodiments, a modified furanosyl sugar moiety is a 2’-substituted sugar moiety. Such modified furanosyl sugar moieties include bicyclic sugars and non-bicyclic sugars. As used herein, "sugar surrogate" means a modified sugar moiety having other than a furanosyl moiety that can link a nucleobase to another group, such as an internucleoside linkage, conjugate group, or terminal group in an oligonucleotide. Modified nucleosides comprising sugar surrogates can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to complementary oligomeric compounds or nucleic acids.
[0074] “Target nucleic acid” means an identified nucleic acid to which an antisense compound is designed to hybridize to at least a portion of the nucleic acid. In certain embodiments, target nucleic acids are endogenous cellular nucleic acids, including, but not limited to RNA transcripts, pre-mRNA, mRNA, microRNA. In certain embodiments, target nucleic acids are viral nucleic acids. In certain embodiments, target nucleic acids are nucleic acids that an antisense compound is designed to affect.
[0075] “Target region” means a portion of a target nucleic acid to which an antisense compound is designed to hybridize.
[0076] “TCA motif” means three nucleosides having the nucleobase sequence TCA (5’-3’). Such nucleosides may have modified sugar moieties and/or modified internucleosides linkages. Unless otherwise indicated, the nucleosides of TCA motifs comprise unmodified 2’-deoxy sugar moieties and unmodified phosphodiester internucleoside linkages.
[0077] "Terminal group" means a chemical group or group of atoms that is covalently linked to a terminus of an oligonucleotide.
[0078] “Acyl” means a -R-C=O group.
[0079] “Alcohol” means a compound with an -OH group bonded to a saturated, alkane-like compound, (ROH).
[0080] “Alkyl” refers to the partial structure that remains when a hydrogen atom is removed from an alkane.
[0081] “Alkane” means a compound of carbon and hydrogen that contains only single bonds. [0082] “Alkene” refers to a hydrocarbon that contains a carbon-carbon double bond, R2C = CR2.
[0083] “Alkyne” refers to a hydrocarbon structure that contains a carbon-carbon triple bond.
[0084] “Alkoxy” refers to a portion of a molecular structure featuring an alkyl group bonded to an oxygen atom.
[0085] “Aryl” refers to any functional group or substituent derived from an aromatic ring.
[0086] “Amine” molecules are compounds containing one or more organic substituents bonded to a nitrogen atom, RNH2, R2NH, or R3N.
[0087] “Amino acid” refers to a difunctional compound with an amino group on the carbon atom next to the carboxyl group, RCH(NH2)CO2H.
[0088] “Azide” refers to N3.
[0089] “Cyanide” refers to CN.
[0090] “Ester” is a compound containing the -CO2R functional group.
[0091] “Ether” refers to a compound that has two organic substituents bonded to the same oxygen atom, i.e. , R-O-R’.
[0092] “Halogen” or “halo” means fluoro (F), chloro (Cl), bromo (Br), or iodo (I).
[0093] “Hydrocarbon” means an organic chemical compound that consists entirely of the elements carbon (C) and hydrogen (H).
[0094] “Phosphate”, “phosphonate”, or “PO” means a compound containing the elements phosphorous (P) and oxygen (O).
[0095] “R” in the molecular formula above and throughout are meant to indicate any suitable organic molecule.
Certain Oligonucleotides
[0096] In certain embodiments, the disclosure provides oligonucleotides, which consist of linked nucleosides. Oligonucleotides may be unmodified oligonucleotides (RNA or DNA) or may be modified oligonucleotides. Modified oligonucleotides comprise at least one modification relative to unmodified RNA or DNA (i.e. , comprise at least one modified nucleoside (comprising a modified sugar moiety and/or a modified nucleobase) and/or at least one modified internucleoside linkage).
A. Certain Modified Nucleosides
[0097] Modified nucleosides comprise a modified sugar moiety or a modified nucleobase or both a modified sugar moiety and a modified nucleobase.
1. Certain Sugar Moieties
[0098] In certain embodiments, modified sugar moieties are non-bicyclic modified sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of other types of modified sugar moieties.
[0099] In certain embodiments, modified sugar moieties are non-bicyclic modified sugar moieties comprising a furanosyl ring with one or more acyclic substituent, including but not limited to substituents at the 2’, 4’, and/or 5’ positions. In certain embodiments one or more acyclic substituent of non-bicyclic modified sugar moieties is branched. Examples of 2’-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 2’-F, 2'-OCH3 (“OMe” or “O-methyl”), and 2'-O(CH2)2OCH3 (“MOE”). In certain embodiments, 2’-substituent groups are selected from among: halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O-C1-C10 alkoxy, O-C1-C10 substituted alkoxy, O-C1-C10 alkyl, O-C1-C10 substituted alkyl, S-alkyl, N(Rm)-alkyl, O-alkenyl, S- alkenyl, N(Rm)-alkenyl, O-alkynyl, S-alkynyl, N(Rm)-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn) or OCH2C(=O)- N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10 alkyl, and the 2’-substituent groups described in Cook et al., U.S. 6,531 ,584; Cook et al., U.S. 5,859,221 ; and Cook et al., U.S. 6,005,087. Certain embodiments of these 2'-substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of 4’-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al., WO 2015/106128. Examples of 5’ -substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 5’-methyl (R or S), 5' -vinyl, and 5’-methoxy. In certain embodiments, non-bicyclic modified sugars comprise more than one non-bridging sugar substituent, for example, 2'-F-5'-methyl sugar moieties and the modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008/101157 and Rajeev et al., US2013/0203836.).
[0100] In certain embodiments, a 2’-substituted nucleoside or 2’- non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2’-substituent group selected from: F, NH2, N3, OCF3, OCH3, O(CH2)3NH2, CH2CH=CH2, OCH2CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2, and N- substituted acetamide (OCH2C(=O)-N(Rm)(Rn)), where each Rm and Rn is, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10 alkyl.
[0101] In certain embodiments, a 2’-substituted nucleoside or 2’- non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2’-substituent group selected from: F, OCF3, OCH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(CH3)2, O(CH2)2O(CH2)2N(CH3)2, and OCH2C(=O)-N(H)CH3 (“NMA”).
[0102] In certain embodiments, a 2’-substituted nucleoside or 2’- non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2’-substituent group selected from: F, OCH3, and OCH2CH2OCH3.
[0103] Nucleosides comprising modified sugar moieties, such as non-bicyclic modified sugar moieties, may be referred to by the position(s) of the substitution(s) on the sugar moiety of the nucleoside. For example, nucleosides comprising 2’-substituted or 2- modified sugar moieties are referred to as 2’-substituted nucleosides or 2-modified nucleosides.
[0104] Certain modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4' and the 2' furanose ring atoms. Examples of such 4’ to 2’ bridging sugar substituents include but are not limited to: 4'- CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' (“LNA”), 4'-CH2-S-2', 4'-(CH2)2-O-2' (“ENA”), 4'-CH(CH3)-O-2' (referred to as “constrained ethyl” or “cEt” when in the S configuration), 4’-CH2-O-CH2-2’, 4’-CH2-N(R)-2’, 4'-CH(CH2OCH3)-O-2' (“constrained MOE” or “cMOE”) and analogs thereof (see, e.g., Seth et al., U.S. 7,399,845, Bhat et al., U.S. 7,569,686, Swayze et al., U.S. 7,741 ,457, and Swayze et al., U.S. 8,022,193), 4'-C(CH3)(CH3)-O-2' and analogs thereof (see, e.g., Seth et al., U.S. 8,278,283), 4'-CH2-N(OCH3)-2' and analogs thereof (see, e.g., Prakash et al., U.S. 8,278,425), 4'-CH2-O-N(CH3)-2' (see, e.g., Allerson et al., U.S. 7,696,345 and Allerson et al., U.S. 8,124,745), 4'-CH2-C(H)(CH3)-2' (see, e.g., Zhou, etal., J. Org. Chem., 2009, 74, 118-134), 4'-CH2-C(=CH2)-2' and analogs thereof (see e.g., Seth et al., U.S. 8,278,426), 4’-C(RaRb)-N(R)-O-2’, 4’-C(RaRb)-O-N(R)- 2’, 4'-CH2-O-N(R)-2', and 4'-CH2-N(R)-O-2', wherein each R, Ra, and Rb is, independently, H, a protecting group, or Ci-Ci2 alkyl (see, e.g. Imanishi et al., U.S. 7,427,672).
[0105] In certain embodiments, such 4’ to 2’ bridges independently comprise from 1 to 4 linked groups independently selected from: -[C(Ra)(Rb)]n-, -[C(Ra)(Rb)]n-O- , -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)-, -C(=O)-, -C(=S)-, -O-, -Si(Ra)2-, -S(=O)x-, and -N(Ra)-; wherein: x is 0, 1 , or 2; n is 1 , 2, 3, or 4; each Ra and Rb is, independently, H, a protecting group, hydroxyl, Ci-Ci2 alkyl, substituted Ci-Ci2 alkyl, C2-Ci2 alkenyl, substituted C2-Ci2 alkenyl, C2-Ci2 alkynyl, substituted C2-Ci2 alkynyl, Cs-C2o aryl, substituted Cs-C2o aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1 J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-Ji), or sulfoxyl (S(=O)-Ji); and each Ji and J2 is, independently, H, Ci-Ci2 alkyl, substituted Ci-Ci2 alkyl, C2-Ci2 alkenyl, substituted C2-Ci2 alkenyl, C2-Ci2 alkynyl, substituted C2-Ci2 alkynyl, Cs-C2o aryl, substituted Cs-C2o aryl, acyl (C(=O)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, Ci-Ci2 aminoalkyl, substituted Ci-Ci2 aminoalkyl, or a protecting group. [0106] Additional bicyclic sugar moieties are known in the art, see, for example: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443, Albaek et al., J. Org. Chem., 2006, 71, 7731-7740, Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219- 2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129, 8362-8379;Wengel et a., U.S. 7,053,207; Imanishi et al., U.S. 6,268,490; Imanishi et al. U.S. 6,770,748; Imanishi et al., U.S. RE44,779; Wengel et al., U.S. 6,794,499; Wengel et al., U.S. 6,670,461 ; Wengel et al., U.S. 7,034,133; Wengel et al., U.S. 8,080,644; Wengel et al., U.S. 8,034,909; Wengel et al., U.S. 8,153,365; Wengel et al., U.S. 7,572,582; and Ramasamy et al., U.S. 6,525,191 ;; Torsten et al., WO 2004/106356; Wengel et al., WO 1999/014226; Seth et al., WO 2007/134181 ; Seth et al., U.S. 7,547,684; Seth et al., U.S. 7,666,854; Seth et al., U.S. 8,088,746; Seth et al., U.S. 7,750,131 ; Seth et al., U.S. 8,030,467; Seth et al., U.S. 8,268,980; Seth et al., U.S. 8,546,556; Seth et al., U.S. 8,530,640; Migawa et al., U.S. 9,012,421 ; Seth et al., U.S. 8,501 ,805; and U.S. Patent Publication Nos. Allerson et al., US2008/0039618 and Migawa et al., US2015/0191727.
[0107] In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, an LNA nucleoside (described herein) may be in the a-L configuration or in the |3-D configuration.
Figure imgf000021_0001
LNA (P-D-configuration) a-Z-LNA (a-Z-configuration) bridge = 4'-CH2-O-2' bridge = 4'-CH2-O-2'
[0108] a-L-methyleneoxy (4’-CH2-O-2’) or a-L-LNA bicyclic nucleosides have been incorporated into antisense compounds that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). Herein, general descriptions of bicyclic nucleosides include both isomeric configurations. When the positions of specific bicyclic nucleosides (e.g., LNA or cEt) are identified in exemplified embodiments herein, they are in the |3-D configuration, unless otherwise specified. [0109] In certain embodiments, modified sugar moieties comprise one or more nonbridging sugar substituent and one or more bridging sugar substituent (e.g., 5’ -substituted and 4’-2’ bridged sugars).
[0110] In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moieties also comprise bridging and/or non-bridging substituents as described herein. For example, certain sugar surrogates comprise a 4’-sulfur atom and a substitution at the 2'- position (see, e.g., Bhat et al., U.S. 7,875,733 and Bhat et al., U.S. 7,939,677) and/or the 5’ position.
[0111] In certain embodiments, sugar surrogates comprise rings having other than 5 atoms. For example, in certain embodiments, a sugar surrogate comprises a sixmembered tetrahydropyran (“THP”). Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include but are not limited to hexitol nucleic acid (“HNA”), anitol nucleic acid (“ANA”), manitol nucleic acid (“MNA”) (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841 -854), fluoro HNA:
Figure imgf000022_0001
F-HNA
[0112] (“F-HNA”, see e.g.Swayze et al., U.S. 8,088,904; Swayze et al., U.S.
8,440,803; Swayze et al., U.S. 8,796,437; and Swayze et al., U.S. 9,005,906; F-HNA can also be referred to as a F-THP or 3' -fluoro tetrahydropyran), and nucleosides comprising additional modified THP compounds having the formula:
Figure imgf000022_0002
wherein, independently, for each of said modified THP nucleoside: Bx is a nucleobase moiety;
T3 and T4 are each, independently, an internucleoside linking group linking the modified THP nucleoside to the remainder of an oligonucleotide or one of T3 and T4 is an internucleoside linking group linking the modified THP nucleoside to the remainder of an oligonucleotide and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or 3'-terminal group; qi, q2, qs, q4, qs, qe and q? are each, independently, H, C-i-Ce alkyl, substituted C1- Ce alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; and each of R1 and R2 is independently selected from among: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(=X)Ji, OC(=X)NJIJ2, NJ3C(=X)NJIJ2, and CN, wherein X is 0, S or NJ1, and each Ji, J2, and J3 is, independently, H or C-i-Ce alkyl.
[0113] In certain embodiments, modified THP nucleosides are provided wherein qi, q2, qs, q4, qs, qe and q? are each H. In certain embodiments, at least one of qi, q2, qs, q4, qs, qe and q? is other than H. In certain embodiments, at least one of qi, q2, qs, q4, qs, qe and q? is methyl. In certain embodiments, modified THP nucleosides are provided wherein one of R1 and R2 is F. In certain embodiments, R1 is F and R2 is H, in certain embodiments, R1 is methoxy and R2 is H, and in certain embodiments, R1 is methoxyethoxy and R2 is H.
[0114] In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligonucleotides have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. 5,698,685; Summerton et al., U.S. 5,166,315; Summerton et al., U.S. 5,185,444; and Summerton et al., U.S. 5,034,506). As used here, the term “morpholino” means a sugar surrogate having the following structure:
Figure imgf000023_0001
[0115] In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modified morpholinos.”
[0116] In certain embodiments, sugar surrogates comprise acyclic moieites. Examples of nucleosides and oligonucleotides comprising such acyclic sugar surrogates include but are not limited to: peptide nucleic acid (“PNA”), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and nucleosides and oligonucleotides described in Manoharan et al., WO2011/133876.
[0117] Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used in modified nucleosides).
2. Certain Modified Nucleobases
[0118] In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising an unmodified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside that does not comprise a nucleobase, referred to as an abasic nucleoside.
[0119] In certain embodiments, modified nucleobases are selected from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6 substituted purines. In certain embodiments, modified nucleobases are selected from: 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2- propyladenine , 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-C^C-CHs) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7- deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N- benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1 ,3-diazaphenoxazine-2-one, 1 ,3- diazaphenothiazine-2-one and 9-(2-aminoethoxy)-1 ,3-diazaphenoxazine-2-one (G- clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in Merigan et al., U.S. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991 , 30, 613; Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, Crooke, S.T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S.T., Ed., CRC Press, 2008, 163-166 and 442-443. [0120] Publications that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, Manohara et al., US2003/0158403; Manoharan et al., US2003/0175906; Dinh et al., U.S. 4,845,205; Spielvogel et al., U.S. 5,130,302; Rogers et al., U.S. 5,134,066; Bischofberger et al., U.S. 5,175,273; Urdea et al., U.S. 5,367,066; Benner et al., U.S. 5,432,272; Matteucci et al., U.S. 5,434,257; Gmeiner et al., U.S. 5,457,187; Cook et al., U.S. 5,459,255; Froehler et al., U.S. 5,484,908; Matteucci et al., U.S. 5,502,177; Hawkins et al., U.S. 5,525,711 ; Haralambidis et al., U.S. 5,552,540; Cook et al., U.S. 5,587,469; Froehler et al., U.S. 5,594,121 ; Switzer et al., U.S. 5,596,091 ; Cook et al., U.S. 5,614,617; Froehler et al., U.S. 5,645,985; Cook et al., U.S. 5,681 ,941 ; Cook et al., U.S. 5,811 ,534; Cook et al., U.S. 5,750,692; Cook et al., U.S. 5,948,903; Cook et al., U.S. 5,587,470; Cook et al., U.S. 5,457,191 ; Matteucci et al., U.S. 5,763,588; Froehler et al., U.S. 5,830,653; Cook et al., U.S. 5,808,027; Cook et al., 6,166,199; and Matteucci et al., U.S. 6,005,096.
B. Certain Modified Internucleoside Linkages
[0121] In certain embodiments, nucleosides of modified oligonucleotides may be linked together using any internucleoside linkage. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include but are not limited to phosphates, which contain a phosphodiester bond (“P=O”) (also referred to as unmodified or naturally occurring linkages), phosphotriesters, methylphosphonates, phosphoram idates, and phosphorothioates (“P=S”), and phosphorodithioates (“HS- P=S”). Representative non-phosphorus containing internucleoside linking groups include but are not limited to methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester , thionocarbamate (-O-C(=O)(NH)-S-); siloxane (-O-SiH2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified internucleoside linkages, compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral internucleoside linkages include but are not limited to alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous- containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.
[0122] Neutral internucleoside linkages include, without limitation, phosphotriesters, methylphosphonates, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), methoxypropyl, and thioformacetal (3'-S-CH2-O-5'). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research', Y.S. Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2 component parts.
C. Certain Motifs
[0123] In certain embodiments, modified oligonucleotides comprise one or more modified nucleoside comprising a modified sugar. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more modified internucleoside linkage. In such embodiments, the modified, unmodified, and differently modified sugar moieties, nucleobases, and/or internucleoside linkages of a modified oligonucleotide define a pattern or motif. In certain embodiments, the patterns of sugar moieties, nucleobases, and internucleoside linkages are each independent of one another. Thus, a modified oligonucleotide may be described by its sugar motif, nucleobase motif and/or internucleoside linkage motif (as used herein, nucleobase motif describes the modifications to the nucleobases independent of the sequence of nucleobases).
2. Certain Nucleobase Motifs
[0124] In certain embodiments, oligonucleotides comprise one or more type of modified sugar and/or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or sugar motif. In certain instances, such sugar motifs include but are not limited to any of the sugar modifications discussed herein.
[0125] In certain embodiments, modified oligonucleotides comprise or consist of a region having a gapmer motif, which comprises two external regions or “wings” and a central or internal region or “gap.” The three regions of a gapmer motif (the 5’-wing, the gap, and the 3’ -wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3’-most nucleoside of the 5’-wing and the 5’-most nucleoside of the 3’-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap (i.e., the wing/gap junction). In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the sugar motif of the 5'-wing differs from the sugar motif of the 3' -wing (asymmetric gapmer).
[0126] In certain embodiments, the wings of a gapmer comprise 1 -5 nucleosides. In certain embodiments, the wings of a gapmer comprise 2-5 nucleosides. In certain embodiments, the wings of a gapmer comprise 3-5 nucleosides. In certain embodiments, the nucleosides of a gapmer are all modified nucleosides. [0127] In certain embodiments, the gap of a gapmer comprises 7-12 nucleosides. In certain embodiments, the gap of a gapmer comprises 7-10 nucleosides. In certain embodiments, the gap of a gapmer comprises 8-10 nucleosides. In certain embodiments, the gap of a gapmer comprises 10 nucleosides. In certain embodiment, each nucleoside of the gap of a gapmer is an unmodified 2’-deoxy nucleoside.
[0128] In certain embodiments, the gapmer is a deoxy gapmer. In such embodiments, the nucleosides on the gap side of each wing/gap junction are unmodified 2’-deoxy nucleosides and the nucleosides on the wing sides of each wing/gap junction are modified nucleosides. In certain such embodiments, each nucleoside of the gap is an unmodified 2’-deoxy nucleoside. In certain such embodiments, each nucleoside of each wing is a modified nucleoside.
[0129] In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif. In such embodiments, each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety. In certain such embodiments, each nucleoside to the entire modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In certain embodiments, a fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each nucleoside of a uniformly modified comprises the same 2’-modification.
3. Certain Internucleoside Linkage Motifs
[0130] In certain embodiments, oligonucleotides comprise modified and/or unmodified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, essentially each internucleoside linking group is a phosphate internucleoside linkage (P=O). In certain embodiments, each internucleoside linking group of a modified oligonucleotide is a phosphorothioate (P=S). In certain embodiments, each internucleoside linking group of a modified oligonucleotide is independently selected from a phosphorothioate and phosphate internucleoside linkage. In certain embodiments, the sugar motif of a modified oligonucleotide is a gapmer and the internucleoside linkages within the gap are all modified. In certain such embodiments, some or all of the internucleoside linkages in the wings are unmodified phosphate linkages. In certain embodiments, the terminal internucleoside linkages are modified.
D. Certain Lengths
[0131] In certain embodiments, oligonucleotides (including modified oligonucleotides) can have any of a variety of ranges of lengths. In certain embodiments, oligonucleotides consist of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number nucleosides in the range. In certain such embodiments, 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 certain embodiments, oligonucleotides consist of 12 to 13, 12 to 14, 12 to 15,
12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21 , 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21 , 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26,
13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21 , 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21 , 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21 , 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to
28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21 , 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21 , 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21 , 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21 , 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to
29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides.
E. Certain Modified Oligonucleotides
[0132] In certain embodiments, the above modifications (sugar, nucleobase, internucleoside linkage) are incorporated into a modified oligonucleotide. In certain embodiments, modified oligonucleotides are characterized by their modification motifs and overall lengths. In certain embodiments, such parameters are each independent of one another. Thus, unless otherwise indicated, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. For example, the internucleoside linkages within the wing regions of a sugar gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region of the sugar motif. Likewise, such sugar gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications. Furthermore, in certain instances, an oligonucleotide is described by an overall length or range and by lengths or length ranges of two or more regions (e.g., a region of nucleosides having specified sugar modifications), in such circumstances it may be possible to select numbers for each range that result in an oligonucleotide having an overall length falling outside the specified range. In such circumstances, both elements must be satisfied. For example, in certain embodiments, a modified oligonucleotide consists if of 15-20 linked nucleosides and has a sugar motif consisting of three regions, A, B, and C, wherein region A consists of 2-6 linked nucleosides having a specified sugar motif, region B consists of 6-10 linked nucleosides having a specified sugar motif, and region C consists of 2-6 linked nucleosides having a specified sugar motif. Such embodiments do not include modified oligonucleotides where A and C each consist of 6 linked nucleosides and B consists of 10 linked nucleosides (even though those numbers of nucleosides are permitted within the requirements for A, B, and C) because the overall length of such oligonucleotide is 22, which exceeds the upper limit of the overall length of the modified oligonucleotide (20). Herein, if a description of an oligonucleotide is silent with respect to one or more parameter, such parameter is not limited. Thus, a modified oligonucleotide described only as having a gapmer sugar motif without further description may have any length, internucleoside linkage motif, and nucleobase motif. Unless otherwise indicated, all modifications are independent of nucleobase sequence.
F. Nucleobase Sequence
[0133] In certain embodiments, oligonucleotides (unmodified or modified oligonucleotides) are further described by their nucleobase sequence. In certain embodiments, oligonucleotides have a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain such embodiments, a region of an oligonucleotide has a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain embodiments, the nucleobase sequence of a region or entire length of an oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% complementary to the second oligonucleotide or nucleic acid, such as a target nucleic acid.
II. Certain Oligomeric Compounds
[0134] In certain embodiments, the disclosure provides oligomeric compounds, which consist of an oligonucleotide (modified or unmodified) and optionally one or more conjugate groups and/or terminal groups. Conjugate groups consist of one or more conjugate moiety and a conjugate linker which links the conjugate moiety to the oligonucleotide. Conjugate groups may be attached to either or both ends of an oligonucleotide and/or at any internal position. In certain embodiments, conjugate groups are attached to the 2'-position of a nucleoside of a modified oligonucleotide. In certain embodiments, conjugate groups that are attached to either or both ends of an oligonucleotide are terminal groups. In certain such embodiments, conjugate groups or terminal groups are attached at the 3’ and/or 5’-end of oligonucleotides. In certain such embodiments, conjugate groups (or terminal groups) are attached at the 3’-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 3’-end of oligonucleotides. In certain embodiments, conjugate groups (or terminal groups) are attached at the 5’-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 5’-end of oligonucleotides.
[0135] Examples of terminal groups include but are not limited to conjugate groups, capping groups, phosphate moieties, protecting groups, modified or unmodified nucleosides, and two or more nucleosides that are independently modified or unmodified.
A. Certain Conjugate Groups
[0136] In certain embodiments, oligonucleotides are covalently attached to one or more conjugate groups. In certain embodiments, conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance. In certain embodiments, conjugate groups impart a new property on the attached oligonucleotide, e.g., fluorophores or reporter groups that enable detection of the oligonucleotide. Certain conjugate groups and conjugate moieties have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991 , 70, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethyl-ammonium 1 ,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651 -3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. The , 1996, 277, 923-937), a tocopherol group (Nishina et al. , Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or a GalNAc cluster (e.g., WO2014/179620).
[0137] Most oligomeric compounds are metabolized in the liver or kidneys, which can reduce the half-life of the oligomeric compound in a subject. For example, in certain embodiments, an oligomeric compound administered to a subject may distribute to the kidneys and then be excreted out in the subject’s urine. In other embodiments, conjugating an oligomeric compound may lead to metabolism in the liver. In certain embodiments, an oligomeric compound administered to a subject is both metabolized by the liver and excreted out through the kidneys. In certain embodiments, the present disclosure provides oligomeric compounds comprising a modified oligonucleotide and a conjugate group, wherein the conjugate group enhances delivery of the modified oligonucleotide. In certain embodiments, the conjugate group enhances delivery of the modified oligonucleotide to a tissue selected from among: skeletal muscle, cardiac muscle, smooth muscle, adipose, white adipose, spleen, bone, intestine, adrenal, testes, ovary, pancreas, pituitary, prostate, skin, uterus, bladder, brain, glomerulus, distal tubular epithelium, breast, lung, heart, kidney, ganglion, frontal cortex, spinal cord, trigeminal ganglia, sciatic nerve, dorsal root ganglion, epididymal fat, diaphragm, and colon.
[0138] Oligomeric compounds typically show good uptake in hepatocytes. In certain embodiments, the present disclosure provides oligomeric compounds comprising a modified oligonucleotide and a conjugate group, wherein the conjugate group enhances uptake in a particular cell type. In certain embodiments, the conjugate group enhances uptake in macrophages. In certain embodiments, the conjugate group enhances uptake in cardiomyocytes. In certain embodiments, the conjugate group enhances uptake in fibroblasts. In certain embodiments, the conjugate group enhances uptake in endothelial cells. In certain embodiments, the conjugate group enhances uptake in heart cells.
1. Conjugate Groups
[0139] Conjugate moieties include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.
[0140] In certain embodiments, a conjugate moiety comprises a compound found endogenously in a subject. For example, in certain embodiments, the conjugate may be a steroid, such as cholesterol. Although cholesterol is endogenously produced in a subject and has certain physiological activities, cholesterol may be used as a conjugate to alter or improve one or more properties of a modified oligonucleotide. For example, cholesterol conjugated to a modified oligonucleotide may increase the modified oligonucleotide’s binding affinity for a given protein, such as HDL.
[0141] In certain embodiments, a conjugate moiety comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5- triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
[0142] In certain embodiments, conjugate moieties impart a new property on the attached oligonucleotide, which may alter the oligonucleotides distribution or pharmacokinetic profile. For example, certain conjugate moieties selected from among lipids, vitamins, steroids, C5-C30 saturated alkyl groups, C5-C30 unsaturated alkyl groups, fatty acids, or lipophilic groups may increase the distribution of an oligonucleotide to various tissues or organs within a subject. In certain embodiments, certain conjugate moieties selected from among lipids, vitamins, steroids, C5-C30 saturated alkyl groups, C5-C30 unsaturated alkyl groups, fatty acids, or lipophilic groups increase affinity for an oligonucleotide with one or more serum proteins, such as albumin. In certain embodiments, certain conjugate moieties selected from among lipids, vitamins, steroids, C5-C30 saturated alkyl groups, C5-C30 unsaturated alkyl groups, fatty acids, or lipophilic groups increase affinity for an oligonucleotide to an extra-hepatic tissue. In certain embodiments, this allows for conjugated oligonucleotides to have longer half-lives because less of the conjugated oligonucleotide is metabolized in the liver. [0143] In certain embodiments, certain conjugate moieties are selected from among lipids, vitamins, steroids, C5-C30 saturated alkyl groups, C5-C30 unsaturated alkyl groups, fatty acids, or lipophilic groups increase affinity for an extra-hepatic tissue selected from among: skeletal muscle, cardiac muscle, smooth muscle, adipose, white adipose, spleen, bone, intestine, adrenal, testes, ovary, pancreas, pituitary, prostate, skin, uterus, bladder, brain, glomerulus, distal tubular epithelium, breast, lung, heart, kidney, ganglion, frontal cortex, spinal cord, trigeminal ganglia, sciatic nerve, dorsal root ganglion, epididymal fat, diaphragm, pancreas, and colon.
2. Conjugate Linkers
[0144] Conjugate moieties are attached to oligonucleotides through conjugate linkers. In certain oligomeric compounds, the conjugate linker is a single chemical bond (i.e. , the conjugate moiety is attached directly to an oligonucleotide through a single bond). In certain embodiments, the conjugate linker comprises a chain structure, such as a hydrocarbyl chain, or an oligomer of repeating units such as ethylene glycol, nucleosides, or amino acid units.
[0145] In certain embodiments, a conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises groups selected from alkyl, amino, oxo, amide and ether groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker includes at least one neutral linking group.
[0146] In certain embodiments, conjugate linkers, including the conjugate linkers described above, are bifunctional linking moieties, e.g., those known in the art to be useful for attaching conjugate groups to parent compounds, such as the oligonucleotides provided herein. In general, a bifunctional linking moiety comprises at least two functional groups. One of the functional groups is selected to bind to a particular site on a parent compound and the other is selected to bind to a conjugate group. Examples of functional groups used in a bifunctional linking moiety include but are not limited to electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In certain embodiments, bifunctional linking moieties comprise one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.
[0147] Examples of conjugate linkers include but are not limited to pyrrolidine, 8- amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include but are not limited to substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
[0148] In certain embodiments, conjugate linkers comprise 1 -10 linker-nucleosides. In certain embodiments, conjugate linkers comprise 2-5 linker-nucleosides. In certain embodiments, conjugate linkers comprise exactly 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise the TCA motif. In certain embodiments, such linker-nucleosides are modified nucleosides. In certain embodiments such linker- nucleosides comprise a modified sugar moiety. In certain embodiments, linker- nucleosides are unmodified. In certain embodiments, linker-nucleosides comprise an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, a cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5- methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is typically desirable for linker-nucleosides to be cleaved from the oligomeric compound after it reaches a target tissue. Accordingly, linker- nucleosides are typically linked to one another and to the remainder of the oligomeric compound through cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.
[0149] Herein, linker-nucleosides are not considered to be part of the oligonucleotide. Accordingly, in embodiments in which an oligomeric compound comprises an oligonucleotide consisting of a specified number or range of linked nucleosides and/or a specified percent complementarity to a reference nucleic acid and the oligomeric compound also comprises a conjugate group comprising a conjugate linker comprising linker-nucleosides, those linker-nucleosides are not counted toward the length of the oligonucleotide and are not used in determining the percent complementarity of the oligonucleotide for the reference nucleic acid. For example, an oligomeric compound may comprise (1 ) a modified oligonucleotide consisting of 8-30 nucleosides and (2) a conjugate group comprising 1-10 linker-nucleosides that are contiguous with the nucleosides of the modified oligonucleotide. The total number of contiguous linked nucleosides in such an oligomeric compound is more than 30. Alternatively, an oligomeric compound may comprise a modified oligonucleotide consisting of 8-30 nucleosides and no conjugate group. The total number of contiguous linked nucleosides in such an oligomeric compound is no more than 30. Unless otherwise indicated conjugate linkers comprise no more than 10 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 5 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 2 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 1 linker-nucleoside.
[0150] In certain embodiments, it is desirable for a conjugate group to be cleaved from the oligonucleotide. For example, in certain circumstances oligomeric compounds comprising a particular conjugate moiety are better taken up by a particular cell type, but once the oligomeric compound has been taken up, it is desirable that the conjugate group be cleaved to release the unconjugated or parent oligonucleotide. Thus, certain conjugate linkers may comprise one or more cleavable moieties. In certain embodiments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety is a group of atoms comprising at least one cleavable bond. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, a cleavable moiety is selectively cleaved inside a cell or subcellular compartment, such as a lysosome. In certain embodiments, a cleavable moiety is selectively cleaved by endogenous enzymes, such as nucleases. [0151] In certain embodiments, a cleavable bond is selected from among: an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. In certain embodiments, a cleavable bond is one or both of the esters of a phosphodiester. In certain embodiments, a cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a phosphate linkage between an oligonucleotide and a conjugate moiety or conjugate group.
[0152] In certain embodiments, a cleavable moiety comprises or consists of one or more linker-nucleosides. In certain such embodiments, the one or more linker- nucleosides are linked to one another and/or to the remainder of the oligomeric compound through cleavable bonds. In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds. In certain embodiments, a cleavable moiety is 2'-deoxy nucleoside that is attached to either the 3' or 5'-terminal nucleoside of an oligonucleotide by a phosphate internucleoside linkage and covalently attached to the remainder of the conjugate linker or conjugate moiety by a phosphate or phosphorothioate linkage. In certain such embodiments, the cleavable moiety is 2'-deoxyadenosine.
III. Certain Antisense Compounds
[0153] In certain embodiments, the present disclosure provides antisense compounds, which comprise or consist of an oligomeric compound comprising an antisense nucleic acid, having a nucleobase sequence complementary to that of a target nucleic acid. In certain embodiments, antisense compounds are single-stranded. Such single-stranded antisense compounds typically comprise or consist of an oligomeric compound that comprises or consists of a modified oligonucleotide and optionally a conjugate group. In certain embodiments, antisense compounds are double-stranded. Such double-stranded antisense compounds comprise a first oligomeric compound having a region complementary to a target nucleic acid and a second oligomeric compound having a region complementary to the first oligomeric compound. The first oligomeric compound of such double stranded antisense compounds typically comprises or consists of a modified oligonucleotide and optionally a conjugate group. The oligonucleotide of the second oligomeric compound of such double-stranded antisense compound may be modified or unmodified. Either or both oligomeric compounds of a double-stranded antisense compound may comprise a conjugate group. The oligomeric compounds of double-stranded antisense compounds may include non-complementary overhanging nucleosides.
[0154] In certain embodiments, oligomeric compounds of antisense compounds are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, antisense compounds selectively affect one or more target nucleic acid. Such selective antisense compounds comprise a nucleobase sequence that hybridizes to one or more target nucleic acid, resulting in one or more desired antisense activity and does not hybridize to one or more non-target nucleic acid or does not hybridize to one or more non-target nucleic acid in such a way that results in significant undesired antisense activity.
[0155] In certain antisense activities, hybridization of an antisense compound to a target nucleic acid results in recruitment of a protein that cleaves the target nucleic acid. For example, certain antisense compounds result in RNase H mediated cleavage of the target nucleic acid. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. The DNA in such an RNA:DNA duplex need not be unmodified DNA. In certain embodiments, the invention provides antisense compounds that are sufficiently “DNA-like” to elicit RNase H activity. Further, in certain embodiments, one or more non-DNA-like nucleoside in the gap of a gapmer is tolerated.
[0156] In certain antisense activities, an antisense compound or a portion of an antisense compound is loaded into an RNA-induced silencing complex (RISC), ultimately resulting in cleavage of the target nucleic acid. For example, certain antisense compounds result in cleavage of the target nucleic acid by Argonaute. Antisense compounds that are loaded into RISC are RNAi compounds. RNAi compounds may be double-stranded (siRNA) or single-stranded (ssRNA).
[0157] In certain embodiments, hybridization of an antisense compound to a target nucleic acid does not result in recruitment of a protein that cleaves that target nucleic acid. In certain such embodiments, hybridization of the antisense compound to the target nucleic acid results in alteration of splicing of the target nucleic acid. In certain embodiments, hybridization of an antisense compound to a target nucleic acid results in inhibition of a binding interaction between the target nucleic acid and a protein or other nucleic acid. In certain such embodiments, hybridization of an antisense compound to a target nucleic acid results in alteration of translation of the target nucleic acid.
[0158] Antisense activities may be observed directly or indirectly. In certain embodiments, observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid, a change in the ratio of splice variants of a nucleic acid or a protein, and/or a phenotypic change in a cell or an animal.
IV. Certain Target Nucleic Acids
[0159] In certain embodiments, antisense compounds comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from: an mRNA and a pre-mRNA, including intronic, exonic and untranslated regions. In certain embodiments, the target RNA is an mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron.
[0160] In certain embodiments, the target nucleic acid is a non-coding RNA. In certain such embodiments, the target non-coding RNA is selected from: a long-non-coding RNA, a short non-coding RNA, an intronic RNA molecule, a snoRNA, a scaRNA, a microRNA (including pre-microRNA and mature microRNA), a ribosomal RNA, and promoter directed RNA. In certain embodiments, the target nucleic acid is a nucleic acid other than a mature mRNA. In certain embodiments, the target nucleic acid is a nucleic acid other than a mature mRNA or a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA or an intronic region of a pre-mRNA. In certain embodiments, the target nucleic acid is a long non-coding RNA. In certain embodiments, the target nucleic acid is a non-coding RNA associated with splicing of other pre-mRNAs. In certain embodiments, the target nucleic acid is a nuclear-retained non-coding RNA.
[0161] In certain embodiments, antisense compounds described herein are complementary to a target nucleic acid comprising a single-nucleotide polymorphism (SNP). In certain such embodiments, the antisense compound is capable of modulating expression of one allele of the SNP-containing target nucleic acid to a greater or lesser extent than it modulates another allele. In certain embodiments, an antisense compound hybridizes to a (SNP)-containing target nucleic acid at the single-nucleotide polymorphism site.
[0162] In certain embodiments, antisense compounds are at least partially complementary to more than one target nucleic acid. For example, antisense compounds of the present invention may mimic microRNAs, which typically bind to multiple targets.
A. Complementarity/Mismatches to the Target Nucleic Acid
[0163] In certain embodiments, antisense compounds comprise an antisense nucleic acid that are complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, such oligonucleotides are 99% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 95% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 90% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 85% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In certain embodiments, an antisense nucleic acid is at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide and comprise a region that is 100% or fully complementary to a target nucleic acid. In certain such embodiments, the region of full complementarity is from 6 to 20 nucleobases in length. In certain embodiments, the region of full complementarity is from 10 to 18 nucleobases in length. In certain embodiments, the region of full complementarity is from 18 to 20 nucleobases in length. [0164] In certain embodiments, the oligomeric compounds of antisense compounds comprise one or more mismatched nucleobases relative to the target nucleic acid. In certain such embodiments, antisense activity against the target is reduced by such mismatch, but activity against a non-target is reduced by a greater amount. Thus, in certain such embodiments selectivity of the antisense compound is improved. In certain embodiments, the mismatch is specifically positioned within an oligonucleotide having a gapmer motif. In certain such embodiments, the mismatch is at position 1 , 2, 3, 4, 5, 6, 7, or 8 from the 5’-end of the gap region. In certain such embodiments, the mismatch is at position 9, 8, 7, 6, 5, 4, 3, 2, 1 from the 3’-end of the gap region. In certain such embodiments, the mismatch is at position 1 , 2, 3, or 4 from the 5’-end of the wing region. In certain such embodiments, the mismatch is at position 4, 3, 2, or 1 from the 3’-end of the wing region.
V. Certain Agonists of Antisense Activity
[0165] In certain embodiments, protein phosphatase 2 (PP2A) agonists are agonists of antisense activity. In certain embodiments, one or more antisense compounds are combined with one or more PP2A agonists to increase the activity of the oligomeric compounds. As discussed herein, PP2A agonists increase intracellular accumulation of oligomeric compounds within a biological cell. PP2A agonists include (but are not limited to) sphingolipids, sphingolipid-like compounds, sphingolipid-like compound 893, sphingolipid-like compound 1090, sphingolipid-like compound 325, FTY720, derivatives of FTY720, ceramide, sphingosine, sphinganine, phytosphingosine, dimethylsphingosine, safingol, perphenazine, perphenazine derivatives (e.g., small molecule activator of PP2A (SMAP), iHAP (2-chloro-10-(4-methoxybenzoyl)-10H-phenothiazine)), SET inhibitors, CIP2a inhibitors, Withaferin A, OSLI-2S, and derivatives thereof. Numerous PP2A agonists are described in the literature and can be utilized in certain embodiments as described herein (see A. R. Clark and M Ohlmeyer, Pharmacol Then 2019;201 :181 -201 ; M. R. Carratu, et al., Curr Med Chem. 2016;23(38):4286-4296; M. Remmerie and V. Janssens, Front Oncol. 2019;9:462; D. B. Kastrinsky, et al., Bioorg Med Chem. 2015;23(19):6528-6534; and K. McClinch, et al., Cancer Res. 2018;78(8):2065-2080; the disclosures of which are incorporated herein by reference). [0166] In certain embodiments, combining PP2A agonists with antisense compounds reduces the amount of oligomeric compound needed to produce the requisite antisense activity. In certain embodiments, PP2A agonists increase antisense activity between 2- fold and 100-fold. In various embodiments, PP2A agonists increase antisense activity 2- fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55- fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, or greater than 100-fold.
[0167] Sphingolipids and PP2A agonists inactivate ADP Ribosylation Factor 6 (ARF6). In certain embodiments, ARF6 antagonists are agonists of antisense activity. In certain embodiments, antisense compounds are combined with ARF6 antagonists. As discussed herein, ARF6 antagonists increase the ability of oligomeric compounds to accumulate within biological cells. ARF6 antagonists include (but are not limited to) sphingolipids, sphingolipid-like compounds, sphingolipid-like compound 893, sphingolipid-like compound 1090, sphingolipid-like compound 325, FTY720, derivatives of FTY720, ceramide, NAV2729, SecinH3, perphenazine, and derivatives thereof. Numerous ARF6 antagonists are described in the literature and can be utilized in certain embodiments as described herein (see B. T. Finicle, et al., J Cell Sci. 2018;131 (12):jcs213314; J. H. Yoo, et al., Cancer Cell. 2016;29(6):889-904; and M. Hafner, et al., Nature. 2006;444(7121 ):941-944; the disclosures of which are incorporated herein by reference). [0168] In certain embodiments, combining ARF6 antagonists with PlKfyve antagonists and with antisense compounds reduces the amount of antisense compound needed to produce the requisite antisense activity. In various embodiments, ARF6 antagonists in combination with PlKfyve antagonists increase antisense activity between 2-fold and 100- fold. In certain embodiments, ARF6 agonists increase antisense activity 2-fold, 5-fold, 10- fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, or greater than 100- fold.
[0169] In certain embodiments, inhibitors of multivesicular body fusion with lysosomes such as Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing (PlKfyve) antagonists are agonists of antisense activity. In certain embodiments, antisense compounds are combined with PlKfyve antagonists to increase the activity of the oligomeric compounds. As discussed herein, PlKfyve antagonists increase the ability of oligomeric compounds to accumulate within biological cells. PlKfyve antagonists include (but are not limited to) sphingolipids, sphingolipid-like compounds, sphingolipid-like compound 893, sphingolipid-like compound 1090, sphingolipid-like compound 325, FTY720, derivatives of FTY720, YM201636, APY0201 , Apilimod, Late Endosome Trafficking Inhibitor EGA, and derivatives thereof. Numerous PlKfyve antagonists are described in the literature and can be utilized in certain embodiments as described herein (see S. M. Kim, et al., J Clin Invest. 2016;126(11 ):4088-4102; H. B. Jefferies, et al., EMBO Rep. 2008;9(2): 164-170; and X. Cai, et al., Chem Biol. 2013;20(7):912-921 ; the disclosures of which are incorporated by reference).
[0170] In certain embodiments, combining PlKfyve antagonists with antisense compounds reduces the amount of oligomeric compound needed to produce the requisite antisense activity. In certain embodiments, PlKfyve antagonists increase antisense activity between 2-fold and 100-fold. In various embodiments, PlKfyve agonists increase antisense activity 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, or greater than 100-fold.
[0171] In certain embodiments, an agonist of antisense activity is applied concurrently with an antisense compound. Accordingly, in certain embodiments, a biological cell is simultaneously contacted with an agonist of antisense activity and an antisense compound. In certain embodiments, an animal is simultaneously administered an agonist of antisense activity and antisense compound.
[0172] In certain embodiments, an agonist of antisense activity is applied as a pretreatment prior to an application of an antisense compound. Accordingly, in certain embodiments, a biological cell is pretreated with an agonist of antisense activity prior to contact with an antisense compound. In certain embodiments, a biological cell is pretreated by contacting the cell with an agonist of antisense activity for a time period of between 1 minute and 300 minutes prior to contact with an antisense compound. In various embodiments, a biological cell is pretreated by contacting the cell with an agonist of antisense activity for a time period of 1 minute, 2 minutes, 3, minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, 240 minutes, 270 minutes, 300 minutes, or more than 300 minutes prior to contact with an antisense compound. In certain embodiments, an animal is pretreated by administration of an agonist of antisense activity for a time period of between 1 minute and 720 minutes prior to administration of an antisense compound. In various embodiments, an animal is pretreated by administration of an agonist antisense activity for a time period of 1 minute, 2 minutes, 3, minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, 240 minutes, 270 minutes, 300 minutes, 330 minutes, 360 minutes, 390 minutes, 420 minutes, 450 minutes, 480 minutes, 510 minutes, 540 minutes, 570 minutes, 600 minutes, 630 minutes, 660 minutes, 690 minutes, 720 minutes, or more than 720 minutes prior to administration of an antisense compound.
[0173] In certain embodiments, an agonist of antisense activity is utilized at concentration between 1 nM to 100 pM. In various embodiments, an agonist of antisense activity is utilized at a concentration less than 1 nM, approximately 1 nM, approximately 10 nM, approximately 100 nM, approximately 1 pM, approximately 10 pM, approximately 100 pM, or greater than 100 pM. When referring to concentration of agonists, approximately is to be interpreted as within an order of magnitude (e.g., approximately 1 nM is 1 nM to less than 10 nM).
I. Sphingolipid-like Compounds
A. Sphingolipid-like Compounds Based on O-benzyl Pyrrolidines
[0174] In certain embodiments, an agonist of antisense activity is based on O-benzyl pyrrolidines. In certain embodiments, an agonist of antisense activity is of formula:
Figure imgf000046_0001
Ri is an optional functional group selected from an alkyl chain, (CH2)nOH, CHOH- alkyl, CHOH-alkyne, (CH2)nO-alkyl, (CH2)nO-alkene, (CH2)nO-alkyne, wherein an akyl, alkyne, or alkene is an aliphatic chain up to ten carbons;
R2 is an aliphatic chain (Ce - C10);
R3 is a mono-, di-, tri- or quad- aromatic substituent comprising H, halogen, alkyl, alkoxy, azide (N3), ether, NO2, or cyanide (CN);
One of R1 and R4 is an alcohol (CH2OH) or H;
L is O-CH2; and n is an independently selected integer selected from 1 , 2, or 3; wherein the phenyl can be moved about the five-carbon ring, e.g., extending from ring positions 3, 4, or 5.
[0175] In certain embodiments of on O-benzyl pyrrolidines, the O-benzyl group can be moved to position 4 (shown above) or 3 as shown below:
Figure imgf000046_0002
[0176] In certain embodiments, alkyl, CH2OH, or (CH2)nOH groups can be added to position 5.
[0177] In certain embodiments, one of R1 or R4 is an alkyl having 1 to 6 carbons.
[0178] It will be understood that compounds described herein may exist as stereoisomers, including enantiomers, diastereomers, cis, trans, syn, anti, solvates (including hydrates), tautomers, and mixtures thereof.
B. Sphingolipid-like Compounds Based on 3- and 4-C-aryl Pyrrolidines [0179] In certain embodiments, an agonist of antisense activity is based on diastereomeric 3- and 4-C-aryl pyrrolidines. In certain embodiments, an agonist of antisense activity is of formula:
Figure imgf000047_0001
R1 is an optional functional group selected from an alkyl chain, (CH2)nOH, CHOH- alkyl, CHOH-alkyne, (CH2)nO-alkyl, (CH2)nO-alkene, (CH2)nO-alkyne, wherein the akyl, the alkyne, or the alkene is an aliphatic chain up to ten carbons;
R2 is an aliphatic chain (Ce - C14);
R3 is a mono-, di-, tri- or tetra- aromatic substituent comprising hydrogen, halogen, alkyl, alkoxy, azide (N3), ether, NO2, or cyanide (CN); n is an independently selected integer selected from 1 , 2, or 3; and wherein the phenyl can be moved about the five-carbon ring, e.g., extending from ring positions 3 or 4, while maintaining the 3- and 4-C-aryl chemistries. [0180] In certain embodiments of diastereomeric 3- and 4-C-aryl 2-hydroxymethyl pyrrolidines, the C-aryl group can be moved to position 3 (shown above) or 4 as shown below:
Figure imgf000048_0001
[0181] In certain embodiments, alkyl, CH2OH, or (CH2)nOH groups can be added to position 5.
[0182] In certain embodiments, R2 is an unsaturated hydrocarbon chain.
[0183] In certain embodiments, the R1 is an alkyl having 1 to 6 carbons.
[0184] It will be understood that compounds described herein may exist as stereoisomers, enantiomers, diastereomers, cis, trans, syn, anti, solvates (including hydrates), tautomers, and mixtures thereof.
[0185] In certain embodiments, an agonist of antisense activity is compound 893, having the formula:
Figure imgf000048_0002
[0186] In certain embodiments, an agonist of antisense activity is compound 1090, having the formula:
Figure imgf000048_0003
C. Sphingolipid-like Compounds Based on Azacycles with Heteroaromatic Appendage
[0187] In certain embodiments, an agonist of antisense activity is based on azacycles with an attached heteroaromatic appendage. In certain embodiments, an agonist of antisense activity is of formula:
Figure imgf000049_0001
or a pharmaceutically acceptable salt thereof;
R is an optionally substituted heteroaromatic moiety such as an optionally substituted pyridazine, optionally substituted pyridine, optionally substituted pyrimidine, phenoxazine, or optionally substituted phenothiazine.
Ri is H, alkyl such as C1-6 alkyl or C1-4 alkyl including methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, etc, Ac, Boc, guanidine moiety.
R2 is an aliphatic chain comprising 6 to 14 carbons.
R3 is a 1 , 2, 3, or 4 substituents, wherein each substituent, independently, is H, halogen, alkyl, alkoxy, N3, NO2, and CN. n is independently 1 , 2, 3, or 4. m is independently 1 or 2.
The phenyl moiety can be attached at any available position of the azacycle core. [0188] In some embodiments, R2 is an unsaturated hydrocarbon chain.
[0189] In some embodiments, R2 is C6-14 alkyl, C6-10 alkyl, C7-9 alkyl, CeH , C7H15, CsHn, C9H19, C10H21, C11H23, C12H25, C13H27, Or Cl4H29.
[0190] In some embodiments R3 is H.
[0191] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4.
[0192] In some embodiments, m is 1. In some embodiments, m is 2.
[0193] In some embodiments, the R2 and R3 substituents can have different combinations around the phenyl ring with regard to their position. [0194] In some embodiments, the Ri is an alkyl having 1 to 6 carbons.
[0195] It will be understood that compounds described herein may exist as stereoisomers, including enantiomers, diastereomers, cis, trans, syn, anti, solvates (including hydrates), tautomers, and mixtures thereof, are contemplated in the compounds described herein.
[0196] In some embodiments, R is a 1 ,2-pyridazine having the formula:
Figure imgf000050_0001
R4 and Rs are functional groups independently selected from: alkyl including methyl, optionally substituted aryl (i.e., unsubstituted aryl or substituted aryl) including optionally substituted phenyl, and optionally substituted heteroaryl including optionally substituted pyridine and optionally substituted pyrimidine.
The pyridazine moiety is connected to the azacycle at the position 4 or 5 of the pyridazine.
[0197] In some embodiments, any substituents of R4 and Rs, if present, are independently a halogen (including F), an alkyl, a terminal alkyne, or an azide.
[0198] In some embodiments, R4 is C1-6 alkyl, such as CH3, C2 alkyl, C3 alkyl, C4 alkyl, Cs alkyl, or Ce alkyl; unsubstituted aryl or substituted aryl, including unsubstituted phenyl, or phenyl having 1 , 2, 3, 4, or 5 substituents; unsubstituted heteroaryl or substituted heteroaryl, including unsubstituted pyridine or pyridine having 1 , 2, 3, or 4 substituents, or unsubstituted pyrimidine or pyrimidine having 1 , 2, or 3 substituents. Any substituent may be used in the substituted aryl (e.g., substituted phenyl) or substituted heteroaryl (e.g., substituted pyridine or substituted pyrimidine). For example, the substituents of the substituted aryl or substituted heteroaryl may independently be, halo (such as F, Cl, Br, I), C1-6 alkyl (such as CH3, C2 alkyl, C3 alkyl, C4 alkyl, Cs alkyl, Ce alkyl), or X-Ra, wherein X is O, -C(=O)-, -NHC(=O)-, or -C(=O)NH- and Ra is C1-6 alkyl (such as CH3, C2 alkyl, C3 alkyl, C4 alkyl, Cs alkyl, Ce alkyl), C2-6 alkenyl (such as -CH=CH2, -CH2CH=CH2, - CH2CH2CH=CH2, -CH2CH2CH2CH=CH2, -CH2CH2CH2CH2CH=CH2, etc.), or C2-6 alkynyl (such as -CH CH2, -CH2CH CH2, -CH2CH2CH CH2, -CH2CH2CH2CH CH2, - CH2CH2CH2CH2CH=CH2, etc.); or azide.
[0199] In some embodiments, Rs is C1-6 alkyl, such as CHs, C2 alkyl, Cs alkyl, C4 alkyl, Cs alkyl, or Ce alkyl; unsubstituted aryl or substituted aryl, including unsubstituted phenyl, or phenyl having 1 , 2, 3, 4, or 5 substituents; unsubstituted heteroaryl or substituted heteroaryl, including unsubstituted pyridine or pyridine having 1 , 2, 3, or 4 substituents, or unsubstituted pyrimidine or pyrimidine having 1 , 2, or 3 substituents. Any substituent may be used in the substituted aryl (e.g., substituted phenyl) or substituted heteroaryl (e.g., substituted pyridine or substituted pyrimidine). For example, the substituents of the substituted aryl or substituted heteroaryl may independently be, halo (such as F, Cl, Br, I), Ci-6 alkyl (such as CH3, C2 alkyl, C3 alkyl, C4 alkyl, Cs alkyl, Ce alkyl), or X-Ra, wherein X is 0, -C(=O)-, -NHC(=O)-, or -C(=O)NH- and Ra is C1-6 alkyl (such as CH3, C2 alkyl, C3 alkyl, C4 alkyl, Cs alkyl, Ce alkyl), C2-6 alkenyl (such as -CH=CH2, -CH2CH=CH2, - CH2CH2CH=CH2, -CH2CH2CH2CH=CH2, -CH2CH2CH2CH2CH=CH2, etc.), or C2-6 alkynyl (such as -CH=CH2, -CH2CH CH2, -CH2CH2CH CH2, -CH2CH2CH2CH CH2, - CH2CH2CH2CH2CH=CH2, etc.); or azide.
[0200] In some embodiments, R4 and Rs are the same functional group. [0201] In some embodiments, R4 and Rs are different functional groups.
[0202] In some embodiments, R4 is C1-6 alkyl, such as methyl, and Rs is optionally substituted phenyl.
[0203] In some embodiments, R4 is C1-6 alkyl, such as methyl, and Rs is optionally substituted pyridine.
[0204] In some embodiments, R4 is C1-6 alkyl, such as methyl, and Rs is optionally substituted pyrimidine.
[0205] In some embodiments, R4 is optionally substituted pyridine and Rs is optionally substituted pyridine.
[0206] In some embodiments, R4 is optionally substituted phenyl and Rs is optionally substituted phenyl.
[0207] In some embodiments, R4 is optionally substituted phenyl and Rs is optionally substituted pyrimidine. [0208] In some embodiments, R is an optionally substituted phenoxazine or an optionally substituted phenothiazine, such as phenoxazine or phenthiazine having the formula:
Figure imgf000052_0001
which may additionally have substituents on any available ring position.
X is selected from: O and S.
R is attached to the azacycle via R’s nitrogen.
[0209] Substituents of R may independently include halogen, alkyl (e.g., C1-6 alkyl, such as CH3, C2 alkyl, C3 alkyl, C4 alkyl, Cs alkyl, or Ce alkyl), alkoxy (e.g., C1-6 alkoxy, such as -OCH3, C2 alkoxy, C3 alkoxy, C4 alkoxy, Cs alkoxy, or Ce alkoxy), N3, NO2, and CN.
[0210] It will be understood that compounds described herein may exist as stereoisomers, including enantiomers, diastereomers, cis, trans, syn, anti, solvates (including hydrates), tautomers, and mixtures thereof.
[0211] In certain embodiments, an agonist of antisense activity is compound 325, having the formula:
Figure imgf000052_0002
D. Sphingolipid-like Compounds Based on 2-C-aryl Azacycles
[0212] In certain embodiments, an agonist of antisense activity is based on diastereomeric 2-C-aryl azacycles. In certain embodiments, an agonist of antisense activity is of formula:
Figure imgf000053_0001
Ri is a functional group selected from H, an alkyl chain, OH, (CH2)nOH, CHOH- alkyl, CHOH-alkyne, (CH2)nOR’, (CH2)nPO(OH)2 and esters thereof, CH=CHPO(OH)2 and esters thereof, (CH2CH2)nPO(OH)2 and esters thereof, and (CH2)nOPO(OH)2 and esters thereof, (CH2)nPOs and esters thereof, where R’ is an alkyl, alkene or alkyne, , wherein the akyl, the alkyne, or the alkene is an aliphatic chain up to ten carbons.
R2 is an aliphatic chain (Ce - C14).
R3 is a mono-, di-, tri- or tetra- aromatic substituent that includes hydrogen, halogen, alkyl, alkoxy, azide (N3), ether, NO2, cyanide (CN), or a combination thereof.
R4 is a functional group selected from H, alkyl including methyl (Me), ester, or acyl.
X’ is an anion of the suitable acid. n is an independently selected integer selected from 1 , 2, or 3. m is an independently selected integer selected from 0, 1 or 2.
The molecule can include an optional functional group of the azacycle’s substituent selected from the following: a polar group in the alpha, beta or gamma position with regard to the azacycle selected from carbonyls (C=O) and alcohols (CHOH); a cyclic carbon chain extending from the alpha, beta or gamma positions with regard to the azacycle back to the N of the azacycle, and a combination thererof.
[0213] In some embodiments, R1 is H, OH, or CH2OH. In some embodiments, R1 is H. In some embodiments, R1 is OH. In some embodiments, R1 is CH2OH.
[0214] In some embodiments, R2 is C6-14 alkyl, C6-10 alkyl, C7-9 alkyl, CeH , C7H15, CsHn, C9H19, CIOH2I , CH H23, CI2H25, CISH27, or Ci4H29. In some embodiments, R2 is CsHn.
[0215] In some embodiments R3 is H.
[0216] In some embodiments, n is 1 . [0217] In some embodiments m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3.
[0218] In some embodiments, the linking group connecting the phenyl ring to the azacycle is C(=O), CH2C(=O), C(=O)CH2, CH2CH2C(=O), CH2,CH2CH2, CH2C(OCH3)H, or CHOHCH2. In some embodiments, the linking group connecting the phenyl ring to the azacycle is C(=O). In some embodiments, the linking group connecting the phenyl ring to the azacycle is CH2C(=O). In some embodiments, the linking group connecting the phenyl ring to the azacycle is C(=O)CH2. In some embodiments, the linking group connecting the phenyl ring to the azacycle is CH2CH2C(=O). In some embodiments, the linking group connecting the phenyl ring to the azacycle is CH2. In some embodiments, the linking group connecting the phenyl ring to the azacycle is CH2CH2. In some embodiments, the linking group connecting the phenyl ring to the azacycle is CH2C(OCH3)H. In some embodiments, the linking group connecting the phenyl ring to the azacycle is CHOHCH2. [0219] In some embodiments, the linking group connecting the phenyl ring to the azacycle includes a cyclic carbon chain extending from the alpha, beta or gamma positions with regard to the azacycle back to the N of the azacycle, so that the azaycle with the linking group form an optionally substituted bicyclic ring of the formula:
Figure imgf000054_0001
[0220] In some embodiments, R4 is H. In some embodiments, R4 is C1-6 alkyl, such as CH3, C2HS, C3H7, C4H9, C5H11, CeH , C1-3 alkyl, etc., C1-6 acyl, or C1-6 ester. In some embodiments, R4 is methyl.
[0221] In still other embodiments, the R2 and R3 substituents can have different combinations around the phenyl ring with regard to their position.
[0222] In still other embodiments, R2 is an unsaturated hydrocarbon chain.
[0223] In still other embodiments, the R1 is an alkyl having 1 to 6 carbons.
[0224] It will be understood that compounds described herein may exist as stereoisomers, including enantiomers, diastereomers, cis, trans, syn, anti, solvates (including hydrates), tautomers, and mixtures thereof. E. Pharmaceutical Salts of Sphingolipid-like Compounds
[0225] Certain sphingolipid-like compounds can also be related to pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” retains the desirable biological activity of the compound without undesired toxicological effects. Salts can be salts with a suitable acid, including, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, benzoic acid, pamoic acid, alginic acid, methanesulfonic acid, naphthalenesulphonic acid, and the like. Also, incorporated cations can include ammonium, sodium, potassium, lithium, zinc, copper, barium, bismuth, calcium, and the like; or organic cations such as tetraalkylammonium and trialkylammonium cations. Also useful are combinations of acidic and cationic salts. Included are salts of other acids and/or cations, such as salts with trifluoroacetic acid, chloroacetic acid, and trichloroacetic acid.
VI. Certain Pharmaceutical Compositions
[0226] In certain embodiments, the present disclosure provides pharmaceutical compositions comprising one or more antisense compounds or a salt thereof. In certain such embodiments, the pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution, one or more antisense compounds, and one or more agonists of antisense activity. In certain embodiments, such pharmaceutical composition consists of a sterile saline solution, one or more antisense compounds, and one or more agonists of antisense activity. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises sterile water, one or more antisense compounds, and one or more agonists of antisense activity. In certain embodiments, the water is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises phosphate-buffered saline (PBS), one or more antisense compounds, and one or more agonists of antisense activity. In certain embodiments, the PBS is pharmaceutical grade PBS. [0227] In certain embodiments, pharmaceutical compositions comprise one or more antisense compound, one or more agonists of antisense activity, and one or more excipients. In certain such embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.
[0228] In certain embodiments, antisense compounds and agonists of antisense activity may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
[0229] In certain embodiments, pharmaceutical compositions comprising an antisense compound encompass any pharmaceutically acceptable salts of the antisense compound, esters of the antisense compound, or salts of such esters. In certain embodiments, pharmaceutical compositions comprising an agonist of antisense activity encompass any pharmaceutically acceptable salts of the agonist of antisense activity. In certain embodiments, pharmaceutical compositions comprising one or more antisense compounds and one or more agonists of antisense activity, upon administration to an animal, including a human, are capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In certain embodiments, prodrugs comprise one or more conjugate group attached to an oligonucleotide, wherein the conjugate group is cleaved by endogenous nucleases within the body.
[0230] Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid, such as an antisense compound, is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.
[0231] In certain embodiments, pharmaceutical compositions comprise a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethyl sulfoxide (DMSO) are used.
[0232] In certain embodiments, pharmaceutical compositions comprise one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissuespecific antibody.
[0233] In certain embodiments, pharmaceutical compositions comprise a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A nonlimiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose. In certain embodiments, dimethyl sulfoxide (DMSO) is utilized as a co-solvent. In certain embodiments, cremophor (or cremophor EL) is utilized as a co-solvent. [0234] In certain embodiments, pharmaceutical compositions comprise one or more compounds that increase bioavailability. For example, 2-hydroxypropyl-beta-cyclodextrin can be utilized in pharmaceutical compositions and may increase bioavailability. In certain embodiment, DMSO, cremophor and 2-hydroxypropyl-beta-cyclodextrin is utilized to increase bioavailability of various agonists, especially sphingolipid-like compounds.
[0235] In certain embodiments, pharmaceutical compositions are prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration. In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multidose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.
[0236] In certain embodiments, a pharmaceutical composition is administered in a therapeutically effective amount as part of a course of treatment. As used in this context, to "treat" means to ameliorate or prevent at least one symptom of the disorder to be treated or to provide a beneficial physiological effect. A therapeutically effective amount can be an amount sufficient to prevent reduce, ameliorate or eliminate the symptoms of diseases or pathological conditions susceptible to such treatment. In certain embodiments, a therapeutically effective amount is an amount sufficient to increase antisense activity of an antisense compound. [0237] Dosage, toxicity and therapeutic efficacy of a pharmaceutical composition can be determined, e.g., by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to non-neoplastic cells and, thereby, reduce side effects.
[0238] Data obtained from cell culture assays or animal studies can be used in formulating a range of dosage for use in humans. If a pharmaceutical composition is provided systemically, the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in a method described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration or within the local environment to be treated in a range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of neoplastic growth) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by liquid chromatography coupled to mass spectrometry.
[0239] An "effective amount" is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a composition depends on the composition selected. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a pharmaceutical composition described herein can include a single treatment or a series of treatments. For example, several divided doses may be administered daily, one dose, or cyclic administration of the compounds to achieve the desired therapeutic result. A single small molecule compound may be administered, or combinations of various small molecule compounds may also be administered.
[0240] It is also possible to add agents that improve the solubility of pharmaceutical compositions. For example, a pharmaceutical composition can be formulated with one or more adjuvants and/or pharmaceutically acceptable carriers according to the selected route of administration. For oral applications, gelatin, flavoring agents, or coating material can be added. In general, for solutions or emulsions, carriers may include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride and potassium chloride, among others. In addition, intravenous vehicles can include fluid and nutrient replenishers, electrolyte replenishers and the like.
[0241] Numerous coating agents can be used in accordance with various embodiments. In certain embodiments, the coating agent is one which acts as a coating agent in conventional delayed release oral formulations, including polymers for enteric coating. Examples include hypromellose phthalate (hydroxy propyl methyl cellulose phthalate; HPMCP); hydroxypropylcellulose (HPC; such as KLLICEL®); ethylcellulose (such as ETHOCEL®); and methacrylic acid and methyl methacrylate (MAA/MMA; such as EU DRAG IT®).
[0242] In certain embodiments, a pharmaceutical composition also includes at least one disintegrating agent, as well as diluent. In some embodiments, a disintegrating agent is a super disintegrant agent. One example of a diluent is a bulking agent such as a polyalcohol. In many embodiments, bulking agents and disintegrants are combined, such as, for example, PEARLITOL FLASH®, which is a ready to use mixture of mannitol and maize starch (mannitol/maize starch). In accordance with a number of embodiments, any polyalcohol bulking agent can be used when coupled with a disintegrant or a super disintegrant agent. Additional disintegrating agents include, but are not limited to, agar, calcium carbonate, maize starch, potato starch, tapioca starch, alginic acid, alginates, certain silicates, and sodium carbonate. Suitable super disintegrating agents include, but are not limited to crospovidone, croscarmellose sodium, AMBERLITE (Rohm and Haas, Philadelphia, Pa.), and sodium starch glycolate.
[0243] In certain embodiments, diluents are selected from the group consisting of mannitol powder, spray dried mannitol, microcrystalline cellulose, lactose, dicalcium phosphate, tricalcium phosphate, starch, pregelatinized starch, compressible sugars, silicified microcrystalline cellulose, and calcium carbonate.
[0244] In certain embodiments, a pharmaceutical composition further utilizes other components and excipients. For example, sweeteners, flavors, buffering agents, and flavor enhancers to make the dosage form more palatable. Sweeteners include, but are not limited to, fructose, sucrose, glucose, maltose, mannose, galactose, lactose, sucralose, saccharin, aspartame, acesulfame K, and neotame. Common flavoring agents and flavor enhancers that may be included in the formulations described herein include, but are not limited to, maltol, vanillin, ethyl vanillin, menthol, citric acid, fumaric acid, ethyl maltol and tartaric acid.
[0245] In certain embodiments, a pharmaceutical composition also includes a surfactant. In certain embodiments, surfactants are selected from the group consisting of Tween 80, sodium lauryl sulfate, and docusate sodium.
[0246] In certain embodiments, a pharmaceutical composition further utilizes a binder. In certain embodiments, binders are selected from the group consisting of povidone (PVP) K29/32, hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC), ethylcellulose (EC), com starch, pregelatinized starch, gelatin, and sugar.
[0247] In certain embodiments, a pharmaceutical composition also includes a lubricant. In certain embodiments, lubricants are selected from the group consisting of magnesium stearate, stearic acid, sodium stearyl fumarate, calcium stearate, hydrogenated vegetable oil, mineral oil, polyethylene glycol, polyethylene glycol 4000- 6000, talc, and glyceryl behenate. [0248] Preservatives and other additives, like antimicrobial, antioxidant, chelating agents, and inert gases, can also be present. (See generally, Remington's Pharmaceutical Sciences, 16th Edition, Mack, (1980), the disclosure of which is incorporated herein by reference.)
EXEMPLARY EMBODIMENTS
[0249] The following examples illustrate certain embodiments of the present disclosure and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular high-affinity modification appears at a particular position, other high- affinity modifications at the same position are considered suitable, unless otherwise indicated.
Example 1: Synthesis of Various compounds
A. Synthesis of Sphingolipid-like Compound 893
Figure imgf000062_0001
sphingolipid-like compound 893
[0250] As illustrated in Fig. 2 synthesis of sphingolipid-like compound 893 (begins with compound 4c. The first intermediate, 5a ((2S,3R)-te/Y-Butyl 2-((fe/t- butyldiphenylsilyloxy)methyl)-3-(4-octylphenyl)pyrrolidine-1 -carboxylate) is synthesized in the following manner: A solution of compound 4c (120 mg, 0.187 mmol) in anhydrous THF (2.4 mL) is cooled to 0 °C. Borane dimethyl sulfide complex (2M in THF, 0.37 mL, 0.748 mmol) is added and the reaction is allowed to warm to room temperature and is stirred overnight. No more starting material is observed by TLC. The solvent is removed under reduced pressure. After the residue is co-evaporated twice with MeOH (2 mL), it is dissolved in CH2CI2, extracted three times with saturated solution of NaHCOs. The organic layer is washed with brine, dried over MgSCM and filtered. The solvent is removed under reduced pressure and the residue was purified by flash chromatography (hexane: EtOAc, 8:1 ) to give 5a (94.6 mg, 81 %) as a slightly yellow oil.
[0251] Synthesis of the next intermediate, compound 5b ((2S,3R)-te/Y-Butyl 2- (hydroxymethyl)-3-(4-octylphenyl)pyrrolidine-1 -carboxylate), begins by cooling a solution of 5a (271 mg, 0.432 mmol) in anhydrous THF (14.3 mL) to 0 °C. tetrabutylammonium fluoride solution (1 M in THF, 0.756 mL, 0.756 mmol) is added and the reaction is allowed to warm to room temperature and is stirred overnight. No more starting material is observed by TLC. The reaction is quenched with saturated solution of NaHCOs extracted three times with CH2CI2. The organic layers are washed with brine, dried over MgSCM and filtered. The solvent is removed under reduced pressure and the residue is purified by flash chromatography (hexane: EtOAc, 6:1 to 4:1 ) to give 5b (155 mg, 92 %) as a colorless oil.
[0252] In the final step in synthesizing sphingolipid-like compound 893 ((2S,3R)-2- (hydroxymethyl)-3-(4-octylphenyl)Pyrrolidinium chloride), HCI (4M in 1 ,4-dioxane, 0.98 mL, 3.9 mmol) is added to a flask with compound 5b (15 mg, 0.039 mmol) and the solution is stirred at room temperature until completion is shown by TLC (24 to 48 hours). The solvent is removed under reduced pressure and 1 ,4-dioxane (2 mL) is added to the flask and evaporated to remove the residual HCI. The crude mixture is purified by flash chromatography (CH2CI2: EtOH, 7:1 to 4:1 ) to give a yellow oil. This oil is dissolved in water, filtered through a plastic syringe filter (pore size: 0.45 pm), lyophilized to give compound 893 (11 .0 mg, 88 %) as a yellow solid.
B. Synthesis of Sphingolipid-like Compound 1090
Figure imgf000064_0001
sphingolipid-like compound 1090
[0253] Sphingolipid-like compound 1090 was obtained according to the procedure for synthesizing Compound 12, which is described below (Fig. 3).
[0254] Synthesis of compound 12 begins with intermediate compound 6b, as illustrated in FIG. 3. To synthesize intermediate compound 12a, triethylamine (22 pL, 0.154 mmol) is added to a solution of compound 6b (30 mg, 0.077 mmol) in anhydrous CH2CI2 (0.30 mL) and the solution is then cooled to 0 °C. Methanesulfonyl chloride (9.0 pL, 0.116 mmol) is added to the solution and the reaction is allowed to warm to room temperature and stirred overnight. The reaction is poured into water and extracted three times with EtOAc. The organic layers are washed with brine, dried over MgSO4 and filtered. The solvent is removed under reduced pressure and the residue is purified by flash chromatography (hexane: EtOAc, 3:1 to 2:1 ) to give 12a (34.0 mg, 94 %) as a colorless oil.
[0255] To synthesize intermediate compound 12b, a solution of compound 12a (29 mg, 0.062 mmol) in anhydrous THF (0.06 mL) is cooled to 0 °C. Lithium triethylborohydride (1.0 M solution in THF, 248 pL, 0.248 mmol) is added to the solution and the reaction is allowed to warm to room temperature and stirred for 5 h. The reaction is poured into water and extracted three times with EtOAc. The organic layers were washed with brine, dried over MgSO4 and filtered. The solvent is removed under reduced pressure and the residue is purified by flash chromatography (hexane: EtOAc, 14:1 ) to give 12b (20.7 mg, 89 %) as a colorless oil. [0256] Finally, to synthesize sphingolipid-like compound 1090 (compound 12), HCI (4M in 1 ,4-dioxane, 0.68 mL, 2.7 mmol) is added to a flask with 12b (10 mg, 0.027 mmol) and the solution is stirred at room temperature overnight. The solvent is removed under reduced pressure and 1 ,4-dioxane (1 mL) is added to the flask and evaporated to remove the residual HCI. The crude mixture is purified by flash chromatography (CH2CI2: EtOH, 9:1 to 3:1 ) to give a yellow oil. This oil was dissolved in water, filtered through a plastic syringe filter (pore size: 0.45 pm), lyophilized to give 12 (8.0 mg, 96 %) as a yellow solid.
C. Synthesis of Compound 325
Figure imgf000065_0001
[0257] [(2S,3R)-2-((3-(6-Methyl-3-(pyrimidin-2-yl)pyridazin-4-yl)propoxy)methyl)-3-(4- octylphenyl)pyrrolidin-1-ium chloride (Compound 325A) and (2S,3R)-2-((3-(3-methyl-6- (pyrimidin-2-yl)pyridazin-4-yl)propoxy)methyl)-3-(4-octylphenyl)pyrrolidin-1 -ium chloride (Compound 325B): Column chromatography (CH2Cl2/MeOH, gradient 99:1 to 95:5). To a stirred solution of 3-methyl-6-(pyrimidin-2-yl)-1 ,2,4,5-tetrazine (50 mg, 0.30 mmol, 1 .0 eq) in DMSO (2.0 mL) was added tert-butyl (2S,3R)-3-(4-octylphenyl)-2-((pent-4-en-1 - yloxy)methyl)pyrrolidine-1 -carboxylate vii (120 mg, 0.30 mmol, 1.0 eq) and the mixture was stirred overnight at 80 °C. The solvent was evaporated to dryness and the crude product was purified by column chromatography (MeOH: dichloromethane, 0:1 to 1 :19) to give the intermediate as a mixture of regioisomers. This mixture was dissolved in dioxane (2.0 mL) and HCI 4M in dioxane (0.5 mL) was added and the mixture was stirred for 2 hours. The solvent was evaporated and the residue was purified by column chromatography (CH2CI2/MeOH, gradient 99:1 to 95:5). In this condition, (2S,3R)-2-((3- (6-methyl-3-(pyrimidin-2-yl)pyridazin-4-yl)propoxy)methyl)-3-(4-octylphenyl)pyrrolidin-1 - ium chloride is eluted for first as a colorless gel; (2S,3R)-2-((3-(3-methyl-6-(pyrimidin-2- yl)pyridazin-4-yl)propoxy)methyl)-3-(4-octylphenyl)pyrrolidin-1 -ium chloride is eluted for last as a colorless gel (20 mg, 14% over 2 steps).
Figure imgf000066_0001
[0258] (2S,3R)-2-((3-(3-methyl-6-(pyrimidin-2-yl)pyridazin-4-yl)propoxy)methyl)-3-(4- octylphenyl)pyrrolidin-1 -ium chloride (Compound 325A): Compound 325A can be separated from compound 325B by careful column chromatography using CH2Cl2/MeOH, gradient 99:1 to 95:5: in this condition compound 325A is eluted for last as a colorless gel.
Figure imgf000066_0002
[0259] (2S,3R)-2-((3-(6-Methyl-3-(pyrimidin-2-yl)pyridazin-4-yl)propoxy)methyl)-3-(4- octylphenyl)pyrrolidin-1 -ium chloride (Compound 325B): Compound 325B can be separated from compound 325A by careful column chromatography using CH2Cl2/MeOH, gradient 99:1 to 95:5: in this condition, compound 325B is eluted for first as a colorless gel.
Example 2: Effect of sphingolipid-like compound pre-treatment on antisense compound activity in vitro
[0260] Antisense oligonucleotides were synthesized using standard solid phase oligonucleotide synthetic methods. Isis No. 353382 targets the mouse Scavenger Receptor Class B, Number 1 (SRB-1 ) transcript (GENBANK accession number NM_016741.1 , SEQ ID NO: 1 ). The sequence of Isis No. 353382 is 5’- GCTTCAGTCATGACTTCCTT-3’ (SEQ ID NO: 2). It is a 5-10-5 MOE gapmer, wherein the five nucleosides at the 5’-end and the five nucleosides at the 3’-end comprise 2’- methoxyethyl (MOE) modifications, and the ten middle nucleosides are 2’- deoxynucleosides. All internucleoside linkages are phosphorothioate linkages. The cytosine bases are 5-methylcytosines. [0261] In order to test the effect of pre-treatment with sphingolipid-like compounds on Isis No. 353382 activity in vitro, mouse hepatocellular SV40 large T-antigen (MHT) carcinoma cells were treated with either no sphingolipid-like compound (control), 3 pM FTY-720, 1 pM COMPOUND 1090, 3 pM COMPOUND 1090, 1 pM COMPOUND 893, or 3 pM COMPOUND 893. Each of 3 pM FTY-720, 1 pM COMPOUND 1090, 3 pM COMPOUND 1090, 1 pM COMPOUND 893, or 3 pM COMPOUND 893 was added 2 hours before Isis No. 353382 at the concentrations shown in the tables below. Cells were lysed 24 hours following oligonucleotide addition, and total RNA was purified. SRB-1 mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results are shown in the tables below as the average SRB-1 mRNA levels in cells treated with Isis No. 353382 relative to control cells that did not receive oligonucleotide treatment. The values were plotted on a dose-response curve and ICso values were then calculated. The results show that pre-treatment with either COMPOUND 893 or COMPOUND 1090 potentiated the effect of Isis No. 353382 in vitro relative to the control (no pretreatment).
Table 1
Figure imgf000067_0001
Table 2 IC50 values of COMPOUND 1090 pre-treatment in vitro
Figure imgf000068_0001
Table 4 IC50 values of COMPOUND 893 pre-treatment in vitro
Figure imgf000068_0002
Example 3: Synthetic Sphingolipid-like Molecules Enhance Activity of Modified Oligomeric Compounds in Murine Embryonic Fibroblasts
[0262] Antisense oligonucleotides were synthesized using standard solid phase oligonucleotide synthetic methods. Isis No. 395251 targets metastasis associated lung adenocarcinoma transcript 1 (MALAT-1 ) (GENBANK Accession No. NR_002847.2; SEQ ID NO: 3). ISIS 395251 has the nucleobase sequence CCAGGCTGGTTATGACTCAG; SEQ ID NO: 4. ISIS 395251 is a 5-10-5 MOE gapmer, wherein the central gap segment comprises ten 2’-deoxynucleosides and is flanked on both sides (in the 5’ and 3’ directions) by wings comprising 5 nucleosides each. Each nucleoside in the 5’ wing segment and each nucleoside in the 3’ wing segment has a 2’-MOE modification. The internucleoside linkages throughout the gapmer are phosphorothioate (P=S) linkages. All cytosine residues throughout the gapmer are 5-methylcytosines. [0263] In order to test the effect of pre-treatment with sphingolipid-like compounds on Isis No. 395251 activity in vitro, control LSL murine embryonic fibroblast cells (LSL MEFs) and murine embryonic fibroblast cells expressing an activated mutant of KRAS (KRAS MEFs) were treated with either no sphingolipid-like compound (control), 2.5 pM COMPOUND 1090, or 5 pM COMPOUND 1090. Each of 2.5 pM COMPOUND 1090, or 5 pM COMPOUND 1090 was added 2 hours before Isis No. 395251 at the following concentrations: 20,000 nM, 6,667 nM, 2222 nM, 741 nM, 246 nM, 82 nM, 27 nM, 9 nM, 3 nM, 1 nM, 0.3 nM, and 0.1 nM. Cells were lysed 24 hours following oligonucleotide addition, and total RNA was purified. MALAT-1 mRNA levels were determined using realtime PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. Percent inhibition vs concentration was plotted on a dose-response curve and IC50 values were then calculated. The results are shown in the tables below. This example shows that pretreatment of a cell with the sphingolipid-like COMPOUND 1090 significantly improves the activity of an antisense oligonucleotide.
Table 5 IC5o values in LSL MEFs
Figure imgf000069_0001
Table 6 IC50 values in KRAS MEFs
Figure imgf000069_0002
Example 4: Synthetic Sphingolipid-like Compounds Enhance Activity of Modified Oligomeric Compounds in Breast Cancer Cells
[0264] In order to test the effect of pre-treatment with sphingolipid-like compounds on Isis No. 395251 (described above) activity in vitro, MDA-MB-231 and MDA-MB-468 cells were treated with either no sphingolipid-like compound (control), 2.5 pM COMPOUND 1090, 5 pM COMPOUND 1090, or 5 pM COMPOUND 893. Each of 2.5 pM COMPOUND 1090, 5 pM COMPOUND 1090, or 5 pM COMPOUND 893 was added 2 hours before Isis No. 395251 at the following concentrations: 20,000 nM, 6,667 nM, 2222 nM, 741 nM, 246 nM, 82 nM, 27 nM, 9 nM, 3 nM, 1 nM, 0.3 nM, and 0.1 nM. Cells were lysed 24 hours following oligonucleotide addition, and total RNA was purified. MALAT-1 mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. Percent inhibition vs concentration was plotted on a dose-response curve and IC50 values were then calculated. The results are shown in the tables below. This example shows that pretreatment of different breast cancer cells with the sphingolipid-like COMPOUND 1090 or COMPOUND 893 significantly improves the activity of an antisense oligonucleotide.
Table 7 IC5o values in MDA-MB-231 cells
Figure imgf000070_0001
Table 8 IC50 values in MDA-MB-468 cells
Figure imgf000070_0002
Example 5: Synthetic Sphingolipid-like Compounds Enhance Activity of Modified Oligomeric Compounds in Pancreatic Cancer Cells
[0265] In order to test the effect of pre-treatment with sphingolipid-like compounds on Isis No. 395251 (described above) activity in vitro, Panc-1 and BxPC3 cells were treated with either no sphingolipid-like compound (control), 2.5 pM COMPOUND 1090, 5 pM COMPOUND 1090, or 5 pM COMPOUND 893. Each of 2.5 pM COMPOUND 1090, 5 pM COMPOUND 1090, or 5 pM COMPOUND 893 was added 2 hours before Isis No. 395251 at the following concentrations: 20,000 nM, 6,667 nM, 2222 nM, 741 nM, 246 nM, 82 nM, 27 nM, 9 nM, 3 nM, 1 nM, 0.3 nM, and 0.1 nM. Cells were lysed 24 hours following oligonucleotide addition, and total RNA was purified. MALAT-1 mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. Percent inhibition vs concentration was plotted on a dose-response curve and ICso values were then calculated. The results are shown in the tables below. This example shows that pretreatment of certain pancreatic cancer cells with the sphingolipid-like COMPOUND 1090 or COMPOUND 893 significantly improves the activity of an antisense oligonucleotide.
Table 9
Figure imgf000071_0001
Table 10
Figure imgf000071_0002
Example 6: Synthetic Sphingolipid-like Compounds Increase the Activity of Antisense Compound Targeting IncRNA MALAT1 in HeLa cells
[0266] HeLa cells were co-treated (i.e., simultaneously treated) with 5 pM of COMPOUND 893 and varying concentration a 5-10-5 2’MOE gapmer targeting the IncRNA MALAT1 (MALAT1 ASO Isis No. 556089) for 24 hours. RNA was then collected and qRT-PCR for MALAT1 RNA levels performed. COMPOUND 893 increases the activity of an ASO targeting IncRNA MALAT1 by 60-fold (Figs. 4A and 4B). The experiment was repeated using a 3-10-3 cET gapmer (Figs. 4C and 4D) and with 5-10-5 2’ MOE gapmer that targets the mRNA ACTN1 (Figs. 4E and 4F). Example 7: Synthetic Sphingolipid-like Compounds Increase Antisense Compound Activity in Multiple Cell Lines
[0267] The ability of COMPOUND 893 and COMPOUND 1090 to increase ASO activity is ubiquitous across various cell lines (Fig. 5A and 5B). Cells were co-treated with 893 or 1090 and varying concentrations of MALAT1 ASO for 24 h. RNA was then collected and qRT-PCR for MALAT1 RNA levels performed.
Example 8: Synthetic Sphingolipid-like Compounds Increase Antisense Compound Accumulation in Non-lysosomal Compartments
[0268] COMPOUND 893 increases accumulation of intracellular ASO in non- lysosomal compartments. HeLa cells were co-treated with untagged 5-10-5 2’MOE ASO in the presence or absence of 5 pM 893 for 6 h and then imaged by confocal microscopy (Fig. 6A). Total intracellular accumulation and colocalization with the lysosome marker LAMP2 was determined using Imaged (Fig. 6B). The experiment was repeated with untagged 3-10-3 cET ASO (Figs. 6C and 6D).
Example 9: Antisense Compound Accumulation in Deacidified Endosomes with Synthetic Sphingolipid-like Compounds
[0269] COMPOUND 893 reduces ASO co-localization with acidified lysosomes (Figs. 7A and 7B). HeLa cells co-treated with 2 pM 5’FAM-conjugated ASO in the presence or absence of 6 pM 893 for 0, 1 , 3, or 6 hours, stained with Lysotracker Red to visualize lysosomes, and then imaged by confocal microscopy. Colocalization of ASO and Lysotracker Red was measured using Imaged. Preventing ASO from reaching the lysosome may increase the time available for ASO escape into the cytosol.
Example 10: Synthetic Sphingolipid-like Compound 893 Outperforms and is Functionally Distinct from Other Small Molecule ASO Potentiators
[0270] COMPOUND 893 improves the effectiveness of ASOs better than the established small molecule ASO potentiators. 893 was compared with the small molecules 6-bromo-indirubin-3'-oxime (6BIO) and AZD8055 for their ability to reduce MALAT 1 levels in HeLa cells treated with the 3-10-3 cET gapmer, as determined by qPCR (Figs. 8A and 8B). As can be seen in the figures, 893 provides a significant improvement of ASO function in HeLa cells as compared to 6BIO and AZD8055.
[0271] COMPOUND 893 was also compared with the ASO enhancer UNC10217938A. To perform this experiment, the 3-10-3 cET gapmer ASO targeting MALAT1 was added to HeLa cells and then 16 hours later treated with 893 (5 pM) or UNC10217938A (10 pM) for two hours (Fig. 8C). RNA was collected 48 hours after ASO addition and quantified (Fig. 8D). The ASO enhancer UNC10217938A dramatically improved the ASOs ability to reduce MALAT1 levels, however UNC10217938A also had a dramatic effect on the healthy appearance of cells. To assess this issue, the 3-10-3 cET gapmer 5’-FAM-ASO targeting MALAT1 and dextran was added to HeLa cells and then 24 hours later treated with 893 (5 pM) or UNC10217938A (10 pM) or left untreated (control) for two hours (Fig. 8E). Cells were fixed and imaged at the conclusion of the 893 or UNC10217938A treatment (Fig. 8F). Treatment with UNC10217938A showed that the dextran was dispersed throughout the cellular soma, suggesting that the endosomal compartments were dramatically disrupted. Treatment with 893, on the other hand, showed the dextran mainly remaining within endosomes, similar to that of the control untreated cells, suggesting that endosomes remained intact and healthy.
Example 11: PlKfyve inhibition blocks delivery of ASOs to lysosomes but fails to increase ASO uptake or activity to the same extent as Synthetic Sphingolipid-like Compound 893
[0272] Inhibition of PlKfyve is known to disrupt lysosomal fusion. The contribution of PlKfyve inhibition on the ASO effect improvement of COMPOUND 893 was assessed. HeLa cells were treated with the 3-10-3 cET 5’FAM-ASO and 893 (5 pM) or with one of the known PlKfyve inhibitors YM201636 (800 nM) and apilimod (100 nM) for 6 hours. Colocalization of the ASO and the lysosomal marker LAMP1 was assessed by confocal microscopy (Figs. 9A and 9B). The images show that 893 significantly reduced the amount ASOs colocalizing with lysosomes almost to the same degree as the PlKfyve inhibitors YM201636 and apilimod. 893 also significantly increased the total amount of ASOs within the cell. The PlKfyve inhibitors YM201636 and apilimod had no affect on the total amount of ASOs in the cell. Thus, unlike the PlKfyve inhibitors, 893 has at least a dual effect to improve ASO function by increasing total amount of ASOs within the cell in addition to reducing the amount of ASO sequestered in the lysosome. This dual function of 893 leads to a greater improvement of ASO function over the PlKfyve inhibitors as assessed by the expression of MALAT1 in HeLa cells treated with the 3-10-3 cEt ASO and 893 or a PlKfyve inhibitor (Figs. 9C and 9D).
Example 12: Inhibition of ARF6 Increases Intracellular Accumulation of ASOs
[0273] The ability of COMPOUND 893 to increase ASO accumulation in cells was assessed by examining its effect on endosomal recycling of ASOs (Fig. 10B). To perform this experiment, HeLa cells were pulsed with 5’FAM-tagged 3-10-3 cEt ASO (2 pM) for 1 h, washed, and then replaced in media with 893 (5 pM) or one of the following ARF6 inhibitors NAV2729 (12.5 pM) and SecinH3 (30 pM), for 2 h prior to imaging (Fig. 10A). The images show that 893, similar to the ARF6 inhibitors retain high levels of the 3-10-3 cEt ASO within cells, suggesting that 893 inhibits recycling in a manner similar to the ARF6 inhibitors (Figs. 10C and 10D).
Example 13: PP2A Activation Increases Antisense Compound Activity
[0274] Structurally distinct COMPOUND 893, small molecule that activates PP2A (SMAP), and perphenazine (PPZ) are each agonists of protein phosphatase 2 (PP2A). Activation of PP2A reduces both endosomal recycling and lysosomal fusion (Fig. 11 A). To assess the effect of 893 and PP2A agonists on ASO activity, HeLa cells were treated with 5’FAM-tagged 3-10-3 cEt ASO (2 pM) and with 893 (5 pM) or one of the following PP2A activators perphenazine (PPZ, 15 pM) and SMAP (15 pM) for 6 hours. Treatment with 893 or a PP2A agonist reduced ASO colocalization with lysosomal marker LAMP1 and increased total intracellular ASOs (Fig. 11 B). Treatment with 893 or a PP2A agonist increased 3-10-3 cEt ASO activity on MALAT 1 in HeLa cells (Fig. 11 C) as well as mouse embryonic fibroblasts (Fig. 11 D). Treatment with 893 or a PP2A agonist also increased the activity of a 5-10-5 2’MOE gapmer targeting MALAT 1 (Fig. 11 E) and ACTN1 (Fig. 11 F) in HeLa cells. Example 14: Dihydro-C2-ceramide Does Not Activate PP2A or Potentiate Antisense Compound Activity
[0275] Dihydro-C2-ceramide has a similar structure to C2-ceramide but lacks the ability to agonize PP2A. Dihydro-C2-ceramide fails to increase ASO activity (Figs. 12A - 12C). An in vitro phosphatase assay was performed using immunoprecipitated PP2A. The results show that ceramide, and COMPOUND 893 increase PP2A activity but dihydroceramide does not (Fig. 12A). HeLa cells were co-treated with 40 pM C2-ceramide or 40 pM dihydro-C2-ceramide with varying concentrations of MALAT1 cEt ASO for 24 hours. RNA was then collected and qRT-PCR for MALAT1 RNA levels performed (Figs 12B and 12C). The data suggest that PP2A activation is necessary for ASO potentiation.
Example 15: Synthetic Sphingolipid-like Compound 893 lowers the does of ASO required in vivo
[0276] The ability of COMPOUND 893 to increase ASO activity in mice was assessed. Mice were treated with 120 mg/kg of compound 893 by oral gavage and 2 hours later were injected subcutaneously with various concentrations of 3-10-3 cEt gapmer ASO targeting MALAT1. Within the liver, 893 lowers the dose required to achieve efficient knockdown approximately 10-fold (Fig. 13A). As exemplified in the data, 5 mg/kg of cEt gapmer ASO with 893 treatment achieved approximately the same MALAT1 reduction as 50 mg/kg of the cEt gapmer ASO without any treatment. Likewise, 0.5 mg/kg of cEt gapmer ASO with 893 treatment achieved approximately the same MALAT1 reduction as 5 mg/kg of the cEt gapmer ASO without any treatment. Improvement of ASO activity was also noticed within the lungs with systemic administration of 50 mg/kg of cEt gapmer ASO with 893 treatment (Fig. 13B) and with 5 mg/kg of cEt gapmer ASO with 893 treatment at 24 and 72 hours (Fig. 13C).
DOCTRINE OF EQUIVALENTS
[0277] While the above description contains many specific embodiments of the invention, these should not be construed as limitations on the scope of the invention, but rather as an example of one embodiment thereof. Accordingly, the scope of the invention should be determined not by the embodiments illustrated, but by the appended claims and their equivalents.

Claims

WHAT IS CLAIMED IS:
1 . A method to enhance oligomeric nucleic acid activity in a biological cell, comprising: contacting a cell with an agonist of antisense activity; and contacting the cell with an oligomeric compound.
2. A method of sensitizing a cell for oligomeric compound modulation, comprising: contacting a biological cell with an agonist of antisense activity for a period of time prior to contacting the biological cell with an oligomeric compound.
3. A method of treating or preventing a disease or condition in a subject by reducing the amount or activity of a target nucleic acid, comprising: administering to a subject an agonist of antisense activity; and administering to the subject an oligomeric compound.
4. The method of any one of claims 1 -3, wherein the agonist of antisense activity is one or more of: a protein phosphatase 2 (PP2A) agonist, an ADP Ribosylation Factor 6 (ARF6) antagonist, or a Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing (PlKfyve) antagonist.
5. The method as in claim 4, wherein the PP2A is agonist is: a sphingolipid, a sphingolipid-like compound, perphenazine, a perphenazine derivative, a SET inhibitor, a CIP2a inhibitor, Withaferin A, OSLI-2S, or a derivative thereof.
6. The method of claim 5, wherein the perphenazine derivative is SMAP or iHAP.
7. The method as in claim 4, wherein the ARF6 antagonist is: a sphingolipid, a sphingolipid-like compound, NAV2729, SecinH3, or a derivative thereof.
-75-
8. The method as in claim 4, wherein the PlKfyve antagonist is: a sphingolipid, a sphingolipid-like compound, YM201636, APY0201 , Apilimod, Late Endosome Trafficking Inhibitor EGA, or a derivative thereof.
9. The method of any one of claims 5 and 6, wherein the sphingolipid is ceramide, sphingosine, sphinganine, phytosphingosine, dimethylsphingosine, safingol, or other sphingolipid that activates PP2A.
10. The method of any one of claims 5-7, wherein the sphingolipid-like compound is based on O-benzyl pyrrolidines having the formula:
Figure imgf000078_0001
Ri is an optional functional group selected from an alkyl chain, (CH2)nOH, CHOH- alkyl, CHOH-alkyne, (CH2)nO-alkyl, (CH2)nO-alkene, (CH2)nO-alkyne, (CH2)nPO(OH)2 and esters thereof, CH=CHPO(OH)2 and esters thereof, (CH2CH2)nPO(OH)2 and esters thereof, and (CH2)nOPO(OH)2 and esters thereof;
R2 is an aliphatic chain (Ce - C10);
Rs is a mono-, di-, tri- or quad- aromatic substituent comprising H, halogen, alkyl, alkoxy, azide (Ns), ether, NO2, or cyanide (CN);
One of Ri R4 is an alcohol (CH2OH) or H;
L is O-CH2;and n is an independently selected integer selected from 1 , 2, or 3.
-76-
11 . The method of any one of claims 5-7, wherein the sphingolipid-like compound is based on diastereomeric 3- and 4-C-aryl pyrrolidines having the formula:
Figure imgf000079_0001
Ri is an optional functional group selected from an alkyl chain, (CH2)nOH, CHOH- alkyl, CHOH-alkyne, (CH2)nO-alkyl, (CH2)nO-alkene, (CH2)nO-alkyne, (CH2)nPO(OH)2 and esters thereof, CH=CHPO(OH)2 and esters thereof, (CH2CH2)nPO(OH)2 and esters thereof, and (CH2)nOPO(OH)2 and esters thereof, (CH2)nPOs and esters thereof;
R2 is an aliphatic chain (Ce - C14);
Rs is a mono-, di-, tri- or tetra- aromatic substituent comprising hydrogen, halogen, alkyl, alkoxy, azide (Ns), ether, NO2, or cyanide (CN); and n is an independently selected integer selected from 1 , 2, or 3.
12. The method of claim 11 , wherein the sphingolipid-like compound is compound 893 having the formula:
Figure imgf000079_0002
13. The method of claim 11 , wherein the sphingolipid-like compound is compound 1090 having the formula:
Figure imgf000080_0001
14. The method of any one of claims 5-7, wherein the sphingolipid-like compound is based on azacycles with an attached heteroaromatic appendage having the formula:
Figure imgf000080_0002
or a pharmaceutically acceptable salt thereof;
R is an optionally substituted heteroaromatic moiety such as an optionally substituted pyridazine, optionally substituted pyridine, optionally substituted pyrimidine, phenoxazine, or optionally substituted phenothiazine;
Ri is H, alkyl such as C1-6 alkyl or C1-4 alkyl including methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, etc, Ac, Boc, guanidine moiety;
R2 is an aliphatic chain comprising 6 to 14 carbons;
Rs is a 1 , 2, 3, or 4 substituents, wherein each substituent, independently, is H, halogen, alkyl, alkoxy, Ns, NO2, and CN; n is independently 1 , 2, 3, or 4; m is independently 1 or 2; the phenyl moiety can be attached at any available position of the azacycle core; and
R is a 1 ,2-pyridazine having the formula:
Figure imgf000081_0001
R4 and Rs are functional groups independently selected from: alkyl including methyl, optionally substituted aryl (i.e., unsubstituted aryl or substituted aryl) including optionally substituted phenyl, and optionally substituted heteroaryl including optionally substituted pyridine and optionally substituted pyrimidine; and the pyridazine moiety is connected to the azacycle at the position 4 or 5 of the pyridazine.
15. The method of claim 14, wherein the sphingolipid-like compound is compound
325 having the formula:
Figure imgf000081_0002
16. The method of any one of claims 5-7, wherein the sphingolipid-like compound is based on diastereomeric 2-C-aryl pyrrolidines having the formula:
Figure imgf000081_0003
Ri is a functional group selected from H, an alkyl chain, OH, (CH2)nOH, CHOH- alkyl, CHOH-alkyne, (CH2)nOR’, (CH2)nPO(OH)2 and esters thereof, CH=CHPO(OH)2 and esters thereof, (CH2CH2)nPO(OH)2 and esters thereof, and (CH2)nOPO(OH)2 and esters thereof, (CH2)nPOs and esters thereof, where R’ is an alkyl, alkene or alkyne;
-79- R2 is an aliphatic chain (Ce - C14);
Rs is a mono-, di-, tri- or tetra- aromatic substituent that includes hydrogen, halogen, alkyl, alkoxy, azide (Ns), ether, NO2, cyanide (CN), or a combination thereof;
R4 is a functional group selected from H, alkyl including methyl (Me), ester, or acyl;
X’ is an anion of the suitable acid; n is an independently selected integer selected from 1 , 2, or 3; and m is an independently selected integer selected from 0, 1 or 2.
17. The method of any one of claims 1 or 2, wherein the cell is a normal cell.
18. The method of any one of claims 1 or 2, wherein the cell is in the lungs.
19. The method of any one of claims 1 or 2, wherein the cell is in the spleen.
20. The method of any one of claims 1 or 2, wherein the cell is a neoplastic cell.
21. The method of any one of claims 1 or 2, wherein the cell is a cancer cell.
22. The method of any one of claims 1 or 2, wherein the cell is a pancreatic cancer cell.
23. The method of any one of claims 1 or 2, wherein the cell is a breast cancer cell.
24. The method of any one of claims 1 or 2 or 17-23, wherein the cell is in vitro.
25. The method of any one of claims 1 or 2 or 17-23, wherein the cell is in an animal.
26. The method of claim 25, wherein the animal is a human.
27. The method of claim 25, wherein the animal is a mouse.
-80-
28. The method of any one of claims 1-27 wherein the oligomeric compound is an antisense compound.
29. The method of claim 28, wherein the antisense compound is an antisense oligomer.
30. The method of claim 28, wherein the antisense compound is an RNase-H-based or RNAi-based antisense compound.
31 . The method of claim 28, wherein the antisense compound comprises at least one modified nucleoside.
32. The method of any one of claims 28-31 , wherein each nucleoside of the antisense compound is a modified nucleoside.
33. The method of any one of claims 28-32, wherein the antisense compound comprises a single-stranded nucleic acid.
34. The method of any one of claims 28-32, wherein the antisense compound comprises double-stranded nucleic acid.
35. The method of any one of claims 31-34, wherein at least one modified nucleoside comprises a modified sugar moiety.
36. The method of any one of claims 29-35, wherein the antisense compound has a sugar motif comprising: a 5’-region consisting of 2-8 linked 5’ -region nucleosides, wherein at least two 5’ -region nucleosides are modified nucleosides and wherein the 3’-most 5’- region nucleoside is a modified nucleoside; a 3’-region consisting of 2-8 linked 3’-region nucleosides, wherein at least two 3’ -region nucleosides are modified nucleosides and wherein the 5’-most 3’- region nucleoside is a modified nucleoside; and
-81- a central region between the 5’-region and the 3’-region consisting of 5-10 linked central region nucleosides, each independently selected from among: a modified nucleoside and an unmodified deoxynucleoside, wherein the 5’-most central region nucleoside is an unmodified deoxynucleoside and the 3’ -most central region nucleoside is an unmodified deoxynucleoside.
37. The method of claim 36, wherein the 5’-region consists of 2 linked 5’ -region nucleosides.
38. The method of claim 36, wherein the 5’-region consists of 3 linked 5’ -region nucleosides.
39. The method of claim 36, wherein the 5’-region consists of 4 linked 5’ -region nucleosides.
40. The method of claim 36, wherein the 5’-region consists of 5 linked 5’ -region nucleosides.
41 . The method of any one of claims 36-40, wherein the 3’-region consists of 2 linked 3’-region nucleosides.
42. The method of any one of claims 36-40, wherein the 3’-region consists of 3 linked 3’-region nucleosides.
43. The method of any one of claims 36-40, wherein the 3’-region consists of 4 linked 3’-region nucleosides.
44. The method of any one of claims 36-40, wherein the 3’-region consists of 5 linked 3’-region nucleosides.
45. The method of any one of claims 36-44, wherein the central region consists of 7 linked central region nucleosides.
46. The method of any one of claims 36-44, wherein the central region consists of 8 linked central region nucleosides.
47. The method of any one of claims 36-44, wherein the central region consists of 9 linked central region nucleosides.
48. The method of any one of claims 36-44, wherein the central region consists of 10 linked central region nucleosides.
49. The method of any one of claims 29-48, wherein the nucleic acid of the antisense compound consists of 14 to 26 linked nucleosides.
50. The method of any one of claims 20-39, wherein the nucleic acid of the antisense compound consists of 16 to 20 linked nucleosides.
51 . The method of any one of claims 31 -50, wherein each modified nucleoside independently comprises a 2’-substituted sugar moiety or a bicyclic sugar moiety.
52. The method of claim 51 , wherein the at least one modified nucleoside comprises a 2’-substituted sugar moiety.
53. The method of claim 52, wherein each modified nucleoside comprising a 2’- substituted sugar moiety comprises a 2’ substituent independently selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)- alkenyl; O, S or N(Rm)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH2)2SCH3, 0- (CH2)2-O-N(Rm)(Rn) or O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl; wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
54. The method of claim 53, wherein each 2’ substituent is independently selected from among: a halogen, OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, O(CH2)2F, OCH2CHF2, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2-SCH3, O(CH2)2-OCF3, O(CH2)3-N(RI)(R2), O(CH2)2-ON(RI)(R2), O(CH2)2-O(CH2)2-N(RI)(R2), OCH2C(=O)- N(RI)(R2), OCH2C(=O)-N(R3)-(CH2)2-N(RI)(R2), and O(CH2)2-N(R3)-C(=NR4)[N(Ri)(R2)]; wherein R1, R2, R3 and R4 are each, independently, H or C-i-Ce alkyl.
55. The method of claim 53, wherein each 2’ substituent is independently selected from among: a halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2-CH=CH2, O(CH2)2- OCH3 (MOE), O(CH2)2-O(CH2)2-N(CH3)2, OCH2C(=O)-N(H)CH3, OCH2C(=O)-N(H)- (CH2)2-N(CH3)2, and OCH2-N(H)-C(=NH)NH2.
56. The method of claim 55, wherein the at least one 2’- substituted sugar moiety comprises a 2’-MOE sugar moiety.
57. The method of claim 55, wherein the at least one 2’- substituted sugar moiety comprises a 2’-OMe sugar moiety.
58. The method of claim 55, wherein the at least one 2’- substituted sugar moiety comprises a 2’-F sugar moiety.
59. The method of any one of claims 31 -58, wherein the antisense compound comprises at least one modified nucleoside comprising a sugar surrogate.
-84-
60. The method of claim 59, wherein the modified nucleoside comprises an F-HNA sugar moiety.
61 . The method of claim 59, wherein the modified nucleoside comprises an HNA sugar moiety.
62. The method of any one of claims 29-61 , wherein the antisense compound comprises at least one modified nucleoside comprising a bicyclic sugar moiety.
63. The method of claim 62, wherein the bicyclic sugar moiety is a cEt sugar moiety.
64. The method of claim 62, wherein bicyclic sugar moiety is an LNA sugar moiety.
65. The method of any one of claims 29-64, wherein the antisense compound comprises at least one modified internucleoside linkage.
66. The method of claim 65, wherein each internucleoside linkage of the antisense compound is a modified internucleoside linkage.
67. The method of claim 65, wherein the antisense compound comprises at least one modified linkage and at least one unmodified phosphodiester internucleoside linkage.
68. The method of claim 65, wherein at least one modified internucleoside linkage is a phosphosphorothioate internucleoside linkage.
69. The method of claim 65, wherein each modified internucleoside linkage is a phosphorothioate internucleoside linkage.
70. The method of any one of claims 28-69, wherein the nucleic acid of the antisense compound has a nucleobase sequence comprising an at least 8 nucleobase portion complementary to an equal length portion of a target nucleic acid.
-85-
71 . The method of any one of claims 28-69, wherein the nucleic acid of the antisense compound has a nucleobase sequence comprising an at least 10 nucleobase portion complementary to an equal length portion of a target nucleic acid.
72. The method of any one of claims 28-69, wherein the nucleic acid of the antisense compound has a nucleobase sequence comprising an at least 12 nucleobase portion complementary to an equal length portion of a target nucleic acid.
73. The method of any one of claims 28-69, wherein the nucleic acid of the antisense compound has a nucleobase sequence comprising an at least 14 nucleobase portion complementary to an equal length portion of a target nucleic acid.
74. The method of any one of claims 28-69, wherein the nucleic acid of the antisense compound has a nucleobase sequence comprising an at least 16 nucleobase portion complementary to an equal length portion of a target nucleic acid.
75. The method of any one of claims 28-69, wherein the nucleic acid of the antisense compound has a nucleobase sequence comprising an at least 18 nucleobase portion complementary to an equal length portion of a target nucleic acid.
76. The method of any one of claims 28-69, wherein the nucleic acid of the antisense compound is at least 90% complementary to a target nucleic acid.
77. The method of any one of claims 28-69, wherein the nucleic acid of the antisense compound is at least 95% complementary to a target nucleic acid.
78. The method of any one of claims 28-69, wherein the nucleic acid of the antisense compound is 100% complementary to a target nucleic acid.
79. The method of any one of claims 28-78, wherein the target nucleic acid is a pre- mRNA.
-86-
80. The method of any one of claims 28-78, wherein the target nucleic acid is an mRNA.
81 . The method of any one of claims 28-78, wherein the target nucleic acid is a target nucleic acid expressed in a cancer cell.
82. The method of any one of claims 28-78, wherein the target nucleic acid is a target nucleic acid expressed in a neoplastic cell.
83. The method of any one of claims 28-78, wherein the target nucleic acid is a target nucleic acid expressed in a pulmonary cell.
84. The method of any one of claims 28-78, wherein the target nucleic acid is a target nucleic acid expressed in a hepatic cell.
85. The method of any one of claims 28-84, wherein the antisense compound comprises a conjugate.
86. The method of any one of claims 28-84, wherein the antisense compound is unconjugated.
-87-
PCT/US2021/072140 2020-10-29 2021-10-29 Compounds and methods for increased antisense activity WO2022094615A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202063107371P 2020-10-29 2020-10-29
US63/107,371 2020-10-29

Publications (1)

Publication Number Publication Date
WO2022094615A1 true WO2022094615A1 (en) 2022-05-05

Family

ID=81383383

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/072140 WO2022094615A1 (en) 2020-10-29 2021-10-29 Compounds and methods for increased antisense activity

Country Status (1)

Country Link
WO (1) WO2022094615A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11479530B2 (en) 2015-09-24 2022-10-25 The Regents Of The University Of California Synthetic sphingolipid-like molecules, drugs, methods of their synthesis and methods of treatment

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016160102A1 (en) * 2015-03-31 2016-10-06 Lam Therapeutics, Inc. Active metabolites of apilimod and uses thereof
WO2016210134A1 (en) * 2015-06-23 2016-12-29 Case Western Reserve University Compositions and methods for treating cancer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016160102A1 (en) * 2015-03-31 2016-10-06 Lam Therapeutics, Inc. Active metabolites of apilimod and uses thereof
WO2016210134A1 (en) * 2015-06-23 2016-12-29 Case Western Reserve University Compositions and methods for treating cancer

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BEIDER ET AL.: "The Sphingosine-1-Phosphate Modulator FTY720 Targets Multiple Myeloma via the CXCR4/CXCL12 Pathway", CLINICAL CANCER RESEARCH, vol. 23, no. 7, 3 October 2016 (2016-10-03), pages 1733 - 1747, XP055767891, DOI: 10.1158/1078-0432.CCR-15-2618 *
FINICLE BRENDAN T., RAMIREZ MANUEL U., LIU GANG, SELWAN ELIZABETH M., MCCRACKEN ALISON N., YU JINGWEN, JOO YOOSUN, NGUYEN JANNETT,: "Sphingolipids inhibit endosomal recycling of nutrient transporters by inactivating ARF6", JOURNAL OF CELL SCIENCE, COMPANY OF BIOLOGISTS LIMITED, CAMBRIDGE, 25 June 2018 (2018-06-25), Cambridge , XP055939068, ISSN: 0021-9533, DOI: 10.1242/jcs.213314 *
GARSI ET AL.: "Design, synthesis and anticancer activity of constrained sphingolipidphenoxazine/ phenothiazine hybrid constructs targeting protein phosphatase 2A", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 29, 19 July 2019 (2019-07-19), pages 2681 - 2685, XP085779597, DOI: 10.1016/j.bmcl.2019.07.023 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11479530B2 (en) 2015-09-24 2022-10-25 The Regents Of The University Of California Synthetic sphingolipid-like molecules, drugs, methods of their synthesis and methods of treatment
US11999693B2 (en) 2015-09-24 2024-06-04 The Regents Of The University Of California Synthetic sphingolipid-like molecules, drugs, methods of their synthesis and methods of treatment

Similar Documents

Publication Publication Date Title
US11149264B2 (en) Modified compounds and uses thereof
US20230020192A1 (en) Compounds and methods for modulating c90rf72
US11116843B2 (en) Conjugated antisense compounds and their use
US11583548B2 (en) Compounds and methods for reducing ATXN3 expression
US11959080B2 (en) Methods and compositions for inhibiting PMP22 expression
EP3353305A1 (en) Conjugated antisense compounds and their use
US11725208B2 (en) Conjugated antisense compounds and their use
AU2019310097A1 (en) Compounds and methods for reducing ATXN2 expression
WO2020061497A1 (en) Compositions and methods for modulation of lmna expression
WO2022094615A1 (en) Compounds and methods for increased antisense activity
WO2020132558A1 (en) Compounds and methods for reducing pmp22 expression
WO2020205463A1 (en) Compounds and methods for modulating ube3a-ats
WO2022246204A2 (en) Compounds for reducing ptbp1 expression
AU2021264010A1 (en) Compounds and methods for modulating ATXN1
WO2021258011A1 (en) Compounds and methods for modulating pmp22
WO2023073661A2 (en) Compounds and methods for reducing psd3 expression
WO2023092057A1 (en) Compounds and methods for modulating progranulin expression
WO2023164656A2 (en) Compounds and methods for modulating atn1 expression

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21887846

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21887846

Country of ref document: EP

Kind code of ref document: A1