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WO2014089146A1 - Compositions and methods for in vivo delivery of antisense compounds - Google Patents

Compositions and methods for in vivo delivery of antisense compounds Download PDF

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Publication number
WO2014089146A1
WO2014089146A1 PCT/US2013/072982 US2013072982W WO2014089146A1 WO 2014089146 A1 WO2014089146 A1 WO 2014089146A1 US 2013072982 W US2013072982 W US 2013072982W WO 2014089146 A1 WO2014089146 A1 WO 2014089146A1
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composition
melittin
antisense compound
certain embodiments
modified
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PCT/US2013/072982
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French (fr)
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Punit P. Seth
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Isis Pharmaceuticals, Inc.
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Publication of WO2014089146A1 publication Critical patent/WO2014089146A1/en

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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
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    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12N2310/11—Antisense
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention provides a compositions and methods useful for enhancing the in vivo delivery of antisense compounds.
  • antisense technology in the treatment of a disease or condition that stems from a disease-causing gene is that it is a direct genetic approach that has the ability to modulate (increase or decrease) the expression of specific disease-causing genes.
  • Another advantage is that validation of a therapeutic target using antisense compounds results in direct and immediate discovery of the drug candidate; the antisense compound is the potential therapeutic agent.
  • RNAi RNA interference
  • microRNAs are small non-coding RNAs that regulate the expression of protein-coding RNAs.
  • the binding of an antisense compound to a microRNA prevents the microRNA from binding to its messenger RNA target, and thus interferes with the function of the microRNA. Regardless of the specific mechanism, this sequence-specificity makes antisense compounds extremely attractive as tools for target validation and gene
  • Antisense technology is an effective means for reducing 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 are routinely incorporated into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target RNA.
  • Vitravene® flamivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, CA
  • FDA U.S. Food and Drug Administration
  • CMV cytomegalovirus
  • RNAi polynucleotides see U.S. Patent Application 20120165393, published on June 28, 2012.
  • compositions comprising an antisense compound and Melittin-[(L)- (ASGPrLig)] x wherein:
  • Melittin is a melittin peptide
  • L is physiologically labile linkage
  • ASGPrLig is an Asialoglycoprotein Receptor ligand
  • x is an integer having a value greater than 80% of the number of primary amines of said melittin peptide
  • antisense compound is selected from among:
  • RNAi compound comprising at least one of:
  • compositions provided herein provide in vivo delivery to a hepatocyte.
  • At least 90% of the amines on each melittin peptide is reversibly linked to the Asialoglycoprotein Receptor ligand.
  • the Melittin peptide comprises an amino acid sequence selected from the list consisting of: Seq. ID 1, Seq. ID 7, Seq. ID 11 , Seq. ID 51, Seq. ID 57, Seq. ID 58, Seq. ID 92, and Seq. ID 96.
  • the Melittin peptide consists of D-form amino acids.
  • L is a disubstituted maleamate. In certain embodiments, L is a disubstituted maleamate and the melittin peptide comprises a polyethyleneglycol (PEG) covalently linked to its amino terminus. In certain embodiments, L is a disubstituted maleamate and the melittin peptide comprises an ASGPrLig-PEG conjugate covalently linked to its amino terminus.
  • PEG polyethyleneglycol
  • L is an amidobenzyl carbamate. In certain embodiments, L is an amidobenzyl carbamate and the melittin peptide comprises a polyethyleneglycol (PEG) covalently linked to its amino terminus. In certain embodiments, L is an amidobenzyl carbamate and the melittin peptide comprises an ASGPrLig-PEG conjugate covalently linked to its amino terminus.
  • PEG polyethyleneglycol
  • the ASGPrLig is selected from the group consisting of lactose, galactose, N-acetylgalactosamine (GalNAc), galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butanoylgalactosamine, and N-iso-butanoyl- galactosamine.
  • the antisense compound is a single stranded RNAi compound.
  • the antisense compound is a single stranded RNAi compound comprising a 5'- phosphate-5'-vinyl modified nucleoside located at the 5' end. In certain embodiments, the antisense compound is a single stranded RNAi compound comprising and two contiguous 2'-0-(CH 2 )2-OCH 3 modified nucleosides located at the 3' end.
  • the antisense compound is a single stranded RNAi compound comprising a 5'-phosphate-5'-vinyl modified nucleoside located at the 5' end and two contiguous 2'-0-(CH 2 )2-OCH 3 modified nucleosides located at the 3' end and each nucleoside located between the 5'-phosphate-5'-vinyl modified nucleoside located at the 5' end and two contiguous 2'-0-(CH 2 )2-OCH 3 modified nucleosides located at the 3' end is independently selected from 2'-F modified nucleosides, 2'-OCH 3 modified nucleosides and ⁇ -D-ribonucleosides.
  • each nucleoside located between the 5'-phosphate-5'-vinyl modified nucleoside located at the 5' end and two contiguous 2'-0-(CH 2 )2-OCH 3 modified nucleosides located at the 3' end is, independently, a 2'-F modified nucleoside or a 2'-OCH 3 modified nucleoside.
  • the nucleosides located between the 5'-phosphate-5'-vinyl modified nucleoside located at the 5' end and two contiguous 2'-0-(CH 2 ) 2 -OCH 3 modified nucleosides located at the 3' alternate between 2'-F modified nucleosides and 2'-OCH 3 modified nucleosides.
  • the antisense compound is a non-RNAi antisense compound.
  • the non-RNAi antisense compound works through an RNaseH mechanism.
  • the non-RNAi antisense compound comprises a first region consisting of from 2 to 5 modified nucleosides, a second region consisting of from 2 to 5 modified nucleosides and a gap region consisting of from 6 to 14 monomer subunits located between the first and second region.
  • each monomer subunit in the gap region is independently, a nucleoside or a modified nucleoside that is different from each of the modified nucleosides in the first and second region.
  • the gap region comprises from about 8 to about 12 monomer subunits. In certain embodiments, the gap region comprises from about 8 to about 10 monomer subunits.
  • each monomer subunit in the gap region is a ⁇ - ⁇ -2'- deoxyribonucleoside. In certain embodiments, one or two of the monomer subunits in the gap region is a modified nucleoside and each of the other monomer subunits in the gap region is a ⁇ -D- 2'-deoxyribonucleoside.
  • each modified nucleoside in the first and second region comprises a modified sugar moiety.
  • each modified nucleoside in the first and second region is, independently, a bicyclic nucleoside comprising a 4'-CH((5)-CH 3 )-0-2' bridge or a 2'-0- (CH 2 ) 2 -OCH 3 modified nucleoside.
  • the antisense compound comprises internucleoside linking groups that are each, independently, a phosphodiester internucleoside linking group or a phosphorothioate internucleoside linking group.
  • the antisense compound comprises internucleoside linking groups that are essentially all phosphorothioate internucleoside linking groups.
  • the antisense compound comprises heterocyclic base moieties that are each, independently, a pyrimidine, substituted pyrimidine, purine or substituted purine.
  • the antisense compound comprises heterocyclic base moieties that are each, independently, uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5- methylcytosine, adenine, 6-N-benzoyladenine, guanine or 2-N-isobutyrylguanine.
  • the antisense compound doesn't include a hydrophobic group having at least 20 carbon atoms or a galactose cluster.
  • the composition is provided in a pharmaceutically acceptable carrier or diluent.
  • methods comprising administering to a subject the composition as provided herein.
  • methods comprising co-administering to a subject an antisense compound and Melittin-[(L)-(ASGPrLig)] x
  • Melittin is a melittin peptide
  • L is physiologically labile linkage
  • ASGPrLig is an Asialoglycoprotein Receptor ligand
  • x is an integer having a value greater than 80% of the number of primary amines of said melittin peptide
  • antisense compound is selected from among:
  • RNAi compound comprising at least one of:
  • the antisense compound and the Melittin-[(L)-(ASGPrLig)] x are administered together. In certain embodiments, the antisense compound and the Melittin- [(L)- (ASGPrLig)] x are administered separately. In certain embodiments, the antisense compound and the Melittin-[(L)-(ASGPrLig)] x are administered at the same time. In certain embodiments, the antisense compound and the Melittin-[(L)-(ASGPrLig)] x are administered at different times.
  • method of manufacturing a composition comprising: a) forming a melittin peptide; b) forming a plurality of uncharged masking agents each comprising an ASGPrLig covalently linked to a disubstituted maleic anhydride or a dipeptide amidobenzyl amine reactive carbonate; c) modifying greater than 80% of primary amines on a population of melittin peptides with the masking agents of step b, d) providing an antisense compound as per claim 1 and the modified melittin peptide in solution suitable for administration in vivo.
  • compositions and methods providing improved in vivo delivery of antisense compounds More particularly the methods include administration of a small delivery peptide selected from the melittin family, or a derivative thereof, with the antisense compound.
  • the small delivery peptide is selected from the melittin family which is functionalized with groups to provide masking and targeting prior to administration. It is expected that administration of the functionalized small delivery peptide with the antisense compound will enhance the delivery and potency of the antisense compound.
  • the methods are expected to provide enhanced delivery of antisense compounds to liver cells in a mammal.
  • the masked delivery peptide and the antisense compound can be coadministered or can be administered separately.
  • concentration of the melittin peptide and the antisense compound and the ratio between them is limited only by their respective solubilities in a selected solvent/mixture.
  • the antisense compound and the melittin peptide may be mixed at any time prior to
  • the components can be stored separately, either in solution or dry.
  • the invention includes a composition comprising an an antisense compound and Melittin-
  • Melittin is a melittin peptide
  • L is physiologically labile linkage
  • ASGPrLig is an Asialoglycoprotein Receptor ligand
  • x is an integer having a value greater than 80% of the number of primary amines of said melittin peptide
  • said antisense compound is selected from among: (a) a single stranded RNAi compound comprising at least one of:
  • Melittin is a bee venom melittin peptide or a derivative as describe herein, and ASGPrLig is covalently linked to Melittin via a physiologically labile reversible linkage L. Cleavage of the physiologically labile reversible linkages restores unmodified amines on Melittin.
  • Melittin can also include an optional polyethyleneglycol (PEG) or ASGPrLig-PEG conjugate group linked to the amino terminus, the carboxy terminus, or an amino or carboxy terminal cysteine of Melittin.
  • a polyethyleneglycol (PEG) or ASGPrLig-PEG conjugate group is covalently attached to the amino terminus or an amino terminal cysteine, x is an integer greater than 1.
  • compositions are provided wherein the antisense compound doesn't include a targeting moiety. In certain embodiments, compositions are provided wherein the antisense compound doesn't include a conjugate group. In certain embodiments, compositions are provided wherein the antisense compound doesn't include a conjugate group or a targeting moiety. In certain embodiments, compositions are provided wherein the antisense compound doesn't include a group that is either a hydrophobic group having 20 or more carbon atoms or a galactose cluster. In certain embodiments, the antisense compound includes a conjugate group that is not a targeting moiety. In certain embodiments, the antisense compound includes a targeting moiety. In certain embodiments, the antisense compound includes a targeting moiety that is not an optionally linked ASGPr ligand.
  • Melittin In its unmodified state, Melittin is membrane active. However, delivery peptide Melittin- [(L)-(ASGPrLig)] x is not membrane active. Reversible modification of Melittin primary amines, by attachment of L- ASGPr ligands reversibly inhibits or inactivates membrane activity of Melittin. A sufficient percentage of Melittin primary amines are modified to inhibit membrane activity of the polymer and also provide for hepatocyte targeting. In certain embodiments, x has a value greater than 80%, of the total percentage of primary amines on Melittin, as determined by the quantity of amines on Melittin in the absence of any masking agents.
  • x has a value greater than 90%, of the primary amines on Melittin. In certain embodiments, x has a value greater than 80% and up to 100% of the primary amines on Melittin. It is noted that melittin typically contains 3-5 primary amines (the amino terminus (if unmodified) and typically 2-4 Lysine residues). Therefore, modification of a percentage of amines is meant to reflect the modification of a percentage on amines in a population of melittin peptides. Upon cleavage of reversible linkages L, unmodified amines are restored thereby reverting Melittin to its unmodified, membrane active state. In certain embodiments, the reversible linkage is a pH labile linkage. In certain embodiments, the reversible linkage is a protease cleavable linkage.
  • the Melittin-[(L)-(ASGPrLig)] x, an ASGPr-targeted reversibly masked membrane active polymer (delivery peptide), and the antisense compound are synthesized or manufactured separately.
  • the antisense compounds are not covalently linked directly or indirectly to the Mellitin- [(L)-(ASGPrLig)] x .
  • Electrostatic or hydrophobic association of the antisense compound with the masked or unmasked polymer is not required for in vivo liver delivery of the antisense compound. In certain embodiments, there are essentially no electrostatic or hydrophobic association of the antisense compound with the masked or unmasked polymer.
  • the masked polymer and the antisense compound can be supplied in the same container or in separate containers. They may be combined prior to administration, co-administered, or administered sequentially.
  • Hydrophilic groups indicate in qualitative terms that the chemical moiety is water-preferring. Typically, such chemical groups are water soluble, and are hydrogen bond donors or acceptors with water.
  • a hydrophilic group can be charged or uncharged. Charged groups can be positively charged (anionic) or negatively charged (cationic) or both (zwitterionic). Examples of hydrophilic groups include the following chemical moieties: carbohydrates, polyoxyethylene, certain peptides, oligonucleotides, amines, amides, alkoxy amides, carboxylic acids, sulfurs, and hydroxyls.
  • Hydrophobic groups indicate in qualitative terms that the chemical moiety is water-avoiding.
  • Lipophilic groups dissolve in fats, oils, lipids, and non-polar solvents and have little to no capacity to form hydrogen bonds.
  • Hydrocarbons containing two or more carbon atoms, certain substituted hydrocarbons, cholesterol, and cholesterol derivatives are examples of hydrophobic groups and compounds.
  • hydrophobic groups are hydrocarbons, containing only carbon and hydrogen atoms. However, non-polar substitutions or non-polar heteroatoms which maintain hydrophobicity, and include, for example fluorine, may be permitted.
  • the term includes aliphatic groups, aromatic groups, acyl groups, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups, each of which may be linear, branched, or cyclic.
  • hydrophobic group also includes: sterols, steroids, cholesterol, and steroid and cholesterol derivatives.
  • membrane active peptides are surface active, amphipathic peptides that are able to induce one or more of the following effects upon a biological membrane: an alteration or disruption of the membrane that allows non-membrane permeable molecules to enter a cell or cross the membrane, pore formation in the membrane, fission of membranes, or disruption or dissolving of the membrane.
  • a membrane, or cell membrane comprises a lipid bilayer.
  • the alteration or disruption of the membrane can be functionally defined by the peptide's activity in at least one the following assays: red blood cell lysis (hemolysis), liposome leakage, liposome fusion, cell fusion, cell lysis, and endosomal release.
  • Membrane active peptides that can cause lysis of cell membranes are also termed membrane lytic peptides. Peptides that preferentially cause disruption of endosomes or lysosomes over plasma membranes are considered endosomo lytic. The effect of membrane active peptides on a cell membrane may be transient. Membrane active peptides possess affinity for the membrane and cause a denaturation or deformation of bilayer structures.
  • the delivery of an antisense compound to a cell is mediated by the melittin peptide disrupting or destabilizing the plasma membrane or an internal vesicle membrane (such as an endosome or lysosome), including forming a pore in the membrane, or disrupting endosomal or lysosomal vesicles thereby permitting release of the contents of the vesicle into the cell cytoplasm.
  • an internal vesicle membrane such as an endosome or lysosome
  • Endosomolytic peptides are peptides that, in response to an endosomal-specific environmental factors, such as reduced pH or the presence of lytic enzymes (proteases), are able to cause disruption or lysis of an endosome or provide for release of a normally cell membrane impermeable compound, such as a nucleic acid or protein, from a cellular internal membrane-enclosed vesicle, such as an endosome or lysosome.
  • Endosomolytic polymers undergo a shift in their physico- chemical properties in the endosome. This shift can be a change in the polymer's solubility or ability to interact with other compounds or membranes as a result in a shift in charge,
  • endosomolytic peptides have pH-labile or enzymatic- sensitive groups or bonds.
  • a reversibly masked membrane active peptide, wherein the masking agents are attached to the polymer via pH labile bonds, can therefore be considered to be an endosomolytic polymer.
  • Melittin is a small amphipathic membrane active peptide, comprising about 23 to about 32 amino acids, derived from the naturally occurring bee venom peptide melittin.
  • the naturally occurring melittin contains 26 amino acids and is predominantly hydrophobic on the amino terminal end and predominantly hydrophilic (cationic) on the carboxy terminal end.
  • Melittin of the invention can be isolated from a biological source or it can be synthetic.
  • a synthetic polymer is formulated or manufactured by a chemical process "by man" and is not created by a naturally occurring biological process.
  • melittin encompasses the naturally occurring bee venom peptides of the melittin family that can be found in, for example, venom of the species: Apis florea, Apis mellifera, Apis cerana, Apis dorsata, Vespula maculifrons, Vespa magnifica, Vespa velutina, Polistes sp. HQL-2001 , and Polistes hebraeus.
  • melittin also encompasses synthetic peptides having amino acid sequence identical to or similar to naturally occurring melittin peptides. Specifically, melittin amino acid sequence encompass those shown in Table 1.
  • 1-8 amino acids can be added to the amino or carboxy terminal ends of the peptide.
  • cysteine residues can be added to the amino or carboxy termini.
  • the list in Table 1, is not meant to be exhaustive as other conservative amino acid substitutions are readily envisioned.
  • Synthetic melittin peptides can contain naturally occurring L form amino acids or the enantiomeric D form amino acids (inverso). However, a melittin peptide should either contain essentially all L form or all D form amino acids but may have amino acids of the opposite stereocenter appended at either the amino or carboxy termini.
  • the melittin amino acid sequence can also be reversed (retro).
  • Retro melittin can have L form amino acids or D form amino acids (retroinverso). Two melittin peptides can also be covalently linked to form a melittin dimer. Melittin can have modifying groups, other than masking agents, that enhance tissue targeting or facilitate in vivo circulation attached to either the amino terminal or carboxy terminal ends of the peptide. However, as used herein, melittin does not include chains or polymers containing more than two melittin peptides covalently linked to one another other or to another polymer or scaffold.
  • the melittin peptides of the invention comprise reversibly modified melittin peptides wherein reversible modification inhibits membrane activity, neutralizes the melittin to reduce positive charge and form a near neutral charge polymer, and provides cell-type specific targeting.
  • the melittin is reversibly modified through reversible modification of primary amines on the peptide.
  • the melittin peptides of the invention are capable of disrupting plasma membranes or lysosomal endocytic membranes.
  • Membrane activity leads to toxicity when the peptide is administered in vivo. Therefore, reversible masking of membrane activity of melittin is necessary for in vivo use.
  • This masking is accomplished through reversible attachment of masking agents to melittin to form a reversibly masked melittin, i.e. a delivery peptide.
  • the masking agents provide cell-specific interactions, i.e. targeting. It is an essential feature of the masking agents that, in aggregate, they inhibit membrane activity of the polymer and provide in vivo hepatocyte targeting.
  • Melittin is membrane active in the unmodified (unmasked) state and not membrane active (inactivated) in the modified (masked) state.
  • a sufficient number of masking agents are linked to the peptide to achieve the desired level of inactivation.
  • the desired level of modification of melittin by attachment of masking agent(s) is readily determined using appropriate peptide activity assays. For example, if melittin possesses membrane activity in a given assay, a sufficient level of masking agent is linked to the peptide to achieve the desired level of inhibition of membrane activity in that assay.
  • modification of the primary amine groups is greater than or equal to 80% or greater than or equal 90% on a population of melittin peptides, as determined by the quantity of primary amines on the peptides in the absence of any masking agents.
  • the attachment of the masking agent(s) to the reduces positive charge of the polymer, thus forming a more neutral delivery peptide.
  • the masked peptide is water soluble.
  • melittin is masked if the modified peptide does not exhibit membrane activity and exhibits cell-specific targeting in vivo. Melittin is reversibly masked if cleavage of bonds linking the masking agents to the peptide results in restoration of amines on the peptide thereby restoring membrane activity. In certain embodiments, melittin is masked and exhibits hepatocyte specific targeting in vivo.
  • the masking agents are covalently bound to melittin through physiologically labile reversible bonds.
  • physiologically labile reversible linkages or bonds the masking agents can be cleaved from the peptide in vivo, thereby unmasking the peptide and restoring activity of the unmasked peptide.
  • an appropriate reversible linkage it is possible to form a conjugate that restores activity of melittin after it has been delivered or targeted to a desired cell type or cellular location. Reversibility of the linkages provides for selective activation of melittin.
  • Reversible covalent linkages contain reversible or labile bonds which may be selected from the group comprising: physiologically labile bonds, cellular physiologically labile bonds, pH labile bonds, very pH labile bonds, extremely pH labile bonds, and proetease cleavable bonds.
  • masking agents as used herein comprise a neutral (uncharged) compound having an ASGPrLig and an amine-reactive group wherein reaction of the amine-reactive group with an amine on a peptide results in linkage of the ASGPrLig to the peptide via a reversible physiologically labile covalent bond.
  • Amine reactive groups are chosen such that cleavage in response to an appropriate physiological condition (e.g., reduced pH such as in an endosome/lysosome, or enzymatic cleavage such as in an endosome/lysosome) results in regeneration of the melittin amine.
  • An ASGPrLig is a group, typically a saccharide, having affinity for the asialoglycoprotein receptor.
  • masking agents as provided herein are able to modify the polymer (form a reversible bond with the polymer) in aqueous solution.
  • the amine-reactive group comprises a disubstituted maleic anhydride.
  • the masking agent is represented by the structure:
  • Ri comprises an asialoglycoprotein receptor ligand (ASGPrLig) and R 2 is an alkyl group such as a methyl (-CH 3 ) group, ethyl (-CH 2 CH 3 ) group, or propyl (-(CH 2 ) 2 CH 3 ) group.
  • ASGPrLig asialoglycoprotein receptor ligand
  • the galactose ligand is linked to the amine-reactive group through a PEG linker as illustrated by the structure:
  • n is an integer between 1 and 19.
  • the amine-reactive group comprises a dipeptide-amidobenzyl reactive carbonate derivative represented by the structure:
  • Ri is the R group of amino acid 1 ;
  • R 2 is the R group of amino acid 2
  • R 3 is -CH 2 -0-C(0)-0-Z, wherein Z is halide
  • R4 comprises the ASGPrLig.
  • Reaction of the activated carbonate with a melittin amine connects the ASGPrLig to the melittin peptide via a peptidase cleavable dipeptide-amidobenzyl carbamate linkage as shown below.
  • Enzymatic cleavage of the dipeptide removes the targeting ligand from the peptide and triggers an elimination reaction which results in regeneration of the peptide amine. While the structure above shows a single masking agent linked to a melittin peptide, in practice, several masking agents are linked to the melittin peptide. In certain embodiments, more than 80% of the amines on a population of melittin peptides are modified.
  • Glu-Gly, Ala-Cit, Phe-Cit (“Cit” is the amino acid citrulline) are shown in Example 14. With respect to the above structure, Glu-Gly, Ala-Cit, Phe-Cit represent R2-R1. While charged amino acids are permissible, in certain embodiments the amino acids are neutral. Other amino acid combinations are possible, provided they are cleaved by an endogenous protease. In addition, 3-5 amino acids may be used as the linker between the ami do benzyl group and the targeting ligand.
  • the ASGPrLig can be linked to the peptidase cleavable dipeptide-amidobenzyl carbonate via a PEG linker.
  • the membrane active polyamine can be conjugated to masking agents in the presence of an excess of masking agents.
  • the excess masking agent may be removed from the conjugated delivery peptide prior to administration of the delivery peptide.
  • the melittin peptides of the invention are further modified, at the amino or carboxyl termini, by covalent attachment of a steric stabilizer or an ASGPrLig-steric stabilizer conjugate.
  • the hydrophobic terminal end is modified; the amino terminal end for melittin having "normal sequence" and the carboxyl terminal end for retro-melittin.
  • the steric stabilizer is a polyethylene glycol.
  • the amino or carboxy terminal modifications may be linked to the peptide during synthesis using methods standard in the art. Alternatively, the amino or carboxy terminal modifications may be done through modification of cysteine residues on melittin peptides having amino or carboxy terminal cysteine residues.
  • the polyethylene glycols have 1-120 ethylene units. In certain embodiments, polyethylene glycols are less than 5 kDa in size. In certain embodiments, for ASGPrLig-steric stabilizer conjugates (NAG-PEG modification), the steric stabilizer is a polyethyleneglycol having 1-24 ethylene units. Terminal PEG modification, when combined with reversible masking, further reduces toxicity of the melittin delivery peptide. Terminal NAG-PEG modification enhances efficacy.
  • a steric stabilizer is a non-ionic hydrophilic polymer (either natural, synthetic, or non-natural) that prevents or inhibits intramolecular or intermolecular interactions of a molecule to which it is attached relative to the molecule containing no steric stabilizer.
  • a steric stabilizer hinders a molecule to which it is attached from engaging in electrostatic interactions. Electrostatic interaction is the non-covalent association of two or more substances due to attractive forces between positive and negative charges.
  • Steric stabilizers can inhibit interaction with blood components and therefore opsonization, phagocytosis, and uptake by the reticuloendothelial system. Steric stabilizers can thus increase circulation time of molecules to which they are attached. Steric stabilizers can also inhibit aggregation of a molecule.
  • the steric stabilizer is a polyethylene glycol (PEG) or PEG derivative.
  • PEG molecules suitable for the invention have about 1-120 ethylene glycol monomers.
  • Targeting moieties or groups enhance the pharmacokinetic or biodistribution properties of a conjugate to which they are attached to improve cell-specific distribution and cell-specific uptake of the conjugate.
  • Galactose and galactose derivates have been used to target molecules to hepatocytes in vivo through their binding to the asialoglycoprotein receptor (ASGPr) expressed on the surface of hepatocytes.
  • ASGPrLig comprises galactose and galactose derivatives having affinity for the ASGPr equal to or greater than that of galactose. Binding of galactose targeting moieties to the ASGPr(s) facilitates cell-specific targeting of the delivery peptide to hepatocytes and endocytosis of the delivery peptide into hepatocytes.
  • ASGPr ligands are selected from the group comprising: lactose, galactose, N-acetylgalactosamine (GalNAc), galactosamine, N-formylgalactosamine, N-acetyl- galactosamine, N-propionylgalactosamine, N-n-butanoylgalactosamine, and N-iso-butanoyl- galactosamine (Iobst, S. T. and Drickamer, K. J.B.C. 1996, 271 , 6686; and Rensen et al, J. Med. Chem., 2004, 47, 5798-5808).
  • ASGPr ligands can be monomeric (e.g., having a single
  • galactosamine or multimeric (e.g., having multiple galactosamines).
  • the melittin peptide is reversibly masked by attachment of ASGPr ligand masking agents to greater than or equal to 80% or greater than or equal to 90% of primary amines on the peptide.
  • the targeting moiety comprises a galactose cluster (galactose cluster targeting moiety).
  • a galactose cluster comprises a molecule having two to four terminal galactose derivatives.
  • galactose derivative includes both galactose and derivatives of galactose having affinity for the asialoglycoprotein receptor equal to or greater than that of galactose.
  • a terminal galactose derivative is attached to a molecule through its C-l carbon.
  • the asialoglycoprotein receptor (ASGPr) is unique to hepatocytes and binds branched galactose-terminal glycoproteins.
  • the galactose cluster has three terminal galactosamines or galactosamine derivatives each having affinity for the asialoglycoprotein receptor.
  • the galactose cluster has three terminal N-acetyl-galactosamines.
  • Other terms common in the art include tri-antennary galactose, tri-valent galactose and galactose trimer.
  • a galactose cluster contains three galactose derivatives each linked to a central branch point.
  • the galactose derivatives are attached to the central branch point through the C-l carbons of the saccharides.
  • the galactose derivative is preferably linked to the branch point via linkers or spacers.
  • the spacer is a flexible hydrophilic spacer (U.S. Pat. No. 5,885,968; Biessen et al. J. Med. Chem., 1995, 39, 1538-1546).
  • the flexible hydrophilic spacer is a PEG spacer.
  • the PEG spacer is a PEG 3 (three ethylene units) spacer.
  • the branch point can be any small molecule which permits attachment of the three galactose derivatives and further permits attachment of the branch point to the desired parent compound.
  • the branch point group is a di-lysine.
  • the di-lysine molecule contains three amine groups through which three galactose derivatives may be attached and a carboxyl reactive group through which the di-lysine may be attached to a parent compound. Attachment of the branch point to the parent compound may occur through a linker or spacer such as a flexible hydrophilic spacer.
  • the flexible hydrophilic spacer is a PEG spacer.
  • the PEG spacer is a PEG 3 spacer.
  • the galactose derivative is an N-acetyl-galactosamine (GalNAc).
  • GalNAc N-acetyl-galactosamine
  • Other saccharides having affinity for the asialoglycoprotein receptor may be selected from the list comprising: galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N- propionyl-galactosamine, N-n-butanoylgalactosamine, and N-iso-butanoylgalactos-amine.
  • affinities of numerous galactose derivatives for the asialoglycoprotein receptor have been studied (see for example: Iobst et ah, J.B.C., 1996, 271, 6686) or are readily determined using methods typical in the art.
  • a linkage or linker is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds.
  • a linkage can connect a masking agent to a peptide. Formation of a linkage may connect two separate molecules into a single molecule or it may connect two atoms in the same molecule. The linkage may be charge neutral or may bear a positive or negative charge.
  • a reversible or labile linkage contains a reversible or labile bond.
  • a linkage may optionally include a spacer that increases the distance between the two joined atoms. A spacer may further add flexibility and/or length to the linkage.
  • Spacers may include, but are not be limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides. Spacer groups are well known in the art and the preceding list is not meant to limit the scope of the invention.
  • linkers and spacers can be used interchangeably.
  • a linker comprises spacer groups.
  • a labile bond is a covalent bond other than a covalent bond to a hydrogen atom that is capable of being selectively broken or cleaved under conditions that will not break or cleave other covalent bonds in the same molecule. More specifically, a labile bond is a covalent bond that is less stable (thermodynamically) or more rapidly broken (kinetically) under appropriate conditions than other non-labile covalent bonds in the same molecule. Cleavage of a labile bond within a molecule may result in the formation of two molecules. For those skilled in the art, cleavage or lability of a bond is generally discussed in terms of half- life (ti/ 2 ) of bond cleavage (the time required for half of the bonds to cleave).
  • labile bonds encompass bonds that can be selectively cleaved more rapidly than other bonds a molecule.
  • Appropriate conditions are determined by the type of labile bond and are well known in organic chemistry.
  • a labile bond can be sensitive to pH, oxidative or reductive conditions or agents, temperature, salt concentration, the presence of an enzyme (such as esterases, including nucleases, and proteases), or the presence of an added agent. For example, increased or decreased pH is the appropriate conditions for a pH-labile bond.
  • the rate at which a labile group will undergo transformation can be controlled by altering the chemical constituents of the molecule containing the labile group. For example, addition of particular chemical moieties (e.g., electron acceptors or donors) near the labile group can affect the particular conditions (e.g., pH) under which chemical transformation will occur.
  • chemical moieties e.g., electron acceptors or donors
  • a physiologically labile bond is a labile bond that is cleavable under conditions normally encountered or analogous to those encountered within a mammalian body.
  • Physiologically labile linkage groups are selected such that they undergo a chemical transformation (e.g., cleavage) when present in certain physiological conditions.
  • a cellular physiologically labile bond is a labile bond that is cleavable under mammalian intracellular conditions.
  • Mammalian intracellular conditions include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt
  • Mammalian intracellular conditions also include the presence of enzymatic activity normally present in a mammalian cell such as from proteolytic or hydrolytic enzymes.
  • a cellular physiologically labile bond may also be cleaved in response to administration of a pharmaceutically acceptable exogenous agent. Physiologically labile bonds that are cleaved under appropriate conditions with a half life of less than 45 minutes are considered very labile. Physiologically labile bonds that are cleaved under appropriate conditions with a half life of less than 15 min are considered extremely labile.
  • Chemical transformation may be initiated by the addition of a pharmaceutically acceptable agent to the cell or may occur spontaneously when a molecule containing the labile bond reaches an appropriate intra- and/or extra-cellular environment.
  • a pH labile bond may be cleaved when the molecule enters an acidified endosome.
  • a pH labile bond may be considered to be an endosomal cleavable bond.
  • Enzyme cleavable bonds may be cleaved when exposed to enzymes such as those present in an endosome or lysosome or in the cytoplasm.
  • a disulfide bond may be cleaved when the molecule enters the more reducing environment of the cell cytoplasm. Thus, a disulfide may be considered to be a cytoplasmic cleavable bond.
  • a pH-labile bond is a labile bond that is selectively broken under acidic conditions (pH ⁇ 7). Such bonds may also be termed endosomally labile bonds, since cell endosomes and lysosomes have a pH less than 7.
  • pH-labile includes bonds that are pH- labile, very pH-labile, and extremely pH-labile.
  • Reaction of an anhydride with an amine forms an amide and an acid.
  • the reverse reaction formation of an anhydride and amine
  • reaction with an amine yields an amide acid, a molecule in which the amide and the acid are in the same molecule.
  • the presence of both reactive groups (the amide and the carboxylic acid) in the same molecule accelerates the reverse reaction.
  • Cis-aconitic acid has been used as such a pH-sensitive linker molecule.
  • the ⁇ -carboxylate is first coupled to a molecule.
  • either the a or ⁇ carboxylate is coupled to a second molecule to form a pH-sensitive coupling of the two molecules.
  • the half life for cleavage of this linker at pH 5 is between 8 and 24 h.
  • the pH at which cleavage occurs is controlled by the addition of chemical constituents to the labile moiety.
  • the rate of conversion of maleamic acids to amines and maleic anhydrides is strongly dependent on substitution (R 2 and R 3 ) of the maleic anhydride system.
  • R 2 is methyl
  • R 3 are hydrogen
  • the rate increase is dramatic: 10,000-fold faster than non-substituted maleic anhydride.
  • the maleamate bond formed from the modification of an amine with 2,3-dimethylmaleic anhydride is cleaved to restore the anhydride and amine with a half-life between 4 and 10 minutes at pH 5. It is anticipated that if R 2 and R 3 are groups larger than hydrogen, the rate of amide-acid conversion to amine and anhydride will be faster than if R 2 and/or R 3 are hydrogen.
  • a very pH-labile bond has a half-life for cleavage at pH 5 of less than 45 minutes.
  • the construction of very pH-labile bonds is well-known in the chemical art.
  • An extremely pH-labile bond has a half-life for cleavage at pH 5 of less than 15 minutes. The construction of extremely pH- labile bonds is well-known in the chemical art.
  • Disubstituted cyclic anhydrides are particularly useful for attachment of masking agents to melittin peptides of the invention. They provide physiologically pH-labile linkages, readily modify amines, and restore those amines upon cleavage in the reduced pH found in cellular endosomes and lysosome. Second, the a or ⁇ carboxylic acid group created upon reaction with an amine, appears to contribute only about l/20 th of the expected negative charge to the polymer (Rozema, et al., Bioconjugate Chemistry, 2003, 14, 51 -57).
  • modification of the peptide with the disubstituted maleic anhydrides effectively neutralizes the positive charge of the peptide rather than creatina a peptide with high negative charge.
  • near neutral delivery peptides are used for in vivo delivery.
  • oligomeric compound refers to a contiguous sequence of linked monomer subunits.
  • Each linked monomer subunit normally includes a heterocyclic base moiety but monomer subunits also include those without a heterocyclic base moiety such as abasic monomer subunits.
  • At least some and generally most if not essentially all of the heterocyclic bases in an oligomeric compound are capable of hybridizing to a nucleic acid molecule, normally a preselected
  • oligomeric compound therefore includes oligonucleotides, oligonucleotide analogs and oligonucleosides. It also includes polymers having one or a plurality of nucleosides having sugar surrogate groups.
  • oligomeric compounds comprise a plurality of monomer subunits independently selected from naturally occurring nucleosides, non-naturally occurring nucleosides, modified nucleosides and nucleosides having sugar surrogate groups.
  • oligomeric compounds are single stranded.
  • oligomeric compounds are double stranded comprising a double-stranded duplex.
  • oligomeric compounds comprise one or more conjugate groups and/or terminal groups.
  • oligomeric compounds having specific motifs as disclosed herein it can be advantageous to mix non-naturally occurring monomer subunits with other non-naturally occurring monomer subunits, naturally occurring monomer subunits (nucleosides) or mixtures thereof.
  • antisense compound refers to an oligomeric compound, at least a portion of which is at least partially complementary and hybridizable to a selected nucleic acid such as a nucleic acid target.
  • an antisense compound modulates (increases or decreases) expression or amount of a target nucleic acid.
  • an antisense compound alters splicing of a target pre-mR A resulting in a different splice variant.
  • an antisense compound modulates expression of one or more different target proteins.
  • Antisense mechanisms contemplated herein include, but are not limited to an R ase H mechanism, R Ai mechanisms, splicing modulation, translational arrest, altering R A processing, inhibiting microRNA function, or mimicking microRNA function.
  • an antisense compound doesn't include a sense region.
  • essentially each monomer subunit in the antisense compound is complementary to a region of a nucleic acid target.
  • antisense activity refers to any detectable and/or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid.
  • such activity may be an increase or decrease in an amount of a nucleic acid or protein.
  • such activity may be a change in the ratio of splice variants of a nucleic acid or protein.
  • Detection and/or measuring of antisense activity may be direct or indirect.
  • antisense activity is assessed by detecting and/or measuring the amount of target protein or the relative amounts of splice variants of a target protein.
  • antisense activity is assessed by detecting and/or measuring the amount of target nucleic acids and/or cleaved target nucleic acids and/or alternatively spliced target nucleic acids. In certain embodiments, antisense activity is assessed by observing a phenotypic change in a cell or animal.
  • single stranded means an oligomeric compound that is not hybridized to its complement and which lacks sufficient self-complementarity to form a stable self-duplex.
  • an antisense compound that has a mechanism of action other than
  • RNAi is referred to as a non-RNAi antisense compound.
  • antisense compounds used in the methods and compositions provided herein are single stranded RNAi compounds. Such antisense compounds are used without a complementary strand to effect activity using an RNAi mechanism. Such single stranded RNAi compounds are known to the art skilled (see for example, published PCT Application WO
  • the term "motif refers to the pattern created by the relative positioning of monomer subunits within an oligomeric compound wherein the pattern is determined by comparing the sugar moieties of the linked monomer subunits.
  • the only determinant for the motif of an oligomeric compound is the differences or lack of differences between the sugar moieties.
  • the internucleoside linkages, heterocyclic bases and further groups such as terminal groups are not considered when determining the motif of an oligomeric compound.
  • Such motifs include without limitation, gapmer motifs, hemimer motifs, blockmer motifs, uniformly fully modified motifs, positionally modified motifs and alternating motifs.
  • internucleoside linkages can also be used including but not limited to phosphodiester and phosphorothioate internucleoside linkages which can be incorporated uniformly or in various combinations.
  • the oligomeric compounds can further include terminal groups at one or both of the 5' and or 3' terminals such as a conjugate or reporter group. The positioning of the monomer subunits, the use of linkage strategies and terminal groups can be easily optimized to enhance a desired activity for a selected target.
  • alternating motif refers to an oligomeric compound comprising a contiguous sequence of linked monomer subunits wherein the monomer subunits have two different types of sugar moieties that alternate for essentially the entire sequence of the oligomeric compound.
  • Oligomeric compounds having an alternating motif can be described by the formula: 5'-A(-L-B-L- A) n (-L-B) nn -3' where A and B are monomer subunits that have different sugar moieties, each L is, independently, an internucleoside linking group, n is from about 4 to about 12 and nn is 0 or 1.
  • the heterocyclic base and internucleoside linkage is independently variable at each position.
  • the motif further optionally includes the use of one or more other groups including but not limited to capping groups, conjugate groups and other 5' and or 3'-terminal groups. This permits alternating oligomeric compounds from about 9 to about 26 monomer subunits in length. This length range is not meant to be limiting as longer and shorter oligomeric compounds are also amenable to oligomeric compounds provided herein.
  • the term "uniformly fully modified motif” refers to an oligomeric compound comprising a contiguous sequence of linked monomer subunits that each have the same type of sugar moiety.
  • the heterocyclic base and internucleoside linkage is independently variable at each position.
  • the motif further optionally includes the use of one or more other groups including but not limited to capping groups, conjugate groups and other 5' and or 3'-terminal groups.
  • hemimer motif refers to an oligomeric compound comprising a contiguous sequence of monomer subunits that each have the same type of sugar moiety with a further short contiguous sequence of monomer subunits located at the 5' or the 3' end that have a different type of sugar moiety.
  • the heterocyclic base and internucleoside linkage is independently variable at each position.
  • the motif further optionally includes the use of one or more other groups including but not limited to capping groups, conjugate groups and other 5' and or 3'-terminal groups.
  • a hemimer is an oligomeric compound of uniform sugar moieties further comprising a short region (1 , 2, 3, 4 or about 5 monomer subunits) having uniform but different sugar moieties located on either the 3' or the 5' end of the oligomeric compound.
  • the hemimer motif comprises a contiguous sequence of from about 10 to about 28 monomer subunits having one type of sugar moiety with from 1 to 5 or from 2 to about 5 monomer subunits having a second type of sugar moiety located at one of the termini.
  • the hemimer is a contiguous sequence of from about 8 to about 20 ⁇ - ⁇ -2'- deoxyribonucleosides having from 1-12 contiguous modified nucleosides located at one of the termini.
  • blockmer motif and “blockmer” refer to an oligomeric compound comprising an otherwise contiguous sequence of monomer subunits wherein the sugar moieties of each monomer subunit is the same except for an interrupting internal block of contiguous monomer subunits having a different type of sugar moiety.
  • the heterocyclic base and internucleoside linkage is independently variable at each position of a blockmer.
  • the motif further optionally includes the use of one or more other groups including but not limited to capping groups, conjugate groups and other 5' or 3 '-terminal groups.
  • a blockmer overlaps somewhat with a gapmer in the definition but typically only the monomer subunits in the block have non-naturally occurring sugar moieties in a blockmer and only the monomer subunits in the external regions have non-naturally occurring sugar moieties in a gapmer with the remainder of monomer subunits in the blockmer or gapmer being ⁇ - D-2'-deoxyribonucleosides or ⁇ -D-ribonucleosides.
  • blockmers are provided herein wherein all of the monomer subunits comprise non-naturally occurring sugar moieties.
  • positionally modified motif is meant to include an otherwise contiguous sequence of monomer subunits having one type of sugar moiety that is interrupted with two or more regions of from 1 to about 5 contiguous monomer subunits having another type of sugar moiety.
  • Each of the two or more regions of from 1 to about 5 contiguous monomer subunits are independently uniformly modified with respect to the type of sugar moiety.
  • each of the two or more regions have the same type of sugar moiety.
  • each of the two or more regions have a different type of sugar moiety.
  • each of the two or more regions independently, have the same or a different type of sugar moiety.
  • the heterocyclic base and internucleoside linkage is independently variable at each position of a positionally modified oligomeric compound.
  • the motif further optionally includes the use of one or more other groups including but not limited to capping groups, conjugate groups and other 5' or 3'- terminal groups.
  • gapmer or “gapped oligomeric compound” refers to an oligomeric compound having two external regions or wings and an internal region or gap. The three regions form a contiguous sequence of monomer subunits with the sugar moieties of the external regions being different than the sugar moieties of the internal region and wherein the sugar moiety of each monomer subunit within a particular region is essentially the same. In certain embodiments, each monomer subunit within a particular region has the same sugar moiety.
  • the gapmer is a symmetric gapmer and when the sugar moiety used in the 5'-external region is different from the sugar moiety used in the 3'-external region, the gapmer is an asymmetric gapmer.
  • the external regions are small (each
  • the monomer subunits independently 1, 2, 3, 4 or about 5 monomer subunits) and the monomer subunits comprise non- naturally occurring sugar moieties with the internal region comprising -D-2'-deoxyribonucleosides.
  • the external regions each, independently, comprise from 1 to about 5 monomer subunits having non-naturally occurring sugar moieties and the internal region comprises from 6 to 18 unmodified nucleosides.
  • the internal region or the gap generally comprises ⁇ - ⁇ -2'- deoxyribonucleosides but can comprise non-naturally occurring sugar moieties.
  • the heterocyclic base and internucleoside linkage is independently variable at each position of a gapped oligomeric compound.
  • the motif further optionally includes the use of one or more other groups including but not limited to capping groups, conjugate groups and other 5' or 3'-terminal groups.
  • gapped oligomeric compounds are provided that are from about 18 to about 21 monomer subunits in length. In certain embodiments, gapped oligomeric compounds are provided that are from about 16 to about 21 monomer subunits in length. In certain embodiments,
  • gapped oligomeric compounds are provided that are from about 10 to about 21 monomer subunits in length. In certain embodiments, gapped oligomeric compounds are provided that are from about 12 to about 16 monomer subunits in length. In certain embodiments, gapped oligomeric compounds are provided that are from about 12 to about 14 monomer subunits in length. In certain embodiments, gapped oligomeric compounds are provided that are from about 14 to about 16 monomer subunits in length.
  • alkyl refers to a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms.
  • alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.
  • Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C1-C12 alkyl) with from 1 to about 6 carbon atoms being more preferred.
  • the term "lower alkyl” as used herein includes from 1 to about 6 carbon atoms.
  • Alkyl groups as used herein may optionally include one or more further substituent groups.
  • alkenyl refers to a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond.
  • alkenyl groups include without limitation, ethenyl, propenyl, butenyl, l-methyl-2- buten-l-yl, dienes such as 1 ,3-butadiene and the like.
  • Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred.
  • Alkenyl groups as used herein may optionally include one or more further substituent groups.
  • alkynyl refers to a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond.
  • alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like.
  • Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred.
  • Alkynyl groups as used herein may optionally include one or more further substituent groups.
  • aliphatic refers to a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond.
  • An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred.
  • the straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus.
  • Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups.
  • alicyclic refers to a cyclic ring system wherein the ring is aliphatic.
  • the ring system can comprise one or more rings wherein at least one ring is aliphatic.
  • Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring.
  • Alicyclic as used herein may optionally include further substituent groups.
  • alkoxy refers to a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule.
  • alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n- butoxy, sec-butoxy, tert-bvXoxy, n-pentoxy, neopentoxy, n-hexoxy and the like.
  • Alkoxy groups as used herein may optionally include further substituent groups.
  • aminoalkyl refers to an amino substituted C1-C12 alkyl radical.
  • the alkyl portion of the radical forms a covalent bond with a parent molecule.
  • the amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.
  • aryl and “aromatic,” refer to a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings.
  • aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like.
  • Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings.
  • Aryl groups as used herein may optionally include further substituent groups.
  • aralkyl and “arylalkyl,” refer to an aromatic group that is covalently linked to a C1-C12 alkyl radical.
  • the alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like.
  • Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.
  • heterocyclic radical refers to a radical mono-, or poly-cyclic ring system that includes at least one heteroatom and is unsaturated, partially saturated or fully saturated, thereby including heteroaryl groups. Heterocyclic is also meant to include fused ring systems wherein one or more of the fused rings contain at least one heteroatom and the other rings can contain one or more heteroatoms or optionally contain no heteroatoms.
  • a heterocyclic radical typically includes at least one atom selected from sulfur, nitrogen or oxygen.
  • heterocyclic radicals include, [l,3]dioxolanyl, pyrrolidinyl, pyrazolinyl, pyrazohdinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and the like.
  • Heterocyclic groups as used herein may optionally include further substituent groups.
  • heteroaryl and “heteroaromatic,” refer to a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms.
  • Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen.
  • heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like.
  • Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom.
  • Heteroaryl groups as used herein may optionally include further substituent groups.
  • heteroarylalkyl refers to a heteroaryl group as previously defined that further includes a covalently attached C1-C12 alkyl radical.
  • the alkyl radical portion of the resulting heteroarylalkyl group is capable of forming a covalent bond with a parent molecule.
  • Examples include without limitation, pyridinylmethylene, pyrimidinylethylene,
  • Heteroarylalkyl groups as used herein may optionally include further substituent groups on one or both of the heteroaryl or alkyl portions.
  • acyl refers to a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula -C(0)-X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.
  • hydrocarbyl includes radical groups that comprise C, O and H. Included are straight, branched and cyclic groups having any degree of saturation. Such hydrocarbyl groups can include one or more additional heteroatoms selected from N and S and can be further mono or poly substituted with one or more substituent groups.
  • the term "mono or polycyclic ring system” is meant to include all ring systems selected from single or polycyclic radical ring systems wherein the rings are fused or linked and is meant to be inclusive of single and mixed ring systems individually selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic and
  • Such mono and poly cyclic structures can contain rings that each have the same level of saturation or each, independently, have varying degrees of saturation including fully saturated, partially saturated or fully unsaturated.
  • Each ring can comprise ring atoms selected from C, N, O and S to give rise to heterocyclic rings as well as rings comprising only C ring atoms which can be present in a mixed motif such as for example benzimidazole wherein one ring has only carbon ring atoms and the fused ring has two nitrogen atoms.
  • Mono or polycyclic ring systems can be attached to parent molecules using various strategies such as directly through a ring atom, fused through multiple ring atoms, through a substituent group or through a bifunctional linking moiety.
  • halo and halogen refer to an atom selected from fluorine, chlorine, bromine and iodine.
  • protecting group refers to a labile chemical moiety which is known in the art to protect reactive groups including without limitation, hydroxyl, amino and thiol groups, against undesired reactions during synthetic procedures.
  • Protecting groups are typically used selectively and/or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group as is or available for further reactions.
  • Protecting groups as known in the art are described generally in Greene's Protective Groups in Organic Synthesis, 4th edition, John Wiley & Sons, New York, 2007.
  • Groups can be selectively incorporated into oligomeric compounds as provided herein as precursors.
  • an amino group can be placed into a compound as provided herein as an azido group that can be chemically converted to the amino group at a desired point in the synthesis.
  • groups are protected or present as precursors that will be inert to reactions that modify other areas of the parent molecule for conversion into their final groups at an appropriate time. Further representative protecting or precursor groups are discussed in Agrawal et ah, Protocols for Oligonucleotide Conjugates, Humana Press; New Jersey, 1994, 26, 1-72.
  • orthogonal protected refers to functional groups which are protected with different classes of protecting groups, wherein each class of protecting group can be removed in any order and in the presence of all other classes (see, Barany et ah, J. Am. Chem. Soc, 1977, 99, 7363- 7365; Barany et ah, J. Am. Chem. Soc, 1980, 102, 3084-3095).
  • Orthogonal protection is widely used in for example automated oligonucleotide synthesis.
  • a functional group is deblocked in the presence of one or more other protected functional groups which is not affected by the deblocking procedure.
  • This deblocked functional group is reacted in some manner and at some point a further orthogonal protecting group is removed under a different set of reaction conditions.
  • the compounds described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-, a or ⁇ , or as (D)- or (L)- such as for amino acids. Included herein are all such possible isomers, as well as their racemic and optically pure forms.
  • Optical isomers may be prepared from their respective optically active precursors by the procedures described above, or by resolving the racemic mixtures.
  • the resolution can be carried out in the presence of a resolving agent, by chromatography or by repeated crystallization or by some combination of these techniques which are known to those skilled in the art. Further details regarding resolutions can be found in Jacques, et ah, Enantiomers, Racemates, and Resolutions, John Wiley & Sons, 198 1 .
  • the compounds described herein contain olefinic double bonds, other unsaturation, or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers or cis- and trans-isomers.
  • all tautomeric forms are also intended to be included.
  • the configuration of any carbon- carbon double bond appearing herein is selected for convenience only and is not intended to limit a particular configuration unless the text so states.
  • substituted and substituted group are meant to include groups that are typically added to a parent compounds or to further substituted substituent groups to enhance one or more desired properties or provide other desired effects.
  • Substituent groups can be protected or unprotected and can be added to one available site or many available sites on a parent compound. As an example if a benzene is substituted with a substituted alky it will not have any overlap with a benzene that is substituted with substituted hydroxyl. In such an example the alkyl portion of the substituted alkyl is covalently linked by one of its carbon atoms to one of the benzene carbon atoms.
  • the alky is Ci and it is substituted with a hydroxyl substituent group (substituted alkyl) then the resultant compound is benzyl alcohol (C 6 H 5 CH 2 OH). If the benzene were substituted with a substituted hydroxyl group and the hydroxyl was substituted with a Ci alkyl group then the resultant compound would be anisole (C 6 H 5 OCH 3 ).
  • each Raa, Rbb and R cc is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree.
  • recursive substituent means that a substituent may recite another instance of itself. Because of the recursive nature of such substituents, theoretically, a large number may be present in any given claim.
  • One of ordinary skill in the art of medicinal chemistry and organic chemistry understands that the total number of such substituents is reasonably limited by the desired properties of the compound intended. Such properties include, by way of example and not limitation, physical properties such as molecular weight, solubility or logP, application properties such as activity against the intended target and practical properties such as ease of synthesis.
  • Recursive substituents are an intended aspect of the invention.
  • One of ordinary skill in the art of medicinal and organic chemistry understands the versatility of such substituents.
  • stable compound and “stable structure” as used herein are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Only stable compounds are contemplated herein.
  • nucleobase generally refers to the nucleobase of a nucleoside or modified nucleoside.
  • heterocyclic base moiety is broader than the term nucleobase in that it includes any heterocyclic base that can be attached to a sugar to prepare a nucleoside or modified nucleoside.
  • heterocyclic base moieties include but are not limited to naturally occurring nucleobases (adenine, guanine, thymine, cytosine and uracil) and protected forms of unmodified nucleobases (4-N-benzoylcytosine, 6-N-benzoyladenine and 2-N-isobutyrylguanine) as well as modified (5-methyl cytosine) or non-naturally occurring heterocyclic base moieties and synthetic mimetics thereof (such as for example phenoxazines).
  • nucleobases adenine, guanine, thymine, cytosine and uracil
  • protected forms of unmodified nucleobases (4-N-benzoylcytosine, 6-N-benzoyladenine and 2-N-isobutyrylguanine)
  • modified (5-methyl cytosine) or non-naturally occurring heterocyclic base moieties and synthetic mimetics thereof such as for example phenoxazines
  • a heterocyclic base moiety is any heterocyclic system that contains one or more atoms or groups of atoms capable of hydrogen bonding to a heterocyclic base of a nucleic acid.
  • nucleobase refers to purines, modified purines, pyrimidines and modified pyrimidines.
  • nucleobase refers to unmodified or naturally occurring nucleobases which include, but are not limited to, the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U) and analogs thereof such as 5-methyl cytosine.
  • nucleobase and heterocyclic base moiety also include optional protection for any reactive functional groups such as 4-N-benzoylcytosine, 4-N-benzoyl-5-methyl- cytosine, 6-N-benzoyladenine or 2-N-isobutyrylguanine.
  • heterocyclic base moieties include without limitation modified nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5- halouracil and cytosine, 5-propynyl (-C ⁇ C-CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8- amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and
  • heterocyclic base moieties include without limitation tricyclic pyrimidines such as l ,3-diazaphenoxazine-2-one, l,3-diazaphenothiazine-2-one and 9-(2- aminoethoxy)-l,3-diazaphenoxazine-2-one (G-clamp).
  • Heterocyclic base moieties 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 heterocyclic base moieties include without limitation those known to the art skilled (see for example: United States Patent No.
  • Modified polycyclic heterocyclic compounds useful as heterocyclic base moieties are disclosed in the above noted U.S. 3,687,808, as well as U.S.: 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187; 5,459,255; 5,484,908; 5,502,177;
  • sugar moiety refers to naturally occurring sugars having a furanose ring system (ribose and 2'-deoxyribose), synthetic and/or non-naturally occurring sugars having a modified furanose ring system and sugar surrogates wherein the furanose ring has been replaced with a mono or polycyclic ring system such as for example a morpholino or hexitol ring system or a non-cyclic sugar surrogate such as that used in peptide nucleic acids.
  • the sugar moiety of a monomer subunit provides the reactive groups that enable the linking of adjacent monomer subunits into an oligomeric compound.
  • sugar moieties useful in the preparation of oligomeric compounds include without limitation, ⁇ -D-ribose, -D-2'-deoxyribose, substituted sugars (such as 2', 5' and bis substituted sugars), 4'-S-sugars (such as 4'-S-ribose, 4'-S-2'-deoxyribose and 4'-S-2'-substituted ribose wherein the ring oxygen atom has been replaced with a sulfur atom), bicyclic modified sugars (such as the 2'-0-CH(CH 3 )-4', 2'-0-CH 2 -4' or 2'-0-(CH 2 ) 2 -4' bridged ribose derived bicyclic sugars) and sugar surrogates (such as for example when the ribose ring has been replaced with a morpholino, a hexitol ring system or an open non-cyclic system).
  • substituted sugars such as 2', 5
  • sugar surrogate refers to replacement of the nucleoside furanose ring with a non-furanose (or 4'-substituted furanose) group with another structure such as another ring system or open system.
  • Such structures can be as simple as a six membered ring as opposed to the five membered furanose ring or can be more complicated such as a bicyclic or tricyclic ring system or a non-ring system such as that used in peptide nucleic acid.
  • sugar surrogates include without limitation sugar surrogate groups such as morpholinos, cyclohexenyls and cyclohexitols. In general the heterocyclic base is maintained even when the sugar moiety is a sugar surrogate so that the resulting monomer subunit will be able to hybridize.
  • sugar substituent group refers to a group that is covalently attached to a sugar moiety.
  • examples of sugar substituent groups include without limitation halogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino, substituted amino, thio, substituted thio and azido.
  • the alkyl and alkoxy groups are Ci to C 6 .
  • the alkenyl and alkynyl groups are C 2 to C 6 .
  • examples of sugar substituent groups include without limitation 2'-F, 2'-allyl, 2'-amino, 2'-azido, 2'-thio, 2'-0-allyl, 2'-OCF 3 , 2'-O-C 1 -C 10 alkyl, 2'-OCH 3 , 2'-0(CH 2 ) n CH 3 , 2'-OCH 2 CH 3 , 2'-0-(CH 2 ) 2 CH 3 , 2'-0-(CH 2 ) 2 -0-CH 3 (MOE), 2'- 0[(CH 2 ) n O] m CH 3 , 2'-0(CH 2 ) 2 SCH 3 , 2'-0-(CH 2 ) 3 -N(R p )(R q ), 2'-0(CH 2 ) n NH 2 , 2'-0-(CH 2 ) 2 -0-
  • Ri and R 2 are each independently, H or Ci-C 2 alkyl.
  • modified sugar moieties include without limitation bicyclic sugars (e.g. bicyclic nucleic acids or bicyclic nucleosides discussed below).
  • examples of "sugar substituent group" or more generally include
  • “substituent group” include without limitation one or two 5'-sugar substituent groups independently selected from Ci-C 6 alkyl, substituted Ci-C 6 alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C 2 -C 6 alkynyl and halogen.
  • examples of sugar substituent groups include without limitation one or two 5'-sugar substituent groups independently selected from vinyl, 5'-methyl, 5'-(5)-methyl and 5'-(i?)-methyl.
  • examples of sugar substituent groups include without limitation one 5 '-sugar substituent group selected from vinyl, 5'-(S methyl and 5'-(i?)-methyl.
  • examples of sugar substituent groups include without limitation substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an oligomeric compound, and other substituents having similar properties.
  • oligomeric compounds include modifed nucleosides comprising 2'-MOE substituent groups (Baker et al, J. Biol. Chem., 1997, 272, 1 1944-12000).
  • Such 2'-MOE substitution has been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2'-0-methyl, 2'-0-propyl, and 2'-0-aminopropyl.
  • Oligonucleotides having the 2'-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, V., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et ah, Chimia, 1996, 50, 168-176; Altmann et ah, Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et ah, Nucleosides Nucleotides , 1997, 16, 917-926).
  • Sugar moieties can be substituted with more than one sugar substituent group including without limitation 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO 2008/101157, published on 8/21/08 for other disclosed 5', 2'-bis substituted nucleosides).
  • Other combinations are also possible, including without limitation, replacement of the ribosyl ring oxygen atom with S and further substitution at the 2'-position (see published U.S. Patent Application
  • a monomer subunit is meant to include all manner of monomers that are amenable to oligomer synthesis.
  • a monomer subunit includes at least a sugar moiety having at least two reactive sites that can form linkages to further monomer subunits.
  • all monomer subunits include a heterocyclic base moiety that is hybridizable to a complementary site on a nucleic acid target.
  • Reactive sites on monomer subunits located on the termini of an oligomeric compound can be protected or unprotected (generally OH) or can form an attachment to a terminal group (conjugate or other group).
  • Monomer subunits include, without limitation, nucleosides and modified nucleosides.
  • monomer subunits include nucleosides such as ⁇ -D- ribonucleosides and -D-2'-deoxyribnucleosides and modified nucleosides including but not limited to substituted nucleosides (such as 2', 5' and bis substituted nucleosides), 4'-S-modified nucleosides (such as 4'-S-ribonucleosides, 4'-S-2'-deoxyribonucleosides and 4'-S-2'-substituted ribonucleosides), bicyclic modified nucleosides (such as bicyclic nucleosides wherein the sugar moiety has a 2'-0-
  • nucleoside refers to a nucleobase- sugar combination.
  • the two most common classes of such nucleobases are purines and pyrimidines.
  • nucleoside includes ⁇ -D-ribonucleosides and p-D-2'-deoxyribonucleosides.
  • nucleotide refers to a nucleoside further comprising a modified or unmodified phosphate internucleoside linking group or a non-phosphate internucleoside linking group.
  • the internucleoside linking group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar.
  • the phosphate and or a non-phosphate internucleoside linking groups are routinely used to covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • modified nucleoside refers to a nucleoside comprising a modified heterocyclic base and or a sugar moiety other than ribose and 2'-deoxyribose.
  • a modified nucleoside comprises a modified heterocyclic base moiety.
  • a modified nucleoside comprises a sugar moiety other than ribose and 2'-deoxyribose.
  • a modified nucleoside comprises a modified heterocyclic base moiety and a sugar moiety other than ribose and 2'-deoxyribose.
  • the term "modified nucleoside” is intended to include all manner of modified nucleosides that can be incorporated into an oligomeric compound using standard oligomer synthesis protocols. Modified nucleosides include abasic nucleosides but in general a heterocyclic base moiety is included for hybridization to a complementary nucleic acid target.
  • modified nucleosides include a furanose ring system or a modified furanose ring system.
  • Modified furanose ring systems include 4'-S analogs, one or more
  • modified nucleosides include without limitation, substituted nucleosides (such as 2', 5', and/or 4' substituted nucleosides) 4'-S- modified nucleosides, (such as 4'-S-ribonucleosides, 4'-S-2'-deoxyribonucleosides and 4'-S-2'- substituted ribonucleosides), bicyclic modified nucleosides (such as 2'-0-CH(CH 3 )-4', 2'-0-CH 2 -4' or 2'-0-(CH 2 ) 2 -4' bridged furanose analogs) and base modified nucleosides.
  • substituted nucleosides such as 2', 5', and/or 4' substituted nucleosides
  • 4'-S- modified nucleosides such as 4'-S-ribonucleosides, 4'-S-2'-deoxyribonucleosides and 4'-S-2'- substituted ribonu
  • the sugar can be modified with more than one of these modifications listed such as for example a bicyclic modified nucleoside further including a 5'-substitution or a 5' or 4' substituted nucleoside further including a 2' substituent.
  • modified nucleoside also includes combinations of these modifications such as base and sugar modified nucleosides. These modifications are meant to be illustrative and not exhaustive as other modifications are known in the art and are also envisioned as possible modifications for the modified nucleosides described herein.
  • modified nucleosides comprise a sugar surrogate wherein the furanose ring has been replaced with a mono or polycyclic ring system or a non-cyclic sugar surrogate such as that used in peptide nucleic acids.
  • sugar moieties for such modified nucleosides includes without limitation morpholino, hexitol, cyclohexenyl, 2.2.2 and 3.2.1 cyclohexose and open non-cyclic groups.
  • modified nucleosides comprise a non-naturally occurring sugar moiety and a modified heterocyclic base moiety.
  • modified nucleosides include without limitation modified nucleosides wherein the heterocyclic base moiety is replaced with a phenoxazine moiety (for example the 9-(2-aminoethoxy)-l ,3-diazaphenoxazine-2-one group, also referred to as a G-clamp which forms four hydrogen bonds when hybridized with a guanosine base) and further replacement of the sugar moiety with a sugar surrogate group such as for example a morpholino, a cyclohexenyl or a bicyclo[3.1.0]hexyl.
  • a sugar surrogate group such as for example a morpholino, a cyclohexenyl or a bicyclo[3.1.0]hexyl.
  • bicyclic nucleoside refers to a nucleoside comprising at least a bicyclic sugar moiety.
  • examples of bicyclic nucleosides include without limitation nucleosides having a furanosyl sugar that comprises a bridge between two of the non-geminal carbons atoms. In certain embodiments, bicyclic nucleosides have a bridge between the 4' and 2' carbon atoms.
  • 4' to 2' bridged bicyclic nucleosides include but are not limited to one of formulae: 4'-(CH 2 )-0-2' (LNA); 4'-(CH 2 )-S-2'; 4'-(CH 2 ) 2 -0-2' (E A); 4'-CH(CH 3 )-0-2' and 4'-C- H(CH 2 OCH 3 )-0-2' (and analogs thereof see U.S.
  • Patent Applications Serial Nos.: 61/099,844; 61/097,787; 61/086,231 ; 61/056,564; 61/026,998; 61/026,995; 60/989,574; International applications WO2009/006478; WO2008/154401 ;
  • bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example a-L-ribofuranose and ⁇ -D-ribofuranose (see PCT international application PCT/DK98/00393, published on March 25, 1999 as WO 99/14226).
  • the bridge of a bicyclic sugar moiety is , -[C(R a )(Rb)] n - , -[C(R a )(Rb)] ceremoni-0-, -C(R a R b )-N(R)-0- or -C(R a R b )-0-N(R)-.
  • the bridge is 4'-CH 2 -2', 4'-(CH 2 ) 2 -2', 4'-(CH 2 ) 3 -2', 4'-CH 2 -0-2', 4'-(CH 2 ) 2 -0-2', 4'-CH 2 -0-N(R)-2' and 4'-CH 2 - N(R)-0-2'- wherein each R is, independently, H, a protecting group or Ci-Ci 2 alkyl.
  • bicyclic nucleosides are further defined by isomeric configuration.
  • a nucleoside comprising a 4'-(CH 2 )-0-2' bridge may be in the a-L configuration or in the ⁇ -D configuration.
  • a-L-methyleneoxy (4'-CH 2 -0-2') BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et ah, Nucleic Acids
  • bicyclic nucleosides include those having a 4' to 2' bridge wherein such bridges include without limitation, a-L-4'-(CH 2 )-0-2', -D-4'-CH 2 -0-2', 4'-(CH 2 ) 2 -0-2', 4'- CH 2 -0-N(R)-2', 4'-CH 2 -N(R)-0-2', 4'-CH(CH 3 )-0-2', 4'-CH 2 -S-2', 4'-CH 2 -N(R)-2', 4'-CH 2 - CH(CH 3 )-2', and 4'-(CH 2 ) 3 -2', wherein R is H, a protecting group or C Ci 2 alkyl.
  • bicyclic nucleosides have the formula:
  • Bx is a heterocyclic base moiety
  • R c is Ci-Ci 2 alkyl or an amino protecting group
  • T a and T b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.
  • cert cyclic nucleosides have the formula:
  • Bx is a heterocyclic base moiety
  • T a and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
  • Z a is Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Ci-C 6 alkyl, substituted C 2 -C 6 alkenyl, substituted C 2 -C 6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thiol.
  • bicyclic nucleosides have the formula:
  • Bx is a heterocyclic base moiety
  • T a and T b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
  • bicyclic nucleosides have the formula:
  • Bx is a heterocyclic base moiety
  • T a and T b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
  • Rd is Ci-C 6 alkyl, substituted Ci-C 6 alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl or substituted C 2 -C 6 alkynyl;
  • each q a , q b , q c and qa is, independently, H, halogen, Ci-C 6 alkyl, substituted Ci-C 6 alkyl, C 2 - C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl or substituted C 2 -C 6 alkynyl, Ci-C 6 alkoxyl, substituted Ci-C 6 alkoxyl, acyl, substituted acyl, Ci-C 6 aminoalkyl or substituted Ci-C 6 aminoalkyl;
  • bicyclic nucleosides have the formula:
  • Bx is a heterocyclic base moiety
  • T a and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
  • q g and q are each, independently, H, halogen, C1-C12 alkyl or substituted C1-C12 alkyl.
  • bicyclic nucleosides have the formula:
  • Bx is a heterocyclic base moiety
  • bicyclic nucleosides include, but are not limited to, (A) a-L- methyleneoxy (4'-CH 2 -0-2') BNA , (B) ⁇ -D-methyleneoxy (4'-CH 2 -0-2') BNA , (C) ethyleneoxy (4'-(CH 2 )2-0-2') BNA , (D) aminooxy (4'-CH 2 -0-N(R)-2') BNA, (E) oxyamino (4'-CH 2 -N(R)-0- 2') BNA, (F) methyl(methyleneoxy) (4'-CH(CH 3 )-0-2') BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4'-CH 2 -S-2') BNA, (H) methylene-amino (4'-CH 2 -N(R)-2') BNA, (I) methyl carbocyclic (4'-CH 2
  • Bx is the base moiety and R is, independently, H, a protecting group, Ci-C 6 alkyl or Ci-C 6 alkoxy.
  • modified nucleosides include nucleosides having sugar surrogate groups that include without limitation, replacement of the ribosyl ring with a sugar surrogate such as a tetrahydropyranyl ring system (also referred to as hexitol) as illustrated below:
  • Bx is a heterocyclic base moiety
  • one of T3 and T 4 is an internucleoside linking group attaching the tetrahydropyran nucleoside analog to the remainder of one of the 5' or 3' end of the oligomeric compound and the other of T3 and T 4 is hydroxyl, a protected hydroxyl, a 5' or 3' terminal group or an internucleoside linking group attaching the tetrahydropyran nucleoside analog to the remainder of the other of the 5' or 3' end of the oligomeric compound;
  • qi, 3 ⁇ 4, q 3 , q 4 , q 5 , q 6 and q 7 are each independently, H, Ci-C 6 alkyl, substituted Ci-C 6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; and
  • qi, q2, q3, q 4 , q 5 , q 6 and q 7 are each H. In certain embodiments, at least one of qi, q2, q3, q 4 , q 5 , q 6 and q 7 is other than H. In certain embodiments, at least one of qi, q2, q3, q 4 , qs, q 6 and q 7 is methyl.
  • THP nucleosides are provided wherein one of Ri and R2 is F. In certain embodiments, Ri is fluoro and R2 is H; Ri is methoxy and R2 is H, and Ri is methoxyethoxy and R2 is H.
  • Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), altritol nucleic acid (ANA), and mannitol nucleic acid (MNA) (see Leumann, C. J., Bioorg. & Med. Chem., 2002, 10, 841-854).
  • HNA hexitol nucleic acid
  • ANA altritol nucleic acid
  • MNA mannitol nucleic acid
  • oligomeric compounds comprise one or more modified
  • cyclohexenyl nucleosides which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides.
  • Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see for example commonly owned, published PCT Application WO 2010/036696, published on April 10, 2010, Robeyns et al, J. Am. Chem. Soc, 2008, 130(6), 1979-1984; Horvath et al, Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al, J. Am. Chem. Soc, 2007, 129(30), 9340-9348; Gu et al. disturb Nucleosides,
  • Bx is a heterocyclic base moiety
  • Additional modified nucleosides can be prepared by any of the applicable techniques of organic synthesis, as, for example, illustrated in the examples below. Many such techniques are well known in the art. However, many of the known techniques are elaborated in Compendium of Organic Synthetic Methods, John Wiley & Sons, New York: Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G. Wade Jr., 1980; Vol. 5, Leroy G. Wade Jr., 1984; and Vol. 6, Michael B. Smith; as well as March, J., Advanced Organic Chemistry, 3rd Edition, John Wiley & Sons, New York, 1985; Comprehensive Organic Synthesis.
  • reactive phosphorus is meant to include groups that are covalently linked to a monomer subunit that can be further attached to an oligomeric compound that are useful for forming internucleoside linkages including for example phosphodiester and phosphorothioate internucleoside linkages.
  • Such reactive phosphorus groups are known in the art and contain phosphorus atoms in P m or P v valence state including, but not limited to, phosphoramidite, H- phosphonate, phosphate triesters and phosphorus containing chiral auxiliaries.
  • reactive phosphorus groups are selected from diisopropylcyanoethoxy
  • a preferred synthetic solid phase synthesis utilizes phosphoramidites (P m chemistry) as reactive phosphites.
  • the intermediate phosphite compounds are subsequently oxidized to the phosphate or thiophosphate (P v chemistry) using known methods to yield, phosphodiester or phosphorothioate internucleoside linkages.
  • Chiral auxiliaries are known in the art (see for example: Wang et al,
  • oligonucleotide refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).
  • RNA ribonucleosides
  • DNA deoxyribonucleosides
  • oligonucleoside refers to a sequence of nucleosides that are joined by
  • Internucleoside linkages that do not have phosphorus atoms.
  • Internucleoside linkages of this type include short chain alkyl, cycloalkyl, mixed heteroatom alkyl, mixed heteroatom cycloalkyl, one or more short chain heteroatomic and one or more short chain heterocyclic.
  • internucleoside linkages include without limitation, siloxane, sulfide, sulfoxide, sulfone, acetyl, formacetyl, thioformacetyl, methylene formacetyl, thioformacetyl, alkeneyl, sulfamate, methyleneimino, methylenehydrazino, sulfonate, sulfonamide, amide and others having mixed N, O, S and CH 2 component parts.
  • internucleoside linkage or "internucleoside linking group” is meant to include all manner of internucleoside linking groups known in the art including but not limited to, phosphorus containing internucleoside linking groups such as phosphodiester and phosphorothioate, and non-phosphorus containing internucleoside linking groups such as formacetyl and methyleneimino.
  • oligomeric compounds as provided herein can be prepared having one or more internucleoside linkages containing modified e.g. non-naturally occurring
  • internucleoside linkages The two main classes of internucleoside linkages are defined by the presence or absence of a phosphorus atom.
  • Modified internucleoside linkages having a phosphorus atom include without limitation, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phos- phoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates,
  • Oligonucleotides having inverted polarity can comprise a single 3' to 3' linkage at the 3 '-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof).
  • Various salts, mixed salts and free acid forms are also included.
  • oligomeric compounds as provided herein can be prepared having one or more non-phosphorus containing internucleoside linkages.
  • Such oligomeric compounds include without limitation, those that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • neutral internucleoside linkage is intended to include internucleoside linkages that are non-ionic.
  • Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antis ens e 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 CH 2 component parts.
  • oligomeric compounds as provided herein can be prepared having one or more optionally protected phosphorus containing internucleoside linkages.
  • Representative protecting groups for phosphorus containing internucleoside linkages such as phosphodiester and phosphorothioate linkages include ⁇ -cyanoethyl, diphenylsilylethyl, ⁇ -cyanobutenyl, cyano p-xylyl (CPX), N-methyl-N-trifluoroacetyl ethyl (META), acetoxy phenoxy ethyl (APE) and butene-4-yl groups. See for example U.S. Patents Nos. 4,725,677 and Re.
  • linking groups and “bifunctional linking moieties” are meant to include groups known in the art that are useful for attachment of chemical functional groups, conjugate groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligomeric compound.
  • a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind to essentially any selected group such as a chemical functional group or a conjugate group.
  • the linker comprises a chain structure or a polymer of repeating units such as ethylene glycols or amino acid units.
  • bifunctional linking moieties examples include without limitation, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups.
  • bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like.
  • Some nonlimiting examples of bifunctional linking moieties include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-( -maleimido methyl) cyclohexane-l-carboxylate (SMCC) and 6- aminohexanoic acid (AHEX or AHA).
  • linking groups include without limitation, substituted Ci-Cio alkyl, substituted or unsubstituted C 2 -Cio alkenyl or substituted or unsubstituted C 2 -Cio 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.
  • the oligomeric compounds as provided herein can be modified by covalent attachment of one or more conjugate groups.
  • conjugate groups modify one or more properties of the oligomeric compounds they are attached to.
  • Such oligonucleotide properties include without limitation, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and clearance.
  • Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional linking moiety or linking group to a parent compound such as an oligomeric compound.
  • conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids,
  • phospholipids biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.
  • the oligomeric compounds as provided herein can be modified by covalent attachment of one or more terminal groups to the 5' or 3'-terminal groups.
  • a terminal group can also be attached at any other position at one of the terminal ends of the oligomeric compound.
  • the terms "5'-terminal group”, “3'-terminal group”, “terminal group” and combinations thereof are meant to include useful groups known to the art skilled that can be placed on one or both of the terminal ends, including but not limited to the 5' and 3'-ends of an oligomeric compound respectively, for various purposes such as enabling the tracking of the oligomeric compound (a fluorescent label or other reporter group), improving the pharmacokinetics or pharmacodynamics of the oligomeric compound (such as for example: uptake and/or delivery) or enhancing one or more other desirable properties of the oligomeric compound (a group for improving nuclease stability or binding affinity).
  • 5' and 3'-terminal groups include without limitation, modified or unmodified nucleosides; two or more linked nucleosides that are independently, modified or unmodified; conjugate groups; capping groups; phosphate moieties; and protecting groups.
  • the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R) or alkyl where R is H, an amino protecting group or unsubstituted or substituted alkyl.
  • the 5' and or 3' terminal group can comprise from 1 to 3 phosphate moieties that are each, independently, unmodified (di or tri-phosphates) or modified.
  • phosphorus moiety refers to a group having the formula:
  • R x and R y are each, independently, hydroxyl, protected hydroxyl group, thiol, protected thiol group, Ci-C 6 alkyl, substituted Ci-C 6 alkyl, Ci-C 6 alkoxy, substituted Ci-C 6 alkoxy, a protected amino or substituted amino; and
  • R z is O or S.
  • the protected phosphorus moiety is preferred to maintain stability during oligomer synthesis.
  • the phosphorus moiety can include deprotected groups.
  • Phosphorus moieties included herein can be attached to a monomer, which can be used in the preparation of oligomeric compounds, wherein the monomer may be attached using O, S, NRa or
  • Ra includes without limitation H, Ci-C 6 alkyl, substituted Ci-C 6 alkyl, Ci-C 6 alkoxy, substituted Ci-C 6 alkoxy, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C 2 -C 6 alkynyl or substituted acyl, and R e and R f each, independently, include without limitation H, halogen, Ci-C 6 alkyl, substituted Ci-C 6 alkyl, Ci-C 6 alkoxy or substituted Ci-C 6 alkoxy.
  • Such linked phosphorus moieties include without limitation, phosphates, modified phosphates, thiophosphates, modified thiophosphates, phosphonates, modified phosphonates, phosphoramidates and modified phosphoramidates.
  • RNA duplexes exist in what has been termed “A Form” geometry while DNA duplexes exist in “B Form” geometry.
  • RNA: RNA duplexes are more stable, or have higher melting temperatures (T m ) than DNA:DNA duplexes (Sanger et ah, Principles of Nucleic Acid Structure,
  • RNA has been attributed to several structural features, most notably the improved base stacking interactions that result from an A-form geometry (Searle et ah, Nucleic Acids Res. , 1993, 21, 2051 -2056).
  • the presence of the 2' hydroxyl in RNA biases the sugar toward a C3' endo pucker, i.e., also designated as Northern pucker, which causes the duplex to favor the A-form geometry.
  • RNA duplex the 2' hydroxyl groups of RNA can form a network of water mediated hydrogen bonds that help stabilize the RNA duplex (Egli et ah, Biochemistry, 1996, 35, 8489-8494).
  • deoxy nucleic acids prefer a C2' endo sugar pucker, i.e., also known as Southern pucker, which is thought to impart a less stable B-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure, Springer- Verlag, New York, NY).
  • the relative ability of a chemically-modified oligomeric compound to bind to complementary nucleic acid strands, as compared to natural oligonucleotides, is measured by obtaining the melting temperature of a hybridization complex of said chemically-modified oligomeric compound with its complementary unmodified target nucleic acid.
  • the melting temperature (T m ) a character- istic physical property of double helixes, denotes the temperature in degrees centigrade at which 50% helical versus coiled (unhybridized) forms are present.
  • T m also commonly referred to as binding affinity
  • Base stacking which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently a reduction in UV absorption indicates a higher T m .
  • RNA target duplex can be modulated through incorporation of chemically-modified nucleosides into the antisense compound.
  • Sugar-modified nucleosides have provided the most efficient means of modulating the T m of an antisense compound with its target RNA.
  • Sugar-modified nucleosides that increase the population of or lock the sugar in the y -endo (Northern, RNA-like sugar pucker) configuration have predominantly provided a per modification T m increase for antisense compounds toward a complementary RNA target.
  • Sugar-modified nucleosides that increase the population of or lock the sugar in the C -endo (Southern, DNA-like sugar pucker) configuration predominantly provide a per modification Tm decrease for antisense compounds toward a complementary RNA target.
  • the sugar pucker of a given sugar-modified nucleoside is not the only factor that dictates the ability of the nucleoside to increase or decrease an antisense compound's T m toward complementary
  • RNA RNA
  • the sugar-modified nucleoside tricycloDNA is predominantly in the CT-endo conformation, however it imparts a 1.9 to 3° C per modification increase in T m toward a complementary R A.
  • a sugar-modified high-affinity nucleoside that does not adopt the C '-endo conformation is a-L-LNA (described in more detail herein).
  • T m means melting temperature which is the temperature at which the two strands of a duplex nucleic acid separate. T m is often used as a measure of duplex stability or the binding affinity of an antisense compound toward a complementary strand such as an RNA molecule.
  • complementarity in reference to nucleobases refers to a nucleobase that is capable of base pairing with another nucleobase.
  • adenine A
  • complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair. Nucleobases or more broadly, heterocyclic base moieties, comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of complementarity.
  • non-complementary in reference to nucleobases refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.
  • complementary in reference to linked nucleosides, oligonucleotides, oligomeric compounds, or nucleic acids, refers to the capacity of an oligomeric compound to hybridize to another oligomeric compound or nucleic acid through nucleobase or more broadly, heterocyclic base, complementarity.
  • an antisense compound and its target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases that can bond with each other to allow stable association between the antisense compound and the target.
  • nucleobases that can bond with each other to allow stable association between the antisense compound and the target.
  • antisense compounds may comprise up to about 20% nucleotides that are mismatched (i.e., are not nucleobase complementary to the corresponding nucleotides of the target).
  • the antisense compounds contain no more than about 15%, more preferably not more than about 10%, most preferably not more than 5% or no mismatches.
  • the remaining nucleotides are nucleobase complementary or otherwise do not disrupt hybridization
  • oligomeric compounds need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligomeric compound may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). In certain embodiments, oligomeric compounds can comprise at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted.
  • an oligomeric compound in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize would represent 90 percent complementarity.
  • the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases.
  • an oligomeric compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within this scope.
  • Percent complementarity of an oligomeric compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et ah, J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649- 656).
  • hybridization refers to the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between
  • nucleobases complementary nucleoside or nucleotide bases
  • the natural base adenine is nucleobase complementary to the natural nucleobases thymidine and uracil which pair through the formation of hydrogen bonds.
  • the natural base guanine is nucleobase complementary to the natural bases cytosine and 5 -methyl cytosine. Hybridization can occur under varying circumstances.
  • target nucleic acid refers to any nucleic acid molecule the expression, amount, or activity of which is capable of being modulated by an antisense compound.
  • the target nucleic acid is DNA or RNA.
  • the target R A is mRNA, pre-mRNA, non-coding RNA, pri-microRNA, pre-microRNA, mature microRNA, promoter-directed RNA, or natural antisense transcripts.
  • the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent.
  • target nucleic acid is a viral or bacterial nucleic acid.
  • oligomeric compounds such as antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid.
  • the oligomeric compounds provided herein may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid.
  • the oligomeric compound may inhibit the activity the target nucleic acid through an occupancy-based method, thus interfering with the activity of the target nucleic acid.
  • RNAse H a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded oligomeric compounds which are "DNA-like" elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide- mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.
  • modulation refers to a perturbation of amount or quality of a function or activity when compared to the function or activity prior to modulation.
  • modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression.
  • modulation of expression can include perturbing splice site selection of pre-mRNA processing, resulting in a change in the amount of a particular splice-variant present compared to conditions that were not perturbed.
  • modulation includes perturbing translation of a protein.
  • pharmaceutically acceptable salts refers to salts that retain the desired activity of the compound and do not impart undesired toxicological effects thereto.
  • pharmaceutically acceptable salt includes a salt prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic or organic acids and bases.
  • the oligomeric compounds described herein are in the form of a sodium salt.
  • oligomeric compounds provided herein comprise from about 8 to about 80 monomer subunits in length.
  • this embodies oligomeric compounds of 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, or 80 monomer subunits in length, or any range therewithin.
  • oligomeric compounds provided herein comprise from about 8 to 40 monomer subunits in length.
  • oligomeric compounds of 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 or 40 monomer subunits in length, or any range therewithin.
  • oligomeric compounds provided herein comprise from about 8 to 20 monomer subunits in length.
  • oligomeric compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 monomer subunits in length, or any range therewithin.
  • oligomeric compounds provided herein comprise from about 8 to 16 monomer subunits in length.
  • oligomeric compounds of 8, 9, 10, 11, 12, 13, 14, 15 or 16 monomer subunits in length, or any range therewithin.
  • oligomeric compounds provided herein comprise from about 10 to 14 monomer subunits in length.
  • oligomeric compounds of 10, 11 , 12, 13 or 14 monomer subunits in length, or any range therewithin.
  • oligomeric compounds provided herein comprise from about 10 to 18 monomer subunits in length.
  • oligomeric compounds of 10, 11 , 12, 13, 14, 15, 16, 17 or 18 monomer subunits in length, or any range therewithin.
  • oligomeric compounds provided herein comprise from about 10 to 21 monomer subunits in length.
  • oligomeric compounds of 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 monomer subunits in length, or any range therewithin.
  • oligomeric compounds provided herein comprise from about 12 to 14 monomer subunits in length.
  • oligomeric compounds of 12, 13 or 14 monomer subunits in length, or any range therewithin.
  • oligomeric compounds provided herein comprise from about 12 to 18 monomer subunits in length.
  • oligomeric compounds of 12, 13, 14, 15, 16, 17 or 18 monomer subunits in length, or any range therewithin.
  • oligomeric compounds provided herein comprise from about 12 to 21 monomer subunits in length.
  • oligomeric compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 monomer subunits in length, or any range therewithin.
  • oligomeric compounds provided herein comprise from about 14 to
  • oligomeric compounds of any of a variety of ranges of lengths of linked monomer subunits are provided.
  • oligomeric compounds are provided consisting of X-Y linked monomer subunits, where X and Y are each independently selected from 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X ⁇ Y.
  • this provides oligomeric compounds comprising: 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-21, 8-22, 8-23, 8-24, 8-25, 8-26, 8-27,
  • the ranges for the oligomeric compounds listed herein are meant to limit the number of monomer subunits in the oligomeric compounds, however such oligomeric compounds may further include 5' and/or 3'-terminal groups including but not limited to protecting groups such as hydroxyl protecting groups, optionally linked conjugate groups and/or other substituent groups.
  • the preparation of oligomeric compounds as disclosed herein is performed according to literature procedures for DNA: Protocols for Oligonucleotides and Analogs, Agrawal, Ed., Humana Press, 1993, and/or RNA: Scaringe, Methods, 2001, 23, 206-217; Gait et al., Applications of Chemically synthesized RNA in RNA:Protein Interactions, Smith, Ed., 1998, 1-36; Gallo et ah, Tetrahedron , 2001, 57, 5707-5713. Additional methods for solid-phase synthesis may be found in Caruthers U.S. Patents Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Patents Nos. 4,725,677 and Re. 34,069.
  • Oligomeric compounds are routinely prepared using solid support methods as opposed to solution phase methods.
  • Commercially available equipment commonly used for the preparation of oligomeric compounds that utilize the solid support method is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed.
  • Suitable solid phase techniques, including automated synthesis techniques, are described in Oligonucleotides and Analogues, a Practical Approach, F. Eckstein, Ed., Oxford University Press, New York, 1991.
  • RNA synthesis strategies that are presently being used commercially include 5'-0-DMT-2'-0-t- butyldimethylsilyl (TBDMS), 5'-0-DMT-2'-0-[ 1 (2-fiuorophenyl)-4-methoxypiperidin-4-yl]
  • FPMP 2'-0-[(triisopropylsilyl)oxy]methyl (2'-0-CH 2 -0-Si(iPr) 3
  • TOM 2'-0-[(triisopropylsilyl)oxy]methyl
  • DOD 5'-0-bis(trimethylsiloxy)cyclododecyloxysilyl ether
  • ACE 5-acetoxyethoxymethyl
  • TBDMS 5'-0-DMT-2'-0-t-butyldimethylsilyl
  • TOM 2'-0-[(triisopropylsilyl)oxy]methyl
  • DOD/ACE (5'-0-bis(trimethylsiloxy)cyclododecyloxysilyl ether-2'-0-bis(2-acetoxyethoxy)methyl
  • FPMP 5'-0-DMT-2'-0-[l(2-fiuorophenyl)-4-ethoxypiperidin-4-yl].
  • each of the aforementioned RNA synthesis strategies can be used herein.
  • the aforementioned RNA synthesis strategies can be performed together in a hybrid fashion e.g. using a 5'-protecting group from one strategy with a 2'-0-protecting from another strategy.
  • suitable target segments may be employed in a screen for additional oligomeric compounds that modulate the expression of a selected protein.
  • Modules are those oligomeric compounds that decrease or increase the expression of a nucleic acid molecule encoding a protein and which comprise at least an 8-nucleobase portion which is complementary to a suitable target segment.
  • the screening method comprises the steps of contacting a suitable target segment of a nucleic acid molecule encoding a protein with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding a protein. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g.
  • the modulator may then be employed herein in further investigative studies of the function of the peptide, or for use as a research, diagnostic, or therapeutic agent.
  • candidate modulators may be evaluated by the extent to which they increase the expression of a microRNA target RNA or protein (as interference with the activity of a microRNA will result in the increased expression of one or more targets of the microRNA).
  • expression refers to the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, splicing, post-transcriptional modification, and translation.
  • oligomeric compounds provided herein can also be applied in the areas of drug discovery and target validation.
  • provided herein is the use of the oligomeric compounds and targets identified herein in drug discovery efforts to elucidate relationships that exist between proteins and a disease state, phenotype, or condition.
  • These methods include detecting or modulating a target peptide comprising contacting a sample, tissue, cell, or organism with one or more oligomeric compounds provided herein, measuring the nucleic acid or protein level of the target and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further oligomeric compound as provided herein.
  • oligomeric compounds are provided for use in therapy.
  • the therapy is reducing target messenger RNA.
  • a dose refers to a specified quantity of a pharmaceutical agent provided in a single administration.
  • a dose may be administered in two or more boluses, tablets, or injections.
  • the desired dose requires a volume not easily accommodated by a single injection.
  • two or more injections may be used to achieve the desired dose.
  • a dose may be administered in two or more injections to minimize injection site reaction in an individual.
  • a route of administration is the path by which a drug, fluid, poison, or other substance is brought into contact with the body.
  • administering drugs and nucleic acids such as antisense compounds for treatment of a mammal are well known in the art and can be applied to administration of the compositions of the invention.
  • the compounds of the present invention can be administered via any suitable route, most preferably parenterally, in a preparation appropriately tailored to that route.
  • the compounds of the present invention can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, subcutaneously, or intraperitoneally.
  • the present invention also provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier or excipient.
  • Parenteral routes of administration include intravascular (intravenous, intraarterial), intramuscular, intraparenchymal, intradermal, subdermal, subcutaneous, intratumor, intraperitoneal, intrathecal, subdural, epidural, and intralymphatic injections that use a syringe and a needle or catheter.
  • Intravascular herein means within a tubular structure called a vessel that is connected to a tissue or organ within the body.
  • a bodily fluid flows to or from the body part. Examples of bodily fluid include blood, cerebrospinal fluid (CSF), lymphatic fluid, or bile.
  • CSF cerebrospinal fluid
  • lymphatic fluid or bile.
  • vessels examples include arteries, arterioles, capillaries, venules, sinusoids, veins, lymphatics, bile ducts, and ducts of the salivary or other exocrine glands.
  • the intravascular route includes delivery through the blood vessels such as an artery or a vein.
  • the blood circulatory system provides systemic spread of the pharmaceutical.
  • compositions are injected in pharmaceutically acceptable carrier solutions.
  • Pharmaceutically acceptable refers to those properties and/or substances which are acceptable to the mammal from a pharmacological toxicological point of view.
  • pharmaceutically acceptable refers to molecular entities, compositions, and properties that are physiologically tolerable and do not typically produce an allergic or other untoward or toxic reaction when administered to a mammal.
  • the term pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S.
  • Pharmacopeia or other generally recognized pharmacopeia for use in animals and more particularly in humans.
  • compositions as provided herein comprising an antisense compound is co-administered with the delivery peptide.
  • co-administered it is meant that the antisense compound and the delivery peptide are administered to the mammal such that both are present in the mammal at the same time.
  • the antisense compound and the delivery peptide may be administered simultaneously or they may be delivered sequentially. For simultaneous administration, they may be mixed prior to administration. For sequential administration, either the antisense compound or the delivery peptide may be administered first.
  • Antisense compounds may be administered up to 30 minutes prior to administration of the delivery peptide. Also the delivery peptide may be administered up to two hours prior to administration of the antisense compound.
  • Antisense compounds may be administered up to 15 minutes prior to administration of the delivery peptide.
  • the delivery peptide may be administered up to 15 minutes prior to administration of the antisense compound.
  • Antisense compounds may be delivered for research purposes or to produce a change in a cell that is therapeutic. In vivo delivery of antisense compounds is useful for research reagents and for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmaco genomic applications.
  • the compositions disclosed herein are expected to be useful for the delivery of antisense compounds resulting in inhibition of endogenous gene expression in hepatocytes.
  • Levels of a reporter (marker) gene expression measured following delivery of an antisense compound indicate a reasonable expectation of similar levels of gene expression following delivery of other antisense compounds.
  • Levels of treatment considered beneficial by a person having ordinary skill in the art differ from disease to disease.
  • Hemophilia A and B are caused by deficiencies of the X-linked clotting factors VIII and IX, respectively. Their clinical course is greatly influenced by the percentage of normal serum levels of factor VIII or ⁇ : ⁇ 2%, severe; 2-5%, moderate; and 5-30% mild. Thus, an increase from 1% to 2% of the normal level of circulating factor in severe patients can be considered beneficial. Levels greater than 6% prevent spontaneous bleeds but not those secondary to surgery or injury. Similarly, inhibition of a gene need not be 100% to provide a therapeutic benefit. A person having ordinary skill in the art of gene therapy would reasonably anticipate beneficial levels of expression of a gene specific for a disease based upon sufficient levels of marker gene results.
  • reporter or marker genes serve as useful paradigms for expression of intracellular proteins in general.
  • liver is one of the most important target tissues for gene therapy given its central role in metabolism (e.g., lipoprotein metabolism in various hypercholesterolemias) and the secretion of circulating proteins (e.g., clotting factors in hemophilia).
  • acquired disorders such as chronic hepatitis (e.g. hepatitis B virus infection) and cirrhosis are common and are also potentially treated by polynucleotide -based liver therapies.
  • a number of diseases or conditions which affect or are affected by the liver are potentially treated through knockdown (inhibition) of gene expression in the liver.
  • Such liver diseases and conditions may be selected from the list comprising: liver cancers (including hepatocellular carcinoma, HCC), viral infections (including hepatitis), metabolic disorders, (including hyperlipidemia and diabetes), fibrosis, and acute liver injury.
  • nucleoside phosphoramidites The preparation of nucleoside phosphoramidites is performed following procedures that are illustrated herein and in the art such as but not limited to US Patent 6,426,220 and published PCT WO 02/36743.
  • oligomeric compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis.
  • Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as alkylated derivatives and those having phosphorothioate linkages.
  • the oligomeric compounds are recovered by precipitating with greater than 3 volumes of ethanol from a 1 M H 4 OAC solution.
  • Phosphinate internucleoside linkages can be prepared as described in U.S. Patent 5,508,270.
  • Alkyl phosphonate internucleoside linkages can be prepared as described in U.S. Patent 4,469,863.
  • 3 '-Deoxy-3 '-methylene phosphonate internucleoside linkages can be prepared as described in U.S. Patents 5,610,289 or 5,625,050.
  • Phosphoramidite internucleoside linkages can be prepared as described in U.S. Patent, 5,256,775 or U.S. Patent 5,366,878.
  • Alkylphosphonothioate internucleoside linkages can be prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively).
  • 3'-Deoxy-3'-amino phosphoramidate internucleoside linkages can be prepared as described in U.S. Patent 5,476,925.
  • Phosphotriester internucleoside linkages can be prepared as described in U.S. Patent 5,023,243.
  • Borano phosphate internucleoside linkages can be prepared as described in U.S. Patents 5,130,302 and 5,177,198.
  • Formacetal and thioformacetal internucleoside linkages can be prepared as described in U.S. Patents 5,264,562 and 5,264,564.
  • Ethylene oxide internucleoside linkages can be prepared as described in U.S. Patent
  • the oligomeric compounds including without limitation oligonucleotides and oligonucleosides, are recovered by precipitation out of 1 M H 4 OAC with >3 volumes of ethanol. Synthesized oligomeric compounds are analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis is determined by the ratio of correct molecular weight relative to the - 16 amu product (+/-32 +/-48).
  • oligomeric compounds are purified by HPLC, as described by Chiang et al, J. Biol. Chem. 1991 , 266, 18162-18171. Results obtained with HPLC-purified material are generally similar to those obtained with non-HP LC purified material.
  • Oligomeric compounds can be synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format.
  • Phosphodiester internucleoside linkages are afforded by oxidation with aqueous iodine.
  • Phosphorothioate internucleoside linkages are generated by sulfurization utilizing 3,H-1 ,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile.
  • Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites can be purchased from commercial vendors (e.g.
  • Non-standard nucleosides are synthesized as per standard or patented methods and can be functionalized as base protected beta-cyanoethyldiisopropyl phosphoramidites.
  • Oligomeric compounds can be cleaved from support and deprotected with concentrated
  • the concentration of oligomeric compounds in each well can be assessed by dilution of samples and UV absorption spectroscopy.
  • the full-length integrity of the individual products can be evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACETM MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACETM 5000, ABI 270). Base and backbone composition is confirmed by mass analysis of the oligomeric compounds utilizing electrospray-mass spectroscopy. All assay test plates are diluted from the master plate using single and multi-channel robotic pipettors. Plates are judged to be acceptable if at least 85% of the oligomeric compounds on the plate are at least 85% full length.
  • Quantitation of target mRNA levels is accomplished by real-time quantitative PCR using the ABI PRISMTM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) according to manufacturer's instructions.
  • ABI PRISMTM 7600, 7700, or 7900 Sequence Detection System PE-Applied Biosystems, Foster City, CA
  • This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time.
  • PCR polymerase chain reaction
  • products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes.
  • a reporter dye e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, CA, Operon Technologies Inc., Alameda, CA or Integrated DNA Technologies Inc., Coralville, IA
  • a quencher dye e.g., TAMRA, obtained from either PE- Applied Biosystems, Foster City, CA, Operon Technologies Inc., Alameda, CA or Integrated DNA Technologies Inc., Coralville, IA
  • TAMRA obtained from either PE- Applied Biosystems, Foster City, CA, Operon Technologies Inc., Alameda, CA or Integrated DNA Technologies Inc., Coralville, IA
  • annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5'-exonuclease activity of Taq polymerase.
  • cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated.
  • additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISMTM Sequence Detection System.
  • a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.
  • primer-probe sets specific to the target gene being measured are evaluated for their ability to be "multiplexed" with a GAPDH amplification reaction.
  • multiplexing both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample.
  • mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only ("single-plexing"), or both (multiplexing).
  • standard curves of GAPDH and target mR A signal as a function of dilution are generated from both the single-plexed and multiplexed samples.
  • the primer-probe set specific for that target is deemed multiplexable.
  • Other methods of PCR are also known in the art.
  • RT and PCR reagents are obtained from Invitrogen Life Technologies (Carlsbad, CA).
  • RT real-time PCR is carried out by adding 20 PCR cocktail (2.5x PCR buffer minus MgCl 2 , 6.6 mM MgCl 2 , 375 ⁇ each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units R Ase inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5x ROX dye) to 96-well plates containing 30 total RNA solution (20- 200 ng). The RT reaction is carried out by incubation for 30 minutes at 48°C.
  • Gene target quantities obtained by RT, real-time PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RIBOGREENTM (Molecular Probes, Inc. Eugene, OR).
  • GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately.
  • Total RNA is quantified using RiboGreenTM RNA quantification reagent (Molecular Probes, Inc. Eugene, OR). Methods of RNA quantification by RIBOGREENTM are taught in Jones, L.J., et al, (Analytical Biochemistry, 1998, 265, 368-374).
  • RIBOGREENTM working reagent 170 ⁇ , of RIBOGREENTM working reagent (RIBOGREENTM reagent diluted 1 :350 in lOmM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 ⁇ , purified, cellular RNA.
  • the plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485nm and emission at 530nm.
  • Antisense modulation of a target expression can be assayed in a variety of ways known in the art.
  • a target mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR.
  • Real-time quantitative PCR is presently desired.
  • RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA.
  • One method of RNA analysis of the present disclosure is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art.
  • Northern blot analysis is also routine in the art.
  • Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISMTM 7600, 7700, or 7900 Sequence Detection System, available from PE- Applied Biosystems, Foster City, CA and used according to manufacturer's instructions.
  • Protein levels of a target can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS).
  • Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp.
  • Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F.M. et al, Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.
  • Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997.
  • Enzyme-linked immunosorbent assays ELISA are standard in the art and can be found at, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 1 1.2.1-1 1.2.22, John Wiley & Sons, Inc., 1991.
  • the oligomeric compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition.
  • Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of a target in health and disease.
  • phenotypic assays which can be purchased from any of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, OR; PerkinElmer, Boston, MA), protein-based assays including enzymatic assays (Panvera, LLC, Madison, WI; BD Biosciences, Franklin Lakes, NJ; Oncogene Research Products, San Diego, CA), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, MI), triglyceride accumulation (Sigma-Aldrich, St. Louis, MO), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, CA; Amersham Biosciences, Piscataway, NJ).
  • cells determined to be appropriate for a particular phenotypic assay i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies
  • a target inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above.
  • treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.
  • Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.
  • Measurement of the expression of one or more of the genes of the cell after treatment is also used as an indicator of the efficacy or potency of the a target inhibitors.
  • Hallmark genes or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.
  • the individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.
  • Poly(A)+ mRNA is isolated according to Miura et al, (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96- well plates, growth medium is removed from the cells and each well is washed with 200 ⁇ L cold PBS. 60 ⁇ iL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) is added to each well, the plate is gently agitated and then incubated at room temperature for five minutes.
  • 60 ⁇ iL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex
  • Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.
  • Total RNA is isolated using an RNEASY 96TM kit and buffers purchased from Qiagen Inc. (Valencia, CA) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium is removed from the cells and each well is washed with 200 ⁇ L cold PBS. 150 Buffer RLT is added to each well and the plate vigorously agitated for 20 seconds.
  • QIAVACTM manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum is applied for 1 minute. 500 ⁇ , of Buffer RW1 is added to each well of the RNEASY 96TM plate and incubated for 15 minutes and the vacuum is again applied for 1 minute. An additional 500 ⁇ L of Buffer RW1 is added to each well of the RNEASY 96TM plate and the vacuum is applied for 2 minutes. 1 mL of Buffer RPE is then added to each well of the RNEASY 96TM plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash is then repeated and the vacuum is applied for an additional 3 minutes. The plate is then removed from the QIAVACTM manifold and blotted dry on paper towels.
  • RNA is then eluted by pipetting 140 iL of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.
  • the repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia CA). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.
  • a QIAGEN Bio-Robot 9604 Qiagen, Inc., Valencia CA.
  • Probes and primers may be designed to hybridize to a target sequence, using published sequence information.
  • primer-probe set was designed using published sequence information (GENBANKTM accession number U92436.1, SEQ ID NO: 97).
  • FAM-TTGCAGCAATTCACTGTAAAGCTGGAAAGG-TAMRA (SEQ ID NO: 100), where FAM is the fluorescent dye and TAMRA is the quencher dye.
  • Suitable melittin peptides are made using peptide synthesis techniques standard in the art. Suitable melittin peptides can be all L-form amino acids, all D-form amino acids (inverso). Independently of L or D form, the melittin peptide sequence can be reversed (retro).
  • CKLK-Melittin (20 mg/mL), TCEP-HC1 (28.7 mg/mL, 100 mM), and MES-Na (21.7 mg/mL, 100 mM) are prepared in deionized H 2 0.
  • CKLK- Melittin (0.030 mmol, 5 mL) is reacted with 1.7 molar equivalents TCEP-HC1 (0.05 ⁇ , 0.51 mL) and left to stir at room temperature for 30 minutes.
  • MES-Na (2 ml) and Water (1.88 mL) are then added in amounts to yield final concentrations of 10 mg/ml Melittin and 20 mM MES-Na.
  • NAG-PEG 2 -Br 100 mg/ml
  • NAG-PEG 2 -Br 4.75 eq, 0.142 mmol, 0.61 mL
  • the solution is left to stir at room temperature for 48 h.
  • Cys-Melittin (0.006 mmol, 1 mL) is reacted with 1.7 molar equivalents TCEP-HC1 (0.010 mmol, 100 ⁇ ) and left to stir at room temperature for 30 minutes.
  • MES-Na 400 ⁇
  • water 390 ⁇
  • the pH is checked and adjusted to pH 6.5-7.
  • a solution of NAG-PEG8-Maleimide (100 mg/mL) is prepared in deionized H 2 0.
  • NAG-PEGs- Maleimide (2 eq, 0.012 mmol, 110 ⁇ ) is added, and the solution is left to stir at room temperature for 48 h.
  • Samples are purified on a Luna 10 ⁇ CI 8 100 A21, 2x250 mm column.
  • Buffer A H.sub.20 0.1% TFA
  • Buffer B MeCN, 10% Isopropyl Alcohol, 0.1% TFA.
  • Flow rate 15 mL/min, 35% A to 62.5% B in 20 min.
  • n is an integer from 1 to 120 (PEG molecular weight up to about 5 kDa).
  • Peptides having acetyl, dimethyl, stearoyl, myristoyl, and PEG amino or carboxyl terminal modifications, but not terminal cysteine residues are generated on resin during peptide synthesis using methods typical in the art.
  • pH labile masking agents Steric stabilizer CDM-PEG and targeting group CDM-NAG (N- acetyl galactosamine) syntheses.
  • CDM 300 mg, 0.16 mmol
  • oxalyl chloride 2 g, 10 wt. eq.
  • dimethylformamide 5 ⁇
  • the reaction is allowed to proceed overnight, after which the excess oxalyl chloride and methylene chloride are removed by rotary evaporation to yield the CDM acid chloride.
  • the acid chloride is dissolved in 1 mL of methylene chloride.
  • Ri comprises a neutral ASGPrLig.
  • the Masking Agent is uncharged.
  • CDM-PEG R is a methyl or ethyl, and n is an integer from 2 to 100.
  • the PEG contains from 5 to 20 ethylene units (n is an integer from 5 to 20). More preferably, PEG contains 10-14 ethylene units (n is an integer from 10 to 14).
  • the PEG may be of variable length and have a mean length of 5-20 or 10-14 ethylene units.
  • the PEG may be monodisperse, uniform or discrete; having, for example, exactly 1 1 or 13 ethylene units.
  • n is an integer from 1 to 10.
  • a PEG spacer may be positioned between the anhydride group and the ASGPrLig.
  • the PEG spacer contains 1-10 ethylene units.
  • an alkyl spacer may be used between the anhydride and the N- acetylgalactosamine.
  • CDM-NAG alkyl spacer
  • n is a integer from 0 to 6.
  • spacers or linkers may be used between the anhydride and the N- Acetylgalactosamine.
  • the spacer or linker is hydrophilic and neutral (preferably uncharged).
  • Protease (Peptidase) Cleavable Masking Agents Melittin peptide can also be reversibly modified using specialized enzyme cleavable linkers. These enzyme cleavable linkers employ a dipeptide connected to an amidobenzyl activated carbonate moiety. Reaction of the activated carbonate with a peptide amine connects a targeting compound, such as asialoglycoprotein receptor ligand, to the melittin peptide via a peptidase cleavable dipeptide-amidobenzyl carbamate linkage. Enzyme cleavage of the dipeptide removes the targeting ligand from the peptide and triggers an elimination reaction which results in regeneration of the peptide amine.
  • a targeting compound such as asialoglycoprotein receptor ligand
  • Dipeptides Glu-Gly, Ala-Cit, Phe-Cit are shown ("Cit" is the amino acid citrulline). Other amino acid combinations are permissible. In addition, 3-5 amino acids may be used as the linker between the amido benzyl group and the targeting ligand. Further, other activated carbonates known in the art are readily substituted for the para-nitrophenol used in the above compounds.
  • R is melittin and Ri comprises a ASGPrLig (e.g. NAG).
  • ASGPrLig e.g. NAG
  • the anhydride carboxyl produced in the reaction between the anhydride and the polymer amine will exhibit about 1/20 ⁇ of the expected charge (Rozema, et ah, Bioconjugate Chemistry, 2003, 14, 51-57). Therefore, the membrane active polymer is effectively neutralized rather than being converted to a highly negatively charged polyanion.
  • protease cleavable masking agents lx mg of peptide and lOx mg HEPES base at 1-10 mg/mL peptide is masked by addition of 2-6x mg of amine-reactive p-nitrophenyl carbonate or N-hydroxysuccinimide carbonate derivatives of the NAG-containing protease cleavable substrate. The solution is then incubated at least 1 hour at RT before injection into animals.

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Abstract

The present invention provides compositions and methods for improved in vivo delivery of oligomeric compounds. In particular, the compositions and methods provide for administration of a small delivery peptide with an antisense compound. A membrane active melittin peptide is functionalized with masking and targeting groups prior to administration to mask its membrane activity until it is inside a targeted cell. It is expected that inclusion of the small delivery peptide will enhance the delivery and potency of antisense compounds. In certain embodiments, the compositions and methods provided herein are expected to enhance the delivery of antisense compounds to liver cells in a mammal.

Description

COMPOSITIONS AND METHODS FOR IN VIVO DELIVERY OF
ANTISENSE COMPOUNDS
FIELD OF THE INVENTION
The present invention provides a compositions and methods useful for enhancing the in vivo delivery of antisense compounds.
SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled CORE0112WOSEQ.txt, created
November 21, 2013, which is 36 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
Targeting disease-causing gene sequences was first suggested more than thirty years ago (Belikova et al., Tet. Lett., 1967, 37, 3557-3562), and antisense activity was demonstrated in cell culture more than a decade later (Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A., 1978, 75, 280- 284). One advantage of antisense technology in the treatment of a disease or condition that stems from a disease-causing gene is that it is a direct genetic approach that has the ability to modulate (increase or decrease) the expression of specific disease-causing genes. Another advantage is that validation of a therapeutic target using antisense compounds results in direct and immediate discovery of the drug candidate; the antisense compound is the potential therapeutic agent.
Generally, the principle behind antisense technology is that an antisense compound hybridizes to a target nucleic acid and modulates gene expression activities or function, such as transcription and/or translation. The modulation of gene expression can be achieved by, for example, target 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 generally refers to antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of targeted endogenous mR A levels.
An additional example of modulation of R A target function by an occupancy-based mechanism is modulation of microRNA function. MicroRNAs are small non-coding RNAs that regulate the expression of protein-coding RNAs. The binding of an antisense compound to a microRNA prevents the microRNA from binding to its messenger RNA target, and thus interferes with the function of the microRNA. Regardless of the specific mechanism, this sequence-specificity makes antisense compounds extremely 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 malignancies and other diseases.
Antisense technology is an effective means for reducing 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 are routinely incorporated into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target RNA. In 1998, the antisense compound, Vitravene® (fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, CA) was the first antisense drug to achieve marketing clearance from the U.S. Food and Drug Administration (FDA), and is currently a treatment of cytomegalovirus (CMV)-induced retinitis in AIDS patients.
One of the challenges to the delivery of antisense compounds into a living cell is passing through the cell membrane. Drugs used in antisense, RNAi, and gene therapies are relatively large hydrophilic polymers and are frequently highly negatively charged. Both of these physical characteristics restrict their direct diffusion across the cell membrane which in turn impedes the delivery of the antisense compounds to the cell cytoplasm and nucleus.
Functionalized small peptides have used for delivery of conjugated double stranded RNAi polynucleotides (see U.S. Patent Application 20120230938, published on September 13, 2012; U.S. Patent Application 20120172412, published on July 5, 2012; U.S. Patent Application 20120157509, published on June 21 , 2012; U.S. Patent Application 20110207799, published on August 25, 2011 ; and U.S. Patent Application 20080152661 , published on June 26, 2008).
Functionalized Melittin peptides have been used for delivery of conjugated double stranded
RNAi polynucleotides (see U.S. Patent Application 20120165393, published on June 28, 2012).
BRIEF SUMMARY OF THE INVENTION
Provided herein are compositions comprising an antisense compound and Melittin-[(L)- (ASGPrLig)]x wherein:
Melittin is a melittin peptide;
L is physiologically labile linkage;
ASGPrLig is an Asialoglycoprotein Receptor ligand;
x is an integer having a value greater than 80% of the number of primary amines of said melittin peptide; and
wherein said antisense compound is selected from among:
(a) a single stranded RNAi compound comprising at least one of:
a 5'-phosphate modified nucleoside located at the 5' end; and
two or more contiguous 2'-0-(CH2)2-OCH3 modified nucleosides located at the 3' end; and
(b) a non-R Ai antisense compound.
In certain embodiments, the compositions provided herein provide in vivo delivery to a hepatocyte.
In certain embodiments, at least 90% of the amines on each melittin peptide is reversibly linked to the Asialoglycoprotein Receptor ligand.
In certain embodiments, the Melittin peptide comprises an amino acid sequence selected from the list consisting of: Seq. ID 1, Seq. ID 7, Seq. ID 11 , Seq. ID 51, Seq. ID 57, Seq. ID 58, Seq. ID 92, and Seq. ID 96.
In certain embodiments, the Melittin peptide consists of D-form amino acids.
In certain embodiments, L is a disubstituted maleamate. In certain embodiments, L is a disubstituted maleamate and the melittin peptide comprises a polyethyleneglycol (PEG) covalently linked to its amino terminus. In certain embodiments, L is a disubstituted maleamate and the melittin peptide comprises an ASGPrLig-PEG conjugate covalently linked to its amino terminus.
In certain embodiments, L is an amidobenzyl carbamate. In certain embodiments, L is an amidobenzyl carbamate and the melittin peptide comprises a polyethyleneglycol (PEG) covalently linked to its amino terminus. In certain embodiments, L is an amidobenzyl carbamate and the melittin peptide comprises an ASGPrLig-PEG conjugate covalently linked to its amino terminus.
In certain embodiments, the ASGPrLig is selected from the group consisting of lactose, galactose, N-acetylgalactosamine (GalNAc), galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butanoylgalactosamine, and N-iso-butanoyl- galactosamine. In certain embodiments, the antisense compound is a single stranded RNAi compound. In certain embodiments, the antisense compound is a single stranded RNAi compound comprising a 5'- phosphate-5'-vinyl modified nucleoside located at the 5' end. In certain embodiments, the antisense compound is a single stranded RNAi compound comprising and two contiguous 2'-0-(CH2)2-OCH3 modified nucleosides located at the 3' end. In certain embodiments, the antisense compound is a single stranded RNAi compound comprising a 5'-phosphate-5'-vinyl modified nucleoside located at the 5' end and two contiguous 2'-0-(CH2)2-OCH3 modified nucleosides located at the 3' end and each nucleoside located between the 5'-phosphate-5'-vinyl modified nucleoside located at the 5' end and two contiguous 2'-0-(CH2)2-OCH3 modified nucleosides located at the 3' end is independently selected from 2'-F modified nucleosides, 2'-OCH3 modified nucleosides and β-D-ribonucleosides. In certain embodiments, each nucleoside located between the 5'-phosphate-5'-vinyl modified nucleoside located at the 5' end and two contiguous 2'-0-(CH2)2-OCH3 modified nucleosides located at the 3' end is, independently, a 2'-F modified nucleoside or a 2'-OCH3 modified nucleoside. In certain embodiments, the nucleosides located between the 5'-phosphate-5'-vinyl modified nucleoside located at the 5' end and two contiguous 2'-0-(CH2)2-OCH3 modified nucleosides located at the 3' alternate between 2'-F modified nucleosides and 2'-OCH3 modified nucleosides.
In certain embodiments, the antisense compound is a non-RNAi antisense compound. In certain embodiments, the non-RNAi antisense compound works through an RNaseH mechanism. In certain embodiments, the non-RNAi antisense compound comprises a first region consisting of from 2 to 5 modified nucleosides, a second region consisting of from 2 to 5 modified nucleosides and a gap region consisting of from 6 to 14 monomer subunits located between the first and second region.
In certain embodiments, each monomer subunit in the gap region is independently, a nucleoside or a modified nucleoside that is different from each of the modified nucleosides in the first and second region. In certain embodiments, the gap region comprises from about 8 to about 12 monomer subunits. In certain embodiments, the gap region comprises from about 8 to about 10 monomer subunits. In certain embodiments, each monomer subunit in the gap region is a β-ϋ-2'- deoxyribonucleoside. In certain embodiments, one or two of the monomer subunits in the gap region is a modified nucleoside and each of the other monomer subunits in the gap region is a β-D- 2'-deoxyribonucleoside.
In certain embodiments, each modified nucleoside in the first and second region comprises a modified sugar moiety. In certain embodiments, each modified nucleoside in the first and second region is, independently, a bicyclic nucleoside comprising a 4'-CH((5)-CH3)-0-2' bridge or a 2'-0- (CH2)2-OCH3 modified nucleoside. In certain embodiments, the antisense compound comprises internucleoside linking groups that are each, independently, a phosphodiester internucleoside linking group or a phosphorothioate internucleoside linking group. In certain embodiments, the antisense compound comprises internucleoside linking groups that are essentially all phosphorothioate internucleoside linking groups.
In certain embodiments, the antisense compound comprises heterocyclic base moieties that are each, independently, a pyrimidine, substituted pyrimidine, purine or substituted purine. In certain embodiments, the antisense compound comprises heterocyclic base moieties that are each, independently, uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5- methylcytosine, adenine, 6-N-benzoyladenine, guanine or 2-N-isobutyrylguanine.
In certain embodiments, the antisense compound doesn't include a hydrophobic group having at least 20 carbon atoms or a galactose cluster.
In certain embodiments, the composition is provided in a pharmaceutically acceptable carrier or diluent.
In certain embodiments, methods are provided comprising administering to a subject the composition as provided herein.
In certain embodiments, methods are provided comprising co-administering to a subject an antisense compound and Melittin-[(L)-(ASGPrLig)]x
wherein:
Melittin is a melittin peptide;
L is physiologically labile linkage;
ASGPrLig is an Asialoglycoprotein Receptor ligand;
x is an integer having a value greater than 80% of the number of primary amines of said melittin peptide; and
wherein said antisense compound is selected from among:
(a) a single stranded RNAi compound comprising at least one of:
a 5'-phosphate modified nucleoside located at the 5' end; and
at least two contiguous 2'-0-(CH2)2-OCH3 modified nucleosides located at the 3' end; and
(b) a non-R Ai antisense compound.
In certain embodiments, the antisense compound and the Melittin-[(L)-(ASGPrLig)]x are administered together. In certain embodiments, the antisense compound and the Melittin- [(L)- (ASGPrLig)]x are administered separately. In certain embodiments, the antisense compound and the Melittin-[(L)-(ASGPrLig)]x are administered at the same time. In certain embodiments, the antisense compound and the Melittin-[(L)-(ASGPrLig)]x are administered at different times.
In certain embodiments, method of manufacturing a composition are provided comprising: a) forming a melittin peptide; b) forming a plurality of uncharged masking agents each comprising an ASGPrLig covalently linked to a disubstituted maleic anhydride or a dipeptide amidobenzyl amine reactive carbonate; c) modifying greater than 80% of primary amines on a population of melittin peptides with the masking agents of step b, d) providing an antisense compound as per claim 1 and the modified melittin peptide in solution suitable for administration in vivo. DETAILED DESCRIPTION OF THE INVENTION
Provided herein are compositions and methods providing improved in vivo delivery of antisense compounds. More particularly the methods include administration of a small delivery peptide selected from the melittin family, or a derivative thereof, with the antisense compound. In particular the small delivery peptide is selected from the melittin family which is functionalized with groups to provide masking and targeting prior to administration. It is expected that administration of the functionalized small delivery peptide with the antisense compound will enhance the delivery and potency of the antisense compound. In certain embodiments, the methods are expected to provide enhanced delivery of antisense compounds to liver cells in a mammal.
The masked delivery peptide and the antisense compound can be coadministered or can be administered separately. The concentration of the melittin peptide and the antisense compound and the ratio between them is limited only by their respective solubilities in a selected solvent/mixture. Also, the antisense compound and the melittin peptide may be mixed at any time prior to
administration, or if administered separately, the components can be stored separately, either in solution or dry.
The invention includes a composition comprising an an antisense compound and Melittin-
[(L)-(ASGPrLig)]x
wherein:
Melittin is a melittin peptide;
L is physiologically labile linkage;
ASGPrLig is an Asialoglycoprotein Receptor ligand;
x is an integer having a value greater than 80% of the number of primary amines of said melittin peptide; and
wherein said antisense compound is selected from among: (a) a single stranded RNAi compound comprising at least one of:
a 5'-phosphate modified nucleoside located at the 5' end; and
two or more contiguous 2'-0-(CH2)2-OCH3 modified nucleosides located at the 3' end; and
(b) a non-R Ai antisense compound.
Melittin is a bee venom melittin peptide or a derivative as describe herein, and ASGPrLig is covalently linked to Melittin via a physiologically labile reversible linkage L. Cleavage of the physiologically labile reversible linkages restores unmodified amines on Melittin. Melittin can also include an optional polyethyleneglycol (PEG) or ASGPrLig-PEG conjugate group linked to the amino terminus, the carboxy terminus, or an amino or carboxy terminal cysteine of Melittin. In certain embodiments, a polyethyleneglycol (PEG) or ASGPrLig-PEG conjugate group is covalently attached to the amino terminus or an amino terminal cysteine, x is an integer greater than 1.
In certain embodiments, compositions are provided wherein the antisense compound doesn't include a targeting moiety. In certain embodiments, compositions are provided wherein the antisense compound doesn't include a conjugate group. In certain embodiments, compositions are provided wherein the antisense compound doesn't include a conjugate group or a targeting moiety. In certain embodiments, compositions are provided wherein the antisense compound doesn't include a group that is either a hydrophobic group having 20 or more carbon atoms or a galactose cluster. In certain embodiments, the antisense compound includes a conjugate group that is not a targeting moiety. In certain embodiments, the antisense compound includes a targeting moiety. In certain embodiments, the antisense compound includes a targeting moiety that is not an optionally linked ASGPr ligand.
In its unmodified state, Melittin is membrane active. However, delivery peptide Melittin- [(L)-(ASGPrLig)]x is not membrane active. Reversible modification of Melittin primary amines, by attachment of L- ASGPr ligands reversibly inhibits or inactivates membrane activity of Melittin. A sufficient percentage of Melittin primary amines are modified to inhibit membrane activity of the polymer and also provide for hepatocyte targeting. In certain embodiments, x has a value greater than 80%, of the total percentage of primary amines on Melittin, as determined by the quantity of amines on Melittin in the absence of any masking agents. In certain embodiments, x has a value greater than 90%, of the primary amines on Melittin. In certain embodiments, x has a value greater than 80% and up to 100% of the primary amines on Melittin. It is noted that melittin typically contains 3-5 primary amines (the amino terminus (if unmodified) and typically 2-4 Lysine residues). Therefore, modification of a percentage of amines is meant to reflect the modification of a percentage on amines in a population of melittin peptides. Upon cleavage of reversible linkages L, unmodified amines are restored thereby reverting Melittin to its unmodified, membrane active state. In certain embodiments, the reversible linkage is a pH labile linkage. In certain embodiments, the reversible linkage is a protease cleavable linkage.
The Melittin-[(L)-(ASGPrLig)] x, an ASGPr-targeted reversibly masked membrane active polymer (delivery peptide), and the antisense compound, are synthesized or manufactured separately. The antisense compounds are not covalently linked directly or indirectly to the Mellitin- [(L)-(ASGPrLig)]x. Electrostatic or hydrophobic association of the antisense compound with the masked or unmasked polymer is not required for in vivo liver delivery of the antisense compound. In certain embodiments, there are essentially no electrostatic or hydrophobic association of the antisense compound with the masked or unmasked polymer. The masked polymer and the antisense compound can be supplied in the same container or in separate containers. They may be combined prior to administration, co-administered, or administered sequentially.
Hydrophilic groups indicate in qualitative terms that the chemical moiety is water-preferring. Typically, such chemical groups are water soluble, and are hydrogen bond donors or acceptors with water. A hydrophilic group can be charged or uncharged. Charged groups can be positively charged (anionic) or negatively charged (cationic) or both (zwitterionic). Examples of hydrophilic groups include the following chemical moieties: carbohydrates, polyoxyethylene, certain peptides, oligonucleotides, amines, amides, alkoxy amides, carboxylic acids, sulfurs, and hydroxyls.
Hydrophobic groups indicate in qualitative terms that the chemical moiety is water-avoiding.
Typically, such chemical groups are not water soluble, and tend not to form hydrogen bonds.
Lipophilic groups dissolve in fats, oils, lipids, and non-polar solvents and have little to no capacity to form hydrogen bonds. Hydrocarbons containing two or more carbon atoms, certain substituted hydrocarbons, cholesterol, and cholesterol derivatives are examples of hydrophobic groups and compounds.
In certain embodiments, hydrophobic groups are hydrocarbons, containing only carbon and hydrogen atoms. However, non-polar substitutions or non-polar heteroatoms which maintain hydrophobicity, and include, for example fluorine, may be permitted. The term includes aliphatic groups, aromatic groups, acyl groups, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups, each of which may be linear, branched, or cyclic. The term hydrophobic group also includes: sterols, steroids, cholesterol, and steroid and cholesterol derivatives. As used herein, membrane active peptides are surface active, amphipathic peptides that are able to induce one or more of the following effects upon a biological membrane: an alteration or disruption of the membrane that allows non-membrane permeable molecules to enter a cell or cross the membrane, pore formation in the membrane, fission of membranes, or disruption or dissolving of the membrane. As used herein, a membrane, or cell membrane, comprises a lipid bilayer. The alteration or disruption of the membrane can be functionally defined by the peptide's activity in at least one the following assays: red blood cell lysis (hemolysis), liposome leakage, liposome fusion, cell fusion, cell lysis, and endosomal release. Membrane active peptides that can cause lysis of cell membranes are also termed membrane lytic peptides. Peptides that preferentially cause disruption of endosomes or lysosomes over plasma membranes are considered endosomo lytic. The effect of membrane active peptides on a cell membrane may be transient. Membrane active peptides possess affinity for the membrane and cause a denaturation or deformation of bilayer structures.
In certain embodiments, the delivery of an antisense compound to a cell is mediated by the melittin peptide disrupting or destabilizing the plasma membrane or an internal vesicle membrane (such as an endosome or lysosome), including forming a pore in the membrane, or disrupting endosomal or lysosomal vesicles thereby permitting release of the contents of the vesicle into the cell cytoplasm.
Endosomolytic peptides are peptides that, in response to an endosomal-specific environmental factors, such as reduced pH or the presence of lytic enzymes (proteases), are able to cause disruption or lysis of an endosome or provide for release of a normally cell membrane impermeable compound, such as a nucleic acid or protein, from a cellular internal membrane-enclosed vesicle, such as an endosome or lysosome. Endosomolytic polymers undergo a shift in their physico- chemical properties in the endosome. This shift can be a change in the polymer's solubility or ability to interact with other compounds or membranes as a result in a shift in charge,
hydrophobicity, or hydrophilicity. Exemplary endosomolytic peptides have pH-labile or enzymatic- sensitive groups or bonds. A reversibly masked membrane active peptide, wherein the masking agents are attached to the polymer via pH labile bonds, can therefore be considered to be an endosomolytic polymer.
Melittin, as used herein, is a small amphipathic membrane active peptide, comprising about 23 to about 32 amino acids, derived from the naturally occurring bee venom peptide melittin. The naturally occurring melittin contains 26 amino acids and is predominantly hydrophobic on the amino terminal end and predominantly hydrophilic (cationic) on the carboxy terminal end. Melittin of the invention can be isolated from a biological source or it can be synthetic. A synthetic polymer is formulated or manufactured by a chemical process "by man" and is not created by a naturally occurring biological process. As used herein, melittin encompasses the naturally occurring bee venom peptides of the melittin family that can be found in, for example, venom of the species: Apis florea, Apis mellifera, Apis cerana, Apis dorsata, Vespula maculifrons, Vespa magnifica, Vespa velutina, Polistes sp. HQL-2001 , and Polistes hebraeus. As used herein, melittin also encompasses synthetic peptides having amino acid sequence identical to or similar to naturally occurring melittin peptides. Specifically, melittin amino acid sequence encompass those shown in Table 1.
Table 1
Seq Sequence (Melittin) Name
Τ 11Γ1»
1 GIGAILKVLATGLPTLISWIKNKRKQ Apis florea
2 AIGAILKVLATGLPTLISWIKNKRKQ G1A
3 CIGAILKVLATGLPTLISWIK KRKQ G1C
4 FIGAILKVLATGLPTLISWIK KRKQ GIF
5 HIGAILKVLATGLPTLISWIKNKRKQ G1H
6 IIGAILKVLATGLPTLISWIK KRKQ Gil
7 LIGAILKVLATGLPTLISWIKNKRKQ GIL
8 NlelGAILKVLATGLPTLISWIKNKRKQ GINle
9 VIGAILKVLATGLPTLISWIKNKRKQ G1V
10 WIGAILKVLATGLPTLISWIK KRKQ G1W
11 YIGAILKVLATGLPTLISWIKNKRKQ G1Y
12 GIGAILKVLACGLPTLISWIK KRKQ Tl lC dMel
13 GIGAILKVLATLLPTLISWIKNKRKQ G12L
14 GIGAILKVLATWLPTLISWIK KRKQ G12W
15 GIGAILKVLATGLPTLISWIKTKRKQ N22T
16 YIGAILNVLATGLPTLISWIKNKRKQ G1Y, K7N
17 YIGAILAVLATGLPTLISWIKNKRKQ G1Y, K7A
18 LIGAILSVLATGLPTLISWIKNKRKQ GIL, K7S
19 LIGAILRVLATGLPTLISWIK KRKQ GIL, K7R
20 LIGAILHVLATGLPTLISWIKNKRKQ GIL, K7H
21 LIGAILKVLACGLPTLISWIK KRKQ G1L. T11C
22 LIGAILKVLATLLPTLISWIKNKRKQ GIL, G12L
23 YIGAILKVLATGLLTLISWIK KRKQ G1Y, P14L
24 LIGAILKVLATGLPCLISWIK KRKQ G1L, T15C
25 LIGAILKVLATGLPTLICWIKNKRKQ GIL, S18C
26 YIGAILKVLATGLPTLISAIKNKRKQ G1Y, W19A
27 GIGAILKVLACGLPTLISWLKNKRKQ T11C, I20L
28 YIGAILKVLATGLPTLISWIANKRKQ G1Y, K21A
29 YIGAILKVLATGLPTLISWIKNARKQ G1Y, K23A
30 LIGAILKVLATGLPTLISWIKNKAKQ GIL, R24A
31 YIGAILKVLATGLPTLISWIKNKRAQ G1Y, K25A
32 YIGAILKVLATGLPTLISWIKNKRKC G1Y, Q26C
33 LLGAILKVLACGLPTLISWIK KRKQ GIL, I2L, T11C
34 LIGALLKVLACGLPTLISWIK KRKQ GIL, I5L, T11C
35 YIGAILAVLATGLPTLISWIANKRKQ G1Y, K7A, K21A
36 YIGAILAVLATGLPTLISWIKNARKQ G1Y, K7A, K23A 37 LIGAILKVLACGLPTLLSWIK KRKQ G1L, T11C, I17L
38 LIGAILKVLACGl PTLICWIK KRKQ G1L, T11C, S18C
39 GIGAILKVLACGLPGLIGWIKNKRKQ T11G, T15G, S18G
40 GIGAILKVLACGLPALIAWIKNKRKQ T11A, T15A, S18A
41 YIGAILAVLATGLPTLISWIANARKQ G1Y, K7A, K21A, K23A
42 YIAAILKVLAAALATLISWIKNKRKQ G1Y, G3A, T11A, G12A, P14A
43 LLGALLKVLATGLPTLLSWLK KRKQ GIL, I2L, 15L, 117L, 120L
44 LNleGANleLKVLATGLPTLNleSWNleKNKRKQ GIL, I2Nle, I5Nle, I17Nle, I20Nle
45 LVGAVLKVLATGLPTLVSWVK KRKQ GIL, I2V, 15 V, II 7V, I20V
46 GLGALLKVLACGLPTLLSWLK KRKQ I2L, I5L, T11C, I17L, I20L
47 GNleGANleLKVLACGLPTLNleSWNleK KRKQ I2Nle, I5Nle, Tl 1C, I17Nle, I20Nle
48 CEDDLLLGAILKVLATGLPTLISWIKNKPvKQ CEDDL-Mel GIL, I2L
49 CLVVLIVVAILKVLATGLPTLISWIKNKRKQ CLWL-Mel GIL I2V, G3V
50 GIGAVLKVLTTGLPALISWIKRKPvQQ Apis mellifera
51 GLIGAILKVLATGLPTLISWIKNKPvKQ C-Mel GIL
52 CNlelGAILKVLATGLPTLISWIKNKRKQ C-Mel GINle
53 GLIGAILKVLATGLPTLISWIKNKRKQ G-Mel GIL
54 LLIGAILKVLATGLPTLISWIKNKRKQ L-Mel GIL
55 KLKLIGAILKVLATGLPTLISWIK KRKQ KLK-Mel GIL
56 KLKYIGAILKVLATGLPTLISWIK KRKQ KLK-Mel G1Y
57 CKLKLIGAILKVLATGLPTLISWIKNKRKQ CKLK-Mel GIL
58 CKLKNlelGAILKVLATGLPTLIS WIKNKRKQ CKLK-Mel GINle
59 GKLKLIGAILKVLATGLPTLISWIK KRKQ GKLK-Mel GIL
60 CPANLIGAILKVLATGLPTLISWIKNKPvKQ CPAN-dMel GIL
61 DEPLRAIGAILKVLATGLPTLIS WIKNKRKQ DEPLR-Mel G1A
62 GIGAILKVLATGLPTLISWIKNKRKQC Mel-Cys
63 LIGAILKVLATGLPTLISWIKNKRKQC GIL Mel-Cys
64 NlelGAILKVLATGLPTLISWIKNKRKQC GINle Mel-C
65 LIGAILKVLATGLPTLISWIKNKRKQKLKC GIL Mel-KLKC
66 YIGAILKVLATGLPTLISWIKNKRKQPLGIAGQC G1Y Mel-PLGIAGQC
67 LIGAILKVLATGLPTLISWIKNKRKQKKKKK GIL Mel-KKKKK
68 YIGAILKVLATGLPTLISWIKNKRKQGFKGC G1Y Mel-GFKGC
69 CFKLIGAILKVLATGLPTLISWIKNKRKQC CFK-G1L Mel-C
70 FGAILKVLATGLPTLISWIKNKRKQ GIF, Ι2Δ
71 LIGAILKVLATGLPTLISWIKNK GIL Mel (1-23)
72 LIGAVLKVLTTGLPALISWIK 1L, L5V, A10T, T15A Mel (1-23)
73 LIGAVLKVLTTGLPALISWIKGE GIL, L5V, A10T, T15A, N22G, K23E
Mel (1-23)
74 QKRKNKIWSILTPLGTALVKLIAGIL GIL retroMel
75 KLKQKRKNKIWSILTPLGTALVKLIAGIL GIL retroMel-KLK
76 GIGAVLKVLTTGLPALISWISRKKRQQ I5V, A10T, T15A, N22R, 1Z24K, K25R
Mel-Q
77 GIGARLKVLTTGLPRISWIKRKRQQ I5R, A10T, T15R, L164, N22R, K25Q
78 GIGAILKVLSTGLPALISWIKRKRQE A10S, T15A, N22R, K25Q, Q26E 79 GIGAVLKVLTTGLPALIGWIKRKRQQ I5V, A10T, T15A, S18G, N22R, K25Q
80 GIGAVLKVLATGLPALISWIKRKRQQ I5V, T15A, N22R, K25Q
81 GIGAVLKVLSTGLPALISWIKRKRQQ I5V, A10S, T15A, N22R, K25Q
82 GIGAILRVLATGLPTLISWIKNKRKQ K7R
83 GIGAILKVLATGLPTLISWIKRKRKQ N22R
84 GIGAILKVLATGLPTLISWIKKKKQQ N22K, R24K, K25Q
85 GIGAILKVLATGLPTLISWIKNKRKQGSKKKK Mel-GSKKKK
86 KKGIGAILKVLATGLPTLISWIKNKRKQ KK-Mel
87 GIGAILEVLATGLPTLIS WIKNKRKQ K7E Mel 88 GIGAVLKVLTTGLPALISWIKRKR I5V. T15A, N22R, 25-26Δ
89 GIGAVLKVLTTGLPALISWIKR I5V. T15A, N22R, 23-26Δ
90 CIGAVLKVLTTGLPALISWIKRKRQQ G1C, I5L, T15A, N22R
91 QQRKRKIWSILAPLGTTLVKLVAGIG I5V, A10T, T15A, N22R retroMel
92 QQRKRKIWSILAPLGTTLVKLVAGIC G1C, I5V, A10T, T15A, N22R retroMel
93 QQKKKKIWSILAPLGTTLVKLVAGIC G1C, I5V, A10T, T15A, N22R, R24K
retroMel
94 QKRK KIWSILTPLGTALVKLIAGIG Q25K reverse Mel
95 QQRKRKIWSILAALGTTLVKLVAGIC G1C, I5V, A10T, P14A, T15A, N22R
retroMel
96 QKRKNKIWSILTPLGTALVKLIAGIG
In addition to the amino acids which retain melittin's inherent high membrane activity, 1-8 amino acids can be added to the amino or carboxy terminal ends of the peptide. Specifically, cysteine residues can be added to the amino or carboxy termini. The list in Table 1, is not meant to be exhaustive as other conservative amino acid substitutions are readily envisioned. Synthetic melittin peptides can contain naturally occurring L form amino acids or the enantiomeric D form amino acids (inverso). However, a melittin peptide should either contain essentially all L form or all D form amino acids but may have amino acids of the opposite stereocenter appended at either the amino or carboxy termini. The melittin amino acid sequence can also be reversed (retro). Retro melittin can have L form amino acids or D form amino acids (retroinverso). Two melittin peptides can also be covalently linked to form a melittin dimer. Melittin can have modifying groups, other than masking agents, that enhance tissue targeting or facilitate in vivo circulation attached to either the amino terminal or carboxy terminal ends of the peptide. However, as used herein, melittin does not include chains or polymers containing more than two melittin peptides covalently linked to one another other or to another polymer or scaffold.
The melittin peptides of the invention comprise reversibly modified melittin peptides wherein reversible modification inhibits membrane activity, neutralizes the melittin to reduce positive charge and form a near neutral charge polymer, and provides cell-type specific targeting. The melittin is reversibly modified through reversible modification of primary amines on the peptide.
The melittin peptides of the invention are capable of disrupting plasma membranes or lysosomal endocytic membranes. Membrane activity, however, leads to toxicity when the peptide is administered in vivo. Therefore, reversible masking of membrane activity of melittin is necessary for in vivo use. This masking is accomplished through reversible attachment of masking agents to melittin to form a reversibly masked melittin, i.e. a delivery peptide. In addition to inhibiting membrane activity, the masking agents provide cell-specific interactions, i.e. targeting. It is an essential feature of the masking agents that, in aggregate, they inhibit membrane activity of the polymer and provide in vivo hepatocyte targeting. Melittin is membrane active in the unmodified (unmasked) state and not membrane active (inactivated) in the modified (masked) state. A sufficient number of masking agents are linked to the peptide to achieve the desired level of inactivation. The desired level of modification of melittin by attachment of masking agent(s) is readily determined using appropriate peptide activity assays. For example, if melittin possesses membrane activity in a given assay, a sufficient level of masking agent is linked to the peptide to achieve the desired level of inhibition of membrane activity in that assay. In certain embodiments, modification of the primary amine groups is greater than or equal to 80% or greater than or equal 90% on a population of melittin peptides, as determined by the quantity of primary amines on the peptides in the absence of any masking agents. In certain embodiments, the attachment of the masking agent(s) to the reduces positive charge of the polymer, thus forming a more neutral delivery peptide. In certain embodiments, the masked peptide is water soluble.
As used herein, melittin is masked if the modified peptide does not exhibit membrane activity and exhibits cell-specific targeting in vivo. Melittin is reversibly masked if cleavage of bonds linking the masking agents to the peptide results in restoration of amines on the peptide thereby restoring membrane activity. In certain embodiments, melittin is masked and exhibits hepatocyte specific targeting in vivo.
It is another essential feature that the masking agents are covalently bound to melittin through physiologically labile reversible bonds. By using physiologically labile reversible linkages or bonds, the masking agents can be cleaved from the peptide in vivo, thereby unmasking the peptide and restoring activity of the unmasked peptide. By choosing an appropriate reversible linkage, it is possible to form a conjugate that restores activity of melittin after it has been delivered or targeted to a desired cell type or cellular location. Reversibility of the linkages provides for selective activation of melittin. Reversible covalent linkages contain reversible or labile bonds which may be selected from the group comprising: physiologically labile bonds, cellular physiologically labile bonds, pH labile bonds, very pH labile bonds, extremely pH labile bonds, and proetease cleavable bonds.
In certain embodiments, masking agents, as used herein comprise a neutral (uncharged) compound having an ASGPrLig and an amine-reactive group wherein reaction of the amine-reactive group with an amine on a peptide results in linkage of the ASGPrLig to the peptide via a reversible physiologically labile covalent bond. Amine reactive groups are chosen such that cleavage in response to an appropriate physiological condition (e.g., reduced pH such as in an endosome/lysosome, or enzymatic cleavage such as in an endosome/lysosome) results in regeneration of the melittin amine. An ASGPrLig is a group, typically a saccharide, having affinity for the asialoglycoprotein receptor. In certain embodiments, masking agents as provided herein are able to modify the polymer (form a reversible bond with the polymer) in aqueous solution.
In certain embodiments, the amine-reactive group comprises a disubstituted maleic anhydride. In certain embodiments, the masking agent is represented by the structure:
Figure imgf000015_0001
wherein in which Ri comprises an asialoglycoprotein receptor ligand (ASGPrLig) and R2 is an alkyl group such as a methyl (-CH3) group, ethyl (-CH2CH3) group, or propyl (-(CH2)2CH3) group.
In certain embodiments, the galactose ligand is linked to the amine-reactive group through a PEG linker as illustrated by the structure:
Figure imgf000015_0002
wherein n is an integer between 1 and 19.
In certain embodiments, the amine-reactive group comprises a dipeptide-amidobenzyl reactive carbonate derivative represented by the structure:
Figure imgf000015_0003
wherein:
Ri is the R group of amino acid 1 ;
R2 is the R group of amino acid 2;
R3 is -CH2-0-C(0)-0-Z, wherein Z is halide,
Figure imgf000015_0004
and R4 comprises the ASGPrLig.
Reaction of the activated carbonate with a melittin amine connects the ASGPrLig to the melittin peptide via a peptidase cleavable dipeptide-amidobenzyl carbamate linkage as shown below.
Figure imgf000016_0001
Enzymatic cleavage of the dipeptide removes the targeting ligand from the peptide and triggers an elimination reaction which results in regeneration of the peptide amine. While the structure above shows a single masking agent linked to a melittin peptide, in practice, several masking agents are linked to the melittin peptide. In certain embodiments, more than 80% of the amines on a population of melittin peptides are modified.
Dipeptides Glu-Gly, Ala-Cit, Phe-Cit ("Cit" is the amino acid citrulline) are shown in Example 14. With respect to the above structure, Glu-Gly, Ala-Cit, Phe-Cit represent R2-R1. While charged amino acids are permissible, in certain embodiments the amino acids are neutral. Other amino acid combinations are possible, provided they are cleaved by an endogenous protease. In addition, 3-5 amino acids may be used as the linker between the ami do benzyl group and the targeting ligand.
As with maleic anhydride -based masking agents, the ASGPrLig can be linked to the peptidase cleavable dipeptide-amidobenzyl carbonate via a PEG linker.
The membrane active polyamine can be conjugated to masking agents in the presence of an excess of masking agents. The excess masking agent may be removed from the conjugated delivery peptide prior to administration of the delivery peptide.
In certain embodiments, the melittin peptides of the invention are further modified, at the amino or carboxyl termini, by covalent attachment of a steric stabilizer or an ASGPrLig-steric stabilizer conjugate. In certain embodiments, the hydrophobic terminal end is modified; the amino terminal end for melittin having "normal sequence" and the carboxyl terminal end for retro-melittin. In certain embodiments, the steric stabilizer is a polyethylene glycol. The amino or carboxy terminal modifications may be linked to the peptide during synthesis using methods standard in the art. Alternatively, the amino or carboxy terminal modifications may be done through modification of cysteine residues on melittin peptides having amino or carboxy terminal cysteine residues. In certain embodiments, the polyethylene glycols have 1-120 ethylene units. In certain embodiments, polyethylene glycols are less than 5 kDa in size. In certain embodiments, for ASGPrLig-steric stabilizer conjugates (NAG-PEG modification), the steric stabilizer is a polyethyleneglycol having 1-24 ethylene units. Terminal PEG modification, when combined with reversible masking, further reduces toxicity of the melittin delivery peptide. Terminal NAG-PEG modification enhances efficacy.
As used herein, a steric stabilizer is a non-ionic hydrophilic polymer (either natural, synthetic, or non-natural) that prevents or inhibits intramolecular or intermolecular interactions of a molecule to which it is attached relative to the molecule containing no steric stabilizer. A steric stabilizer hinders a molecule to which it is attached from engaging in electrostatic interactions. Electrostatic interaction is the non-covalent association of two or more substances due to attractive forces between positive and negative charges. Steric stabilizers can inhibit interaction with blood components and therefore opsonization, phagocytosis, and uptake by the reticuloendothelial system. Steric stabilizers can thus increase circulation time of molecules to which they are attached. Steric stabilizers can also inhibit aggregation of a molecule. In certain embodiments, the steric stabilizer is a polyethylene glycol (PEG) or PEG derivative. PEG molecules suitable for the invention have about 1-120 ethylene glycol monomers.
Targeting moieties or groups enhance the pharmacokinetic or biodistribution properties of a conjugate to which they are attached to improve cell-specific distribution and cell-specific uptake of the conjugate. Galactose and galactose derivates have been used to target molecules to hepatocytes in vivo through their binding to the asialoglycoprotein receptor (ASGPr) expressed on the surface of hepatocytes. In certain embodiments, the asialoglycoprotein receptor ligand (ASGPrLig) comprises galactose and galactose derivatives having affinity for the ASGPr equal to or greater than that of galactose. Binding of galactose targeting moieties to the ASGPr(s) facilitates cell-specific targeting of the delivery peptide to hepatocytes and endocytosis of the delivery peptide into hepatocytes.
In certain embodiments, ASGPr ligands are selected from the group comprising: lactose, galactose, N-acetylgalactosamine (GalNAc), galactosamine, N-formylgalactosamine, N-acetyl- galactosamine, N-propionylgalactosamine, N-n-butanoylgalactosamine, and N-iso-butanoyl- galactosamine (Iobst, S. T. and Drickamer, K. J.B.C. 1996, 271 , 6686; and Rensen et al, J. Med. Chem., 2004, 47, 5798-5808). ASGPr ligands can be monomeric (e.g., having a single
galactosamine) or multimeric (e.g., having multiple galactosamines).
In certain embodiments, the melittin peptide is reversibly masked by attachment of ASGPr ligand masking agents to greater than or equal to 80% or greater than or equal to 90% of primary amines on the peptide. In certain embodiments, the targeting moiety comprises a galactose cluster (galactose cluster targeting moiety). As used herein, a galactose cluster comprises a molecule having two to four terminal galactose derivatives. As used herein, the term galactose derivative includes both galactose and derivatives of galactose having affinity for the asialoglycoprotein receptor equal to or greater than that of galactose. A terminal galactose derivative is attached to a molecule through its C-l carbon. The asialoglycoprotein receptor (ASGPr) is unique to hepatocytes and binds branched galactose-terminal glycoproteins. In certain embodiments, the galactose cluster has three terminal galactosamines or galactosamine derivatives each having affinity for the asialoglycoprotein receptor. In certain embodiments, the galactose cluster has three terminal N-acetyl-galactosamines. Other terms common in the art include tri-antennary galactose, tri-valent galactose and galactose trimer. It is known that tri-antennary galactose derivative clusters are bound to the ASGPr with greater affinity than bi-antennary or mono-antennary galactose derivative structures (Baenziger and Fiete, Cell, 1980, 22, 611-620; Connolly et al, J. Biol. Che ., 1982, 257, 939-945).
In certain embodiments, a galactose cluster contains three galactose derivatives each linked to a central branch point. The galactose derivatives are attached to the central branch point through the C-l carbons of the saccharides. The galactose derivative is preferably linked to the branch point via linkers or spacers. In certain embodiments, the spacer is a flexible hydrophilic spacer (U.S. Pat. No. 5,885,968; Biessen et al. J. Med. Chem., 1995, 39, 1538-1546). In certain embodiments, the flexible hydrophilic spacer is a PEG spacer. In certain embodiments, the PEG spacer is a PEG3 (three ethylene units) spacer. The branch point can be any small molecule which permits attachment of the three galactose derivatives and further permits attachment of the branch point to the desired parent compound. In certain embodiments, the branch point group is a di-lysine. In certain embodiments, the di-lysine molecule contains three amine groups through which three galactose derivatives may be attached and a carboxyl reactive group through which the di-lysine may be attached to a parent compound. Attachment of the branch point to the parent compound may occur through a linker or spacer such as a flexible hydrophilic spacer. In certain embodiments, the flexible hydrophilic spacer is a PEG spacer. In certain embodiments, the PEG spacer is a PEG3 spacer.
In certain embodiments, the galactose derivative is an N-acetyl-galactosamine (GalNAc). Other saccharides having affinity for the asialoglycoprotein receptor may be selected from the list comprising: galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N- propionyl-galactosamine, N-n-butanoylgalactosamine, and N-iso-butanoylgalactos-amine. The affinities of numerous galactose derivatives for the asialoglycoprotein receptor have been studied (see for example: Iobst et ah, J.B.C., 1996, 271, 6686) or are readily determined using methods typical in the art.
A linkage or linker is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. For example, a linkage can connect a masking agent to a peptide. Formation of a linkage may connect two separate molecules into a single molecule or it may connect two atoms in the same molecule. The linkage may be charge neutral or may bear a positive or negative charge. A reversible or labile linkage contains a reversible or labile bond. A linkage may optionally include a spacer that increases the distance between the two joined atoms. A spacer may further add flexibility and/or length to the linkage. Spacers may include, but are not be limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides. Spacer groups are well known in the art and the preceding list is not meant to limit the scope of the invention. In certain embodiments, linkers and spacers can be used interchangeably. In certain embodiments, a linker comprises spacer groups.
A labile bond is a covalent bond other than a covalent bond to a hydrogen atom that is capable of being selectively broken or cleaved under conditions that will not break or cleave other covalent bonds in the same molecule. More specifically, a labile bond is a covalent bond that is less stable (thermodynamically) or more rapidly broken (kinetically) under appropriate conditions than other non-labile covalent bonds in the same molecule. Cleavage of a labile bond within a molecule may result in the formation of two molecules. For those skilled in the art, cleavage or lability of a bond is generally discussed in terms of half- life (ti/2) of bond cleavage (the time required for half of the bonds to cleave). Thus, labile bonds encompass bonds that can be selectively cleaved more rapidly than other bonds a molecule. Appropriate conditions are determined by the type of labile bond and are well known in organic chemistry. A labile bond can be sensitive to pH, oxidative or reductive conditions or agents, temperature, salt concentration, the presence of an enzyme (such as esterases, including nucleases, and proteases), or the presence of an added agent. For example, increased or decreased pH is the appropriate conditions for a pH-labile bond.
The rate at which a labile group will undergo transformation can be controlled by altering the chemical constituents of the molecule containing the labile group. For example, addition of particular chemical moieties (e.g., electron acceptors or donors) near the labile group can affect the particular conditions (e.g., pH) under which chemical transformation will occur.
As used herein, a physiologically labile bond is a labile bond that is cleavable under conditions normally encountered or analogous to those encountered within a mammalian body. Physiologically labile linkage groups are selected such that they undergo a chemical transformation (e.g., cleavage) when present in certain physiological conditions.
As used herein, a cellular physiologically labile bond is a labile bond that is cleavable under mammalian intracellular conditions. Mammalian intracellular conditions include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt
concentration found in or analogous to those encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activity normally present in a mammalian cell such as from proteolytic or hydrolytic enzymes. A cellular physiologically labile bond may also be cleaved in response to administration of a pharmaceutically acceptable exogenous agent. Physiologically labile bonds that are cleaved under appropriate conditions with a half life of less than 45 minutes are considered very labile. Physiologically labile bonds that are cleaved under appropriate conditions with a half life of less than 15 min are considered extremely labile.
Chemical transformation (cleavage of the labile bond) may be initiated by the addition of a pharmaceutically acceptable agent to the cell or may occur spontaneously when a molecule containing the labile bond reaches an appropriate intra- and/or extra-cellular environment. For example, a pH labile bond may be cleaved when the molecule enters an acidified endosome. Thus, a pH labile bond may be considered to be an endosomal cleavable bond. Enzyme cleavable bonds may be cleaved when exposed to enzymes such as those present in an endosome or lysosome or in the cytoplasm. A disulfide bond may be cleaved when the molecule enters the more reducing environment of the cell cytoplasm. Thus, a disulfide may be considered to be a cytoplasmic cleavable bond.
In certain embodiments, a pH-labile bond is a labile bond that is selectively broken under acidic conditions (pH<7). Such bonds may also be termed endosomally labile bonds, since cell endosomes and lysosomes have a pH less than 7. The term pH-labile includes bonds that are pH- labile, very pH-labile, and extremely pH-labile.
Reaction of an anhydride with an amine forms an amide and an acid. For many anhydrides, the reverse reaction (formation of an anhydride and amine) is very slow and energetically unfavorable. However, if the anhydride is a cyclic anhydride, reaction with an amine yields an amide acid, a molecule in which the amide and the acid are in the same molecule. The presence of both reactive groups (the amide and the carboxylic acid) in the same molecule accelerates the reverse reaction. In particular, the product of primary amines with maleic anhydride and maleic 9 13 anhydride derivatives, maleamic acids, revert back to amine and anhydride 1x10 to 1x10 times faster than its noncyclic analogues (Kirby, Adv. Phys. Org. Chem., 1980, 183-278).
Reaction of an amine with an anhydride to form an amide and an acid.
Figure imgf000021_0001
Reaction of an amine with a cyclic anhydride to form an acid amide
Figure imgf000021_0002
Cleavage of the amide acid to form an amine and an anhydride is pH-dependent and is greatly accelerated at acidic pH. This pH-dependent reactivity can be exploited to form reversible pH-labile bonds and linkers. Cis-aconitic acid has been used as such a pH-sensitive linker molecule. The γ-carboxylate is first coupled to a molecule. In a second step, either the a or β carboxylate is coupled to a second molecule to form a pH-sensitive coupling of the two molecules. The half life for cleavage of this linker at pH 5 is between 8 and 24 h.
Structures of cis-aconitic anhydride and maleic anhydride.
Figure imgf000021_0003
aconic acid maleic anhydride
The pH at which cleavage occurs is controlled by the addition of chemical constituents to the labile moiety. The rate of conversion of maleamic acids to amines and maleic anhydrides is strongly dependent on substitution (R2 and R3) of the maleic anhydride system. When R2 is methyl, the rate of conversion is 50-fold higher than when R2 and R3 are hydrogen. When there are alkyl substitutions at both R2 and R3 (e.g., 2,3-dimethylmaleicanhydride) the rate increase is dramatic: 10,000-fold faster than non-substituted maleic anhydride. The maleamate bond formed from the modification of an amine with 2,3-dimethylmaleic anhydride is cleaved to restore the anhydride and amine with a half-life between 4 and 10 minutes at pH 5. It is anticipated that if R2 and R3 are groups larger than hydrogen, the rate of amide-acid conversion to amine and anhydride will be faster than if R2 and/or R3 are hydrogen. A very pH-labile bond has a half-life for cleavage at pH 5 of less than 45 minutes. The construction of very pH-labile bonds is well-known in the chemical art. An extremely pH-labile bond has a half-life for cleavage at pH 5 of less than 15 minutes. The construction of extremely pH- labile bonds is well-known in the chemical art.
Disubstituted cyclic anhydrides are particularly useful for attachment of masking agents to melittin peptides of the invention. They provide physiologically pH-labile linkages, readily modify amines, and restore those amines upon cleavage in the reduced pH found in cellular endosomes and lysosome. Second, the a or β carboxylic acid group created upon reaction with an amine, appears to contribute only about l/20th of the expected negative charge to the polymer (Rozema, et al., Bioconjugate Chemistry, 2003, 14, 51 -57). Thus, modification of the peptide with the disubstituted maleic anhydrides effectively neutralizes the positive charge of the peptide rather than creatina a peptide with high negative charge. In certain embodiments, near neutral delivery peptides are used for in vivo delivery.
As used herein, the term "oligomeric compound" refers to a contiguous sequence of linked monomer subunits. Each linked monomer subunit normally includes a heterocyclic base moiety but monomer subunits also include those without a heterocyclic base moiety such as abasic monomer subunits. At least some and generally most if not essentially all of the heterocyclic bases in an oligomeric compound are capable of hybridizing to a nucleic acid molecule, normally a preselected
RNA target. The term "oligomeric compound" therefore includes oligonucleotides, oligonucleotide analogs and oligonucleosides. It also includes polymers having one or a plurality of nucleosides having sugar surrogate groups.
In certain embodiments, oligomeric compounds comprise a plurality of monomer subunits independently selected from naturally occurring nucleosides, non-naturally occurring nucleosides, modified nucleosides and nucleosides having sugar surrogate groups. In certain embodiments, oligomeric compounds are single stranded. In certain embodiments, oligomeric compounds are double stranded comprising a double-stranded duplex. In certain embodiments, oligomeric compounds comprise one or more conjugate groups and/or terminal groups.
When preparing oligomeric compounds having specific motifs as disclosed herein it can be advantageous to mix non-naturally occurring monomer subunits with other non-naturally occurring monomer subunits, naturally occurring monomer subunits (nucleosides) or mixtures thereof.
As used herein, "antisense compound" refers to an oligomeric compound, at least a portion of which is at least partially complementary and hybridizable to a selected nucleic acid such as a nucleic acid target. In certain embodiments, an antisense compound modulates (increases or decreases) expression or amount of a target nucleic acid. In certain embodiments, an antisense compound alters splicing of a target pre-mR A resulting in a different splice variant. In certain embodiments, an antisense compound modulates expression of one or more different target proteins. Antisense mechanisms contemplated herein include, but are not limited to an R ase H mechanism, R Ai mechanisms, splicing modulation, translational arrest, altering R A processing, inhibiting microRNA function, or mimicking microRNA function. In certain embodiments, an antisense compound doesn't include a sense region. In certain embodiments, essentially each monomer subunit in the antisense compound is complementary to a region of a nucleic acid target.
As used herein, "antisense activity" refers to any detectable and/or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, such activity may be an increase or decrease in an amount of a nucleic acid or protein. In certain embodiments, such activity may be a change in the ratio of splice variants of a nucleic acid or protein. Detection and/or measuring of antisense activity may be direct or indirect. For example, in certain embodiments, antisense activity is assessed by detecting and/or measuring the amount of target protein or the relative amounts of splice variants of a target protein. In certain embodiments, antisense activity is assessed by detecting and/or measuring the amount of target nucleic acids and/or cleaved target nucleic acids and/or alternatively spliced target nucleic acids. In certain embodiments, antisense activity is assessed by observing a phenotypic change in a cell or animal.
As used herein, "single stranded" means an oligomeric compound that is not hybridized to its complement and which lacks sufficient self-complementarity to form a stable self-duplex.
In certain embodiments, an antisense compound that has a mechanism of action other than
RNAi is referred to as a non-RNAi antisense compound.
In certain embodiments, antisense compounds used in the methods and compositions provided herein are single stranded RNAi compounds. Such antisense compounds are used without a complementary strand to effect activity using an RNAi mechanism. Such single stranded RNAi compounds are known to the art skilled (see for example, published PCT Application WO
2011/139702, published on November 10, 2011; and Filed PCT Application Serial Number PCT
US2012/052874, filed August 29, 2012, which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety, see also Lima et ah, Cell, 2012,
150, 883-894 and Dongbo et al, Cell, 2012, 150, 895-908).
As used herein the term "motif refers to the pattern created by the relative positioning of monomer subunits within an oligomeric compound wherein the pattern is determined by comparing the sugar moieties of the linked monomer subunits. The only determinant for the motif of an oligomeric compound is the differences or lack of differences between the sugar moieties. The internucleoside linkages, heterocyclic bases and further groups such as terminal groups are not considered when determining the motif of an oligomeric compound.
The preparation of motifs has been disclosed in various publications including without limitation, representative U.S. patents 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;
5,403,71 1; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922; and published international applications WO 2005/121371 and WO 2005/121372 (both published on December 22, 2005), certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
Such motifs include without limitation, gapmer motifs, hemimer motifs, blockmer motifs, uniformly fully modified motifs, positionally modified motifs and alternating motifs. In conjunction with these motifs a wide variety of internucleoside linkages can also be used including but not limited to phosphodiester and phosphorothioate internucleoside linkages which can be incorporated uniformly or in various combinations. The oligomeric compounds can further include terminal groups at one or both of the 5' and or 3' terminals such as a conjugate or reporter group. The positioning of the monomer subunits, the use of linkage strategies and terminal groups can be easily optimized to enhance a desired activity for a selected target.
As used herein the term "alternating motif refers to an oligomeric compound comprising a contiguous sequence of linked monomer subunits wherein the monomer subunits have two different types of sugar moieties that alternate for essentially the entire sequence of the oligomeric compound. Oligomeric compounds having an alternating motif can be described by the formula: 5'-A(-L-B-L- A)n(-L-B)nn-3' where A and B are monomer subunits that have different sugar moieties, each L is, independently, an internucleoside linking group, n is from about 4 to about 12 and nn is 0 or 1. The heterocyclic base and internucleoside linkage is independently variable at each position. The motif further optionally includes the use of one or more other groups including but not limited to capping groups, conjugate groups and other 5' and or 3'-terminal groups. This permits alternating oligomeric compounds from about 9 to about 26 monomer subunits in length. This length range is not meant to be limiting as longer and shorter oligomeric compounds are also amenable to oligomeric compounds provided herein.
As used herein the term "uniformly fully modified motif refers to an oligomeric compound comprising a contiguous sequence of linked monomer subunits that each have the same type of sugar moiety. The heterocyclic base and internucleoside linkage is independently variable at each position. The motif further optionally includes the use of one or more other groups including but not limited to capping groups, conjugate groups and other 5' and or 3'-terminal groups.
As used herein the term "hemimer motif refers to an oligomeric compound comprising a contiguous sequence of monomer subunits that each have the same type of sugar moiety with a further short contiguous sequence of monomer subunits located at the 5' or the 3' end that have a different type of sugar moiety. The heterocyclic base and internucleoside linkage is independently variable at each position. The motif further optionally includes the use of one or more other groups including but not limited to capping groups, conjugate groups and other 5' and or 3'-terminal groups. In general, a hemimer is an oligomeric compound of uniform sugar moieties further comprising a short region (1 , 2, 3, 4 or about 5 monomer subunits) having uniform but different sugar moieties located on either the 3' or the 5' end of the oligomeric compound.
In certain embodiments, the hemimer motif comprises a contiguous sequence of from about 10 to about 28 monomer subunits having one type of sugar moiety with from 1 to 5 or from 2 to about 5 monomer subunits having a second type of sugar moiety located at one of the termini. In certain embodiments, the hemimer is a contiguous sequence of from about 8 to about 20 β-ϋ-2'- deoxyribonucleosides having from 1-12 contiguous modified nucleosides located at one of the termini.
As used herein the terms "blockmer motif and "blockmer" refer to an oligomeric compound comprising an otherwise contiguous sequence of monomer subunits wherein the sugar moieties of each monomer subunit is the same except for an interrupting internal block of contiguous monomer subunits having a different type of sugar moiety. The heterocyclic base and internucleoside linkage is independently variable at each position of a blockmer. The motif further optionally includes the use of one or more other groups including but not limited to capping groups, conjugate groups and other 5' or 3 '-terminal groups. A blockmer overlaps somewhat with a gapmer in the definition but typically only the monomer subunits in the block have non-naturally occurring sugar moieties in a blockmer and only the monomer subunits in the external regions have non-naturally occurring sugar moieties in a gapmer with the remainder of monomer subunits in the blockmer or gapmer being β- D-2'-deoxyribonucleosides or β-D-ribonucleosides. In certain embodiments, blockmers are provided herein wherein all of the monomer subunits comprise non-naturally occurring sugar moieties.
As used herein the term "positionally modified motif is meant to include an otherwise contiguous sequence of monomer subunits having one type of sugar moiety that is interrupted with two or more regions of from 1 to about 5 contiguous monomer subunits having another type of sugar moiety. Each of the two or more regions of from 1 to about 5 contiguous monomer subunits are independently uniformly modified with respect to the type of sugar moiety. In certain embodiments, each of the two or more regions have the same type of sugar moiety. In certain embodiments, each of the two or more regions have a different type of sugar moiety. In certain embodiments, each of the two or more regions, independently, have the same or a different type of sugar moiety. The heterocyclic base and internucleoside linkage is independently variable at each position of a positionally modified oligomeric compound. The motif further optionally includes the use of one or more other groups including but not limited to capping groups, conjugate groups and other 5' or 3'- terminal groups.
As used herein the term "gapmer" or "gapped oligomeric compound" refers to an oligomeric compound having two external regions or wings and an internal region or gap. The three regions form a contiguous sequence of monomer subunits with the sugar moieties of the external regions being different than the sugar moieties of the internal region and wherein the sugar moiety of each monomer subunit within a particular region is essentially the same. In certain embodiments, each monomer subunit within a particular region has the same sugar moiety. When the sugar moieties of the external regions are the same the gapmer is a symmetric gapmer and when the sugar moiety used in the 5'-external region is different from the sugar moiety used in the 3'-external region, the gapmer is an asymmetric gapmer. In certain embodiments, the external regions are small (each
independently 1, 2, 3, 4 or about 5 monomer subunits) and the monomer subunits comprise non- naturally occurring sugar moieties with the internal region comprising -D-2'-deoxyribonucleosides. In certain embodiments, the external regions each, independently, comprise from 1 to about 5 monomer subunits having non-naturally occurring sugar moieties and the internal region comprises from 6 to 18 unmodified nucleosides. The internal region or the gap generally comprises β-ϋ-2'- deoxyribonucleosides but can comprise non-naturally occurring sugar moieties. The heterocyclic base and internucleoside linkage is independently variable at each position of a gapped oligomeric compound. The motif further optionally includes the use of one or more other groups including but not limited to capping groups, conjugate groups and other 5' or 3'-terminal groups.
In certain embodiments, gapped oligomeric compounds are provided that are from about 18 to about 21 monomer subunits in length. In certain embodiments, gapped oligomeric compounds are provided that are from about 16 to about 21 monomer subunits in length. In certain
embodiments, gapped oligomeric compounds are provided that are from about 10 to about 21 monomer subunits in length. In certain embodiments, gapped oligomeric compounds are provided that are from about 12 to about 16 monomer subunits in length. In certain embodiments, gapped oligomeric compounds are provided that are from about 12 to about 14 monomer subunits in length. In certain embodiments, gapped oligomeric compounds are provided that are from about 14 to about 16 monomer subunits in length.
As used herein the term "alkyl," refers to a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C1-C12 alkyl) with from 1 to about 6 carbon atoms being more preferred. The term "lower alkyl" as used herein includes from 1 to about 6 carbon atoms. Alkyl groups as used herein may optionally include one or more further substituent groups.
As used herein the term "alkenyl," refers to a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, propenyl, butenyl, l-methyl-2- buten-l-yl, dienes such as 1 ,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.
As used herein the term "alkynyl," refers to a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond.
Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substituent groups.
As used herein the term "aliphatic," refers to a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups. As used herein the term "alicyclic" refers to a cyclic ring system wherein the ring is aliphatic. The ring system can comprise one or more rings wherein at least one ring is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring. Alicyclic as used herein may optionally include further substituent groups.
As used herein the term "alkoxy," refers to a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n- butoxy, sec-butoxy, tert-bvXoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups.
As used herein the term "aminoalkyl" refers to an amino substituted C1-C12 alkyl radical.
The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.
As used herein the terms "aryl" and "aromatic," refer to a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substituent groups.
As used herein the terms "aralkyl" and "arylalkyl," refer to an aromatic group that is covalently linked to a C1-C12 alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.
As used herein the term "heterocyclic radical" refers to a radical mono-, or poly-cyclic ring system that includes at least one heteroatom and is unsaturated, partially saturated or fully saturated, thereby including heteroaryl groups. Heterocyclic is also meant to include fused ring systems wherein one or more of the fused rings contain at least one heteroatom and the other rings can contain one or more heteroatoms or optionally contain no heteroatoms. A heterocyclic radical typically includes at least one atom selected from sulfur, nitrogen or oxygen. Examples of heterocyclic radicals include, [l,3]dioxolanyl, pyrrolidinyl, pyrazolinyl, pyrazohdinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and the like. Heterocyclic groups as used herein may optionally include further substituent groups. As used herein the terms "heteroaryl," and "heteroaromatic," refer to a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms.
Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen.
Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include further substituent groups.
As used herein the term "heteroarylalkyl," refers to a heteroaryl group as previously defined that further includes a covalently attached C1-C12 alkyl radical. The alkyl radical portion of the resulting heteroarylalkyl group is capable of forming a covalent bond with a parent molecule.
Examples include without limitation, pyridinylmethylene, pyrimidinylethylene,
napthyridinylpropylene and the like. Heteroarylalkyl groups as used herein may optionally include further substituent groups on one or both of the heteroaryl or alkyl portions.
As used herein the term "acyl," refers to a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula -C(0)-X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.
As used herein the term "hydrocarbyl" includes radical groups that comprise C, O and H. Included are straight, branched and cyclic groups having any degree of saturation. Such hydrocarbyl groups can include one or more additional heteroatoms selected from N and S and can be further mono or poly substituted with one or more substituent groups.
As used herein the term "mono or polycyclic ring system" is meant to include all ring systems selected from single or polycyclic radical ring systems wherein the rings are fused or linked and is meant to be inclusive of single and mixed ring systems individually selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic and
heteroarylalkyl. Such mono and poly cyclic structures can contain rings that each have the same level of saturation or each, independently, have varying degrees of saturation including fully saturated, partially saturated or fully unsaturated. Each ring can comprise ring atoms selected from C, N, O and S to give rise to heterocyclic rings as well as rings comprising only C ring atoms which can be present in a mixed motif such as for example benzimidazole wherein one ring has only carbon ring atoms and the fused ring has two nitrogen atoms. The mono or polycyclic ring system can be further substituted with substituent groups such as for example phthalimide which has two =0 groups attached to one of the rings. Mono or polycyclic ring systems can be attached to parent molecules using various strategies such as directly through a ring atom, fused through multiple ring atoms, through a substituent group or through a bifunctional linking moiety.
As used herein the terms "halo" and "halogen," refer to an atom selected from fluorine, chlorine, bromine and iodine.
As used herein the term "oxo" refers to the group (=0).
As used herein the term "protecting group," refers to a labile chemical moiety which is known in the art to protect reactive groups including without limitation, hydroxyl, amino and thiol groups, against undesired reactions during synthetic procedures. Protecting groups are typically used selectively and/or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group as is or available for further reactions. Protecting groups as known in the art are described generally in Greene's Protective Groups in Organic Synthesis, 4th edition, John Wiley & Sons, New York, 2007.
Groups can be selectively incorporated into oligomeric compounds as provided herein as precursors. For example an amino group can be placed into a compound as provided herein as an azido group that can be chemically converted to the amino group at a desired point in the synthesis. Generally, groups are protected or present as precursors that will be inert to reactions that modify other areas of the parent molecule for conversion into their final groups at an appropriate time. Further representative protecting or precursor groups are discussed in Agrawal et ah, Protocols for Oligonucleotide Conjugates, Humana Press; New Jersey, 1994, 26, 1-72.
The term "orthogonally protected" refers to functional groups which are protected with different classes of protecting groups, wherein each class of protecting group can be removed in any order and in the presence of all other classes (see, Barany et ah, J. Am. Chem. Soc, 1977, 99, 7363- 7365; Barany et ah, J. Am. Chem. Soc, 1980, 102, 3084-3095). Orthogonal protection is widely used in for example automated oligonucleotide synthesis. A functional group is deblocked in the presence of one or more other protected functional groups which is not affected by the deblocking procedure. This deblocked functional group is reacted in some manner and at some point a further orthogonal protecting group is removed under a different set of reaction conditions. This allows for selective chemistry to arrive at a desired compound or oligomeric compound. The compounds described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-, a or β, or as (D)- or (L)- such as for amino acids. Included herein are all such possible isomers, as well as their racemic and optically pure forms. Optical isomers may be prepared from their respective optically active precursors by the procedures described above, or by resolving the racemic mixtures. The resolution can be carried out in the presence of a resolving agent, by chromatography or by repeated crystallization or by some combination of these techniques which are known to those skilled in the art. Further details regarding resolutions can be found in Jacques, et ah, Enantiomers, Racemates, and Resolutions, John Wiley & Sons, 198 1 . When the compounds described herein contain olefinic double bonds, other unsaturation, or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers or cis- and trans-isomers. Likewise, all tautomeric forms are also intended to be included. The configuration of any carbon- carbon double bond appearing herein is selected for convenience only and is not intended to limit a particular configuration unless the text so states.
The terms "substituent" and "substituent group," as used herein, are meant to include groups that are typically added to a parent compounds or to further substituted substituent groups to enhance one or more desired properties or provide other desired effects. Substituent groups can be protected or unprotected and can be added to one available site or many available sites on a parent compound. As an example if a benzene is substituted with a substituted alky it will not have any overlap with a benzene that is substituted with substituted hydroxyl. In such an example the alkyl portion of the substituted alkyl is covalently linked by one of its carbon atoms to one of the benzene carbon atoms. If the alky is Ci and it is substituted with a hydroxyl substituent group (substituted alkyl) then the resultant compound is benzyl alcohol (C6H5CH2OH). If the benzene were substituted with a substituted hydroxyl group and the hydroxyl was substituted with a Ci alkyl group then the resultant compound would be anisole (C6H5OCH3).
Substituent groups amenable herein include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (-C(O)Raa), carboxyl (-C(O)O-Raa), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (-O-Raa), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (-N(Rbb)(Rcc)), imino(=NRbb), amido (-C(0)N(Rbb)(Rcc) or -N(Rbb)C(0)Raa), azido (-N3), nitro
(-NO2), cyano (-CN), carbamido (-OC(0)N(Rbb)(Rcc) or -N(Rbb)C(0)ORaa), ureido (-N(Rbb)C(0)-
N(Rbb)(Rcc)), thioureido (-N(Rbb)C(S)N(Rbb)(Rcc)), guanidinyl (-N(Rbb)C(=NRbb)N(Rbb)(Rcc)), amidinyl
Figure imgf000031_0001
thiol (-SRbb), sulfinyl (-S(0)Rbb), sulfonyl (-S(0)2Rbb) and sulfonamidyl (-S(0)2N(Rbb)(Rcc) or -N(Rbb)S(0)2Rbb). Wherein each Raa, Rbb and Rcc is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree.
In this context, "recursive substituent" means that a substituent may recite another instance of itself. Because of the recursive nature of such substituents, theoretically, a large number may be present in any given claim. One of ordinary skill in the art of medicinal chemistry and organic chemistry understands that the total number of such substituents is reasonably limited by the desired properties of the compound intended. Such properties include, by way of example and not limitation, physical properties such as molecular weight, solubility or logP, application properties such as activity against the intended target and practical properties such as ease of synthesis.
Recursive substituents are an intended aspect of the invention. One of ordinary skill in the art of medicinal and organic chemistry understands the versatility of such substituents. To the degree that recursive substituents are present in a claim of the invention, the total number will be determined as set forth above.
The terms "stable compound" and "stable structure" as used herein are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Only stable compounds are contemplated herein.
As used herein the term "nucleobase" generally refers to the nucleobase of a nucleoside or modified nucleoside. The term "heterocyclic base moiety" is broader than the term nucleobase in that it includes any heterocyclic base that can be attached to a sugar to prepare a nucleoside or modified nucleoside. Such heterocyclic base moieties include but are not limited to naturally occurring nucleobases (adenine, guanine, thymine, cytosine and uracil) and protected forms of unmodified nucleobases (4-N-benzoylcytosine, 6-N-benzoyladenine and 2-N-isobutyrylguanine) as well as modified (5-methyl cytosine) or non-naturally occurring heterocyclic base moieties and synthetic mimetics thereof (such as for example phenoxazines).
In certain embodiments, a heterocyclic base moiety is any heterocyclic system that contains one or more atoms or groups of atoms capable of hydrogen bonding to a heterocyclic base of a nucleic acid. In certain embodiments, nucleobase refers to purines, modified purines, pyrimidines and modified pyrimidines. In certain embodiments, nucleobase refers to unmodified or naturally occurring nucleobases which include, but are not limited to, the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U) and analogs thereof such as 5-methyl cytosine. The terms nucleobase and heterocyclic base moiety also include optional protection for any reactive functional groups such as 4-N-benzoylcytosine, 4-N-benzoyl-5-methyl- cytosine, 6-N-benzoyladenine or 2-N-isobutyrylguanine.
In certain embodiments, heterocyclic base moieties include without limitation modified nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5- halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8- amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7- methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8- azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein.
In certain embodiments, heterocyclic base moieties include without limitation tricyclic pyrimidines such as l ,3-diazaphenoxazine-2-one, l,3-diazaphenothiazine-2-one and 9-(2- aminoethoxy)-l,3-diazaphenoxazine-2-one (G-clamp). Heterocyclic base moieties 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 heterocyclic base moieties include without limitation those known to the art skilled (see for example: United States Patent No. 3,687,808; Swayze et al., The Medicinal Chemistry of Oligonucleotides in Antisense a Drug Technology, Chapter 6, pages 143-182, Crooke, S.T., ed., 2008); The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J.I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et ah, 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- 302). Modified polycyclic heterocyclic compounds useful as heterocyclic base moieties are disclosed in the above noted U.S. 3,687,808, as well as U.S.: 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187; 5,459,255; 5,484,908; 5,502,177;
5,525,71 1; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269;
5,681,941; 5,750,692; 5,763,588; 5,830,653; 6,005,096; and U.S. Patent Application Publication
20030158403, each of which is incorporated herein by reference in its entirety. As used herein the term "sugar moiety" refers to naturally occurring sugars having a furanose ring system (ribose and 2'-deoxyribose), synthetic and/or non-naturally occurring sugars having a modified furanose ring system and sugar surrogates wherein the furanose ring has been replaced with a mono or polycyclic ring system such as for example a morpholino or hexitol ring system or a non-cyclic sugar surrogate such as that used in peptide nucleic acids. The sugar moiety of a monomer subunit provides the reactive groups that enable the linking of adjacent monomer subunits into an oligomeric compound. Illustrative examples of sugar moieties useful in the preparation of oligomeric compounds include without limitation, β-D-ribose, -D-2'-deoxyribose, substituted sugars (such as 2', 5' and bis substituted sugars), 4'-S-sugars (such as 4'-S-ribose, 4'-S-2'-deoxyribose and 4'-S-2'-substituted ribose wherein the ring oxygen atom has been replaced with a sulfur atom), bicyclic modified sugars (such as the 2'-0-CH(CH3)-4', 2'-0-CH2-4' or 2'-0-(CH2)2-4' bridged ribose derived bicyclic sugars) and sugar surrogates (such as for example when the ribose ring has been replaced with a morpholino, a hexitol ring system or an open non-cyclic system).
As used herein the term "sugar surrogate" refers to replacement of the nucleoside furanose ring with a non-furanose (or 4'-substituted furanose) group with another structure such as another ring system or open system. Such structures can be as simple as a six membered ring as opposed to the five membered furanose ring or can be more complicated such as a bicyclic or tricyclic ring system or a non-ring system such as that used in peptide nucleic acid. In certain embodiments, sugar surrogates include without limitation sugar surrogate groups such as morpholinos, cyclohexenyls and cyclohexitols. In general the heterocyclic base is maintained even when the sugar moiety is a sugar surrogate so that the resulting monomer subunit will be able to hybridize.
As used herein the term "sugar substituent group" refers to a group that is covalently attached to a sugar moiety. In certain embodiments, examples of sugar substituent groups include without limitation halogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino, substituted amino, thio, substituted thio and azido. In certain embodiments the alkyl and alkoxy groups are Ci to C6. In certain embodiments, the alkenyl and alkynyl groups are C2 to C6. In certain embodiments, examples of sugar substituent groups include without limitation 2'-F, 2'-allyl, 2'-amino, 2'-azido, 2'-thio, 2'-0-allyl, 2'-OCF3, 2'-O-C1-C10 alkyl, 2'-OCH3, 2'-0(CH2)nCH3, 2'-OCH2CH3, 2'-0-(CH2)2CH3, 2'-0-(CH2)2-0-CH3 (MOE), 2'- 0[(CH2)nO]mCH3, 2'-0(CH2)2SCH3, 2'-0-(CH2)3-N(Rp)(Rq), 2'-0(CH2)nNH2, 2'-0-(CH2)2-0-
N(Rp)(Rq), 0(CH2)nON[(CH2)nCH3]2, 2'-0(CH2)nONH2, 2'-0-(CH2)2-0-(CH2)2-N(Rp)(Rq), 2'-0-
CH2C(=0)-N(Rp)(Rq), 2'-OCH2C(=0)N(H)CH3, 2'-0-CH2C(=0)-N(H)-(CH2)2-N(Rp)(Rq) and 2'-0-
CH2-N(H)-C(=NRr)[N(Rp)(Rq)], wherein each Rp, Rq and Rr is, independently, H, substituted or unsubstituted Ci-Cio alkyl or a protecting group and where n and m are from 1 to about 10.
In certain embodiments, examples of sugar substituent groups include without limitation 2'- F, 2'-allyl, 2'-amino, 2'-azido, 2'-thio, 2'-0-allyl, 2'-OCF3, 2'-0-Ci-Cio alkyl, 2'-0-CH3, OCF3, 2'-0- CH2CH3, 2'-0-(CH2)2CH3, 2'-0-(CH2)2-0-CH3 (MOE), 2'-0(CH2)2SCH3, 2'-0-CH2-CH=CH2, 2'-0- (CH2)3-N(Rm)(Rn), 2'-0-(CH2)2-0-N(Rm)(Rn), 2'-0-(CH2)2-0-(CH2)2-N(Rm)(R„), 2'-0-CH2C(=0)- N(Rm)(Rn), 2'-0-CH2C(=0)-N(H)-(CH2)2-N(Rm)(Rn) and 2'-0-CH2-N(H)-C(=NRm)[N(Rm)(Rn)] wherein each Rm and Rn is, independently, H, substituted or unsubstituted Ci-Cio alkyl or a protecting group. In certain embodiments, examples of 2,-sugar substituent groups include without limitation fiuoro, -0-CH3, -0-CH2CH3, -0-(CH2)2CH3, -0-(CH2)2-0-CH3, -0-CH2-CH=CH2, -O- (CH2)3-N(R (R2), O-(CH2)2-O-N(R (R2), -0-(CH2)2-0-(CH2)2-N(R1)(R2), -0-CH2C(=0)-
N(Ri)(R2), -0-CH2C(=0)-N(H)-(CH2)2-N(Ri)(R2) and -0-CH2-N(H)-C(=NRi)[N(Ri)(R2)] wherein Ri and R2 are each independently, H or Ci-C2 alkyl. In certain embodiments, examples of sugar substituent groups include without limitation fluoro, -0-CH3, -0-(CH2)2-0-CH3, -0-CH2C(=0)- N(H)(CH3), -0-CH2C(=0)-N(H)-(CH2)2-N(CH3)2 and -0-CH2-N(H)-C(=NCH3)[N(CH3)2]. In certain embodiments, examples of sugar substituent groups include without limitation fluoro, -O- CH3, -0-(CH2)2-0-CH3, -0-CH2C(=0)-N(H)(CH3) and -0-CH2C(=0)-N(H)-(CH2)2-N(CH3)2. Further examples of modified sugar moieties include without limitation bicyclic sugars (e.g. bicyclic nucleic acids or bicyclic nucleosides discussed below).
In certain embodiments, examples of "sugar substituent group" or more generally
"substituent group" include without limitation one or two 5'-sugar substituent groups independently selected from Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl and halogen. In certain embodiments, examples of sugar substituent groups include without limitation one or two 5'-sugar substituent groups independently selected from vinyl, 5'-methyl, 5'-(5)-methyl and 5'-(i?)-methyl. In certain embodiments, examples of sugar substituent groups include without limitation one 5 '-sugar substituent group selected from vinyl, 5'-(S methyl and 5'-(i?)-methyl.
In certain embodiments, examples of sugar substituent groups include without limitation substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an oligomeric compound, and other substituents having similar properties. In certain embodiments, oligomeric compounds include modifed nucleosides comprising 2'-MOE substituent groups (Baker et al, J. Biol. Chem., 1997, 272, 1 1944-12000). Such 2'-MOE substitution has been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2'-0-methyl, 2'-0-propyl, and 2'-0-aminopropyl. Oligonucleotides having the 2'-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, V., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et ah, Chimia, 1996, 50, 168-176; Altmann et ah, Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et ah, Nucleosides Nucleotides , 1997, 16, 917-926).
Sugar moieties can be substituted with more than one sugar substituent group including without limitation 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO 2008/101157, published on 8/21/08 for other disclosed 5', 2'-bis substituted nucleosides). Other combinations are also possible, including without limitation, replacement of the ribosyl ring oxygen atom with S and further substitution at the 2'-position (see published U.S. Patent Application
US2005-0130923, published on June 16, 2005) and 5'-substitution of a bicyclic nucleoside (see PCT International Application WO 2007/134181, published on 1 1/22/07 wherein a 4'-CH2-0-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or a 5'-vinyl group).
As used herein the term "monomer subunit" is meant to include all manner of monomers that are amenable to oligomer synthesis. In general a monomer subunit includes at least a sugar moiety having at least two reactive sites that can form linkages to further monomer subunits. Essentially all monomer subunits include a heterocyclic base moiety that is hybridizable to a complementary site on a nucleic acid target. Reactive sites on monomer subunits located on the termini of an oligomeric compound can be protected or unprotected (generally OH) or can form an attachment to a terminal group (conjugate or other group). Monomer subunits include, without limitation, nucleosides and modified nucleosides. In certain embodiments, monomer subunits include nucleosides such as β-D- ribonucleosides and -D-2'-deoxyribnucleosides and modified nucleosides including but not limited to substituted nucleosides (such as 2', 5' and bis substituted nucleosides), 4'-S-modified nucleosides (such as 4'-S-ribonucleosides, 4'-S-2'-deoxyribonucleosides and 4'-S-2'-substituted ribonucleosides), bicyclic modified nucleosides (such as bicyclic nucleosides wherein the sugar moiety has a 2'-0-
CHRa-4' bridging group, wherein Ra is H, alkyl or substituted alkyl), other modified nucleosides and nucleosides having sugar surrogates. As used herein, the term "nucleoside" refers to a nucleobase- sugar combination. The two most common classes of such nucleobases are purines and pyrimidines. The term nucleoside includes β-D-ribonucleosides and p-D-2'-deoxyribonucleosides.
As used herein, the term "nucleotide" refers to a nucleoside further comprising a modified or unmodified phosphate internucleoside linking group or a non-phosphate internucleoside linking group. For nucleotides that include a pentofuranosyl sugar, the internucleoside linking group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. The phosphate and or a non-phosphate internucleoside linking groups are routinely used to covalently link adjacent nucleosides to one another to form a linear polymeric compound.
As used herein the term "modified nucleoside" refers to a nucleoside comprising a modified heterocyclic base and or a sugar moiety other than ribose and 2'-deoxyribose. In certain
embodiments, a modified nucleoside comprises a modified heterocyclic base moiety. In certain embodiments, a modified nucleoside comprises a sugar moiety other than ribose and 2'-deoxyribose. In certain embodiments, a modified nucleoside comprises a modified heterocyclic base moiety and a sugar moiety other than ribose and 2'-deoxyribose. The term "modified nucleoside" is intended to include all manner of modified nucleosides that can be incorporated into an oligomeric compound using standard oligomer synthesis protocols. Modified nucleosides include abasic nucleosides but in general a heterocyclic base moiety is included for hybridization to a complementary nucleic acid target.
In certain embodiments, modified nucleosides include a furanose ring system or a modified furanose ring system. Modified furanose ring systems include 4'-S analogs, one or more
substitutions at any position such as for example the 2', 3', 4' and 5' positions and addition of bridges for form additional rings such as a 2'-0-CH(CH3)-4' bridge. Such modified nucleosides include without limitation, substituted nucleosides (such as 2', 5', and/or 4' substituted nucleosides) 4'-S- modified nucleosides, (such as 4'-S-ribonucleosides, 4'-S-2'-deoxyribonucleosides and 4'-S-2'- substituted ribonucleosides), bicyclic modified nucleosides (such as 2'-0-CH(CH3)-4', 2'-0-CH2-4' or 2'-0-(CH2)2-4' bridged furanose analogs) and base modified nucleosides. The sugar can be modified with more than one of these modifications listed such as for example a bicyclic modified nucleoside further including a 5'-substitution or a 5' or 4' substituted nucleoside further including a 2' substituent. The term modified nucleoside also includes combinations of these modifications such as base and sugar modified nucleosides. These modifications are meant to be illustrative and not exhaustive as other modifications are known in the art and are also envisioned as possible modifications for the modified nucleosides described herein.
In certain embodiments, modified nucleosides comprise a sugar surrogate wherein the furanose ring has been replaced with a mono or polycyclic ring system or a non-cyclic sugar surrogate such as that used in peptide nucleic acids. Illustrative examples of sugar moieties for such modified nucleosides includes without limitation morpholino, hexitol, cyclohexenyl, 2.2.2 and 3.2.1 cyclohexose and open non-cyclic groups.
In certain embodiments, modified nucleosides comprise a non-naturally occurring sugar moiety and a modified heterocyclic base moiety. Such modified nucleosides include without limitation modified nucleosides wherein the heterocyclic base moiety is replaced with a phenoxazine moiety (for example the 9-(2-aminoethoxy)-l ,3-diazaphenoxazine-2-one group, also referred to as a G-clamp which forms four hydrogen bonds when hybridized with a guanosine base) and further replacement of the sugar moiety with a sugar surrogate group such as for example a morpholino, a cyclohexenyl or a bicyclo[3.1.0]hexyl.
As used herein the term "bicyclic nucleoside" refers to a nucleoside comprising at least a bicyclic sugar moiety. Examples of bicyclic nucleosides include without limitation nucleosides having a furanosyl sugar that comprises a bridge between two of the non-geminal carbons atoms. In certain embodiments, bicyclic nucleosides have a bridge between the 4' and 2' carbon atoms.
Examples of such 4' to 2' bridged bicyclic nucleosides, include but are not limited to one of formulae: 4'-(CH2)-0-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-0-2' (E A); 4'-CH(CH3)-0-2' and 4'-C- H(CH2OCH3)-0-2' (and analogs thereof see U.S. Patent 7,399,845, issued on July 15, 2008); 4'- C(CH3)(CH3)-0-2' (and analogs thereof see published International Application WO/2009/006478, published January 8, 2009); 4'-CH2-N(OCH3)-2' (and analogs thereof see published International Application WO2008/150729, published December 11, 2008); 4'-CH2-0-N(CH3)-2' (see U.S. Patent 7,96,345, issued on April 13, 2010,); 4'-CH2-N(R)-0-2', wherein R is H, C Ci2 alkyl, or a protecting group (see U.S. Patent 7,427,672, issued on September 23, 2008); 4'-CH2-C(H)(CH3)-2' (see Chattopadhyaya, et al, J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-CH2-2' and 4'-CH2-C- (=CH2)-2' (and analogs thereof see published International Application WO 2008/154401, published on December 8, 2008). Further bicyclic nucleosides have been reported in published literature (see for example: Srivastava et al, J. Am. Chem. Soc, 2007, 129(26) 8362-8379; Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372; Elayadi et al., Curr. Opinion Invens. Drugs, 2001 , 2, 558- 561; Braasch et al, Chem. Biol, 2001, 8, 1-7; Oram et al, Curr. Opinion Mol. Ther., 2001, 3, 239- 243; Wahlestedt et al, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 5633-5638; 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; U.S. Patents Nos.: 7,741,457; 7,696,345; 7,547,684,;7,399,845; 7,053,207; 7,034,133; 6,794,499; 6,770,748; 6,670,461 ; 6,525,191; 6,268,490; U.S. Patent Publication Nos.: US2008-0039618; U.S. Patent Applications, Serial Nos.: 61/099,844; 61/097,787; 61/086,231 ; 61/056,564; 61/026,998; 61/026,995; 60/989,574; International applications WO2009/006478; WO2008/154401 ;
WO2008/150729; WO 2007/134181; WO 2005/021570; WO 2004/106356; WO 94/14226). Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example a-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on March 25, 1999 as WO 99/14226).
In certain embodiments, bicyclic nucleosides comprise a bridge between the 4' and the 2' carbon atoms of the pentofuranosyl sugar moiety including without limitation, bridges comprising 1 or from 1 to 4 linked groups (generally forming a 4 to 6 membered ring with the parent sugar moiety) independently selected from -[C(Ra)(Rb)]n-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)-, -C(=0)- , -C(=S)-, -0-, -Si(Ra)2-, -S(=0)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, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-Ci2 alkenyl, C2-Ci2 alkynyl, substituted C2-Ci2 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJi, NJiJ2, SJi, N3, COOJi, acyl (C(=0)-H), substituted acyl, CN, sulfonyl (S(=0)2-Ji), or sulfoxyl (S(=0)-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(=0)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, Ci-Ci2 aminoalkyl, substituted Ci-Ci2 aminoalkyl or a protecting group.
In certain embodiments, the bridge of a bicyclic sugar moiety is , -[C(Ra)(Rb)]n- , -[C(Ra)(Rb)]„-0-, -C(RaRb)-N(R)-0- or -C(RaRb)-0-N(R)-. In certain embodiments, the bridge is 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-0-2', 4'-(CH2)2-0-2', 4'-CH2-0-N(R)-2' and 4'-CH2- N(R)-0-2'- wherein each R is, independently, H, a protecting group or Ci-Ci2 alkyl.
In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4'-(CH2)-0-2' bridge, may be in the a-L configuration or in the β-D configuration. Previously, a-L-methyleneoxy (4'-CH2-0-2') BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et ah, Nucleic Acids
Research, 2003, 21, 6365-6372).
In certain embodiments, bicyclic nucleosides include those having a 4' to 2' bridge wherein such bridges include without limitation, a-L-4'-(CH2)-0-2', -D-4'-CH2-0-2', 4'-(CH2)2-0-2', 4'- CH2-0-N(R)-2', 4'-CH2-N(R)-0-2', 4'-CH(CH3)-0-2', 4'-CH2-S-2', 4'-CH2-N(R)-2', 4'-CH2- CH(CH3)-2', and 4'-(CH2)3-2', wherein R is H, a protecting group or C Ci2 alkyl.
In certain embodiments, bicyclic nucleosides have the formula:
Figure imgf000040_0001
wherein:
Bx is a heterocyclic base moiety;
-Qa-Qb-Qc- is -CH2-N(RC)-CH2-, -C(=0)-N(Rc)-CH2-, -CH2-0-N(Rc)-, -CH2-N(Rc)-0- or - N(Rc)-0-CH2;
Rc is Ci-Ci2 alkyl or an amino protecting group; and
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.
In cert cyclic nucleosides have the formula:
Figure imgf000040_0002
wherein:
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Za is Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thiol.
In certain embodiments, each of the substituted groups, is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJc, NJcJa, SJC, N3, OC(=X)Jc, and NJeC(=X)NJcJd, wherein each Jc, Jd and Je is, independently, H, Ci-C6 alkyl, or substituted Ci-C6 alkyl and X is O or NJC.
In certain embodiments, bicyclic nucleosides have the formula:
Figure imgf000041_0001
wherein:
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Zb is Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl or substituted acyl (C(=0)-).
In certain embodiments, bicyclic nucleosides have the formula:
Figure imgf000041_0002
wherein:
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Rd is Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
each qa, qb, qc and qa is, independently, H, halogen, Ci-C6 alkyl, substituted Ci-C6 alkyl, C2- C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, Ci-C6 alkoxyl, substituted Ci-C6 alkoxyl, acyl, substituted acyl, Ci-C6 aminoalkyl or substituted Ci-C6 aminoalkyl;
In certain embodiments, bicyclic nucleosides have the formula:
Figure imgf000041_0003
wherein:
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium; qa, qb, qe and qf are each, independently, hydrogen, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C1-C12 alkoxy, substituted C1-C12 alkoxy, OJj, SJj, SOJj, S02Jj, NJjJk, N3, CN, C(=0)OJj, C(=0)NJjJk, C(=0)Jj, 0-C(=0)NJjJk, N(H)C(=NH)NJjJk, N(H)C(=0)NJjJk orN(H)C(=S)NJjJk;
or qe and qf together are =C(qg)(qh);
qg and q are each, independently, H, halogen, C1-C12 alkyl or substituted C1-C12 alkyl.
The synthesis and preparation of adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil bicyclic nucleosides having a 4'-CH2-0-2' bridge, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). The synthesis of bicyclic nucleosides has also been described in WO 98/39352 and WO 99/14226.
Analogs of various bicyclic nucleosides that have 4' to 2' bridging groups such as 4'-CH2-0- 2' and 4'-CH2-S-2', have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219- 2222). Preparation of oligodeoxyribonucleotide duplexes comprising bicyclic nucleosides for use as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226 ). Furthermore, synthesis of 2'-amino-BNA, a novel conformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035- 10039). In addition, 2'-amino- and 2'-methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.
In certain embodiments, bicyclic nucleosides have the formula:
Figure imgf000042_0001
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium; each qi, c , qk and qi is, independently, H, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2- C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C1-C12 alkoxyl, substituted C1-C12 alkoxyl, OJj, SJj, SOJj, S02Jj, NJjJk, N3, CN, C(=0)OJj, C(=0)NJjJk, C(=0)Jj, O- C(=0)NJj Jk, N(H)C(=NH)NJj Jk, N(H)C(=0)NJj Jk or N(H)C(=S)NJj Jk; and
qi and qj or qi and qk together are =C(qg)(qh), wherein qg and qh are each, independently, H, halogen, C1-C12 alkyl or substituted C1-C12 alkyl.
One carbocyclic bicyclic nucleoside having a 4'-(CH2)3-2' bridge and the alkenyl analog bridge 4'-ΟΗ=ΟΗ-ΟΗ2-2' have been described (Frier et ah, Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et ah, J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et ah, J. Am. Chem. Soc. 2007, 129(26), 8362-8379).
In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) a-L- methyleneoxy (4'-CH2-0-2') BNA , (B) β-D-methyleneoxy (4'-CH2-0-2') BNA , (C) ethyleneoxy (4'-(CH2)2-0-2') BNA , (D) aminooxy (4'-CH2-0-N(R)-2') BNA, (E) oxyamino (4'-CH2-N(R)-0- 2') BNA, (F) methyl(methyleneoxy) (4'-CH(CH3)-0-2') BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4'-CH2-S-2') BNA, (H) methylene-amino (4'-CH2-N(R)-2') BNA, (I) methyl carbocyclic (4'-CH2-CH(CH3)-2') BNA, (J) propylene carbocyclic (4'-(CH2)3-2') BNA, and (K) vinyl BNA as depicted below.
Figure imgf000043_0001
wherein Bx is the base moiety and R is, independently, H, a protecting group, Ci-C6 alkyl or Ci-C6 alkoxy.
In certain embodiments, modified nucleosides include nucleosides having sugar surrogate groups that include without limitation, replacement of the ribosyl ring with a sugar surrogate such as a tetrahydropyranyl ring system (also referred to as hexitol) as illustrated below:
Figure imgf000044_0001
In cert surrogates are selected having the formula:
Figure imgf000044_0002
wherein:
Bx is a heterocyclic base moiety;
one of T3 and T4 is an internucleoside linking group attaching the tetrahydropyran nucleoside analog to the remainder of one of the 5' or 3' end of the oligomeric compound and the other of T3 and T4 is hydroxyl, a protected hydroxyl, a 5' or 3' terminal group or an internucleoside linking group attaching the tetrahydropyran nucleoside analog to the remainder of the other of the 5' or 3' end of the oligomeric compound;
qi, ¾, q3, q4, q5, q6 and q7 are each independently, H, Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; and
one of Ri and R2 is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ1J2, SJi, N3, OC(=X)Ji, OC(=X)NJiJ2, NJ3C(=X)NJiJ2 and CN, wherein X is O, S or Ji and each Ji, J2 and J3 is, independently, H or Ci-C6 alkyl.
In certain embodiments, qi, q2, q3, q4, q5, q6 and q7 are each H. In certain embodiments, at least one of qi, q2, q3, q4, q5, q6 and q7 is other than H. In certain embodiments, at least one of qi, q2, q3, q4, qs, q6 and q7 is methyl. In certain embodiments, THP nucleosides are provided wherein one of Ri and R2 is F. In certain embodiments, Ri is fluoro and R2 is H; Ri is methoxy and R2 is H, and Ri is methoxyethoxy and R2 is H.
Such sugar surrogates can be referred to as a "modified tetrahydropyran nucleoside" or "modified THP nucleoside". Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), altritol nucleic acid (ANA), and mannitol nucleic acid (MNA) (see Leumann, C. J., Bioorg. & Med. Chem., 2002, 10, 841-854).
In certain embodiments, oligomeric compounds comprise one or more modified
cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides. Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see for example commonly owned, published PCT Application WO 2010/036696, published on April 10, 2010, Robeyns et al, J. Am. Chem. Soc, 2008, 130(6), 1979-1984; Horvath et al, Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al, J. Am. Chem. Soc, 2007, 129(30), 9340-9348; Gu et al.„ Nucleosides,
Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al, Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al, Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al, Tetrahedron, 2004, 60(9), 2111- 2123; Gu et al, Oligonucleotides, 2003, 13(6), 479-489; Wang et al, J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al, Nucleic Acids Research, 2001 , 29(24), 4941-4947; Wang et al, J. Org. Chem., 2001 , 66, 8478-82; Wang et al, Nucleosides, Nucleotides & Nucleic Acids , 2001, 20(4-7), 785-788; Wang et al, J. Am. Chem., 2000, 122, 8595-8602; Published PCT application, WO 06/047842; and Published PCT Application WO 01/049687; the text of each is incorporated by referen irety). Certain modified cyclohexenyl nucleosides have Formula X.
Figure imgf000045_0001
X
wherein independently for each of said at least one cyclohexenyl nucleoside analog of
Formula X:
Bx is a heterocyclic base moiety;
one of T3 and T4 is an internucleoside linking group attaching the cyclohexenyl nucleoside to the remainder of one of the 5' or 3' end of the oligomeric compound and the other of T3 and T4 is hydroxyl, a protected hydroxyl, a 5' or 3' terminal group or an internucleoside linking group attaching the cyclohexenyl nucleoside to the remainder of the other of the 5' or 3' end of the oligomeric compound; and qi, q2, q3, q4, q5, q6, q7, qs and q9 are each, independently, H, Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or other sugar substituent group.
Many other monocyclic, bicyclic and tricyclic ring systems are known in the art and are suitable as sugar surrogates that can be used to modify nucleosides for incorporation into oligomeric compounds as provided herein (see for example review article: Leumann, Christian J. Bioorg. & Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to further enhance their activity.
Some representative U.S. patents that teach the preparation of such modified sugars include without limitation, U.S.: 4,981 ,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;
5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591 ,722; 5,597,909; 5,610,300;
5,627,053; 5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and International Application PCT/US2005/019219, filed June 2, 2005 and published as WO 2005/121371 on
December 22, 2005 certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
Additional modified nucleosides can be prepared by any of the applicable techniques of organic synthesis, as, for example, illustrated in the examples below. Many such techniques are well known in the art. However, many of the known techniques are elaborated in Compendium of Organic Synthetic Methods, John Wiley & Sons, New York: Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G. Wade Jr., 1980; Vol. 5, Leroy G. Wade Jr., 1984; and Vol. 6, Michael B. Smith; as well as March, J., Advanced Organic Chemistry, 3rd Edition, John Wiley & Sons, New York, 1985; Comprehensive Organic Synthesis. Selectivity, Strategy & Efficiency in Modern Organic Chemistry, in 9 Volumes, Barry M. Trost, Editor-in-Chief, Pergamon Press, New York, 1993; Advanced Organic Chemistry, Part B: Reactions and Synthesis, 4th Edition; Carey and Sundberg, Kluwer Academic/Plenum Publishers, New York, 2001; Advanced Organic Chemistry, Reactions, Mechanisms, and Structure, 2nd Edition, March, McGraw Hill, 1977; Greene, T.W., and Wutz, P.G.M., Protecting Groups in Organic Synthesis, 4th Edition, John Wiley & Sons, New York, 1991 ; and Larock, R.C., Comprehensive Organic Transformations, 2nd Edition, John Wiley & Sons, New York, 1999.
As used herein the term "reactive phosphorus" is meant to include groups that are covalently linked to a monomer subunit that can be further attached to an oligomeric compound that are useful for forming internucleoside linkages including for example phosphodiester and phosphorothioate internucleoside linkages. Such reactive phosphorus groups are known in the art and contain phosphorus atoms in Pm or Pv valence state including, but not limited to, phosphoramidite, H- phosphonate, phosphate triesters and phosphorus containing chiral auxiliaries. In certain embodiments, reactive phosphorus groups are selected from diisopropylcyanoethoxy
phosphoramidite (-0*-P[N[(CH(CH3)2]2]0(CH2)2CN) and H-phosphonate (-0*-P(=0)(H)OH), wherein the O* is normally attached to the 3'-position of the Markush group of Formula I. A preferred synthetic solid phase synthesis utilizes phosphoramidites (Pm chemistry) as reactive phosphites. The intermediate phosphite compounds are subsequently oxidized to the phosphate or thiophosphate (Pv chemistry) using known methods to yield, phosphodiester or phosphorothioate internucleoside linkages. Chiral auxiliaries are known in the art (see for example: Wang et al,
Tetrahedron Letters, 1997, 38(5), 705-708; Jin et al, J. Org. Chem, 1997, 63, 3647-3654; Wang et al, Tetrahedron Letters, 1997, 38(22), 3797-3800; and U.S. patent 6,867,294, issued March 15, 2005). Additional reactive phosphates and phosphites are disclosed in Tetrahedron Report Number 309 (Beaucage and Iyer, Tetrahedron, 1992, 48, 2223-231 1).
As used herein, "oligonucleotide" refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).
The term "oligonucleoside" refers to a sequence of nucleosides that are joined by
internucleoside linkages that do not have phosphorus atoms. Internucleoside linkages of this type include short chain alkyl, cycloalkyl, mixed heteroatom alkyl, mixed heteroatom cycloalkyl, one or more short chain heteroatomic and one or more short chain heterocyclic. These internucleoside linkages include without limitation, siloxane, sulfide, sulfoxide, sulfone, acetyl, formacetyl, thioformacetyl, methylene formacetyl, thioformacetyl, alkeneyl, sulfamate, methyleneimino, methylenehydrazino, sulfonate, sulfonamide, amide and others having mixed N, O, S and CH2 component parts.
As used herein the term "internucleoside linkage" or "internucleoside linking group" is meant to include all manner of internucleoside linking groups known in the art including but not limited to, phosphorus containing internucleoside linking groups such as phosphodiester and phosphorothioate, and non-phosphorus containing internucleoside linking groups such as formacetyl and methyleneimino. Internucleoside linkages also includes neutral non-ionic internucleoside linkages such as amide-3 (3*-CH2-C(=0)-N(H)-5*), amide-4 (3*-CH2-N(H)-C(=0)-5*) and methylphosphonate wherein a phosphorus atom is not always present. In certain embodiments, oligomeric compounds as provided herein can be prepared having one or more internucleoside linkages containing modified e.g. non-naturally occurring
internucleoside linkages. The two main classes of internucleoside linkages are defined by the presence or absence of a phosphorus atom. Modified internucleoside linkages having a phosphorus atom include without limitation, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phos- phoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Oligonucleotides having inverted polarity can comprise a single 3' to 3' linkage at the 3 '-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.
Representative U.S. patents that teach the preparation of the above phosphorus containing linkages include without limitation, U.S.: 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;
5,188,897; 5,194,599; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676;
5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,527,899; 5,536,821 ;
5,541,306; 5,550,111 ; 5,563,253; 5,565,555; 5,571 ,799; 5,587,361; 5,625,050; 5,672,697 and
5,721 ,218, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.
In certain embodiments, oligomeric compounds as provided herein can be prepared having one or more non-phosphorus containing internucleoside linkages. Such oligomeric compounds include without limitation, those that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. Representative U.S. patents that teach the preparation of the above oligo nucleosides include without limitation, U.S.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;
5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541 ,307;
5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;
5,633,360; 5,677,437; 5,677,439; 5,646,269 and 5,792,608, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.
As used herein "neutral internucleoside linkage" is intended to include internucleoside linkages that are non-ionic. Neutral internucleoside linkages include without limitation, phosphotriesters, methylphosphonates, MMI (3*-CH2-N(CH3)-0-5*), amide-3 (3*-CH2-C(=0)-N(H)- 5*), amide-4 (3*-CH2-N(H)-C(=0)-5*), formacetal (3*-0-CH2-0-5*), and thioformacetal (3*-S-CH2-0- 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 Antis ens e 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.
In certain embodiments, oligomeric compounds as provided herein can be prepared having one or more optionally protected phosphorus containing internucleoside linkages. Representative protecting groups for phosphorus containing internucleoside linkages such as phosphodiester and phosphorothioate linkages include β-cyanoethyl, diphenylsilylethyl, δ-cyanobutenyl, cyano p-xylyl (CPX), N-methyl-N-trifluoroacetyl ethyl (META), acetoxy phenoxy ethyl (APE) and butene-4-yl groups. See for example U.S. Patents Nos. 4,725,677 and Re. 34,069 (β-cyanoethyl); Beaucage et ah, Tetrahedron, 1993, 49(10), 1925-1963; Beaucage et ah, Tetrahedron, 1993, 49(46), 10441- 10488; Beaucage et al, Tetrahedron, 1992, 48(12), 2223-231 1.
As used herein the terms "linking groups" and "bifunctional linking moieties" are meant to include groups known in the art that are useful for attachment of chemical functional groups, conjugate groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligomeric compound. In general, a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind to essentially any selected group such as a chemical functional group or a conjugate group. In some embodiments, the linker comprises a chain structure or a polymer of repeating units such as ethylene glycols or amino acid units. Examples of functional groups that are routinely used in bifunctional linking moieties include without limitation, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like. Some nonlimiting examples of bifunctional linking moieties include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-( -maleimido methyl) cyclohexane-l-carboxylate (SMCC) and 6- aminohexanoic acid (AHEX or AHA). Other linking groups include without limitation, substituted Ci-Cio alkyl, substituted or unsubstituted C2-Cio alkenyl or substituted or unsubstituted C2-Cio 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.
In certain embodiments, the oligomeric compounds as provided herein can be modified by covalent attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the oligomeric compounds they are attached to. Such oligonucleotide properties include without limitation, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional linking moiety or linking group to a parent compound such as an oligomeric compound. A preferred list of conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids,
phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.
In certain embodiments, the oligomeric compounds as provided herein can be modified by covalent attachment of one or more terminal groups to the 5' or 3'-terminal groups. A terminal group can also be attached at any other position at one of the terminal ends of the oligomeric compound. As used herein the terms "5'-terminal group", "3'-terminal group", "terminal group" and combinations thereof are meant to include useful groups known to the art skilled that can be placed on one or both of the terminal ends, including but not limited to the 5' and 3'-ends of an oligomeric compound respectively, for various purposes such as enabling the tracking of the oligomeric compound (a fluorescent label or other reporter group), improving the pharmacokinetics or pharmacodynamics of the oligomeric compound (such as for example: uptake and/or delivery) or enhancing one or more other desirable properties of the oligomeric compound (a group for improving nuclease stability or binding affinity). In certain embodiments, 5' and 3'-terminal groups include without limitation, modified or unmodified nucleosides; two or more linked nucleosides that are independently, modified or unmodified; conjugate groups; capping groups; phosphate moieties; and protecting groups. As used herein the term "phosphate moiety" refers to a terminal phosphate group that includes phosphates as well as modified phosphates. The phosphate moiety can be located at either terminus but is preferred at the 5'-terminal nucleoside. In one aspect, the terminal phosphate is unmodified having the formula -0-P(=0)(OH)OH. In another aspect, the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R) or alkyl where R is H, an amino protecting group or unsubstituted or substituted alkyl. In certain
embodiments, the 5' and or 3' terminal group can comprise from 1 to 3 phosphate moieties that are each, independently, unmodified (di or tri-phosphates) or modified.
As use e term "phosphorus moiety" refers to a group having the formula:
Figure imgf000051_0001
wherein:
Rx and Ry are each, independently, hydroxyl, protected hydroxyl group, thiol, protected thiol group, Ci-C6 alkyl, substituted Ci-C6 alkyl, Ci-C6 alkoxy, substituted Ci-C6 alkoxy, a protected amino or substituted amino; and
Rz is O or S.
As a monomer such as a phosphoramidite or H-phosphonate the protected phosphorus moiety is preferred to maintain stability during oligomer synthesis. After incorporation into an oligomeric compound the phosphorus moiety can include deprotected groups.
Phosphorus moieties included herein can be attached to a monomer, which can be used in the preparation of oligomeric compounds, wherein the monomer may be attached using O, S, NRa or
CReRf, wherein Ra includes without limitation H, Ci-C6 alkyl, substituted Ci-C6 alkyl, Ci-C6 alkoxy, substituted Ci-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or substituted acyl, and Re and Rf each, independently, include without limitation H, halogen, Ci-C6 alkyl, substituted Ci-C6 alkyl, Ci-C6 alkoxy or substituted Ci-C6 alkoxy. Such linked phosphorus moieties include without limitation, phosphates, modified phosphates, thiophosphates, modified thiophosphates, phosphonates, modified phosphonates, phosphoramidates and modified phosphoramidates.
RNA duplexes exist in what has been termed "A Form" geometry while DNA duplexes exist in "B Form" geometry. In general, RNA: RNA duplexes are more stable, or have higher melting temperatures (Tm) than DNA:DNA duplexes (Sanger et ah, Principles of Nucleic Acid Structure,
1984, Springer- Verlag; New York, NY.; Lesnik et ah, Biochemistry, 1995, 34, 10807-10815; Conte et ah, Nucleic Acids Res. , 1997, 25, 2627-2634). The increased stability of RNA has been attributed to several structural features, most notably the improved base stacking interactions that result from an A-form geometry (Searle et ah, Nucleic Acids Res. , 1993, 21, 2051 -2056). The presence of the 2' hydroxyl in RNA biases the sugar toward a C3' endo pucker, i.e., also designated as Northern pucker, which causes the duplex to favor the A-form geometry. In addition, the 2' hydroxyl groups of RNA can form a network of water mediated hydrogen bonds that help stabilize the RNA duplex (Egli et ah, Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleic acids prefer a C2' endo sugar pucker, i.e., also known as Southern pucker, which is thought to impart a less stable B-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure, Springer- Verlag, New York, NY).
The relative ability of a chemically-modified oligomeric compound to bind to complementary nucleic acid strands, as compared to natural oligonucleotides, is measured by obtaining the melting temperature of a hybridization complex of said chemically-modified oligomeric compound with its complementary unmodified target nucleic acid. The melting temperature (Tm), a character- istic physical property of double helixes, denotes the temperature in degrees centigrade at which 50% helical versus coiled (unhybridized) forms are present. Tm (also commonly referred to as binding affinity) is measured by using the UV spectrum to determine the formation and breakdown (melting) of hybridization. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently a reduction in UV absorption indicates a higher Tm.
It is known in the art that the relative duplex stability of an antisense compound:RNA target duplex can be modulated through incorporation of chemically-modified nucleosides into the antisense compound. Sugar-modified nucleosides have provided the most efficient means of modulating the Tm of an antisense compound with its target RNA. Sugar-modified nucleosides that increase the population of or lock the sugar in the y -endo (Northern, RNA-like sugar pucker) configuration have predominantly provided a per modification Tm increase for antisense compounds toward a complementary RNA target. Sugar-modified nucleosides that increase the population of or lock the sugar in the C -endo (Southern, DNA-like sugar pucker) configuration predominantly provide a per modification Tm decrease for antisense compounds toward a complementary RNA target. The sugar pucker of a given sugar-modified nucleoside is not the only factor that dictates the ability of the nucleoside to increase or decrease an antisense compound's Tm toward complementary
RNA. For example, the sugar-modified nucleoside tricycloDNA is predominantly in the CT-endo conformation, however it imparts a 1.9 to 3° C per modification increase in Tm toward a complementary R A. Another example of a sugar-modified high-affinity nucleoside that does not adopt the C '-endo conformation is a-L-LNA (described in more detail herein).
As used herein, "Tm" means melting temperature which is the temperature at which the two strands of a duplex nucleic acid separate. Tm is often used as a measure of duplex stability or the binding affinity of an antisense compound toward a complementary strand such as an RNA molecule.
As used herein, "complementarity" in reference to nucleobases refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is
complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair. Nucleobases or more broadly, heterocyclic base moieties, comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of complementarity.
As used herein, "non-complementary" in reference to nucleobases refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.
As used herein, "complementary" in reference to linked nucleosides, oligonucleotides, oligomeric compounds, or nucleic acids, refers to the capacity of an oligomeric compound to hybridize to another oligomeric compound or nucleic acid through nucleobase or more broadly, heterocyclic base, complementarity. In certain embodiments, an antisense compound and its target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases that can bond with each other to allow stable association between the antisense compound and the target. One skilled in the art recognizes that the inclusion of mismatches is possible without eliminating the ability of the oligomeric compounds to remain in association. Therefore, described herein are antisense compounds that may comprise up to about 20% nucleotides that are mismatched (i.e., are not nucleobase complementary to the corresponding nucleotides of the target). Preferably the antisense compounds contain no more than about 15%, more preferably not more than about 10%, most preferably not more than 5% or no mismatches.
The remaining nucleotides are nucleobase complementary or otherwise do not disrupt hybridization
(e.g., universal bases). One of ordinary skill in the art would recognize the compounds provided herein are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to a target nucleic acid.
It is understood in the art that the sequence of an oligomeric compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligomeric compound may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). In certain embodiments, oligomeric compounds can comprise at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted. For example, an oligomeric compound in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an oligomeric compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within this scope. Percent complementarity of an oligomeric compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et ah, J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649- 656).
As used herein, "hybridization" refers to the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between
complementary nucleoside or nucleotide bases (nucleobases). For example, the natural base adenine is nucleobase complementary to the natural nucleobases thymidine and uracil which pair through the formation of hydrogen bonds. The natural base guanine is nucleobase complementary to the natural bases cytosine and 5 -methyl cytosine. Hybridization can occur under varying circumstances.
As used herein, "target nucleic acid" refers to any nucleic acid molecule the expression, amount, or activity of which is capable of being modulated by an antisense compound. In certain embodiments, the target nucleic acid is DNA or RNA. In certain embodiments, the target R A is mRNA, pre-mRNA, non-coding RNA, pri-microRNA, pre-microRNA, mature microRNA, promoter-directed RNA, or natural antisense transcripts. For example, the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In certain embodiments, target nucleic acid is a viral or bacterial nucleic acid.
Further included herein are oligomeric compounds such as antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid. Once introduced to a system, the oligomeric compounds provided herein may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid. Alternatively, the oligomeric compound may inhibit the activity the target nucleic acid through an occupancy-based method, thus interfering with the activity of the target nucleic acid.
One non-limiting example of such an enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded oligomeric compounds which are "DNA-like" elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide- mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.
As used herein, "modulation" refers to a perturbation of amount or quality of a function or activity when compared to the function or activity prior to modulation. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression. As a further example, modulation of expression can include perturbing splice site selection of pre-mRNA processing, resulting in a change in the amount of a particular splice-variant present compared to conditions that were not perturbed. As a further example, modulation includes perturbing translation of a protein.
As used herein, the term "pharmaceutically acceptable salts" refers to salts that retain the desired activity of the compound and do not impart undesired toxicological effects thereto. The term "pharmaceutically acceptable salt" includes a salt prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic or organic acids and bases.
Pharmaceutically acceptable salts of the oligomeric compounds and or compositions described herein may be prepared by methods well-known in the art. For a review of
pharmaceutically acceptable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts:
Properties, Selection and Use (Wiley- VCH, Weinheim, Germany, 2002). Sodium salts of antisense oligonucleotides are useful and are well accepted for therapeutic administration to humans.
Accordingly, in certain embodiments the oligomeric compounds described herein are in the form of a sodium salt.
In certain embodiments, oligomeric compounds provided herein comprise from about 8 to about 80 monomer subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, or 80 monomer subunits in length, or any range therewithin.
In certain embodiments, oligomeric compounds provided herein comprise from about 8 to 40 monomer subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 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 or 40 monomer subunits in length, or any range therewithin.
In certain embodiments, oligomeric compounds provided herein comprise from about 8 to 20 monomer subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 monomer subunits in length, or any range therewithin.
In certain embodiments, oligomeric compounds provided herein comprise from about 8 to 16 monomer subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 8, 9, 10, 11, 12, 13, 14, 15 or 16 monomer subunits in length, or any range therewithin.
In certain embodiments, oligomeric compounds provided herein comprise from about 10 to 14 monomer subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 10, 11 , 12, 13 or 14 monomer subunits in length, or any range therewithin.
In certain embodiments, oligomeric compounds provided herein comprise from about 10 to 18 monomer subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 10, 11 , 12, 13, 14, 15, 16, 17 or 18 monomer subunits in length, or any range therewithin.
In certain embodiments, oligomeric compounds provided herein comprise from about 10 to 21 monomer subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 monomer subunits in length, or any range therewithin.
In certain embodiments, oligomeric compounds provided herein comprise from about 12 to 14 monomer subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 12, 13 or 14 monomer subunits in length, or any range therewithin.
In certain embodiments, oligomeric compounds provided herein comprise from about 12 to 18 monomer subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 12, 13, 14, 15, 16, 17 or 18 monomer subunits in length, or any range therewithin.
In certain embodiments, oligomeric compounds provided herein comprise from about 12 to 21 monomer subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 monomer subunits in length, or any range therewithin.
In certain embodiments, oligomeric compounds provided herein comprise from about 14 to
18 monomer subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 14, 15, 16, 17 or 18 monomer subunits in length, or any range therewithin.
In certain embodiments, oligomeric compounds of any of a variety of ranges of lengths of linked monomer subunits are provided. In certain embodiments, oligomeric compounds are provided consisting of X-Y linked monomer subunits, where X and Y are each independently selected from 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X < Y. For example, in certain embodiments, this provides oligomeric compounds comprising: 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-21, 8-22, 8-23, 8-24, 8-25, 8-26, 8-27,
8- 28, 8-29, 8-30, 9-10, 9-1 1, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23,
9- 24, 9-25, 9-26, 9-27, 9-28, 9-29, 9-30, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10- 19, 10-20, 10-21, 10-22, 10-23, 10-24, 10-25, 10-26, 10-27, 10-28, 10-29, 10-30, 1 1-12, 11-13, 11- 14, 1 1-15, 11-16, 11-17, 11-18, 11-19, 11-20, 1 1-21, 11-22, 11-23, 1 1-24, 11-25, 1 1-26, 11-27, 11- 28, 1 1-29, 11-30, 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 12-21, 12-22, 12-23, 12-
24, 12-25, 12-26, 12-27, 12-28, 12-29, 12-30, 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 13-
21, 13-22, 13-23, 13-24, 13-25, 13-26, 13-27, 13-28, 13-29, 13-30, 14-15, 14-16, 14-17, 14-18, 14-
19, 14-20, 14-21, 14-22, 14-23, 14-24, 14-25, 14-26, 14-27, 14-28, 14-29, 14-30, 15-16, 15-17, 15- 18, 15-19, 15-20, 15-21, 15-22, 15-23, 15-24, 15-25, 15-26, 15-27, 15-28, 15-29, 15-30, 16-17, 16-
18, 16-19, 16-20, 16-21, 16-22, 16-23, 16-24, 16-25, 16-26, 16-27, 16-28, 16-29, 16-30, 17-18, 17-
19, 17-20, 17-21, 17-22, 17-23, 17-24, 17-25, 17-26, 17-27, 17-28, 17-29, 17-30, 18-19, 18-20, 18- 21, 18-22, 18-23, 18-24, 18-25, 18-26, 18-27, 18-28, 18-29, 18-30, 19-20, 19-21, 19-22, 19-23, 19- 24, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 20-21, 20-22, 20-23, 20-24, 20-25, 20-26, 20-27, 20- 28, 20-29, 20-30, 21-22, 21-23, 21-24, 21-25, 21-26, 21-27, 21-28, 21-29, 21-30, 22-23, 22-24, 22-
25, 22-26, 22-27, 22-28, 22-29, 22-30, 23-24, 23-25, 23-26, 23-27, 23-28, 23-29, 23-30, 24-25, 24-
26, 24-27, 24-28, 24-29, 24-30, 25-26, 25-27, 25-28, 25-29, 25-30, 26-27, 26-28, 26-29, 26-30, 27- 28, 27-29, 27-30, 28-29, 28-30, or 29-30 linked monomer subunits.
In certain embodiments, the ranges for the oligomeric compounds listed herein are meant to limit the number of monomer subunits in the oligomeric compounds, however such oligomeric compounds may further include 5' and/or 3'-terminal groups including but not limited to protecting groups such as hydroxyl protecting groups, optionally linked conjugate groups and/or other substituent groups.
In certain embodiments, the preparation of oligomeric compounds as disclosed herein is performed according to literature procedures for DNA: Protocols for Oligonucleotides and Analogs, Agrawal, Ed., Humana Press, 1993, and/or RNA: Scaringe, Methods, 2001, 23, 206-217; Gait et al., Applications of Chemically synthesized RNA in RNA:Protein Interactions, Smith, Ed., 1998, 1-36; Gallo et ah, Tetrahedron , 2001, 57, 5707-5713. Additional methods for solid-phase synthesis may be found in Caruthers U.S. Patents Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Patents Nos. 4,725,677 and Re. 34,069.
Oligomeric compounds are routinely prepared using solid support methods as opposed to solution phase methods. Commercially available equipment commonly used for the preparation of oligomeric compounds that utilize the solid support method is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. Suitable solid phase techniques, including automated synthesis techniques, are described in Oligonucleotides and Analogues, a Practical Approach, F. Eckstein, Ed., Oxford University Press, New York, 1991.
The synthesis of RNA and related analogs relative to the synthesis of DNA and related analogs has been increasing as efforts in RNA interference and micro RNA increase. The primary
RNA synthesis strategies that are presently being used commercially include 5'-0-DMT-2'-0-t- butyldimethylsilyl (TBDMS), 5'-0-DMT-2'-0-[ 1 (2-fiuorophenyl)-4-methoxypiperidin-4-yl]
(FPMP), 2'-0-[(triisopropylsilyl)oxy]methyl (2'-0-CH2-0-Si(iPr)3 (TOM) and the 5'-0-silyl ether- 2'-ACE (5'-0-bis(trimethylsiloxy)cyclododecyloxysilyl ether (DOD)-2'-0-bis(2- acetoxyethoxy)methyl (ACE). A current list of some of the major companies currently offering R A products include Pierce Nucleic Acid Technologies, Dharmacon Research Inc., Ameri Biotechnologies Inc., and Integrated DNA Technologies, Inc. One company, Princeton Separations, is marketing an RNA synthesis activator advertised to reduce coupling times especially with TOM and TBDMS chemistries. The primary groups being used for commercial RNA synthesis are:
TBDMS: 5'-0-DMT-2'-0-t-butyldimethylsilyl; TOM: 2'-0-[(triisopropylsilyl)oxy]methyl;
DOD/ACE: (5'-0-bis(trimethylsiloxy)cyclododecyloxysilyl ether-2'-0-bis(2-acetoxyethoxy)methyl; and FPMP: 5'-0-DMT-2'-0-[l(2-fiuorophenyl)-4-ethoxypiperidin-4-yl]. In certain embodiments, each of the aforementioned RNA synthesis strategies can be used herein. In certain embodiments, the aforementioned RNA synthesis strategies can be performed together in a hybrid fashion e.g. using a 5'-protecting group from one strategy with a 2'-0-protecting from another strategy.
In some embodiments, "suitable target segments" may be employed in a screen for additional oligomeric compounds that modulate the expression of a selected protein. "Modulators" are those oligomeric compounds that decrease or increase the expression of a nucleic acid molecule encoding a protein and which comprise at least an 8-nucleobase portion which is complementary to a suitable target segment. The screening method comprises the steps of contacting a suitable target segment of a nucleic acid molecule encoding a protein with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding a protein. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding a peptide, the modulator may then be employed herein in further investigative studies of the function of the peptide, or for use as a research, diagnostic, or therapeutic agent. In the case of oligomeric compounds targeted to microRNA, candidate modulators may be evaluated by the extent to which they increase the expression of a microRNA target RNA or protein (as interference with the activity of a microRNA will result in the increased expression of one or more targets of the microRNA).
As used herein, "expression" refers to the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, splicing, post-transcriptional modification, and translation.
The oligomeric compounds provided herein can also be applied in the areas of drug discovery and target validation. In certain embodiments, provided herein is the use of the oligomeric compounds and targets identified herein in drug discovery efforts to elucidate relationships that exist between proteins and a disease state, phenotype, or condition. These methods include detecting or modulating a target peptide comprising contacting a sample, tissue, cell, or organism with one or more oligomeric compounds provided herein, measuring the nucleic acid or protein level of the target and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further oligomeric compound as provided herein. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype. In certain embodiments, oligomeric compounds are provided for use in therapy. In certain
embodiments, the therapy is reducing target messenger RNA.
As used herein, the term "dose" refers to a specified quantity of a pharmaceutical agent provided in a single administration. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual.
In pharmacology and toxicology, a route of administration is the path by which a drug, fluid, poison, or other substance is brought into contact with the body. In general, methods of
administering drugs and nucleic acids such as antisense compounds for treatment of a mammal are well known in the art and can be applied to administration of the compositions of the invention. The compounds of the present invention can be administered via any suitable route, most preferably parenterally, in a preparation appropriately tailored to that route. Thus, the compounds of the present invention can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, subcutaneously, or intraperitoneally. Accordingly, the present invention also provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier or excipient.
Parenteral routes of administration include intravascular (intravenous, intraarterial), intramuscular, intraparenchymal, intradermal, subdermal, subcutaneous, intratumor, intraperitoneal, intrathecal, subdural, epidural, and intralymphatic injections that use a syringe and a needle or catheter. Intravascular herein means within a tubular structure called a vessel that is connected to a tissue or organ within the body. Within the cavity of the tubular structure, a bodily fluid flows to or from the body part. Examples of bodily fluid include blood, cerebrospinal fluid (CSF), lymphatic fluid, or bile. Examples of vessels include arteries, arterioles, capillaries, venules, sinusoids, veins, lymphatics, bile ducts, and ducts of the salivary or other exocrine glands. The intravascular route includes delivery through the blood vessels such as an artery or a vein. The blood circulatory system provides systemic spread of the pharmaceutical.
The described compositions are injected in pharmaceutically acceptable carrier solutions. Pharmaceutically acceptable refers to those properties and/or substances which are acceptable to the mammal from a pharmacological toxicological point of view. The phrase pharmaceutically acceptable refers to molecular entities, compositions, and properties that are physiologically tolerable and do not typically produce an allergic or other untoward or toxic reaction when administered to a mammal. Preferably, as used herein, the term pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S.
Pharmacopeia or other generally recognized pharmacopeia for use in animals and more particularly in humans.
The compositions as provided herein comprising an antisense compound is co-administered with the delivery peptide. By co-administered it is meant that the antisense compound and the delivery peptide are administered to the mammal such that both are present in the mammal at the same time. The antisense compound and the delivery peptide may be administered simultaneously or they may be delivered sequentially. For simultaneous administration, they may be mixed prior to administration. For sequential administration, either the antisense compound or the delivery peptide may be administered first.
Antisense compounds may be administered up to 30 minutes prior to administration of the delivery peptide. Also the delivery peptide may be administered up to two hours prior to administration of the antisense compound.
Antisense compounds may be administered up to 15 minutes prior to administration of the delivery peptide. The delivery peptide may be administered up to 15 minutes prior to administration of the antisense compound.
Antisense compounds may be delivered for research purposes or to produce a change in a cell that is therapeutic. In vivo delivery of antisense compounds is useful for research reagents and for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmaco genomic applications. The compositions disclosed herein are expected to be useful for the delivery of antisense compounds resulting in inhibition of endogenous gene expression in hepatocytes. Levels of a reporter (marker) gene expression measured following delivery of an antisense compound indicate a reasonable expectation of similar levels of gene expression following delivery of other antisense compounds. Levels of treatment considered beneficial by a person having ordinary skill in the art differ from disease to disease. For example, Hemophilia A and B are caused by deficiencies of the X-linked clotting factors VIII and IX, respectively. Their clinical course is greatly influenced by the percentage of normal serum levels of factor VIII or ΓΧ: <2%, severe; 2-5%, moderate; and 5-30% mild. Thus, an increase from 1% to 2% of the normal level of circulating factor in severe patients can be considered beneficial. Levels greater than 6% prevent spontaneous bleeds but not those secondary to surgery or injury. Similarly, inhibition of a gene need not be 100% to provide a therapeutic benefit. A person having ordinary skill in the art of gene therapy would reasonably anticipate beneficial levels of expression of a gene specific for a disease based upon sufficient levels of marker gene results. In the hemophilia example, if marker genes were expressed to yield a protein at a level comparable in volume to 2% of the normal level of factor VIII, it can be reasonably expected that the gene coding for factor VIII would also be expressed at similar levels. Thus, reporter or marker genes serve as useful paradigms for expression of intracellular proteins in general.
The liver is one of the most important target tissues for gene therapy given its central role in metabolism (e.g., lipoprotein metabolism in various hypercholesterolemias) and the secretion of circulating proteins (e.g., clotting factors in hemophilia). In addition, acquired disorders such as chronic hepatitis (e.g. hepatitis B virus infection) and cirrhosis are common and are also potentially treated by polynucleotide -based liver therapies. A number of diseases or conditions which affect or are affected by the liver are potentially treated through knockdown (inhibition) of gene expression in the liver. Such liver diseases and conditions may be selected from the list comprising: liver cancers (including hepatocellular carcinoma, HCC), viral infections (including hepatitis), metabolic disorders, (including hyperlipidemia and diabetes), fibrosis, and acute liver injury.
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 in their entirety for any purpose. While in certain embodiments, oligomeric compounds provided herein can be utilized as described, the following examples serve only to illustrate and are not intended to be limiting.
Examples (General)
In general, !H and 13C NMR spectra are recorded on a 300 MHz and 75 MHz Bruker spectrometer, respectively. Example 1
Synthesis of Nucleoside Phosphoramidites
The preparation of nucleoside phosphoramidites is performed following procedures that are illustrated herein and in the art such as but not limited to US Patent 6,426,220 and published PCT WO 02/36743.
Example 2
Synthesis of Oligomeric Compounds
The oligomeric compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as alkylated derivatives and those having phosphorothioate linkages.
Oligomeric compounds: Unsubstituted and substituted phosphodiester (P=0) oligomeric compounds, including without limitation, oligonucleotides can be synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.
In certain embodiments, phosphorothioate internucleoside linkages (P=S) are synthesized similar to phosphodiester internucleoside linkages with the following exceptions: thiation is effected by utilizing a 10% w/v solution of 3,H-l ,2-benzodithiole-3-one 1,1 -dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time is increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55°C (12-16 hr), the oligomeric compounds are recovered by precipitating with greater than 3 volumes of ethanol from a 1 M H4OAC solution. Phosphinate internucleoside linkages can be prepared as described in U.S. Patent 5,508,270.
Alkyl phosphonate internucleoside linkages can be prepared as described in U.S. Patent 4,469,863.
3 '-Deoxy-3 '-methylene phosphonate internucleoside linkages can be prepared as described in U.S. Patents 5,610,289 or 5,625,050.
Phosphoramidite internucleoside linkages can be prepared as described in U.S. Patent, 5,256,775 or U.S. Patent 5,366,878. Alkylphosphonothioate internucleoside linkages can be prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively).
3'-Deoxy-3'-amino phosphoramidate internucleoside linkages can be prepared as described in U.S. Patent 5,476,925.
Phosphotriester internucleoside linkages can be prepared as described in U.S. Patent 5,023,243.
Borano phosphate internucleoside linkages can be prepared as described in U.S. Patents 5,130,302 and 5,177,198.
Oligomeric compounds having one or more non-phosphorus containing internucleoside linkages including without limitation methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone oligomeric compounds having, for instance, alternating MMI and P=0 or P=S linkages can be prepared as described in U.S. Patents 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289.
Formacetal and thioformacetal internucleoside linkages can be prepared as described in U.S. Patents 5,264,562 and 5,264,564.
Ethylene oxide internucleoside linkages can be prepared as described in U.S. Patent
5,223,618.
Example 3
Isolation and Purification of Oligomeric Compounds
After cleavage from the controlled pore glass solid support or other support medium and deblocking in concentrated ammonium hydroxide at 55°C for 12-16 hours, the oligomeric compounds, including without limitation oligonucleotides and oligonucleosides, are recovered by precipitation out of 1 M H4OAC with >3 volumes of ethanol. Synthesized oligomeric compounds are analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis is determined by the ratio of correct molecular weight relative to the - 16 amu product (+/-32 +/-48). For some studies oligomeric compounds are purified by HPLC, as described by Chiang et al, J. Biol. Chem. 1991 , 266, 18162-18171. Results obtained with HPLC-purified material are generally similar to those obtained with non-HP LC purified material.
Example 4
Synthesis of Oligomeric Compounds using the 96 Well Plate Format
Oligomeric compounds, including without limitation oligonucleotides, can be synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleoside linkages are afforded by oxidation with aqueous iodine. Phosphorothioate internucleoside linkages are generated by sulfurization utilizing 3,H-1 ,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites can be purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, CA, or Pharmacia, Piscataway, NJ). Non-standard nucleosides are synthesized as per standard or patented methods and can be functionalized as base protected beta-cyanoethyldiisopropyl phosphoramidites.
Oligomeric compounds can be cleaved from support and deprotected with concentrated
NH4OH at elevated temperature (55-60 °C) for 12-16 hours and the released product then dried in vacuo. The dried product is then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors. Example 5
Analysis of Oligomeric Compounds using the 96-Well Plate Format
The concentration of oligomeric compounds in each well can be assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products can be evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition is confirmed by mass analysis of the oligomeric compounds utilizing electrospray-mass spectroscopy. All assay test plates are diluted from the master plate using single and multi-channel robotic pipettors. Plates are judged to be acceptable if at least 85% of the oligomeric compounds on the plate are at least 85% full length. Example 6
Real-time Quantitative PCR Analysis of target mRNA Levels
Quantitation of target mRNA levels is accomplished by real-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, CA, Operon Technologies Inc., Alameda, CA or Integrated DNA Technologies Inc., Coralville, IA) is attached to the 5'-end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE- Applied Biosystems, Foster City, CA, Operon Technologies Inc., Alameda, CA or Integrated DNA Technologies Inc., Coralville, IA) is attached to the 3' end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3' quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5'-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.
Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be "multiplexed" with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only ("single-plexing"), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mR A signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.
RT and PCR reagents are obtained from Invitrogen Life Technologies (Carlsbad, CA). RT, real-time PCR is carried out by adding 20 PCR cocktail (2.5x PCR buffer minus MgCl2, 6.6 mM MgCl2, 375 μΜ each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units R Ase inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5x ROX dye) to 96-well plates containing 30 total RNA solution (20- 200 ng). The RT reaction is carried out by incubation for 30 minutes at 48°C. Following a 10 minute incubation at 95 °C to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol are carried out: 95°C for 15 seconds (denaturation) followed by 60°C for 1.5 minutes (annealing/- extension).
Gene target quantities obtained by RT, real-time PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RIBOGREEN™ (Molecular Probes, Inc. Eugene, OR). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, OR). Methods of RNA quantification by RIBOGREEN™ are taught in Jones, L.J., et al, (Analytical Biochemistry, 1998, 265, 368-374).
In this assay, 170 μΐ, of RIBOGREEN™ working reagent (RIBOGREEN™ reagent diluted 1 :350 in lOmM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μΐ, purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485nm and emission at 530nm.
Example 7
Analysis of oligonucleotide inhibition of target expression
Antisense modulation of a target expression can be assayed in a variety of ways known in the art. For example, a target mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR. Real-time quantitative PCR is presently desired. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. One method of RNA analysis of the present disclosure is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System, available from PE- Applied Biosystems, Foster City, CA and used according to manufacturer's instructions.
Protein levels of a target can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 1 1.12.1-1 1.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F.M. et al, Current Protocols in Molecular Biology, Volume 2, pp.
11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.
Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F.M. et al, Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 1 1.2.1-1 1.2.22, John Wiley & Sons, Inc., 1991.
Example 8
Design of phenotypic assays and in vivo studies for the use of target inhibitors
Phenotypic assays
Once target inhibitors have been identified by the methods disclosed herein, the oligomeric compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition.
Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of a target in health and disease.
Representative phenotypic assays, which can be purchased from any of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, OR; PerkinElmer, Boston, MA), protein-based assays including enzymatic assays (Panvera, LLC, Madison, WI; BD Biosciences, Franklin Lakes, NJ; Oncogene Research Products, San Diego, CA), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, MI), triglyceride accumulation (Sigma-Aldrich, St. Louis, MO), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, CA; Amersham Biosciences, Piscataway, NJ).
In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with a target inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.
Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.
Measurement of the expression of one or more of the genes of the cell after treatment is also used as an indicator of the efficacy or potency of the a target inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.
In vivo studies
The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.
Example 9
RNA Isolation
Poly(A)+ mRNA isolation
Poly(A)+ mRNA is isolated according to Miura et al, (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96- well plates, growth medium is removed from the cells and each well is washed with 200 μL cold PBS. 60 \iL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) is added to each well, the plate is gently agitated and then incubated at room temperature for five minutes. 55 iL of lysate is transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine CA). Plates are incubated for 60 minutes at room temperature, washed 3 times with 200 μΐ. of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate is blotted on paper towels to remove excess wash buffer and then air- dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70°C, is added to each well, the plate is incubated on a 90°C hot plate for 5 minutes, and the eluate is then transferred to a fresh 96-well plate.
Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.
Total RNA Isolation
Total RNA is isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia, CA) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium is removed from the cells and each well is washed with 200 μL cold PBS. 150 Buffer RLT is added to each well and the plate vigorously agitated for 20 seconds.
150 of 70% ethanol is then added to each well and the contents mixed by pipetting three times up and down. The samples are then transferred to the RNEASY 96™ well plate attached to a
QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum is applied for 1 minute. 500 μΐ, of Buffer RW1 is added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum is again applied for 1 minute. An additional 500 μL of Buffer RW1 is added to each well of the RNEASY 96™ plate and the vacuum is applied for 2 minutes. 1 mL of Buffer RPE is then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash is then repeated and the vacuum is applied for an additional 3 minutes. The plate is then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate is then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA is then eluted by pipetting 140 iL of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.
The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia CA). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out. Example 10
Target-specific primers and probes
Probes and primers may be designed to hybridize to a target sequence, using published sequence information.
For example, for human PTEN, the following primer-probe set was designed using published sequence information (GENBANK™ accession number U92436.1, SEQ ID NO: 97).
Forward primer: AATGGCTAAGTGAAGATGACAATCAT (SEQ ID NO: 98)
Reverse primer: TGCACATATCATTACACCAGTTCGT (SEQ ID NO: 99
And the PCR probe:
FAM-TTGCAGCAATTCACTGTAAAGCTGGAAAGG-TAMRA (SEQ ID NO: 100), where FAM is the fluorescent dye and TAMRA is the quencher dye.
Example 11
Western blot analysis of target protein levels
Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 μΐ/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to a target is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™
(Molecular Dynamics, Sunnyvale CA).
Example 12
Melittin synthesis
Melittin peptides are made using peptide synthesis techniques standard in the art. Suitable melittin peptides can be all L-form amino acids, all D-form amino acids (inverso). Independently of L or D form, the melittin peptide sequence can be reversed (retro).
Example 13
Amino terminal Modification of Melittin Derivatives
Solutions of CKLK-Melittin (20 mg/mL), TCEP-HC1 (28.7 mg/mL, 100 mM), and MES-Na (21.7 mg/mL, 100 mM) are prepared in deionized H20. In a 20 mL scintillation vial, CKLK- Melittin (0.030 mmol, 5 mL) is reacted with 1.7 molar equivalents TCEP-HC1 (0.05 μηιοΐ, 0.51 mL) and left to stir at room temperature for 30 minutes. MES-Na (2 ml) and Water (1.88 mL) are then added in amounts to yield final concentrations of 10 mg/ml Melittin and 20 mM MES-Na. The pH is checked and adjusted to pH 6.5-7. A solution of NAG-PEG2-Br (100 mg/ml) is prepared in deionized H20, NAG-PEG2-Br (4.75 eq, 0.142 mmol, 0.61 mL) is added, and the solution is left to stir at room temperature for 48 h.
Alternatively, in a 20 mL scintillation vial, Cys-Melittin (0.006 mmol, 1 mL) is reacted with 1.7 molar equivalents TCEP-HC1 (0.010 mmol, 100 μί) and left to stir at room temperature for 30 minutes. MES-Na (400 μί) and water (390 μϋ) are added in amounts to yield final concentrations of 10 mg/mL Melittin and 20 mM MES-Na. The pH is checked and adjusted to pH 6.5-7. A solution of NAG-PEG8-Maleimide (100 mg/mL) is prepared in deionized H20. NAG-PEGs- Maleimide (2 eq, 0.012 mmol, 110 μί) is added, and the solution is left to stir at room temperature for 48 h.
Samples are purified on a Luna 10μ CI 8 100 A21, 2x250 mm column. Buffer A: H.sub.20 0.1% TFA and Buffer B: MeCN, 10% Isopropyl Alcohol, 0.1% TFA. Flow rate of 15 mL/min, 35% A to 62.5% B in 20 min.
Other amino terminal modifications are made using similar means. Carboxyl terminal modifications are made substituting melittin peptides having carboxyl terminal cysteines for melittins having amino terminal cysteines.
Compounds used to modify Cys-Melittin or Melittin-Cys:
Figure imgf000072_0001
n is an integer from 1 to 120 (PEG molecular weight up to about 5 kDa).
Figure imgf000072_0002
H3 NAG-PEG maleimide
Figure imgf000073_0001
ide
Peptides having acetyl, dimethyl, stearoyl, myristoyl, and PEG amino or carboxyl terminal modifications, but not terminal cysteine residues, are generated on resin during peptide synthesis using methods typical in the art.
Example 14
Synthesis of Masking Agents
pH labile masking agents: Steric stabilizer CDM-PEG and targeting group CDM-NAG (N- acetyl galactosamine) syntheses. To a solution of CDM (300 mg, 0.16 mmol) in 50 mL methylene chloride is added oxalyl chloride (2 g, 10 wt. eq.) and dimethylformamide (5 μΕ). The reaction is allowed to proceed overnight, after which the excess oxalyl chloride and methylene chloride are removed by rotary evaporation to yield the CDM acid chloride. The acid chloride is dissolved in 1 mL of methylene chloride. To this solution is added 1.1 molar equivalents polyethylene glycol monomethyl ether (MW average 550) for CDM-PEG or (aminoethoxy)ethoxy-2-(acetylamino)-2- deoxy-13-D-galactopyranoside (i.e. amino bisethoxyl-ethyl NAG) for CDM-NAG, and pyridine (200 μΕ, 1.5 eq) in 10 mL of methylene chloride. The solution is then stirred for 1.5 hours. The solvent is removed and the resulting solid is dissolved into 5 mL of water and is purified using reverse-phase gradient.
Figure imgf000073_0002
CDM Generic Disubtituted Maleic
Anhydride Masking Agent
Ri comprises a neutral ASGPrLig. Preferably the Masking Agent is uncharged.
Figure imgf000073_0003
CDM-PEG R is a methyl or ethyl, and n is an integer from 2 to 100. Preferably, the PEG contains from 5 to 20 ethylene units (n is an integer from 5 to 20). More preferably, PEG contains 10-14 ethylene units (n is an integer from 10 to 14). The PEG may be of variable length and have a mean length of 5-20 or 10-14 ethylene units. Alternatively, the PEG may be monodisperse, uniform or discrete; having, for example, exactly 1 1 or 13 ethylene units.
Figure imgf000074_0001
CDM-NAG
n is an integer from 1 to 10. As shown above, a PEG spacer may be positioned between the anhydride group and the ASGPrLig. In certain embodiments, the PEG spacer contains 1-10 ethylene units.
Alternatively an alkyl spacer may be used between the anhydride and the N- acetylgalactosamine.
Figure imgf000074_0002
CDM-NAG (alkyl spacer)
n is a integer from 0 to 6.
Other spacers or linkers may be used between the anhydride and the N- Acetylgalactosamine. In certain embodiments, the spacer or linker is hydrophilic and neutral (preferably uncharged).
Protease (Peptidase) Cleavable Masking Agents: Melittin peptide can also be reversibly modified using specialized enzyme cleavable linkers. These enzyme cleavable linkers employ a dipeptide connected to an amidobenzyl activated carbonate moiety. Reaction of the activated carbonate with a peptide amine connects a targeting compound, such as asialoglycoprotein receptor ligand, to the melittin peptide via a peptidase cleavable dipeptide-amidobenzyl carbamate linkage. Enzyme cleavage of the dipeptide removes the targeting ligand from the peptide and triggers an elimination reaction which results in regeneration of the peptide amine.
The following enzymatically cleavable linkers are synthesized:
Figure imgf000076_0001
-Gle-Gly-PABC-PNP
Figure imgf000076_0002
Dipeptides Glu-Gly, Ala-Cit, Phe-Cit are shown ("Cit" is the amino acid citrulline). Other amino acid combinations are permissible. In addition, 3-5 amino acids may be used as the linker between the amido benzyl group and the targeting ligand. Further, other activated carbonates known in the art are readily substituted for the para-nitrophenol used in the above compounds.
Example 15
Reversible Modification/Masking of Melittin
Modification with maleic anhydride-based masking agents: Prior to modification, 5x mg of disubstituted maleic anhydride masking agent (e.g. CDM-NAG) is lyophilized from a 0.1% aqueous solution of glacial acetic acid. To the dried disubstituted maleic anhydride masking agent is added a solution of x mg melittin in 0.2x mL of isotonic glucose and lOx mg of HEPES free base.
Following complete dissolution of anhydride, the solution is incubated for at least 30 minutes at RT prior to animal administration. Reaction of disubstituted maleic anhydride masking agent with the peptide provides the compound:
Figure imgf000077_0001
wherein R is melittin and Ri comprises a ASGPrLig (e.g. NAG). The anhydride carboxyl produced in the reaction between the anhydride and the polymer amine will exhibit about 1/20ώ of the expected charge (Rozema, et ah, Bioconjugate Chemistry, 2003, 14, 51-57). Therefore, the membrane active polymer is effectively neutralized rather than being converted to a highly negatively charged polyanion.
Modification with protease cleavable masking agents: lx mg of peptide and lOx mg HEPES base at 1-10 mg/mL peptide is masked by addition of 2-6x mg of amine-reactive p-nitrophenyl carbonate or N-hydroxysuccinimide carbonate derivatives of the NAG-containing protease cleavable substrate. The solution is then incubated at least 1 hour at RT before injection into animals.

Claims

What is Claimed is:
1. A composition comprising an antisense compound and Melittin-[(L)-(ASGPrLig)]x wherein:
Melittin is a melittin peptide;
L is physiologically labile linkage;
ASGPrLig is an Asialoglycoprotein Receptor ligand;
x is an integer having a value greater than 80% of the number of primary amines of said melittin peptide; and
wherein said antisense compound is selected from among:
(a) a single stranded RNAi compound comprising at least one of:
a 5'-phosphate modified nucleoside located at the 5' end; and
two or more contiguous 2'-0-(CH2)2-OCH3 modified nucleosides located at the 3' end; and
(b) a non-RNAi antisense compound.
2. The composition of claim 1 for in vivo delivery to a hepatocyte.
3. The composition of any of claims 1 or 2 wherein at least 90% of the amines on each melittin peptide are reversibly linked to Asialoglycoprotein Receptor ligands.
4. The composition of any of claims 1 to 3 wherein the Melittin peptide comprises an amino acid sequence selected from the list consisting of: Seq. ID 1, Seq. ID 7, Seq. ID 11, Seq. ID 51, Seq. ID 57, Seq. ID 58, Seq. ID 92, and Seq. ID 96.
5. The composition of any of claims 1 to 4 wherein the Melittin peptide consists of D-form amino acids.
6. The composition of any of claims 1 to 5 wherein L is a disubstituted maleamate.
7. The composition of claim 6, wherein the melittin peptide comprises a polyethyleneglycol (PEG) covalently linked to its amino terminus.
8. The composition of claim 6 further comprising an ASGPrLig-PEG conjugate covalently linked to the amino terminus of the melittin peptide.
9. The composition of any of claims 1 to 5 wherein L is an amidobenzyl carbamate.
10. The composition of claim 9 further comprising a polyethyleneglycol (PEG) covalently linked to the amino terminus of the melittin peptide.
11. The composition of claim 9 further comprising an ASGPrLig-PEG conjugate covalently linked to the amino terminus of the melittin peptide.
12. The composition of any of claims 1 to 1 1 wherein the ASGPrLig is selected from the group consisting of lactose, galactose, N-acetylgalactosamine (GalNAc), galactosamine, N-formyl- galactosamine, N-acetyl-galactosamine, N-propionylgalactosamine, N-n-butanoylgalactosamine, and N-iso-butanoyl-galactosamine.
13. The composition of any of claims 1 to 12 wherein the antisense compound is a single stranded RNAi compound.
14. The composition of any of claims 1 to 13 wherein the antisense compound is a single stranded RNAi compound comprising a 5'-phosphate-5'-vinyl modified nucleoside located at the 5' end.
15. The composition of any of claims 1 to 14 wherein the antisense compound is a single stranded RNAi compound comprising two contiguous 2'-0-(CH2)2-OCH3 modified nucleosides located at the 3' end.
16. The composition of any of claims 1 to 15 wherein the antisense compound is a single stranded RNAi compound comprising a 5'-phosphate-5'-vinyl modified nucleoside located at the 5' end and two contiguous 2'-0-(CH2)2-OCH3 modified nucleosides located at the 3' end and each nucleoside located between the 5'-phosphate-5'-vinyl modified nucleoside located at the 5' end and two contiguous 2'-0-(CH2)2-OCH3 modified nucleosides located at the 3' end is independently selected from 2'-F modified nucleosides, 2'-OCH3 modified nucleosides and β-D-ribonucleosides.
17. The composition of claim 16 wherein each nucleoside located between the 5'-phosphate-5'- vinyl modified nucleoside located at the 5' end and two contiguous 2'-0-(CH2)2-OCH3 modified nucleosides located at the 3' end is, independently, a 2'-F modified nucleoside or a 2'-OCH3 modified nucleoside.
18. The composition of claim 16 wherein the nucleosides located between the 5'-phosphate-5'- vinyl modified nucleoside located at the 5' end and two contiguous 2'-0-(CH2)2-OCH3 modified nucleosides located at the 3' alternate between 2'-F modified nucleosides and 2'-OCH3 modified nucleosides.
19. The composition of any of claims 1 to 12 wherein the antisense compound is a non-R Ai antisense compound.
20. The composition of claim 19 wherein the non-RNAi antisense compound works through an RNaseH mechanism.
21. The composition of any of claims 19 or 20 wherein the non-RNAi antisense compound comprises a first region consisting of from 2 to 5 modified nucleosides, a second region consisting of from 2 to 5 modified nucleosides and a gap region consisting of from 6 to 14 monomer subunits located between the first and second region.
22. The composition of claim 21 wherein each monomer subunit in the gap region is independently, a nucleoside or a modified nucleoside that is different from each of the modified nucleosides in the first and second region.
23. The composition of any of claims 21 or 22 wherein the gap region comprises from about 8 to about 12 monomer subunits.
24. The composition of any of claims 21 or 22 wherein the gap region comprises from about 8 to about 10 monomer subunits.
25. The composition of any of claims 21 to 24 wherein each monomer subunit in the gap region is a -D-2'-deoxyribonucleoside.
26. The composition of any of claims 21 to 24 wherein one or two of the monomer subunits in the gap region is a modified nucleoside and each of the other monomer subunits in the gap region is a -D-2'-deoxyribonucleoside.
27. The composition of any of claims 21 to 26 wherein each modified nucleoside in the first and second region comprises a modified sugar moiety.
28. The composition of any of claims 21 to 27 wherein each modified nucleoside in the first and second region is, independently, a bicyclic nucleoside comprising a 4'-CH((5)-CH3)-0-2' bridge or a 2'-0-(CH2)2-OCH3 modified nucleoside.
29. The composition of any of claims 1 to 28 wherein the antisense compound comprises internucleoside linking groups that are each, independently, a phosphodiester internucleoside linking group or a phosphorothioate internucleoside linking group.
30. The composition of any of claims 1 to 29 wherein the antisense compound comprises internucleoside linking groups that are essentially all phosphorothioate internucleoside linking groups.
31. The composition of any of claims 1 to 30 wherein the antisense compound comprises heterocyclic base moieties that are each, independently, a pyrimidine, substituted pyrimidine, purine or substituted purine.
32. The composition of any of claims 1 to 31 wherein the antisense compound comprises heterocyclic base moieties that are each, independently, uracil, thymine, cytosine, 4-N-benzoyl- cytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine or 2-N-isobutyrylguanine.
33. The composition of any of claims 1 to 32 wherein the antisense compound doesn't include a hydrophobic group having at least 20 carbon atoms or a galactose cluster.
34. The composition of claim 1 wherein in a pharmaceutically acceptable carrier or diluent.
35. A method comprising administering to a subject the composition of any of claims 1-34.
36. A method comprising co-administering to a subject an antisense compound and Melittin- [(L)-(ASGPrLig)]x
wherein:
Melittin is a melittin peptide;
L is physiologically labile linkage;
ASGPrLig is an Asialoglycoprotein Receptor ligand;
x is an integer having a value greater than 80% of the number of primary amines of said melittin peptide; and
wherein said antisense compound is selected from among:
(a) a single stranded RNAi compound comprising at least one of:
a 5'-phosphate modified nucleoside located at the 5' end; and
at least two contiguous 2'-0-(CH2)2-OCH3 modified nucleosides located at the 3' end; and
(b) a non-RNAi antisense compound.
37. The method of claim 36, wherein the antisense compound and the Melittin-[(L)- (ASGPrLig)]x are administered together.
38. The method of claim 36, wherein the antisense compound and the Melittin-[(L)- (ASGPrLig)]x are administered separately.
39. The method of any of claims 36-38, wherein the antisense compound and the Melittin- [(L)- (ASGPrLig)]x are administered at the same time.
40. The method of any of claims 36 or 38, wherein the antisense compound and the Melittin- [(L)-(ASGPrLig)]x are administered at different times.
41. A method of manufacturing a composition comprising: a) forming a melittin peptide; b) forming a plurality of uncharged masking agents each comprising an ASGPrLig covalently linked to a disubstituted maleic anhydride or a dipeptide amidobenzyl amine reactive carbonate; c) modifying greater than 80% of primary amines on a population of melittin peptides with the masking agents of step b, d) providing an antisense compound as per claim 1 and the modified melittin peptide in solution suitable for administration in vivo.
PCT/US2013/072982 2012-12-04 2013-12-04 Compositions and methods for in vivo delivery of antisense compounds WO2014089146A1 (en)

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
WO2014197816A1 (en) * 2013-06-06 2014-12-11 Massachusetts Institute Of Technology Stimulus responsive nanocomplexes and methods of use thereof
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