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Definitions

• This invention relates to oligonucleotide reagents, oligonucleotide therapeutics, and methods of making and using thereof.
• oligonucleotides into clinical medicines and their use as basic research tools are an ongoing endeavor. For example, the use of antisense oligonucleotides for gene silencing was described as early as 1978. Since this time other oligonucleotide based approaches have emerged for regulating gene expression, including RNA interference, microRNAs, and, recently, targeted inhibition or inactivation of long non-coding RNAs.
• multimeric oligonucleotide compounds are provided that are useful for regulating gene expression and function.
• Some aspects of the invention are based on the discovery that relatively high levels of a monomeric oligonucleotides can be achieved in a target tissue or cell when monomeric units are connected by a cleavable linker (e.g., an endonuclease-sensitive linker) and administered as a multimer.
• the properties of a linker are selected to modulate the pharmacokinetic and pharmacodynamic properties of the multimeric oligonucleotide compounds.
• linker properties can be tuned to control the extent to which monomeric units are released in a particular tissue-type or cell-type to be targeted.
• an advantage of using multimers is that it allows simultaneous knockdown of multiple targets, while exploiting the pharmacokinetic and/or pharmacodynamic advantages of the administered oligonucleotide.
• a sequence-specific concomitant knockdown of two or more targets may be achieved with a heteromultimer containing targeting oligonucleotides directed against several target gene combinations.
• multimeric oligonucleotides compounds provided herein comprise two or more targeting oligonucleotides linked together by a cleavable linker.
• each targeting oligonucleotide has a region complementary to a target region of a genomic target sequence.
• the targeting oligonucleotides hybridize to a target nucleic acid encoded by a genomic target sequence and inhibit the function and/or effect degradation of the target nucleic acid.
• the target nucleic acid may be, for example, a long non- coding RNA (IncRNA), microRNA, or mRNA.
• the targeting oligonucleotide is an antisense
• oligonucleotide ASO
• siRNA e.g. , a single stranded siRNA
• miRNA sponge e.g. a single stranded siRNA
• AMO anti- microRNA antisense oligonucleotide
• the oligonucleotide binds specifically to a target nucleic acid in a cell and brings about degradation of the target nucleic acid.
• the degradation is mediated by RNAse H.
• the degradation is mediated by an RNAi pathway.
• the targeting oligonucleotide binds specifically to its target nucleic acid in a cell and inhibits the function of the target nucleic acid.
• the targeting oligonucleotide binds to a target IncRNA and inhibits interaction of the IncRNA with one or more interacting proteins (e.g. , a subunit of Polycomb Repressor Complex 2 (PRC2)).
• interacting proteins e.g. , a subunit of Polycomb Repressor Complex 2 (PRC2)
• compounds that comprise the general formula: X-L-[X-L] ; -X, in which i is an integer from 0 to 9, the value of which indicates the number of units of [X-L], present in the compound, in which each X is independently a targeting oligonucleotide having a region of complementarity comprising at least 7 contiguous nucleotides complementary to a target region of a genomic target sequence, and each L is a linker that links at least two Xs and that is more susceptible to cleavage in a mammalian extract than each X.
• the 5 '-end of the target region complementary to the first X and the 3 '-end of the target region complementary to the second X are not within a distance of 0 to 4 nucleotides in the genomic target sequence. In some embodiments, the 5 '-end of the target region complementary to the first X and the 3 '-end of the target region
• the targeting oligonucleotides are 8 to 15, 10 to 16, 10 to 20, 10 to 25, 15 to 30, 8 to 50, 10 to 100 or more nucleotides in length.
• the targeting oligonucleotides are 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides in length.
• At least one L does not comprise an oligonucleotide having a self-complementary nucleotide sequence. In some embodiments, all Ls do not comprise an oligonucleotide having a self-complementary nucleotide sequence. In some embodiments, at least one L does not comprise an oligonucleotide having a nucleotide sequence that is complementary to a region of the genomic target sequence that is contiguous with the target regions complementary to two immediately flanking Xs of the at least one L. In some embodiments, the compound does not comprise a ribozyme. In some embodiments, all Ls do not comprise an oligonucleotide having a nucleotide sequence that is complementary to a region of the genomic target sequence that is contiguous with the target regions complementary to two immediately flanking Xs.
• i is an integer from 0 to 3, 1 to 3, 1 to 5, 1 to 9, 1 to 15, 1 to 20. In some embodiments, i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more. In some
• the at least one L linker comprises an oligonucleotide that is more susceptible to cleavage by an endonuclease in the mammalian extract than the targeting oligonucleotides.
• at least one L is a linker having a nucleotide sequence comprising from 1 to 10 thymidines or uridines.
• at least one L is a linker having a nucleotide sequence comprising deoxyribonucleotides linked through phosphodiester internucleotide linkages.
• At least one L is a linker having a nucleotide sequence comprising from 1 to 10 thymidines linked through phosphodiester internucleotide linkages. In some embodiments, at least one L is a linker having a nucleotide sequence comprising from 1 to 10 uridines linked through phosphorothioate intemucleotide linkages. In certain embodiments, at least one L is a linker having the formula:
• Z is an oligonucleotide.
• Z has a nucleotide sequence comprising from 1 to 10 thymidines or uridines.
• at least one L does not comprise an oligonucleotide having a self- complementary nucleotide sequence and does not comprise an oligonucleotide having a nucleotide sequence that is complementary to a region of the genomic target sequence that is contiguous with two flanking target regions.
• at least one L is a linker that does not comprise an oligonucleotide having an abasic site.
• the linker comprises a polypeptide that is more susceptible to cleavage by an endopeptidase in the mammalian extract than the targeting oligonucleotides.
• the endopeptidase is trypsin, chymotrypsin, elastase, thermolysin, pepsin, or endopeptidase V8.
• the endopeptidase is cathepsin B, cathepsin D, cathepsin L, cathepsin C, papain, cathepsin S or endosomal acidic insulinase.
• At least one L is a linker comprising a peptide having an amino acid sequence selected from: ALAL (SEQ ID NO: 125), APISFFELG (SEQ ID NO: 126), FL, GFN, R/KXX, GRWHTVGLRWE (SEQ ID NO: 127), YL, GF, and FF, in which X is any amino acid.
• At least one L is a linker comprising the formula - (CH 2 ) w S-S(CH 2 ) m -, wherein n and m are independently integers from 0 to 10. In certain embodiments, at least one L the linker comprises a low pH-labile bond.
• the low pH-labile bond comprises an amine, an imine, an ester, a benzoic imine, an amino ester, a diortho ester, a polyphosphoester, a polyphosphazene, an acetal, a vinyl ether, a hydrazone, an azidomethyl-methylmaleic anhydride, a thiopropionate, a masked endosomolytic agent or a citraconyl group.
• At least one L is a branched linker.
• the branched linker comprises a phosphoramidite linkage.
• the compound is a non- symmetrical branched trimer. In certain embodiments, the compound is a symmetrical branched trimer. In some embodiments, at least one L is a linker that is at least 2-fold more sensitive to cleavage in the presence of a mammalian extract than the targeting oligonucleotides.
• the compound may have the following general formula:
• the compound has the following general formula:
• the compound has the following general formula:
• the compound has the following general formula:
• j and k are independently 0 or 1, the value of which indicates, respectively, the number of Xy and X 3 ⁇ 4 present in the compound, and at least one of Xy and X 3 ⁇ 4 are present in the compound.
• compounds comprise at least two targeting oligonucleotides linked through a linker that is at least 2-fold more sensitive to enzymatic cleavage in the presence of a mammalian extract than the at least two targeting oligonucleotides, wherein each targeting oligonucleotide has a region of complementarity comprising at least 7 contiguous nucleotides complementary to a target region of a genomic target sequence.
• the targeting oligonucleotides are 8 to 15, 10 to 16, 12 to 16, 10 to 20, 10 to 25, 15 to 30, 8 to 50, 10 to 100 or more nucleotides in length.
• the targeting oligonucleotides are 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides in length.
• the linker is at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold or more sensitive to enzymatic cleavage in the presence of a mammalian extract than the two targeting oligonucleotides.
• the linker is an oligonucleotide.
• the oligonucleotide has a sequence that is not complementary to the genomic target sequence at a position immediately adjacent to the target region.
• the mammalian extract is an extract from kidney, liver, intestinal or tumor tissue.
• the mammalian extract is a cell extract.
• the mammalian extract is an endosomal extract.
• At least one targeting oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.
• the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.
• at least one targeting oligonucleotide comprises at least one a 2'-fluoro-deoxyribonucleotide.
• At least one targeting oligonucleotide comprises deoxyribonucleotides flanked by at least one bridged nucleotide on each of the 5' and 3' ends of the deoxyribonucleotides. In some embodiments, at least one targeting oligonucleotide comprises phosphorothioate internucleotide linkages between at least two nucleotides. In certain embodiments, at least one targeting oligonucleotide comprises a 2' O-methyl. In some embodiments, at least one targeting oligonucleotide comprises a G-clamp, 5- propynyl, or 5-octadienyl-pyrimidine. In certain embodiments, at least one targeting
• oligonucleotide is a gapmer comprising RNase H recruiting nucleotides.
• at least one targeting oligonucleotide is a single stranded siRNA.
• the compound is linked to a functional moiety (e.g., a lipophilic moiety or targeting moiety that binds to a cell surface receptor).
• a functional moiety e.g., a lipophilic moiety or targeting moiety that binds to a cell surface receptor.
• the functional moiety is linked to a targeting oligonucleotide.
• the functional moiety is linked to a linker.
• At least two targeting oligonucleotides are in the same 5' to 3' orientation relative to the linker. In some embodiments, at least two targeting oligonucleotides are in opposite 5' to 3' orientations relative to the linker. In certain embodiments,
• At least one targeting oligonucleotide is linked to the linker through a terminal nucleotide. In certain embodiments, at least one targeting oligonucleotide is linked to the linker through an internal nucleotide. In some embodiments, at least one targeting oligonucleotide is a single- stranded oligonucleotide.
• the target region complementary to at least one targeting oligonucleotide is present in the sense strand of a gene.
• the gene is an non-coding RNA gene.
• the non-coding RNA gene is a long non-coding RNA gene.
• the non-coding RNA gene is an miRNA gene.
• the gene is a protein coding gene.
• the genomic target sequence of at least one targeting oligonucleotide is the sequence of a PRC-2 associated region.
• at least two target regions are present in the sense strand of different genes.
• at least two target regions are present in the sense strand of the same gene.
• at least two target regions are different.
• at least two target regions are identical.
• the product of the gene mediates gene expression through an epigenetic mechanism.
• compositions are provided that comprise any of the compounds disclosed herein and a carrier.
• the compositions comprise a buffered solution.
• the compound is conjugated to the carrier.
• pharmaceutical compositions are provided that comprise any of the compounds disclosed herein and a pharmaceutically acceptable carrier.
• kits are provided that comprise a container housing any of the compounds or compositions disclosed herein.
• methods of increasing expression of a target gene in a cell comprise: contacting the cell with any of the compounds disclosed herein, and maintaining the cell under conditions in which the compound enters into the cell.
• the genomic target sequence of at least one targeting oligonucleotide of the compound is present in the sense strand of an IncRNA gene, the product of which is an IncRNA that inhibits expression of the target gene.
• presence of the compound in the cell results in a level of expression of the target gene that is at least 50% greater, at least 60% greater, at least 70% greater, at least 80%, or at least 90% greater than a level of expression of the target gene in a control cell that does not contain the compound.
• methods of increasing levels of a target gene in a subject are provided.
• the methods comprise
• the genomic target sequence of at least one targeting oligonucleotide of the compound is present in the sense strand of an IncRNA gene, the product of which inhibits expression of the target gene.
• a condition associated with altered levels of expression of a target gene in a subject are provided.
• the condition is associated with decreased or increased levels of expression of the target gene compared to a control subject who does not have the condition.
• the methods comprise administering the compound to the subject.
• the genomic target sequence of at least one targeting oligonucleotide of the compound is present in the sense strand of an IncRNA gene, the product of which inhibits expression of the target gene. Accordingly, in some embodiments, the at least one targeting oligonucleotide hybridizes to the IncRNA and inhibits its function or brings about its
• methods of modulating activity of a target gene in a cell comprise contacting the cell with any of the compounds disclosed herein, and maintaining the cell under conditions in which the compound enters into the cell. In some embodiments, presence of the compound in the cell results in reduced expression or activity of the target gene in the cell. According to some aspects of the invention, methods of modulating levels of a target gene in a subject are provided. In some embodiments, the methods comprise administering any of the compounds disclosed herein to the subject. In some embodiments the genomic target sequence of at least one targeting oligonucleotide is present in the sense strand of the target gene. In some embodiments, the target gene is a protein coding gene or non-coding gene.
• multimeric oligonucleotide compounds comprise two or more targeting oligonucleotides (e.g., ASOs), each having a nuclease-resistant modified backbone, wherein the targeting oligonucleotides are linked to each other by one or more degradable linkers.
• the backbone contains inter-nucleoside linkages.
• the individual linked targeting oligonucleotides, contained in a compound may be directed to the same target, or to multiple targets.
• the multimeric compounds can be homodimers, homotrimers, etc., heterodimers, heterotrimers, etc. They can be linear, branched, or circular.
• the invention is based, in part, on the discovery that multimeric oligonucleotide compounds (e.g., a 14-mer ASO linked to another 14-mer ASO) show significantly higher levels of the corresponding monomeric oligonucleotide compounds in the liver when the monomer units are connected by a rapidly degradable linker (e.g., a nuclease- sensitive linker or a disulfide linker), as opposed to a linker that is nuclease-resistant and, therefore, slowly degradable.
• a rapidly degradable linker e.g., a nuclease- sensitive linker or a disulfide linker
• the detected liver levels of the dimer-derived monomeric units were five to ten times higher than that of the corresponding monomers administered in the monomeric form.
• rapidly degradable linkers are referred as "cleavable" (such as, e.g., a nuclease- sensitive, phosphodiester, linkage or a linker comprising a disulfide bond), while more stable linkages, such as, e.g., nuclease-resistant phosphorothioates, as referred to as "noncleavable.”
• the compounds are directed to one or more hepatic targets
• ASOs are directed to hepatic targets, including but not limited to ApoC3 and ApoB.
• targeting oligonucleotides contain 12 to 16 nucleotide bases, wherein one or more targeting oligonucleotides are gapmers.
• Targeting oligonucleotides e.g., ASOs
• including gapmers can comprise a 2' modification in the sugar residues (e.g., locked-nucleic acid (LNA) modification), 2'-O-methyl and 2'-fluoro
• nucleotide modification such as G-clamp, 5-propynyl, and 5-octadienyl- pyrimidine.
• the invention further provides pharmaceutical compositions, comprising compounds of the invention along with pharmaceutically acceptable excipients.
• the pharmaceutical composition is characterized by one or more of the following properties when administered in vivo:
• the invention further provides methods of inhibiting mRNA levels of one or more targets, comprising administering to a cell or a subject the compound of the invention in an amount effective to inhibit the expression of the target(s).
• the methods provide a therapeutically effective knockdown of the target(s) persists for two weeks or longer following the administration.
• the method can be used with targets that are associated with a metabolic disease, cancer, cardiovascular disease, and other conditions.
• the multimeric targeting oligonucleotides may be referred to by the respective target names only, e.g., "ApoC3-ApoC3 dimer” stands as a short hand for "ApoC3-ApoC3 ASO dimer.”
• Figure 1A shows a schematic representation of an exemplary construct, in which two 14-mer gapmers (e.g., 3LNA-8DNA-3LNA as illustrated) are connected via a linker (represented light shaded circles).
• Figure IB shows examples of various configurations of dimers and multimers (homopolymers or heteropolymers).
• Figures 1C and ID show details of the chemical structures of certain multimeric ASOs.
• Figure 2 demonstrates in vitro stability of dimers in plasmas and their degradation in liver homogenates, as determined by liquid chromatography-mass spectrometry (LC-MS).
• Figures 2A and 2B demonstrate slow degradation of both ApoC3 ASO monomer (SEQ ID NO: l, designated as per Example 2(E)) and cleavable ApoC3-ApoC3 ASO dimers (SEQ ID NO:2 and SEQ ID NO:4) in murine and monkey plasmas respectively.
• Figure 2C demonstrates efficient cleavage into monomers of the cleavable ApoC3-ApoC3 ASO dimers (SEQ ID NO:2 and SEQ ID NO:4) and the relative stability ApoC3 ASO monomer (SEQ ID NO: l) in mouse liver homogenate.
• Figure 2D shows cleavable SEQ ID NO: 18) and
• Figure 3 addresses various aspects of linker designs in homodimers.
• Hep3B cells were treated at various concentrations (0.001, 0.006, 0.03, 0.2, 0.8, 4.0, 20 and 100 nM) of the indicated oligonucleotides formulated with a lipotransfection agent. mRNA content and cell viability was determined 48 hours after treatment.
• Hep3B cells were treated at eight concentrations (0.1, 0.6, 3.0, 20, 80, 400, 2000 and 10,000 nM) of the indicated oligonucleotides without any transfection agent ("gymnotic delivery").
• mRNA content and cell viability were determined after 8 days of treatment. In all cases, the graphs depict percentage effect relative to a non-specific oligonucleotide (negative control).
• Figure 4 addresses various aspects of the design of various heterodimers (di- and trimers).
• Hep3B cells were treated at various concentrations (0.001, 0.006, 0.03, 0.2, 0.8, 4.0, 20 and 100 nM) of the indicated
• oligonucleotides formulated with a lipotransfection agent were determined 48 hours after treatment.
• mRNA content and cell viability were determined 48 hours after treatment.
• Hep3B cells were treated at eight concentrations (0.1, 0.6, 3.0, 20, 80, 400, 2000 and 10,000 nM) of the indicated oligonucleotides without any transfection agent ("gymnotic delivery").
• mRNA content and cell viability were determined after 8 days of treatment. In all cases, the graphs depict percentage effect relative to a non-specific oligonucleotide (negative control).
• Figures 5A-5C demonstrate that under the conditions tested, the time course of knock-down depended on the type of linker used to connect the two antisense moieties in the dimeric ASOs.
• Human ApoC3 transgenic mice were administered a single subcutaneous dose of homodimers SEQ ID NO: 5 or 3 (which are disulphide-linked homodimers of the same monomer) at 10 mg/kg, or vehicle.
• Figure 5A demonstrates an associated increased reduction of the liver ApoC3 mRNA levels in human ApoC3 transgenic mice following treatment with the endonuclease-sensitive, phosphodiester-linked, homodimers (SEQ ID NO:4 and SEQ ID NO:2).
• Homodimers SEQ ID NO:4 and 2 exhibited an increased reduction of liver ApoC3 mRNA levels compared to the monomer (SEQ ID NO: l) after 14 days.
• Figures 5B and 5C show ApoC3 protein knockdown 7 days (Figure 5B) and 14 days (Figure 5C) after a single 10 mg/kg dose of the SEQ ID NO: l monomer and dimeric LNA gapmers SEQ ID NO:2 - SEQ ID NO:5 in human ApoC3 transgenic mice.
• the figures demonstrate increased duration in the reduction of serum ApoC3 protein levels in human ApoC3 transgenic mice following treatment with the endonuclease-sensitive phosphodiester-linked homodimers, SEQ ID NO: l, SEQ ID NO:4 and SEQ ID NO:2.
• SEQ ID NO:4 and SEQ ID NO:2 exhibited a reduction of serum ApoC3 levels similar to monomer SEQ ID NO: 1 after 7 days, but in contrast to the monomer, the reduction the reduction in target gene expression in cells treated with the cleavable dimers (SEQ ID NO:2 or 4) was sustained and, as a result, increased compared to SEQ ID NO: l after 14 days.
• Figures 6A-6C show illustrative LC-MS results for samples extracted from liver for the following ASOs respectively SEQ ID NO:2 ( Figure 6A), SEQ ID NO:3 ( Figure 6B), and SEQ ID NO:4 ( Figure 6C).
• "IS" designates an internal standard.
• FIGS 7A and 7B illustrate that SEQ ID NO: 21, an ApoC3/ApoB
• FIG. 8A and 8B illustrate the effects of these treatments on in vivo target mRNAs in the liver. Data in these figures are plotted as % knockdown of the target mRNAs with knockdown of mouse apoB mRNA plotted on the x axis and knockdown of human ApoC3 (i.e., the transgene) plotted on the y axis.
• Figures 9A and 9B illustrate differences in concentrations of ApoB monomer after overnight incubation at 37 °C or under frozen conditions of heterodimers and ApoB monomer ASOs in liver and kidney homogenates.
• BLQ is "Beneath Limit of Quantification.”
• Figure 10 illustrate differences in concentrations of ApoB monomer detected in plasma 3 days post-treatment with heterodimers and ApoB monomer ASOs.
• Figures 11A and 11B illustrate measured concentrations of ApoB monomer metabolite in kidneys at Day3 and Day 14 following administration of heterodimers and ApoB monomer ASOs.
• Figures 12A and 12B illustrate measured concentrations of ApoB monomer metabolite in liver at Day3 and Day 14 following administration of heterodimers and ApoB monomer ASOs.
• Figures 13A and 13B illustrate that dimer oligonucleotides significantly decreased miR-122 (lOmg/kg dose, mouse liver).
• Figures 14A and 14B illustrate that dimer oligonucleotides significantly decreased miR-122 (50mg/kg dose, mouse liver).
• Figures 15 illustrates that dimer oligonucleotides are ⁇ 5x more active than monomer (in vivo Id study).
• Figures 16A, 16B, and 16C illustrate that dimer oligonucleotides robustly decreased Malat-1 IncRNA expression.
• Multimeric oligonucleotide compounds are provided that are useful for regulating gene expression and/or function.
• the multimeric oligonucleotides compounds provided herein comprise two or more targeting oligonucleotides linked together by a cleavable linker.
• the multimeric oligonucleotides are useful for regulating the expression or function of a wide range of target nucleic acids including, for example, a long non-coding RNA (IncRNA), microRNA, or mRNA.
• the targeting oligonucleotide of the multimer is an antisense oligonucleotide (ASO), siRNA (e.g. , a single stranded siRNA), miRNA sponge, or anti-microRNA antisense oligonucleotide (AMO).
• ASO antisense oligonucleotide
• siRNA e.g. , a single stranded siRNA
• miRNA sponge e.g. a single strand
• Multimeric oligonucleotide compounds comprise the general formula: X-L-[X-L] ; -X, in which i is an integer, the value of which indicates the number of units of [X-L], present in the compound, and in which each X is a targeting oligonucleotide and each L is a linker that links at least two Xs and that is more susceptible to cleavage in a mammalian extract than each X.
• i is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
• a mammalian extract refers to a sample extracted from a mammalian tissue, cell or subcellular compartment (e.g., an endosome).
• a mammalian extract comprises one or more biomolecules (e.g. , enzymes) from the tissue, cell or subcellular compartment.
• a mammalian extract comprises one or more of a nuclease, peptidase, protease, phosphatase, oxidase, and reductase.
• the mammalian extract may be an extract from any tissue, including, for example, kidney, liver, intestinal or tumor tissue.
• the mammalian extract may be a cell extract or an extract from a subcellular component, such as a nuclear extract, or an endosomal extract.
• cleavage refers to the the breaking of one or more chemical bonds in a relatively large molecule in a manner that produces two or more relatively small molecules. Cleavage in the mammalian extract may be mediated by a nuclease, peptidase, protease, phosphatase, oxidase, or reductase, for example.
• cleavable refers to rapidly degradable linkers, such as, e.g., phosphodiester and disulfides, while the term “noncleavable” refer to more stable linkages, such as, e.g., nuclease -resistant phosphorothioates (e.g., a racemic mixture of Sp and Rp diastereoisomers, as used in the Examples below, or a backbone enriched in Sp form).
• nuclease -resistant phosphorothioates e.g., a racemic mixture of Sp and Rp diastereoisomers, as used in the Examples below, or a backbone enriched in Sp form.
• the compound has the following general formula:
• i is an integer indicating the number of units of [X-L], present in the compound; j and k are independently 0 or 1, the value of which indicates, respectively, the number of X and X ⁇ present in the compound; and / and m are integers the value of which indicate, respectively, the number of units of [X-L] / and [L-X] m present in the compound.
• i is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more.
• / and m are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more.
• at least one of [X-L] / and [L-X] m are present in the compound.
• i, j, k, I, and m are 0.
• i is 1, and j, k, I, and m are 0.
• the compound may have the following general formula:
• i 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.
• the compound may have the following general formula:
• i 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.
• the compound may have the following general formula:
• j and k are independently 0 or 1, the value of which indicates, respectively, the number of Xy and X 3 ⁇ 4 present, and at least one of Xy and X 3 ⁇ 4 are present in the compound.
• the targeting oligonucleotide has a region of complementarity comprising at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 contiguous nucleotides complementary to a target region of a genomic target sequence.
• the targeting oligonucleotide may have a region of complementarity comprising 4, 5, 6, 7, 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, 45, or 50 contiguous nucleotides complementary to a target region of a genomic target sequence.
• the region of complementary may have one or more mismatches compared with the nucleotide sequence of the target region provided that the targeting oligonucleotide is still capable of hybridizing with the target region. In some embodiments, the region of complementary has no mismatches compared with the nucleotide sequence of the target region. It should also be appreciated that a targeting oligonucleotide may hybridize with a target region through Watson- Crick base pairing, Hoogsteen base pairing, reverse-Hoogsteen binding, or other binding mechanism. In some embodiments, the targeting oligonucleotide is an aptamer, e.g. , an aptamer that binds to an intracellular or nuclear protein.
• the 5 '-end of the target region complementary to the first X and the 3 '-end of the target region complementary to the second X are not within a distance of 0 to 1, 0 to 2, 0 to 3, 0 to 4, 0 to 5, 0 to 10, 0 to 15, 0 to 20, 0 to 25, 0 to 50, nucleotides in the genomic target sequence when the target regions complementary to the first X and second X do not overlap in the genomic target sequence.
• the different X's have
• nucleic acid sequence of the X's may be identical with one another or overlapping or completely distinct.
• multimeric oligonucleotide compounds comprises ASOs.
• the invention provides in some embodiments multimeric oligonucleotide compounds, comprising two or more target- specific antisense oligonucleotides (ASOs), each ASO having a nuclease -resistant modified backbone, in which the targeting oligonucleotides are linked to each other by one or more degradable linkers.
• ASOs target-specific antisense oligonucleotides
• oligonucleotide in the context of targeting oligonucleotides (e.g., ASOs), refers to an targeting oligonucleotide that (i) is directed to a single site or a single contiguous stretch of nucleotides on a target and (ii) is not covalently linked to the another targeting oligonucleotide directed to the same or another site on the same or another target.
• Multimeric oligonucleotide compounds are not monomeric because they contain targeting oligonucleotides (e.g., ASOs) that are covalently linked to each other.
• the number of targeting oligonucleotides (e.g., ASOs) in a multimeric oligonucleotide compound of the invention may be two or more, three or more, four or more, etc.
• a multimeric oligonucleotide compound may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more individual Targeting oligonucleotides (e.g., ASOs) directed to one or more targets.
• the individual Targeting oligonucleotides e.g., ASOs
• the targeting oligonucleotide is a dimer comprising two targeting oligonucleotides specific to the same target, or a dimer comprising two targeting oligonucleotides specific to two different targets, or alternatively, a trimer comprising three targeting oligonucleotides specific to the same target, or a trimer comprising three targeting oligonucleotides specific three different targets, etc.
• the individual targeting oligonucleotides can be specific to the same target, yet directed to distinct target sites on the target, such as two sites on the target sequence that are separated by at least 10, 20, 50, 100, 300 or more nucleotides.
• the target sites can be directly adjacent to each other and not separated by any intervening sequences.
• the multimers can be linear or branched or a combination thereof.
• two ASO may be connected head-to-tail (5 '-to-3' -linear) (type A) or as in type B, tail-to-tail (3 '-to-3' -branched); the ASOs could also be connected head- to-head (5 '-to-5' -branched).
• three or more antisense molecules can be connected (examples C, D, E in Figure IB).
• the multimer can be in the form of a circular nucleic acid.
• multimeric oligonucleotides provided herein comprise two or more targeting oligonucleotides linked together by a cleavable linker.
• each targeting oligonucleotide has a region complementary to a target region of a genomic target sequence.
• the targeting oligonucleotide is an antisense oligonucleotide (ASO), siRNA (e.g. , a single stranded siRNA), miRNA sponge, or anti- microRNA antisense oligonucleotide (AMO).
• ASO antisense oligonucleotide
• siRNA e.g. , a single stranded siRNA
• miRNA sponge e.g. a single stranded siRNA
• AMO anti- microRNA antisense oligonucleotide
• oligonucleotide binds specifically to a target RNA in a cell and brings about degradation of the RNA.
• the degradation is mediated by RNAse H.
• the degradation is mediated by an RNAi pathway.
• genomic target sequence refers to a nucleotide sequence of clinical, therapeutic or research interest in a genome (e.g., a mammalian genome, e.g. , a human or mouse genome).
• a genomic target sequence is a sequence of a genome that comprises a gene coding or regulatory region, or that is present within a gene coding or regulatory region.
• a genomic target sequence is a sequence that encodes at least a portion of a gene.
• the gene may be an non-coding RNA gene or a protein coding gene.
• the non-coding RNA gene may be a long non-coding RNA gene or an miRNA gene, for example.
• a genomic target sequence is a sequence positioned in a regulatory region of one or more genes, such as a promoter, enhancer, silencer region, locus control region and other functional region of a genome.
• the genomic target sequence is present in the sense strand of a gene.
• the sense strand or coding strand is the segment of double stranded DNA running from 5' - 3' that is complementary to the antisense strand or template strand of a gene.
• the sense strand is the strand of DNA that has the same sequence as the RNA transcribed from the gene (e.g., mRNA, IncRNA, or miRNA), which takes the antisense strand as its template during transcription.
• the "target region" of a genomic target sequence is a sequence of nucleotides that consitutes a hybridization site of a targeting oligonucleotide.
• the actual target is a sequence of nucleotides that consitutes a hybridization site of a targeting oligonucleotide.
• oligonucleotide may hybridize with the genomic target itself (e.g. , a promoter element) or an nucleic acid encoded by the genomic target sequence or containing the genomic target sequence (e.g., an IncRNA, miRNA, or mRNA).
• the target region encodes a site on a transcribed RNA, and hybridization of a targeting oligonucleotide to the site results in inactivation or degradation of the transcribed RNA.
• the targeting oligonucleotides hybridize to a transcribed RNA encoded by a genomic target sequence and inhibit the function and/or effect degradation of the transcribed RNA.
• the RNA may be, for example, a long non-coding RNA (IncRNA), microRNA, or mRNA.
• multimeric oligonucleotides compounds provided herein may comprise two or more targeting oligonucleotides that are each complementary to the same or different genomic target sequences, and thus that may regulate the same or different genes.
• the genomic target sequences is present in the sense strand of different genes. In some embodiments, the genomic target sequences is present in the sense strand of the same gene.
• the genomic target sequence of at least one targeting oligonucleotide is or comprises the sequence of a PRC-2 associated region.
• PRC2-associated region refers to a region of a nucleic acid that comprises or encodes a sequence of nucleotides that interact directly or indirectly with a component of PRC2.
• a PRC2- associated region may be present in a RNA (e.g., a long non-coding RNA (IncRNA)) that interacts with a PRC2.
• IncRNA long non-coding RNA
• a PRC2-associated region may be present in a DNA that encodes an RNA that interacts with PRC2.
• a PRC2-associated region is a region of an RNA that crosslinks to a component of PRC2 in response to in situ ultraviolet irradiation of a cell that expresses the RNA, or a region of genomic DNA that encodes that RNA region.
• a PRC2-associated region is a region of an RNA that immunoprecipitates with an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that RNA region.
• a PRC2-associated region is a region of an RNA that
• RNA region immunoprecipitates with an antibody that binds specifically to SUZ12, EED, EZH2 or RBBP4 (which as noted above are components of PRC2), or a region of genomic DNA that encodes that RNA region.
• a PRC2-associated region is a region of an RNA that is protected from nucleases (e.g., RNases) in an RNA-immunoprecipitation assay that employs an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that protected RNA region.
• a PRC2-associated region is a region of an RNA that is protected from nucleases (e.g., RNases) in an RNA-immunoprecipitation assay that employs an antibody that targets SUZ12, EED, EZH2 or RBBP4, or a region of genomic DNA that encodes that protected RNA region.
• a PRC2-associated region is a region of an RNA within which occur a relatively high frequency of sequence reads in a sequencing reaction of products of an RNA-immunoprecipitation assay that employs an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that RNA region.
• a PRC2-associated region is a region of an RNA within which occur a relatively high frequency of sequence reads in a sequencing reaction of products of an RNA-immunoprecipitation assay that employs an antibody that binds specifically to SUZ12, EED, EZH2 or RBBP4, or a region of genomic DNA that encodes that protected RNA region.
• the PRC2- associated region may be referred to as a "peak.”
• a PRC2-associated region comprises a sequence of 40 to 60 nucleotides that interact with PRC2 complex. In some embodiments, a PRC2-associated region comprises a sequence of 40 to 60 nucleotides that encode an RNA that interacts with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of up to 5kb in length that comprises a sequence (e.g., of 40 to 60 nucleotides) that interacts with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of up to 5kb in length within which an RNA is encoded that has a sequence (e.g., of 40 to 60 nucleotides) that is known to interact with PRC2.
• a PRC2-associated region comprises a sequence of about 4kb in length that comprise a sequence (e.g., of 40 to 60 nucleotides) that interacts with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of about 4kb in length within which an RNA is encoded that includes a sequence (e.g., of 40 to 60 nucleotides) that is known to interact with PRC2.
• a PRC2-associated region has a sequence as set forth in SEQ ID NOS: 632,564, 1 to 916,209, or 916,626 to 934,931 of International Patent Appl. Pub. No.: WO/2012/087983, or SEQ ID NOS: 1 to 193,049 of International Patent Appl. Pub. No.: WO/2012/065143, each of which is entitled, POLYCOMB -ASSOCIATED NON-CODING RNAS, and the contents of each of which are incorporated by reference herein in their entireties.
• the targeting oligonucleotides interfere with the binding of and function of PRC2 by preventing recruitment of PRC2 to a specific chromosomal locus through IncRNAs.
• administration of multimeric oligonucleotide compounds comprising targeting oligonucleotides designed to specifically bind a PRC2-associated region of a IncRNA can stably displace not only the IncRNA, but also the PRC2 that binds to the IncRNA, from binding chromatin.
• IncRNA can recruit PRC2 in a cis fashion, repressing gene expression at or near the specific chromosomal locus from which the IncRNA was transcribed.
• the compounds disclosed herein may be used to inhibit cis mediated gene repression by IncRNAs.
• targeting oligonucleotides may comprise at least one ribonucleotide, at least one deoxyribonucleotide, and/or at least one bridged nucleotide.
• the oligonucleotide may comprise a bridged nucleotide, such as a locked nucleic acid (LNA) nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridged nucleic acid (ENA) nucleotide. Examples of such nucleotides are disclosed herein and known in the art.
• the oligonucleotide comprises a nucleotide analog disclosed in one of the following United States Patent or Patent Application Publications: US 7,399,845, US 7,741,457, US 8,022,193, US 7,569,686, US 7,335,765, US 7,314,923, US 7,335,765, and US 7,816,333, US 20110009471, the entire contents of each of which are incorporated herein by reference for all purposes.
• the targeting oligonucleotide may have one or more 2' O-methyl nucleotides.
• the oligonucleotide may consist entirely of 2' O-methyl nucleotides.
• the targeting oligonucleotide may contain one or more nucleotide analogues.
• the targeting oligonucleotide may have at least one nucleotide analogue that results in an increase in T m of the oligonucleotide in a range of 1°C, 2 °C, 3°C, 4 °C, or 5°C compared with an oligonucleotide that does not have the at least one nucleotide analogue.
• the targeting oligonucleotide may have a plurality of nucleotide analogues that results in a total increase in T m of the oligonucleotide in a range of 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C or more compared with an oligonucleotide that does not have the nucleotide analogue.
• the targeting oligonucleotide may be of up to 50 nucleotides in length or up to 100 nucleotides in length, in which 2 to 10, 2 to 15. , 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45, 2 to 75, 2 to 95, or more nucleotides of the oligonucleotide are nucleotide analogues.
• the oligonucleotide may be of 8 to 30 nucleotides in length in which 2 to 10, 2 to 15 4 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide are nucleotide analogues.
• the oligonucleotide may be of 8 to 15 nucleotides in length in which 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides of the oligonucleotide are nucleotide analogues.
• the oligonucleotides may have every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified.
• the targeting oligonucleotide may consist entirely of bridged nucleotides (e.g. , LNA nucleotides, cEt nucleotides, ENA nucleotides).
• the oligonucleotide may comprise alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides.
• the oligonucleotide may comprise alternating deoxyribonucleotides and 2'-0-methyl nucleotides.
• the oligonucleotide may comprise alternating deoxyribonucleotides and ENA nucleotide analogues.
• oligonucleotide may comprise alternating deoxyribonucleotides and LNA nucleotides.
• the oligonucleotide may comprise alternating LNA nucleotides and 2'-0-methyl nucleotides.
• the oligonucleotide may have a 5' nucleotide that is a bridged nucleotide (e.g. , a LNA nucleotide, cEt nucleotide, ENA nucleotide).
• the oligonucleotide may have a 5' nucleotide that is a deoxyribonucleotide.
• the targeting oligonucleotide may comprise deoxyribonucleotides flanked by at least one bridged nucleotide (e.g. , a LNA nucleotide, cEt nucleotide, ENA nucleotide) on each of the 5' and 3' ends of the deoxyribonucleotides.
• the oligonucleotide may comprise deoxyribonucleotides flanked by 1, 2, 3, 4, 5, 6, 7, 8 or more bridged nucleotides (e.g. , LNA nucleotides, cEt nucleotides, ENA nucleotides) on each of the 5' and 3' ends of the
• the 3' position of the oligonucleotide may have a 3' hydroxyl group.
• the 3' position of the oligonucleotide may have a 3' thiophosphate.
• the targeting oligonucleotide may be conjugated with a label.
• the oligonucleotide may be conjugated with a biotin moiety, cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end.
• the targeting oligonucleotide may comprise one or more modifications comprising: a modified sugar moiety, and/or a modified internucleoside linkage, and/or a modified nucleotide and/or combinations thereof. It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the modifications described herein may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide. [0086] In some embodiments, the targeting oligonucleotides are chimeric oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide.
• oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
• Chimeric targeting oligonucleotides of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or
• the targeting oligonucleotide comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-0-alkyl, 2'-0-alkyl-0- alkyl or 2'-fluoro-modified nucleotide.
• RNA modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA.
• modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
• modified backbones for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
• oligonucleotides with phosphorothioate backbones and those with heteroatom backbones particularly CH 2 -NH-0-CH 2 , CH, ⁇ N(CH 3 ) ⁇ 0 ⁇ CH 2 (known as a
• methylene(methylimino) or MMI backbone CH 2 -0--N (CH 3 )-CH 2 , CH 2 -N (CH 3 )-N (CH 3 )- CH 2 and O-N (CH 3 )- CH 2 -CH 2 backbones, wherein the native phosphodiester backbone is represented as O- P— O- CH,); amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbone structures (see Summerton and Weller, U.S. Pat. No.
• PNA peptide nucleic acid
• Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral
• phosphonates phosphinates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates,
• Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41 (14), 4503-4510); Genesis, volume 30, issue 3, 2001 ; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No.
• the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g. , as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001 ; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties).
• PMO phosphorodiamidate morpholino oligomer
• Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc, 2000, 122, 8595-8602.
• Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones 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 comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts; see US patent nos.
• Modified oligonucleotides are also known that include oligonucleotides that are based on or constructed from arabinonucleotide or modified arabinonucleotide residues.
• Arabinonucleosides are stereoisomers of ribonucleosides, differing only in the configuration at the 2'-position of the sugar ring.
• a 2'-arabino modification is 2'-F arabino.
• the modified oligonucleotide is 2'-fluoro-D-arabinonucleic acid (FANA) (as described in, for example, Lon et al., Biochem., 41 :3457-3467, 2002 and Min et al., Bioorg. Med. Chem. Lett., 12:2651-2654, 2002; the disclosures of which are incorporated herein by reference in their entireties). Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on a 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
• PCT Publication No. WO 99/67378 discloses arabinonucleic acids (ANA) oligomers and their analogues for improved sequence specific inhibition of gene expression via association to complementary messenger RNA.
• ANA arabinonucleic acids
• ENAs ethylene-bridged nucleic acids
• Preferred ENAs include, but are not limited to, 2'-0,4'-C-ethylene-bridged nucleic acids.
• LNAs are described in WO/2008/043753 and include compounds of the following general formula.
• the LNA used in the oligonucleotides described herein comprises at least one LNA unit according any of the formulas
• Y is -0-, -S-, -NH-, or N(R ); Z and Z* are independently selected among an intemucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non- natural nucleotide base moiety, and RH is selected from hydrogen and Ci-4-alkyl.
• the Locked Nucleic Acid (LNA) used in the oligonucleotides described herein comprises at least one nucleotide comprises a Locked Nucleic Acid (LNA) unit according any of the formulas shown in Scheme 2 of PCT/DK2006/000512.
• LNA Locked Nucleic Acid
• the LNA used in the oligomer of the invention comprises intemucleoside linkages selected from -0-P(O) 2 -O-, -0-P(0,S)-0-, -0-P(S) 2 -O-, -S-P(0) 2 -0-, -S- P(0,S)-0-, -S-P(S) 2 -0-, -0-P(O) 2 -S-, -0-P(0,S)-S-, -S-P(0) 2 -S-, -0-PO(R H )-0-, 0-PO(OCH 3 )- 0-, -0-PO(NR H )-0-, -0-PO(OCH 2 CH 2 S-R)-O-, -0-PO(BH 3 )-0-, -0-PO(NHR H )-0-, -0-P(0) 2 - NR H -, -NR H -P(0) 2 -0-, -NR
• thio-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from S or -CH 2 -S-.
• Thio-LNA can be in both beta- D and alpha-L-configuration.
• amino-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from -N(H)-, N(R)-, CH 2 -N(H)-, and -CH 2 - N(R)- where R is selected from hydrogen and Ci-4-alkyl.
• Amino-LNA can be in both beta-D and alpha-L-configuration.
• oxygen-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above represents -O- or -CH 2 -0-. Oxy-LNA can be in both beta-D and alpha-L-configuration.
• ena-LNA comprises a locked nucleotide in which Y in the general formula above is -CH 2 -0- (where the oxygen atom of -CH 2 -0- is attached to the 2'-position relative to the base B).
• One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, OCH 3 OCH 3 , OCH 3 0(CH 2 )n CH 3 , 0(CH 2 )n NH 2 or 0(CH 2 )n CH 3 where n is from 1 to about 10; Ci to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF 3 ; OCF 3 ; 0-, S-, or N- alkyl; 0-, S-, or N-alkenyl; SOCH 3 ; S0 2 CH 3 ; ON0 2 ; N0 2 ; N 3 ; NH2; heterocyclo alkyl;
• heterocyclo alkaryl aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an
• oligonucleotide and other substituents having similar properties A preferred modification includes 2'-methoxyethoxy [2'-0-CH 2 CH 2 OCH 3 , also known as 2'-0-(2-methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486). Other preferred modifications include 2'-methoxy (2'-0- CH ), 2'-propoxy (2'-OCH 2 CH 2 CH ) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
• Targeting oligonucleotides can also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
• nucleobase often referred to in the art simply as “base”
• “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U).
• Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g.
• hypoxanthine 6-methyladenine
• 5-Me pyrimidines particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, isocytosine, pseudoisocytosine, as well as synthetic nucleobases, e.g.
• 2-aminoadenine 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other hetero substituted alkyladenines
• 2-thiouracil 2-thiothymine
• 5-bromouracil 5- hydroxymethyluracil, 5-propynyluracil, 8-azaguanine, 7-deazaguanine
• N6 (6- aminohexyl)adenine, 6-aminopurine, 2-aminopurine, 2-chloro-6-aminopurine and 2,6- diaminopurine or other diaminopurines.
• both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
• the base units are maintained for hybridization with an appropriate nucleic acid target compound.
• an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
• PNA peptide nucleic acid
• the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone.
• the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
• PNA compounds include, but are not limited to, US patent nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497- 1500.
• nucleobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in "The Concise Encyclopedia of Polymer Science And
• 5-substituted pyrimidines 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.
• 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 ⁇ 0>C
• nucleobases are described in US patent nos. 3,687,808, as well as 4,845,205; 5,130,302; 5,134,066; 5,175, 273; 5, 367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091;
• the targeting oligonucleotides are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
• one or more targeting oligonucleotides, of the same or different types can be conjugated to targeting moieties with enhanced specificity for a cell type or tissue type.
• Such moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.
• a thioether e.g. , hexyl- S- tritylthiol (Manoharan et al, Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.
• a phospholipid e.g. , di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl- rac- glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain
• conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
• Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine,
• Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence- specific hybridization with the target nucleic acid.
• Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference.
• Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g. , hexyl-5- tritylthiol, a thiocholesterol, an aliphatic chain, e.g. , dodecandiol or undecyl residues, a phospholipid, e.g.
• di-hexadecyl-rac- glycerol or triethylammonium 1,2-di-O-hexadecyl-rac- glycero-3-H-phosphonate a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety. See, e.g. , U.S. Pat. Nos.
• targeting oligonucleotide modification include modification of the 5' or 3' end of the oligonucleotide.
• the 3' end of the oligonucleotide comprises a hydroxyl group or a thiophosphate.
• additional molecules e.g. a biotin moiety or a fluorophor
• the targeting oligonucleotide comprises a biotin moiety conjugated to the 5' nucleotide.
• the targeting oligonucleotide comprises locked nucleic acids (LNA), ENA modified nucleotides, 2'-0-methyl nucleotides, or 2'-fluoro- deoxyribonucleotides.
• LNA locked nucleic acids
• the targeting oligonucleotide comprises alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides.
• the targeting oligonucleotide comprises alternating deoxyribonucleotides and 2'-0-methyl nucleotides.
• the targeting oligonucleotide comprises alternating deoxyribonucleotides and ENA modified nucleotides.
• the targeting oligonucleotide comprises alternating deoxyribonucleotides and locked nucleic acid nucleotides. In some embodiments, the targeting oligonucleotide comprises alternating locked nucleic acid nucleotides and 2'-0-methyl nucleotides.
• the 5' nucleotide of the oligonucleotide is a
• the 5' nucleotide of the oligonucleotide is a locked nucleic acid nucleotide.
• the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one locked nucleic acid nucleotide on each of the 5' and 3' ends of the deoxyribonucleotides.
• the nucleotide at the 3' position of the oligonucleotide has a 3' hydroxyl group or a 3' thiophosphate.
• the targeting oligonucleotide comprises
• the targeting oligonucleotide comprises phosphorothioate intemucleotide linkages between at least two nucleotides. In some embodiments, the targeting oligonucleotide comprises phosphorothioate intemucleotide linkages between all nucleotides.
• targeting oligonucleotide can have any combination of modifications as described herein.
• oligonucleotide based linkers may also include any of the modifications disclosed herein.
• the targeting oligonucleotides are targeting oligonucleotides that contain locked nucleic acid 3-8-3 gapmers which have a phosphorothioate backbone.
• the chemistry of the oligonucleotide is not limited to LNA (2'- 0,4' -C-methylene -bridged nucleic acids described, e.g., in PCT patent application WO
• chemistries include, for instance, 2'-O,4'-C-ethylene-bridged nucleic acids (ENA; European patent No.
• HNA hexitol nucleic acids
• FANA fluoro- HNA
• 2'-deoxy-2'-fluoro-13-D-arabino nucleic acids FANA; EP 1088066
• 2'-modified analogs such as 2'-O-methyl (2'-OMe) and 2'-O-(2-methoxyethyl) (MOE) modified nucleic acids
• CeNA EP 1210347 and EP 1244667
• phosphate-modified analogs such as phosphoroamidate, morpholinos, base-modified analogs, such as G-clamps (WO 99/24452) and 5-alkynyl-pyrimidines.
• LNA other gapmers are described in PCT patent applications published as WO 01/25248, WO 01/48190, WO 2003/085110, WO 2004/046160, WO 2008/113832, WO 2005/023825 and WO 2007/14651; examples of FANA/DNA/FANA gapmers are described in EP 1315807; examples of 2'-OMe/FANA/2'-OMe gapmers are described in US patent No. 6,673,611. [00119]
• the backbone may be stabilized by other modifications, for example, methylphosphonate or other chemistries.
• the antisense oligonucleotides of this invention can work via an RNase H mechanism, but can also work by steric blocking only, which also includes transcriptional gene silencing and transcriptional gene activation (see, e.g., Hawkins et al., 2009, Nucl. Acids Res., 37(9):2984-2995 and Schwartz et al., 2008, Nature Struct. Mol. Biol., 15:842-848).
• the dimer/multimer approach can also be combined with any modification which increases the delivery into cells, including lipophilic modifications, conjugates to cell surface receptors or ligands (e.g., folate), aptamers, etc.
• RNA:RNA, RNA:2'-0-methyl-RNA, RNA:PNA or RNA:LNA duplexes (without a DNA gap) may be used.
• the ASO chemistry may be adjusted based on the intended use. Any chemistry suitable for the antisense
• oligonucleotides should be applicable to the dimer/multimer approach of the invention (for the state-of-the-art chemistries, see, e.g., Bennett and Swaize, 2009, Ann. Rev. Pharmacol. Toxicol., 50:259-293; Yokota at al., 2010, Arch. Neurol., 66:32-38; Aboul-Fadl, 2005, Curr. Med. Chem., 12:2193-2214; Kurreck, 2003, Eur. J. Biochem., 270: 1628-1644).
• the targeting oligonucleotides are 14- nucleotide long, but could be generally longer or shorter.
• the targeting oligonucleotides are 14- nucleotide long, but could be generally longer or shorter.
• the targeting oligonucleotides are 14- nucleotide long, but could be generally longer or shorter.
• oligonucleotide could be 8-50-nucleotide long, or 10-40, 10-25, 8-20, 10-25, 12-25, 12-20, 12- 16, 12-15, 12-14, 12-13, 13-16, 13-15, or 13-14 nucleotides long.
• targeting oligonucleotides are so-called tiny LNAs, containing as few as 8 or fewer nucleotides (see, e.g., Obad et al. (2011) Nature Genetics, 43:371).
• a targeting oligonucleotide comprises at least 7 contiguous nucleotides complementary to the target sequence.
• targeting oligonucleotide e.g., ASO
• ASO may additionally comprise 1, 2, 3 or more non-complementary nucleotides, either within the contiguous sequences or flanking them.
• at least one or all of the targeting oligonucleotides are gapmers.
• the targeting oligonucleotides are X-N-Y gapmers, wherein at X or Y contains 0, 1, 2, 3, 4, 5 or more modified nucleotides, e.g., LNA, ENA, FANA, G-clamp, and N is 3, 4, 5, 6, 7, 8, 9, or 10 deoxynucleotides with non-modified sugars.
• ASO can be a 3-8-3, 2-10-2, 3-9-2, 2-9-3, 2-8-2, 3-7-2, 2-7-3 gapmer or another type of gapmer or mixmer.
• linker generally refers to a chemical moiety that is capable of covalently linking two or more targeting oligonucleotides, in which at least one bond comprised within the linker is capable of being cleaved (e.g., in a biological context, such as in a mammalian extract, such as an endosomal extract), such that at least two targeting oligonucleotides, in which at least one bond comprised within the linker is capable of being cleaved (e.g., in a biological context, such as in a mammalian extract, such as an endosomal extract), such that at least two targeting
• oligonucleotides are no longer covalently linked to one another after bond cleavage.
• a provided linker may include a region that is non-cleavable, as long as the linker also comprises at least one bond that is cleavable.
• the linker comprises a polypeptide that is more susceptible to cleavage by an endopeptidase in the mammalian extract than the targeting oligonucleotides.
• the endopeptidase may be a trypsin, chymotrypsin, elastase, thermolysin, pepsin, or endopeptidase V8.
• the endopeptidase may be a cathepsin B, cathepsin D, cathepsin L, cathepsin C, papain, cathepsin S or endosomal acidic insulinase.
• the linker comprise a peptide having an amino acid sequence selected from: ALAL (SEQ ID NO: 125), APISFFELG (SEQ ID NO: 126), FL, GFN, R/KXX, GRWHTVGLRWE (SEQ ID NO: 127), YL, GF, and FF, in which X is any amino acid.
• the linker comprises the formula -(CH2),jS-S(CH2)m-, wherein n and m are independently integers from 0 to 10.
• the linker of a multimeric oligonucleotide may comprise an oligonucleotide that is more susceptible to cleavage by an endonuclease in the mammalian extract than the targeting oligonucleotides.
• the linker may have a nucleotide sequence comprising from 1 to 10 thymidines or uridines.
• the linker may have a nucleotide sequence comprising deoxyribonucleotides linked through phosphodiester intemucleotide linkages.
• the linker may have a nucleotide sequence comprising from 1 to 10 thymidines linked through phosphodiester intemucleotide linkages.
• the linker may have a nucleotide sequence comprising from 1 to 10 uridines linked through phosphorothioate intemucleotide linkages.
• the linker may have the formula: 0— ? P— o— z— o— ? P— o—
• Z is an oligonucleotide.
• Z may have a nucleotide sequence comprising from 1 to 10 thymidines or uridines.
• the linker does not comprise an oligonucleotide having a self-complementary nucleotide sequence. In some embodiments, the linker does not comprise an oligonucleotide having a nucleotide sequence that is complementary to a region of the genomic target sequence that is contiguous with two flanking target regions. In some embodiments, the linker does not comprise an oligonucleotide having a self-complementary nucleotide sequence and does not comprise an oligonucleotide having a nucleotide sequence that is complementary to a region of the genomic target sequence that is contiguous with two flanking target regions of the particular linker. In some embodiments, the at least one L is a linker that does not comprise an oligonucleotide having an abasic site.
• multimeric oligonucleotide compounds comprise at least two targeting oligonucleotides each of which is linked to one or two other targeting oligonucleotides through a linker.
• at least one linker is 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more sensitive to enzymatic cleavage in the presence of a mammalian extract than at least two targeting oligonucleotides. It should be appreciated that different linkers can be designed to be cleaved at different rates and/or by different enzymes in compounds comprising two or more linkers.
• different linkers can be designed to be sensitive to cleavage in different tissues, cells or subcellular compartments in compounds comprising two or more linkers. This can advantageously permit compounds to have targeting oligonucleotides that are released from compounds at different rates, by different enzymes, or in different tissues, cells or subcellular compartments thereby controlling release of the monomeric oligonucleotides to a desired in vivo location or at a desired time following administration.
• the invention also provides ASO multimers comprising targeting oligonucleotides having nuclease -resistant backbone (e.g., phosphorothioate), wherein the targeting oligonucleotides are linked to each other by one or more cleavable linkers.
• ASO multimers comprising targeting oligonucleotides having nuclease -resistant backbone (e.g., phosphorothioate), wherein the targeting oligonucleotides are linked to each other by one or more cleavable linkers.
• linkers are stable in plasma, blood or serum which are richer in exonucleases, and less stable in the intracellular environments which are relatively rich in endonucleases.
• the intracellular stability of linkers can be assessed in vitro or in vivo as described in the Examples.
• a linker is considered “non-cleavable” if the linker's half-life is at least 24, or 28, 32, 36, 48, 72, 96 hours or longer under the conditions described here, such as in liver homogenates.
• a linker is considered “cleavable” if the half-life of the linker is at most 10, or 8, 6, 5 hours or shorter.
• the linker is a nuclease-cleavable oligonucleotide linker.
• the nuclease-cleavable linker contains one or more phosphodiester bonds in the oligonucleotide backbone.
• the linker may contain a single phosphodiester bridge or 2, 3, 4, 5, 6, 7 or more phosphodiester linkages, for example as a string of 1-10 deoxynucleotides, e.g., dT, or ribonucleotides, e.g., rU, in the case of RNA linkers.
• the cleavable linker contains one or more phosphodiester linkages.
• the cleavable linker may consist of phosphorothioate linkages only.
• phosphorothioate-linked deoxynucleotides which are only cleaved slowly by nucleases (thus termed "noncleavable")
• phosphorothioate-linked rU undergoes relatively rapid cleavage by ribonucleases and therefore is considered cleavable herein. It is also possible to combine dN and rN into the linker region, which are connected by phosphodiester or
• the linker can also contain chemically modified nucleotides, which are still cleavable by nucleases, such as, e.g., 2'-0-modified analogs.
• 2'-O-methyl or 2'-fluoro nucleotides can be combined with each other or with dN or rN nucleotides.
• the linker is a part of the multimer that is usually not complementary to a target, although it could be. This is because the linker is generally cleaved prior to targeting oligonucleotides action on the target, and therefore, the linker identity with respect to a target is inconsequential. Accordingly, in some aspects of nucleotides, which are still cleavable by nucleases, such as, e.g., 2'-0-modified analogs.
• 2'-O-methyl or 2'-fluoro nucleotides can be combined with each other or with dN or rN nucleotides.
• a linker is an (oligo)nucleotide linker that is not complementary to any of the targets against which the targeting oligonucleotides are designed.
• the cleavable linker is oligonucleotide linker that contains a continuous stretch of deliberately introduced Rp phosphorothioate stereoisomers (e.g., 4, 5, 6, 7 or longer stretches).
• the Rp stereoisoform unlike Sp isoform, is known to be susceptible to nuclease cleavage (Krieg et al., 2003, Oligonucleotides, 13:491-499).
• Such a linker would not include a racemic mix of PS linkages oligonucleotides since the mixed linkages are relatively stable and are not likely to contain long stretches of the Rp stereoisomers, and therefore, considered “non-cleavable" herein.
• a linker comprises a stretch of 4, 5, 6, 7 or more phosphorothioated nucleotides, wherein the stretch does not contain a substantial amount or any of the Sp stereoisoform. The amount could be considered substantial if it exceeds 10% on per- mole basis.
• the linker is a non-nucleotide linker, for example, a single phosphodiester bridge.
• the cleavable linkers may be present in other linear or branched multimers.
• the cleavable linker comprises a "doubler,” “trebler,” or another branching chemical group with multiple “arms” that link phosphodiester linked nucleotides, as for example, illustrated in Figures 1C and ID and Formulas IV, V, and VIII.
• cleavable linkers can be incorporated as shown in Formulas I and II.
• the linker can be designed so as to undergo a chemical or enzymatic cleavage reaction.
• Chemical reactions involve, for example, cleavage in acidic environment (e.g., endosomes), reductive cleavage (e.g., cytosolic cleavage) or oxidative cleavage (e.g., in liver microsomes).
• the cleavage reaction can also be initiated by a rearrangement reaction.
• Enzymatic reactions can include reactions mediated by nucleases, peptidases, proteases, phosphatases, oxidases, reductases, etc.
• a linker can be pH-sensitive, cathepsin- sensitive, or predominantly cleaved in endosomes and/or cytosol.
• the linker comprises a peptide.
• the linker comprises a peptide which includes a sequence that is cleavable by an endopeptidase.
• the linker may comprise additional amino acid residues and/or non-peptide chemical moieties, such as an alkyl chain.
• the linker comprises Ala-Leu-Ala-Leu (SEQ ID NO.: 125), which is a substrate for cathepsin B.
• a cathepsin B-cleavable linker is cleaved in tumor cells.
• the linker comprises Ala-Pro-Ile-Ser-Phe-Phe-Glu-Leu-Gly (SEQ ID NO.: 126), which is a substrate for cathepsins D, L, and B (see, for example, Fischer et al, Chembiochem 2006, 7, 1428-1434).
• a cathepsin-cleavable linker is cleaved in HeLA cells.
• the linker comprises Phe-Lys, which is a substrate for cathepsin B.
• the linker comprises Phe-Lys-p-aminobenzoic acid (PABA). See, e.g., the maleimidocaproyl-Phe-Lys-PABA linker described in Walker et al, Bioorg. Med. Chem. Lett. 2002, 12, 217-219.
• the linker comprises Gly-Phe-2- naphthylamide, which is a substrate for cathepsin C (see, for example, Berg et al. Biochem. J. 1994, 300, 229-235).
• a cathepsin C-cleavable linker is cleaved in hepatocytes, In some embodiments, the linker comprises a cathepsin S cleavage site.
• the linker comprises Gly-Arg-Trp-His-Thr-Val-Gly-Leu-Arg- Trp-Glu (SEQ ID NO.: 127), Gly-Arg-Trp-Pro-Pro-Met-Gly-Leu-Pro-Trp-Glu (SEQ ID NO.: 137), or Gly-Arg-Trp-His-Pro-Met-Gly-Ala-Pro-Trp-Glu (SEQ ID NO.: 138), for example, as described in Lutzner et al, J.
• a cathepsin S-cleavable linker is cleaved in antigen presenting cells.
• the linker comprises a papain cleavage site. Papain typically cleaves a peptide having the sequence -R/K-X-X (see Chapman et al, Annu. Rev. Physiol 1997, 59, 63-88).
• a papain-cleavable linker is cleaved in endosomes.
• the linker comprises an endosomal acidic insulinase cleavage site.
• the linker comprises Tyr-Leu, Gly-Phe, or Phe-Phe (see, e.g., Authier et al, FEBS Lett. 1996, 389, 55-60).
• an endosomal acidic insulinase-cleavable linker is cleaved in hepatic cells.
• the linker is pH sensitive.
• the linker comprises a low pH-labile bond.
• a low-pH labile bond is a bond that is selectively broken under acidic conditions (pH ⁇ 7). Such bonds may also be termed endosomally labile bonds, because cell endosomes and lysosomes have a pH less than 7.
• the linker comprises an amine, an imine, an ester, a benzoic imine, an amino ester, a diortho ester, a polyphosphoester, a polyphosphazene, an acetal, a vinyl ether, a hydrazone, an azidomethyl-methylmaleic anhydride, a thiopropionate, a masked endosomolytic agent or a citraconyl group.
• the linker comprises a low pH-labile bond selected from the following: ketals that are labile in acidic environments (e.g., pH less than 7, greater than about 4) to form a diol and a ketone; acetals that are labile in acidic environments (e.g., pH less than 7, greater than about 4) to form a diol and an aldehyde; imines or iminiums that are labile in acidic environments (e.g., pH less than 7, greater than about 4) to form an amine and an aldehyde or a ketone; silicon-oxygen-carbon linkages that are labile under acidic condition; silicon-nitrogne (silazane) linkages; silicon-carbon linkages (e.g., arylsilanes, vinylsilanes, and allylsilanes); maleamates (amide bonds synthesized from maleic anhydride derivatives and amines); ortho esters; hydra
• Organosilanes e.g., silyl ethers, silyl enol ethers
• Silicon-oxygen-carbon linkages are susceptible to hydrolysis under acidic conditions to form silanols and an alcohol (or enol).
• the substitution on both the silicon atom and the alcohol carbon can affect the rate of hydrolysis due to steric and electronic effects. This allows for the possibility of tuning the rate of hydrolysis of the silicon-oxygen-carbon linkage by changing the substitution on either the organosilane, the alcohol, or both the organosilane and alcohol.
• charged or reactive groups such as amines or carboxylate, may be attached to the silicon atom, which confers the labile compound with charge and/or reactivity.
• Silazanes are inherently more susceptible to hydrolysis than is the silicon-oxygen-carbon linkage, however, the rate of hydrolysis is increased under acidic conditions.
• the substitution on both the silicon atom and the amine can affect the rate of hydrolysis due to steric and electronic effects. This allows for the possibility of tuning the rate of hydrolysis of the silazane by changing the substitution on either the silicon or the amine.
• pH labile bond is an acid labile enol ether bond.
• the rate at which this labile bond is cleaved depends on the structures of the carbonyl compound formed and the alcohol released.
• analogs of ethyl isopropenyl ether, which may be synthesized from ⁇ -haloethers, generally have shorter half lives than analogs of ethyl
• reaction of an anhydride with an amine forms an amide and an acid.
• the reverse reaction formation of an anhydride and amine
• the anhydride is a cyclic anhydride
• reaction with an amine yields a molecule in which the amide and the acid are in the same molecule, an amide acid.
• the presence of both reactive groups (the amide and the carboxylic acid) in the same molecule accelerates the reverse reaction.
• the linker comprises maleamic acid. Cleavage of the amide acid to form an amine and an anhydride is pH-dependent, and is greatly accelerated at acidic pH.
• 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 linker comprises a benzoic imine as a low-pH labile bond. See, for example, the conjugates described in Zhu et al, Langmuir 2012, 28, 11988-96; Ding et al, Bioconjug. Chem. 2009, 20, 1163-70.
• the linker comprises a low pH-labile hydrazone bond.
• acid-labile bonds have been extensively used in the field of conjugates, e.g., antibody-drug conjugates. See, for example, Zhou et al, Biomacromolecules 2011, 12, 1460-7; Yuan et al, Acta Biomater. 2008, 4, 1024-37; Zhang et al, Acta Biomater. 2007, 6, 838-50; Yang et al, J. Pharmacol. Exp. Ther. 2007, 321, 462-8; Reddy et al, Cancer Chemother. Pharmacol. 2006, 58, 229-36; Doronina et al, Nature Biotechnol. 2003, 21, 778-84.
• the linker comprises a low pH-labile vinyl ether. See, for example, Shin et al, J. Control. Release 2003, 91, 187-200. In some embodiments, the linker comprises a low pH-labile phosphoamine bond. In some embodiments, the linker comprises a low pH-labile traceless click linker. For example, in certain embodiments, the linker comprises azidomethyl-methylmaleic anhydride (see Maier et al, J. Am. Chem. Soc. 2012 134, 10169-73. In some embodiments, the linker comprises a low pH-labile 4-hydrazinosulfonyl benzoic acid linker. See, for example, Kaminskas et al, Mol. Pharm.
• the linker comprises a low pH-labile para-phenylpropionic acid linker (see, e.g., Indira Chandran et al, Cancer Lett. 2012 316, 151-6).
• the linker comprises a low pH-labile ⁇ -thiopropionate linker (see, e.g., Dan et al, Langmuir 2011, 27, 612- 7).
• the linker comprises a low pH-labile ester (see, for example, Zhu et al, Bioconjug. Chem. 2010, 21, 2119-27).
• the linker comprises a low pH-labile ketal (see, e.g., Abraham et al, J. Biomater. Sci. Polym. Ed. 2011, 22, 1001-22) or acetal (see, e.g., Liu et al, J. Am. Chem. Soc. 2010, 132, 1500).
• the linker comprises a low pH-labile 4-(4'-acetylphenoxy)butanoic acid linker (see, e.g., DiJoseph et al, Blood 2004, 103, 1807-14).
• the linker comprises a low pH-labile cis- aconityl linker (see, e.g., Haas et al, J. Drug Target 2002, 10, 81-9; Ahmad et al, Anticancer Res. 1990, 10, 837-43; Dillman et al, Cancer Res. 1988, 48, 6097-102).
• the linker comprises a low pH-labile diortho ester (see, e.g, Guo et al, Bioconjug. Chem. 2001, 12, 291-300).
• the linker comprises a masked endosomolytic agent.
• Endosomolytic polymers are polymers that, in response to a change in pH, are able to cause disruption or lysis of an endosome or provide for escape of a normally membrane-impermeable compound, such as a polynucleotide or protein, from a cellular internal membrane-enclosed vesicle, such as an endosome or lysosome.
• a subset of endosomolytic compounds is fusogenic compounds, including fusogenic peptides. Fusogenic peptides can facilitate endosomal release of agents such as oligomeric compounds to the cytoplasm. See, for example, US Patent
• the linker can also be designed to undergo an organ/ tissue-specific cleavage.
• organ/ tissue-specific cleavage For example, for certain targets, which are expressed in multiple tissues, only the knock-down in liver may be desirable, as knock-down in other organs may lead to undesired side effects.
• linkers susceptible to liver- specific enzymes such as pyrrolase (TPO) and glucose-6- phosphatase (G-6-Pase)
• TPO pyrrolase
• G-6-Pase glucose-6- phosphatase
• linkers not susceptible to liver enzymes but susceptible to kidney- specific enzymes, such as gamma-glutamyltranspeptidase can be engineered, so that the antisense effect is limited to the kidneys mainly.
• intestine-specific peptidases cleaving Phe-Ala and Leu- Ala could be considered for orally administered multimeric targeting oligonucleotides.
• an enzyme recognition site into the linker, which is recognized by an enzyme over-expressed in tumors, such as plasmin (e.g., PHEA-D-Val-Leu-Lys recognition site)
• tumor-specific knock-down should be feasible.
• specific cleavage and knock-down should be achievable in many organs.
• the linker can also contain a targeting signal, such as /V-acetyl galactosamine for liver targeting, or folate, vitamin A or RGD-peptide in the case of tumor or activated macrophage targeting.
• a targeting signal such as /V-acetyl galactosamine for liver targeting, or folate, vitamin A or RGD-peptide in the case of tumor or activated macrophage targeting.
• the cleavable linker is organ- or tissue-specific, for example, liver- specific, kidney-specific, intestine- specific, etc.
• the targeting oligonucleotides can be linked through any part of the individual targeting oligonucleotide, e.g., via the phosphate, the sugar (e.g., ribose, deoxyribose), or the nucleobase.
• the linker when linking two oligonucleotides together, can be attached e.g.
• the linker when linking two oligonucleotides together, can attach internal residues of each
• oligonucleotides e.g., via a modified nucleobase.
• a modified nucleobase e.g., a modified nucleobase
• the linkers described herein can also be used to attach other moieties to an oligonucleotide.
• moieties include lipophilic moieties, targeting moieties (e.g., a ligand of a cell surface receptor), and tags (e.g., a fluorescent moiety for imaging or an affinity tag such as biotin).
• the linker is attached to an oligonucleotide via click chemistry (for a review of using click chemistry with DNA, see El-Sagheer et al, Chem. Soc. Rev. 2010, 39, 1388-1405).
• click chemistry is used to describe any facile reaction that occurs in high yields, under mild conditions, and in the presence of diverse functional groups, but it is most commonly used to refer to a [3+2] azide-alkyne cycloaddition reaction. Such reactions are generally catalyzed by Cu 1 and proceed in the presence of functional groups typically encountered in biological molecules.
• an unnatural base is introduced into the oligonucleotide, wherein the base is modified to comprise an alkyne or azide. See below for exemplary base modifications:
• R' is, for example, hydrogen, a suitable protecting group or coupling moiety (e.g., 4,4'-dimethoxytrityl (DMT), or a phosphoramidite group), a triphosphate, or R' denotes the point of connection to the rest of an oligonucleotide.
• a suitable protecting group or coupling moiety e.g., 4,4'-dimethoxytrityl (DMT), or a phosphoramidite group
• DMT 4,4'-dimethoxytrityl
• R' denotes the point of connection to the rest of an oligonucleotide.
• an oligonucleotide is modified such that the ribose moiety comprises an alkyne or azide for coupling the linker.
• the ribose moiety comprises an alkyne or azide for coupling the linker.
• an oligonucleotide is modified on the 5' or 3' end with an alkyne or azide for coupling the linker via click chemistry.
• an alkyne or azide for coupling the linker via click chemistry.
• the nucleosides shown below can be used to synthesize such oligonucleotides:
• Exemplary reagents which allow linking targeting oligonucleotides through a nucleobase include protected amino functionality at the base that can then be coupled to other suitable functional groups.
• Fmoc Amino-Modifier C6 dT (Glen Research catalog number 10-1536-xx) is used as a starting material:
• exemplary reagents which allow linking targeting oligonucleotides through a nucleobase include protected thiol functionality at the base that can then be coupled to other suitable functional groups or used to form a disulfide bond.
• S-Bz- Thiol-Modifier C6 dT (Glen Research catalog number 10-1039-xx) is used as a starting material:
• Thiol-Modifier C6 S-S (Glen Research catalog number 10-1936-xx), 3 '-Thiol-Modifier C3 S-S CPG (Glen Research catalog number 20-2933-xx), or 5'-Maleimide-Modifier Phosphoramidite (Glen Research catalog number 10-1938-xx) is used to introduce a linker: Thiol-Modifier C6 S-S
• Cholesteryl-TEG Phosphoramidite (Glen Research catalog number 10-1975-xx) or a-Tocopherol-TEG Phosphoramidite (Glen Research catalog number 10-1977-xx) is used in phosphoramidite synthesis to add a lipophilic moiety to an targeting oligonucleotide:
• one or more of the following starting materials are used in oligonucleotide synthesis to introduce an alkyne into an targeting oligonucleotide that can be reacted via click chemistry with an azide to attach another targeting oligonucleotide or another moiety such as a lipophilic group or targeting group:
• one or more of the following starting materials are used to attach a lipophilic group or targeting group via click chemistry to an targeting oligonucleotide functionalized with an alkyne, such as the ones described above:
• the disclosure provides a method of inhibiting target expression levels of one or more targets, comprising administering to a cell or a subject the compounds of the invention in an amount effective to inhibit the expression of the target(s).
• the target is an mRNA.
• the target could be a microRNA, as described above.
• the individual targeting oligonucleotides may be referred to as "antagomiRs.”
• the target can be a non-coding RNA naturally expressed in the cells.
• the subjects treated according to the methods of the invention can be animals, including humans, primates, and rodents.
• Cells can be present in vitro, or treated ex vivo. In some cases, ex vivo treated cells are re-administered to the subject.
• the invention also encompasses dual and multiple target antisense inhibitors, in particular those to treat liver diseases, metabolic diseases, cardiovascular diseases, inflammatory diseases, neurological diseases, viral, bacterial, parasitic, or prion infections and cancer.
• target antisense inhibitors include the use of dimeric antisense inhibitors to inhibit liver targets (also referred to as "hepatic targets"), such as ApoB and ApoC3 dual inhibition. Since knock-down of ApoB has been reported to lead to undesired lipid deposition in the liver, the simultaneous knock-down of ApoC3 can decrease this side effect.
• the present invention is also suitable for cancer treatment.
• multimeric oligonucleotides compounds of the invention that comprise targeting
• oligonucleotides which are directed 1) against targets responsible for the differentiation, development, or growth of cancers, such as: oncoproteins or transcription factors, e.g., c-myc, N-myc, c-myb, c-fos, c-fos/jun, PCNA, pl20, EJ-ras, c-Ha-ras, N-ras, rrg, bcl-2, bcl-x, bcl-w, cdc-2, c-raf-1, c-mos, c-src, c-abl, c-ets; 2) against cellular receptors, such as EGF receptor, Her- 2, c-erbA, VEGF receptor (KDR-1), retinoid receptors; 3) against protein kinases, c-fms, Tie-2, c-raf-1 kinase, PKC-alpha, protein kinase A
• antisense or directed against 8 components of spindle formation, such as eg5 and PLKl, or 9) against targets to suppress metastasis, such as CXCR4.
• antisense sequences directed against 10 factors which suppress apoptosis, such as survivin, stat3 and hdm2, or which suppress the expression of multiple drug resistance genes, such as MDR1 (P-glycoprotein).
• the dimer/multimer can also degrade or antagonize microRNA (miRNA) which are single- stranded RNA molecules of about 21-23 nucleotides in length regulating gene expression.
• miRNAs are encoded by genes that are transcribed from DNA but not translated into protein (non-coding RNA); instead they are processed from primary transcripts known as pri- miRNA to short stem-loop structures called pre-miRNA and finally to functional miRNA.
• Mature miRNA molecules are partially complementary to one or more messenger RNA
• miRNA mRNA molecules
• TGF-P2 receptor e.g., TGF-P2 receptor, RBI and PLAG1
• An miRNA expression signature of human solid tumors defining cancer gene targets was reported, for example, by Volinia et al., PNAS, 2006, 103, 2257-61.
• compositions comprising a compound of the invention and one or more pharmaceutically acceptable excipients.
• Methods of formulating and administering oligonucleotides to a cell or a subject are known in the art (see, e.g., Hardee, Gregory E.; Tillman, Lloyd G.; Geary, Richard S. Routes and Formulations For Delivery of Antisense Oligonucleotides. Antisense Drug Technology (2nd Edition) 2008, 217-236.
• the compounds of the invention possess favorable pharmacokinetic and/or pharmacodynamic properties.
• a pharmaceutically acceptable carrier for example, in some case, a pharmaceutically acceptable carrier, or a pharmaceutically acceptable carrier, or a pharmaceutically acceptable carrier, or a pharmaceutically acceptable carrier.
• compositions of the invention are characterized by one or more of the following properties when administered in vivo:
• a composition that includes a multimeric oligonucleotide compound can be delivered to a subject by a variety of routes.
• routes include: intravenous, intradermal, topical, rectal, parenteral, anal, intravaginal, intranasal, pulmonary, ocular.
• terapéuticaally effective amount is the amount of multimeric oligonucleotide compound present in the composition that is needed to provide the desired level of target gene modulation (e.g. , inhibition or activation) in the subject to be treated to give the anticipated physiological response.
• physiologically effective amount is that amount delivered to a subject to give the desired palliative or curative effect.
• pharmaceutically acceptable carrier means that the carrier can be administered to a subject with no significant adverse toxicological effects to the subject.
• the multimeric oligonucleotide compound molecules of the invention can be incorporated into pharmaceutical compositions suitable for administration.
• Such compositions typically include one or more species of multimeric oligonucleotide compounds and a pharmaceutically acceptable carrier.
• pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
• pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
• the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated.
• Supplementary active compounds can also be incorporated into the compositions.
• compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration. [00170] The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the multimeric oligonucleotide compound in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the multimeric oligonucleotide compound and mechanically introducing the oligonucleotide.
• Topical administration refers to the delivery to a subject by contacting the formulation directly to a surface of the subject.
• the most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g., to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface.
• the most common topical delivery is to the skin.
• the term encompasses several routes of administration including, but not limited to, topical and transdermal. These modes of administration typically include penetration of the skin's permeability barrier and efficient delivery to the target tissue or stratum.
• Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition.
• Topical administration can also be used as a means to selectively deliver oligonucleotides to the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.
• Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
• Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
• Coated condoms, gloves and the like may also be useful.
• Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics.
• the dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin.
• Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal adsorption. Absorption via this route may be enhanced by the use of an oily vehicle (inunction) or through the use of one or more penetration enhancers.
• Other effective ways to deliver a composition disclosed herein via the transdermal route include hydration of the skin and the use of controlled release topical patches.
• the transdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or local therapy.
• iontophoresis transfer of ionic solutes through biological membranes under the influence of an electric field
• phonophoresis or sonophoresis use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the cornea
• optimization of vehicle characteristics relative to dose position and retention at the site of administration may be useful methods for enhancing the transport of topically applied compositions across skin and mucosal sites.
• oligonucleotides administered through these membranes may have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the oligonucleotides to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the oligonucleotide can be applied, localized and removed easily.
• GI gastrointestinal
• compositions can be targeted to a surface of the oral cavity, e.g., to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek.
• the sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many agents. Further, the sublingual mucosa is convenient, acceptable and easily accessible.
• a pharmaceutical composition of multimeric oligonucleotide compound may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a mixed micellar pharmaceutical formulation as described above and a propellant.
• the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity.
• compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, slurries, emulsions, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches.
• carriers that can be used include lactose, sodium citrate and salts of phosphoric acid.
• Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets.
• useful diluents are lactose and high molecular weight polyethylene glycols.
• the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.
• Parenteral administration includes intravenous drip, subcutaneous,
• parental administration involves administration directly to the site of disease (e.g. injection into a tumor).
• Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
• Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir.
• the total concentration of solutes should be controlled to render the preparation isotonic.
• any of the multimeric oligonucleotide compounds described herein can be administered to ocular tissue.
• the compositions can be applied to the surface of the eye or nearby tissue, e.g., the inside of the eyelid.
• ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers.
• Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers.
• the multimeric oligonucleotide compound can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure.
• Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably multimeric oligonucleotide compounds, within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.
• Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self-contained. Dry powder dispersion devices, for example, deliver agents that may be readily formulated as dry powders. A multimeric oligonucleotide compound may be stably stored as lyophilized or spray- dried powders by itself or in combination with suitable powder carriers.
• the delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
• a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
• the term "powder” means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the alveoli.
• the powder is said to be "respirable.”
• the average particle size is less than about 10 ⁇ in diameter preferably with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 ⁇ m and most preferably less than about 5.0 ⁇ m.
• the particle size distribution is between about 0.1 ⁇ m and about 5 ⁇ m in diameter, particularly about 0.3 ⁇ m to about 5 ⁇ m.
• dry means that the composition has a moisture content below about 10% by weight (% w) water, usually below about 5% w and preferably less it than about 3% w.
• a dry composition can be such that the particles are readily dispersible in an inhalation device to form an aerosol.
• the types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.
• HSA human serum albumin
• bulking agents such as carbohydrates, amino acids and polypeptides
• pH adjusters or buffers such as sodium chloride
• salts such as sodium chloride
• Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.
• Pulmonary administration of a micellar multimeric oligonucleotide compound formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFC and CFC propellants.
• Exemplary devices include devices which are introduced into the vasculature, e.g., devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g., catheters or stents, can be placed in the vasculature of the lung, heart, or leg.
• Other devices include non-vascular devices, e.g., devices implanted in the peritoneum, or in organ or glandular tissue, e.g., artificial organs.
• the device can release a therapeutic substance in addition to a multimeric oligonucleotide compound, e.g., a device can release insulin.
• unit doses or measured doses of a composition that includes multimeric oligonucleotide compound are dispensed by an implanted device.
• the device can include a sensor that monitors a parameter within a subject.
• the device can include pump, e.g., and, optionally, associated electronics.
• Tissue e.g., cells or organs can be treated with a multimeric oligonucleotide compound, ex vivo and then administered or implanted in a subject.
• the tissue can be autologous, allogeneic, or xenogeneic tissue.
• tissue can be treated to reduce graft v. host disease .
• the tissue is allogeneic and the tissue is treated to treat a disorder characterized by unwanted gene expression in that tissue.
• tissue e.g., hematopoietic cells, e.g., bone marrow hematopoietic cells, can be treated to inhibit unwanted cell proliferation.
• the multimeric oligonucleotide compound treated cells are insulated from other cells, e.g., by a semi-permeable porous barrier that prevents the cells from leaving the implant, but enables molecules from the body to reach the cells and molecules produced by the cells to enter the body.
• the porous barrier is formed from alginate.
• a contraceptive device is coated with or contains a multimeric oligonucleotide compound.
• exemplary devices include condoms, diaphragms, IUD (implantable uterine devices, sponges, vaginal sheaths, and birth control devices.
• the invention features a method of administering a multimeric oligonucleotide compound to a subject (e.g., a human subject).
• a subject e.g., a human subject.
• the unit dose is between about 10 mg and 25 mg per kg of bodyweight. In one embodiment, the unit dose is between about 1 mg and 100 mg per kg of bodyweight. In one embodiment, the unit dose is between about 0.1 mg and 500 mg per kg of bodyweight. In some embodiments, the unit dose is more than 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 25, 50 or 100 mg per kg of bodyweight.
• the defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with a particular target gene.
• the unit dose for example, can be administered by injection (e.g., intravenous or intramuscular), an inhaled dose, or a topical application.
• the unit dose is administered daily. In some embodiments, the unit dose is administered daily.
• the unit dose is not administered with a frequency (e.g., not a regular frequency).
• the unit dose may be administered a single time.
• the unit dose is administered more than once a day, e.g., once an hour, two hours, four hours, eight hours, twelve hours, etc.
• a subject is administered an initial dose and one or more maintenance doses of a multimeric oligonucleotide compound.
• the maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose.
• a maintenance regimen can include treating the subject with a dose or doses ranging from 0.0001 to 100 mg/kg of body weight per day, e.g., 100, 10, 1, 0.1, 0.01, 0.001, or 0.0001 mg per kg of bodyweight per day.
• the maintenance doses may be administered no more than once every 1, 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient.
• the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once for every 5 or 8 days.
• the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state.
• the dosage of the oligonucleotide may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
• the effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
• a delivery device e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
• the concentration of the multimeric oligonucleotide compound is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans.
• the concentration or amount of multimeric oligonucleotide compound administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, pulmonary. For example, nasal formulations may tend to require much lower concentrations of some ingredients in order to avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10-100 times in order to provide a suitable nasal formulation.
• treatment of a subject with a therapeutically effective amount of a multimeric oligonucleotide compound can include a single treatment or, preferably, can include a series of treatments.
• the effective dosage of a multimeric oligonucleotide compound used for treatment may increase or decrease over the course of a particular treatment.
• the subject can be monitored after administering a multimeric oligonucleotide compound. Based on information from the monitoring, an additional amount of the multimeric oligonucleotide compound can be administered.
• Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved.
• Optimal dosing schedules can be calculated from measurements of target gene expression levels in the body of the patient.
• Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models.
• the animal models include transgenic animals that express a human target gene.
• the composition for testing includes a multimeric oligonucleotide compound that is complementary, at least in an internal region, to a sequence that is conserved between target gene in the animal model and the target gene in a human.
• the administration of the multimeric oligonucleotide compound is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular.
• Administration can be provided by the subject or by another person, e.g., a health care provider.
• the composition can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.
• kits comprising a container housing a composition comprising a multimeric oligonucleotide compound.
• the composition is a pharmaceutical composition comprising a multimeric oligonucleotide compound and a pharmaceutically acceptable carrier.
• the individual components of the pharmaceutical composition may be provided in one container.
• the kit may be packaged in a number of different configurations such as one or more containers in a single box.
• the different components can be combined, e.g., according to instructions provided with the kit.
• the components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition.
• the kit can also include a delivery device.
• Antisense oligonucleotides against ApoC3, ApoB, Hif-1 alpha, survivin and B2M were either selected using a series of bioinformatics filters and computational design algorithms or were derived from the literature. They were selected to be 13, 14 or more nucleotides in length and tested using one or multiple chemical modification design patterns (for example, 3LNAs-8DNAs-3LNAs). The list of all targeting oligonucleotide sequences is given in Table 1 and specific chemical modification patterns are explicitly specified when data is presented. Factors taken into account during the design include species homology, alignment to multiple human transcripts, off-target matches, SNPs, exon-exon boundaries, coverage of the transcript, and statistical models of efficacy and polyA regions.
• Figures 1A and IB show schematic representation of exemplary
• two 14-mer gapmers e.g., 3LNA- 8DNA-3LNA
• a cleavable linker which can be cleaved by enzymes, such as nucleases, peptidases or by reduction or oxidation. It could also be a linker which is cleaved by a pH shift within the cells (e.g. acidic pH in endosomes).
• the two antisense gapmers can be identical (homo-dimer), which leads to suppression of a single target mRNAl.
• the two antisense gapmers can also have different sequences (hetero-dimer) which are complementary to two or more different targets and which will lead to inhibition of two targets (mRNAl and mRNA2) or trimers or tetramers, etc., specific to 3, 4, or more different targets.
• hetero-dimer complementary to two or more different targets and which will lead to inhibition of two targets (mRNAl and mRNA2) or trimers or tetramers, etc., specific to 3, 4, or more different targets.
• RNA, LNA, 2'-0-Methyl, 2'-Fluoro, 5-Propynyl and G-Clamps were coupled with extended coupling times (e.g. 8 to 15 min for RNA, LNA, 2'-0-Methyl, 2'-Fluoro, 5-Propynyl and G-Clamps).
• extended coupling times e.g. 8 to 15 min for RNA, LNA, 2'-0-Methyl, 2'-Fluoro, 5-Propynyl and G-Clamps.
• AMA a 50:50 mixture of ammonium hydroxide and aqueous methylamine
• the crude DMTr-on oligonucleotides were purified via DMTr- selective cartridge purification techniques and if necessary further purified via RP HPLC and desalted via cartridge-based
• oligonucleotides were characterized using LC-MS.
• a C Technologies Solo VP Slope (Bridgewater, NJ) reader equipped with "Quick Slope" software was used to determine the concentration of oligonucleotides. Fifty ⁇ of sample was required for the measurement in a micro quartz vessel. The instrument measured the change in absorbance at varying path lengths, utilizing Beer's Law to determine final concentrations. Extinction coefficients were calculated using the nearest neighbor model.
• the ASO synthesized first is connected to the linker X via its 5 '-end whereas the finally synthesized ASO is connected via Y. This might lead to 3'- and 5'- modified metabolites after cleavage. Due to its linearity, a trimer, tetramer, or other multimer could be synthesized by adding a second, third, or more cleavable linker(s) followed by another ASO (1, 2 or 3).
• X is e.g. dT-dT-dT-dT or rUrlPrlPrll or (CH2)6-S-S-(CH 2 )6
• SED ID NO:2 (ApoC3-ApoC3 homodimer ASO) contains ASOl (BA*BA*BG*dC*dA*dA*dC*dC*dT*dA*dC*BA*BG*BG
• X is dT-dT-dT-dTU or (CH 2 ) 6 -S-S-(CH 2 ) 6
• symmetric dimers synthesis was performed using a triethylene glycol (teg) derivatized solid support and a symmetric doubler (brancher) phosphoramidite from Glen Research (catalog number 10-1920) illustrated below
• Oligonucleotide dimers synthesized with this doubler (brancher) have the following structure:
• the first ASOl is synthesized by linear addition of all monomers until the full length sequence of ASOl was obtained on solid support. Then, the cleavable (or noncleavable) linker X (tetrathymidyl, tetrauridyl, disulfide etc.) is synthesized followed by addition of the doubler using a symmetric doubler phosphoramidite (Glen Research, 10-1920) the solid phase synthesis of the linker X and ASO 1 was performed in parallel as indicated in the figure below.
• the non-symmetric brancher phosphoramidite is coupled after first synthesis of ASOl, followed by sequential synthesis of AS02 and AS03.
• the non-symmetrical phosphoramidite structure is shown in Formula IX.
• the resulting trimer has the structure show in Figure ID.
• Table 2 provides a descriptive legend for chemical structure designations used throughout the specification, including in Table 1.
• A is adenosine
• G is guanosine
• T thymidine
• U uridine
• APC is a G-clamp
• PU is 5-propynyl-uridine
• PC is 5-propynyl-cytidine
• oligos were incubated in 95 % plasma of mouse or monkey and in 5 % liver homogenate at a concentration of 30 ⁇ and at 37 °C. Samples for measurement were taken after 0, 7, 24 and 48 h of incubation. Samples were subjected to a phenol/chloroform extraction and analyzed using LC-MS.
• Plasmas used were a Na-Citrate plasma from female NMRI mice (Charles River) and K-EDTA plasma from male Cynomolgous monkeys (Seralab International).
• each oligo was prepared representing each individual incubation time point (0, 7, 24 and 48 h) in mouse and monkey plasma and in mouse liver homogenate, respectively.
• a blank sample and a recovery sample were prepared of each oligo and incubation matrix.
• plasma samples were prepared by adding 5 ⁇ of the 600 ⁇ oligo stock solution to 95 ⁇ of mouse or monkey plasma, respectively, with a final oligo concentration of 30 ⁇ .
• Recovery samples were prepared by adding 5 ⁇ of water to 95 ⁇ of plasma. Blank samples are oligo in water with a final concentration of 100 ⁇ .
• Liver samples and recoveries were prepared in the same way except that liver homogenate in PBS was used instead of plasma.
• the dried samples were dissolved in water (100 ⁇ ).
• the recovery samples were dissolved in water (95 ⁇ ) and spiked with 5 ⁇ of the respective oligo stock solution (600 ⁇ ).
• Samples were analyzed by LC-MS (Agilent 1200, Bruker Esquire 3000) using a Waters Acquity UPLC OST C18 column (1.7 ⁇ , 2.1x50) with HFIP/TEA/water (385 mM 1,1,1,3,3,3-hexafluoroisopropanol, 14.4 mM triethylamine in water) as buffer A and methanol as buffer B at a flow rate of 0.3 ml/min and a column temperature of 60 °C.
• the following gradient was used: 3 min at 5% B, 5-15 % B in 2.5 min (10 /min), 15-23 % B in 5.5 min, 23-30 % B in 3 min, 30-100 % B in 0.5 min, 5 min at 100 % B, 100-5 % B in 0.5 min, 5 min at 5 % B.
• Samples were analyzed in 96-well plate format. A standard curve with 8 standards (5, 10, 15, 20, 50, 75, 90, 100 ⁇ g/ml; 25 ⁇ g/ml IS), standard 0 (0 ⁇ g/ml; 25 ⁇ g/ml IS) and three recovery samples (20, 50, 100 ⁇ g/ml; 25 ⁇ g/ml IS) were prepared for each oligo. Samples related to one oligo were analyzed together on the same plate.
• Samples were processed as follows. A piece of approximately 50 mg of tissue was cut from the respective organ tissue, weighed and placed into the respective well of a 96- deepwell plate. Two steel balls were placed into each well and 500 ⁇ homogenization buffer 20 ⁇ DTT (1 M), 50 ⁇ of proteinase K solution was added. The plate was sealed with STAR lab foils (StarLab E 2796 3070) and samples were homogenized using a Qiagen Tissue Lyzer 3x 30 s at 17 Hz. Subsequently, the plate was incubated in a water bath for 2 hours at 55 °C followed by transfer of the samples to a new 96-deepwell plate using an automated liquid- handling system (TomTec Quadra 3). After the addition of 200 ⁇ ammonium hydroxide (25 %) and 500 ⁇ phenol/chloroform/isoamyl alcohol (25:24: 1) the plate was vortexed using a
• Multitubevortex for 5 min. Subsequently, the plate was incubated for 10 min at 4 °C and centrifuged at 4 °C for another 10 min at 3500 RCF. The plate was then passed to the TomTec system which was used to remove the aqueous layer. The remaining organic layer was washed by adding 500 ⁇ water. The aqueous phase was again removed using the TomTec system. The aqueous phases were combined, 50 ⁇ HCl (1 N), 500 ⁇ SAX Load High buffer (see below) and 300 ⁇ acetonitrile were added, and the resulting solution was mixed thoroughly by up-and- down pipetting using the TomTec system. The program "SPE extraction of tissue samples 100416" was used for the subsequent solid-phase extraction procedure.
• VARIAN Bond Elut 96 square- well SAX 100 mg (Cat. No.: A396081C) were equilibrated with acetonitrile, water and SAX load buffer (see below), samples were loaded and washed with SAX load buffer. The samples were eluted with SAX elute buffer (vide infra) and subsequently diluted with SAX/RP dilution buffer (vide infra).
• WATERS Oasis HLB LP 96- well Plate 60 ⁇ 60 mg (Part No. 186000679) were equilibrated with acetonitrile, water and SAX dilution buffer (see below). The samples were loaded and the cartridge washed with water.
• the samples were eluted with RP elute buffer (vide infra). Freeze the elution plate for 1 hour at - 80 °C and lyophilize to dryness. The dried samples were reconstituted in 50 ⁇ water and dialyzed for 60 min against water using Thermo Slide- A-Lyzer. The samples were then subjected to CGE analysis on a Beckman Coulter PACE/MDQ system. The conditions were: (i) Capillary: eCAP DNA, neutral, 21 cm, 100 ⁇ I.D.
• FIG. 2 illustrates in vitro dimer stability in murine or monkey plasmas and degradation of dimer in liver homogenates as determined by LC-MS.
• Figures 2 A and 2B demonstrate slow degradation of both ApoC3 ASO monomer (SEQ ID NO: l, designated as per Example 2(E)) and cleavable ApoC3-ApoC3 ASO dimers (SEQ ID NO:2 and SEQ ID NO:4) in murine and monkey plasmas respectively.
• Figure 2C demonstrates efficient degradation of the cleavable ApoC3-ApoC3 ASO dimers (SEQ ID NO:2 and SEQ ID NO:4) and the relative stability ApoC3 ASO monomer (SEQ ID NO: l) in mouse liver homogenate.
• Figure 2D shows cleavable SEQ ID NO: 18) and noncleavable SEQ ID NO: 19) ApoB-ApoB ASO homodimers incubated in murine plasma or liver homogenate, demonstrating stability of both types of molecules in plasma, and a more efficient degradation of the cleavable version in the liver homogenate.
• Hep3B Human hepatocarcinoma cells
• DSMZ Deutsche Sammlung von Mirkoorganismen und Zellkulturen GmbH
• Lipofectamine 2000 (L2k) for 48 hr in Earle's Balanced Salt Solution (Lonza, Verviers, Belgium) with L-glutamine (2 mM).
• mRNA levels of target and reference (a housekeeping gene) mRNA was determined by the Quanti Gene Assay (Affymetrix, Santa Clara, CA, USA) according to the manufactures standard protocol. Prior to lysis, cell viability was analyzed by Cell Titer Blue Assay (Promega, Madison, WI, USA). Inactive, scrambled, ASO was used as negative control and reference (SEQ ID NO:31). The QuantiGene 2.0 assay (Affymetrix, Santa Clara, CA) was utilized to measure the expression level of target genes in Hep3B cells before and after the incubation with the ASOs . Human ApoB/ApoC3 probes and housekeeping gene PPIB probes were purchased from Affymetrix.
• Standard assay procedures were carried out according to the manufacturer's recommendations. On the day of harvesting, 200 ⁇ /well of lysis buffer (with 1: 100 protease K) was added to the cells. A total of 60 ⁇ of lysate was used for human ApoC3 probes, while 20 ⁇ lysate was used for human ApoB and PPIB probes respectively. Assay plates were read on the GloRunner Microplate Luminometer (Promega Corp, Sunnyvale, CA). The data were normalized against housekeeping gene PPIB.
• Hep3B cells were treated with 8 consecutive concentrations (0.001, 0.006, 0.03, 0.2, 0.8, 4.0, 20 and 100 nM) of oligonucleotide were formulated with the Lipotransfection agent. mRNA content and cell viability were determined after 48 hr of treatment.
• Example 5 In Vitro Comparisons of Cleavable vs. Noncleavable Linker Designs With ApoC3 Homodimers ( Figures 3B, 3C, 3J, 3K)
• SEQ ID NO: 18 is better compared to the same sequence used as a monomer (SEQ ID NO: 13).
• Figure 31 shows that the ApoB homodimer, if connected via a metabolically unstable linker (SEQ ID NO: 18), is much more effective than its counterpart connected by a stable linker (SEQ ID NO: 19).
• Example 9 In Vitro Activity Assessment By Gymnotic Delivery For Knock-Down Activity Of Cleavable ApoB/ApoC3 Heterodimers With Various Chemical Modifications ( Figures 4C, 4D, 4E, 4F, 4G, 4H, 41, and 4J)
• SEQ ID NO:55 increases in knock-down activity toward both targets.
• the heterodimer (SEQ ID NO:34) assembled from SEQ ID NO: 13 and SEQ ID NO:56 increased in potency in lower concentration only for the ApoB target.
• the heterodimer (SEQ ID NO:35) assembled from SEQ ID NO: 13 and SEQ ID NO:57 increased in potency in lower concentrations for ApoB, while losing activity for ApoC3.
• the heterodimer (SEQ ID NO:36) assembled from SEQ ID NO: 13 and SEQ ID NO:58 increased in knock-down potency in lower concentrations for ApoB, while losing activity for ApoC3.
• SEQ ID NO: 13 and SEQ ID NO:30 showed no modification of knock-down for ApoB, while ApoC3 knock-down activity decreased.
• Example 10 Direct Comparison of Knock-Down Activity Of A Cleavable Hif- lalpha/Survivin Heterodimer Versus Its Parent Monomers Using Gymnotic Delivery (Figure 4K) [00249] Cell culture, knock-down analysis and transfection procedures were performed as described in Example 5. The diagram in Figure 4K depicts that the assembled HiF- la/Survivin heterodimer (SEQ ID NO:23) inherits the individual knock-down potentials of both parent sequences (SEQ ID NOs:27 and 28).
• Example 12 Direct Comparison Knock-Down Activity Of A Cleavable HIF- lalpha/ApoB/ApoC3 Heterotrimers Versus Its Parent Monomers By Using Gymnotic Delivery (Figure 4M)
• Acute in vivo activity assessments were performed in male and female human ApoC3 transgenic mice (Jackson Labs Stock 905918, B6; CBA Tg (APOC3) 3707Bres/J), which are on a C57BL/6 background and express the human apoC3 gene including the human promoter.
• Male (22-30 g) and female mice (20-25 g) employed in this study were 10 weeks old and fed regular chow diet.
• ApoC3 ASO homodimers SEQ ID NOs:4, 5, 2, or 3
• ApoC3 ASO monomer SEQ ID NO: l were formulated in sterile PBS pH7.0 (Gibco) for each dose immediately before subcutaneous (sc) injection. Animals were administered equal volumes (100 ⁇ ) of the homodimers or monomer via sc route between the shoulder blades. A control group was treated using equal volumes of PBS in parallel. Each treatment group consisted of 3 male and 4 female transgenic mice.
• Mouse blood was collected at Day 0 and Day 7 via submandibular puncture (50-75 ⁇ ), as well as at study termination (Day 14) by cardiac puncture, post-euthanasia. Blood was collected in serum separator tubes at room temperature and allowed to clot for 30 minutes. Tubes were spun at 1000 rpm for 5 min at room temperature and serum above separator layer was collected and immediately aliquotted and frozen at -80 °C for future analysis. ApoC3 protein was determined using an ELISA (Wang et al., J. Lipid Res., 2011, 52(6): 1265-71).
• Figure 5A demonstrates an associated increased reduction of liver ApoC3 mRNA levels in human ApoC3 transgenic mice following treatment with the endonuclease-sensitive phosphodiester- linked homodimers (SEQ ID NO:4 and SEQ ID NO:2).
• Human ApoC3 transgenic mice were administered a single subcutaneous dose of homodimers SEQ ID NO:5 or 3, which are disulphide-linked homodimers of the same monomer (each at 10 mg/kg), or vehicle.
• SEQ ID NO:4 and 2 exhibited an increased reduction of liver ApoC3 mRNA levels compared to the monomer (SEQ ID NO: l) after 14 days.
• Figures 5B and 5C show ApoC3 protein knockdown 7 days (Figure 5B) and 14 days (Figure 5C) after a single 10 mg/kg dose of the monomer and dimeric LNA gapmers (SEQ ID NO:4 and 3) in human ApoC3 transgenic mice.
• the 3'3'- phosphodiester-linked dimer with a total of eight phosphodiester linkages (SEQ ID NO:4) shows the fastest onset of knockdown after a single 10 mg/kg dose. This demonstrates that the pharmacokinetic/pharmacodynamic properties can be modulated by selecting a desirable linker.
• Example 14 Biodistribution of Dimers
• SEQ ID NOs:4, 2 and 3 was investigated in mice.
• the cleavage products were analyzed by capillary gel electrophoresis (CGE) which was performed on a PACE/MDQ system (Beckman Coulter) equipped with the Karat 7.0 software (Beckman Coulter). Further parts were: eCAP DNA capillary, neutral, 21 cm, 100 ⁇ I.D. (Beckman # 477477); ssDNA 100 R gel (Beckman # 477621); buffer: Tris/boric acid/EDTA buffer containing 7 M urea (Beckman # 338481).
• cleavage products were further characterized using LC-ESTTOF experiments which were performed on a Bruker Esquire 6000 and an Agilent 1200 HPLC system, together with Waters ACQUITY UPLC OST CI 8 1.7 ⁇ (part # 186003949) column.
• Tissue homogenization was done with a Multi-Tube Vortexer (VWR) and Lysing Matrix D (MP Biomedicals). Plate shaking was done using a VarioMag monoshaker. Deep-well plates were from VWR (2.2 ml, cat. No. 732-0585) and were sealed with Clear seal diamond foil (Thermo, cat. No. 732-4890) prior to tissue homogenization and were resealed for phenol/chloroform-extraction using Re-Seal (3M Empore 98-0604-0472-4 adhesive). Acetonitrile was purchased from Merck.
• Phenol/chloroform/isoamyl alcohol (25:24: 1, P2069-100ML) and dithiothreitol (DTT, cat. No. 43816) were from Sigma, Proteinase K was from Qiagen (cat. No. 19133), Slide-A-lyzer (200 ⁇ , 10 kDa cut-off) were purchased from Fisher Scientific. High-grade 18 MOhm "1 water (Millipore Milli-Q system) was used for reagent and sample preparations. A TomTec Quadra3 system was used for all liquid handling steps.
• Plasma was Na-Citrate plasma from female NMRI mouse (Charles River) K- EDTA plasma from male Cynomolgous monkey (Seralab International).
• each oligo was prepared representing each individual incubation time point (0, 7, 24 and 48 h) in mouse and monkey plasma and in mouse liver homogenate, respectively.
• a blank sample and a recovery sample were prepared of each oligo and incubation matrix.
• plasma samples were prepared by adding 5 ⁇ of the 600 ⁇ oligo stock solution to 95 ⁇ of mouse or monkey plasma, respectively, with a final oligo concentration of 30 ⁇ .
• Recovery samples were prepared by adding 5 ⁇ of water to 95 ⁇ of plasma. Blank samples are oligo in water with a final concentration of 100 ⁇ .
• Liver samples and recoveries were prepared equally; apart from the fact that liver homogenate in PBS was used instead of plasma.
• Samples were analyzed in 96-well plate format. A standard curve with 8 standards (5, 10, 15, 20, 50, 75, 90, 100 ⁇ ; 25 ⁇ IS), a standard 0 (0 ⁇ ; 25 ⁇ IS) and three recovery samples (20, 50, 100 ⁇ ; 25 ⁇ g/ml IS) has been prepared for each oligo. Samples and standards of one particular oligo were analyzed together on the same plate.
• a piece of approximately 50 mg of tissue was cut from the respective organ tissue, weighted and placed into the respective well of a 96-deepwell plate. Two steel balls were placed into each well and 500 ⁇ homogenization buffer 20 ⁇ DTT (1 M), 50 ⁇ of proteinase K solution was added. The plate was sealed with STAR lab foils (StarLab E 2796 3070) and samples are homogenized using a Qiagen Tissue Lyzer for 3x 30 s at 17 Hz. Subsequently the plate was incubated in a water bath for 2 h at 55 °C followed by transfer of the samples to a new 96-deepwell plate using an automated liquid-handling system (TomTec Quadra 3).
• the plate was vortexed using a Multitubevortex for 5 min. Subsequently, the plate was incubated for 10 min at 4 °C and centrifuged at 4 °C for another 10 min at 3500 RCF. The plate was then handled to the TomTec System which was used to remove the aqueous layer. The remaining organic layer was washed by adding 500 ⁇ water. The aqueous phase was again removed using the TomTec system.
• the aqueous phases were combined, 50 ⁇ HC1 (1 N), 500 ⁇ SAX Load High buffer (vide infra) and 300 ⁇ acetonitrile was added and the resulting solution was mixed thoroughly by up and down pipetting using the TomTec system. ( The program "SPE extraction of tissue samples 100416" was used for the subsequent solid-phase extraction procedure).
• A396081C were equilibrated with acetonitrile, water and SAX load buffer (see below), samples were load and washed with SAX load buffer.
• the samples were eluted with SAX elute buffer (vide infra) and subsequently diluted with SAX/RP dilution buffer (vide infra).
• WATERS Oasis HLB LP 96-well Plate 60 ⁇ 60 mg (Part No.: 186000679) were equilibrated with acetonitrile, water and SAX dilution buffer (vide infra). The samples were load and the cartridge washed with water. The samples were eluted with RP elute buffer (vide infra).
• the sample was centrifuged at 3500 RFC for 20 min at 4 °C and 400 ⁇ of the aqueous layer were removed and dried in a lyophilizer.
• the dried samples were dissolved in water (100 ⁇ ).
• the recovery samples were dissolved in water (95 ⁇ ) and spiked with 5 ⁇ of the respective oligo stock solution (600 ⁇ ).
• Samples were analyzed by LC-MS (Agilent 1200, Bruker Esquire 3000) using a Waters Acquity UPLC OST C18 column (1.7 ⁇ , 2.1x50) with HFIP/TEA/water (385 mM 1,1,1,3,3,3- hexafluoroisopropanol, 14.4 mM triethylamine in water) as buffer A and methanol as buffer B and a flow rate of 0.3 ml/min at a column temperature of 60 °C.
• LC-MS Waters Acquity UPLC OST C18 column (1.7 ⁇ , 2.1x50) with HFIP/TEA/water (385 mM 1,1,1,3,3,3- hexafluoroisopropanol, 14.4 mM triethylamine in water) as buffer A and methanol as buffer B and a flow rate of 0.3 ml/min at a column temperature of 60 °C.
• the following gradient was used: 3 min at 5% B, 5-15% B in 2.5 min (10%/min), 15-23% B in 5.5 min, 23-30% B in 3 min, 30-100% B in 0.5 min, 5 min at 100 % B, 100-5% B in 0.5 min, 5 min at 5 % B.
• Table 5 shows organ-distribution of antisense dimers SEQ ID NO:2, 3 and 4 as percent of total administered dose 24 hrs after a single i.v. bolus injection into mice.
• Peak 1 refers to the degradation product, whereas Peak 2 is remaining dimer starting material. The sum of both components represents the percentage of total dose in the corresponding organ.
• Organ-distribution of monomeric SEQ ID NO: 1 as percent of total administered in mice and monkeys in previous studies as compared to the dimers (last row) is shown Table 6. Percent total dose calculation based on: 5 kg monkey, 135 g liver, 30 g kidney (Davies et al., Pharm. Res., 1993, 10 (7): 1093). Table 5
• the monomeric cleavage product was slightly larger than monomer resulting from reductive disulfide cleavage and is indicated as "#1 plus X" in the figure, where X is a yet unidentified organic radical with the molecular weight of less 100 Da. It could be hypothesized that that "#1 plus X" results from oxidative cleavage rather than reductive cleavage of the disulfide bond. If the dimers had been already cleaved in the serum, the bio-distribution to liver and kidneys should not have increased so dramatically as compared to monomers. Thus, the stability of the dimers in plasma and in liver homogenates was investigated. It was demonstrated in Example 3 that plasma stability of the dimers is relatively high over 48 hours, while the dimers are rapidly cleaved in liver
• Example 15 In Vivo Activity Assessment of a Cleavable ApoC3/ApoB ASO Heterodimer
• the ApoC3/ApoB ASO heterodimer (SEQ ID NO: 21) or a non-targeting ASO (SEQ ID NO: 119) were formulated in sterile saline (pH7.0) immediately before intravenous (iv) injection via the tail vein. Animals were administered heterodimer (0.3, 1, 3, or 10 mg/kg) or negative control ASO (10 mg/kg) or saline (0 mg/kg) as a vehicle control in a volume of 5 ml/kg.
• mice Groups of mice consisted of 2 male and 2 female transgenic mice which were terminated on days 1, 3, 7, 14 and 29 after treatment administration. After euthanasia by C0 2 inhalation, blood was obtained by cardiac puncture (0.5-1 ml). Livers were dissected, weighed, and a fragment saved in a labeled histology cassette snap frozen by immersion in liquid nitrogen. Liver samples were maintained at -80°C for subsequent analyses. [00273] Each blood sample was divided in half. Serum was prepared in serum separator tubes which were allowed to clot for 4 hours on ice. Plasma was prepared in EDTA-containing tubes which were maintained on ice until processed. Tubes were spun at 10,000 rpm for 5 min at 4°C and supernatants collected and frozen at -80°C for future analyses.
• Samples were amplified as per manufacturer's instructions (Qiagen, Quantitect Probe RT-PCR kit). Quantitative real-time PCR (qRT-PCR) was performed in a 7900HT Fast Real-Time PCR System (Applied Biosystems). All samples were analyzed in triplicate in Microamp Optical 384well reaction plates (Applied Biosystems) and normalized with Gapdh signal as the internal control. Primers were Apolipoprotein C-III (Applied Biosystems, Mm00445670_ml and Hs00163644_ml), Apolipoprotein B (Applied Biosystems,
• Results are expressed as fold induction relative to vehicle-treated samples.
• An ApoC3/ApoB ASO heterodimer linked with four diester bases (cleavable; SEQ ID NO: 21), or an ApoC3/ApoB ASO heterodimer linked with four phosphothioate bases (stable; SEQ ID NO: 59), or an ApoC3/ApoB ASO heterodimer linked with PEG-6 (stable; SEQ ID NO: 60), or the ApoC3 monomer ASO (SEQ ID NO: 30), or the ApoB monomer ASO (SEQ ID NO: 13), or the physical combination of the ApoC3 and ApoB monomers (SEQ ID NO: 30 plus SEQ ID NO: 13), or a non-targeting ASO (SEQ ID NO: 119) were formulated in sterile SALINE (pH7.0) immediately before intravenous (iv) injection via the tail vein.
• Groups consisted of 6-7 male transgenic mice which were terminated 3 or 14 days after treatment administration. After euthanasia by CO 2 inhalation, blood was obtained by cardiac puncture (0.5-1 ml). Livers were dissected, weighed, and a fragment put in a labeled histology cassette snap frozen by immersion in liquid nitrogen. Whole kidneys were also stored in labeled histology cassettes and snap frozen in liquid nitrogen. Liver and kidney samples were maintained at -80°C for subsequent analyses.
• Serum was prepared in serum separator tubes which were allowed to clot for 4 hours on ice.
• Plasma was prepared in EDTA-containing tubes which were maintained on ice until processed. Tubes were spun at 10,000 rpm for 5 min at 4°C and supernatants collected and frozen at -80°C for future analyses.
• Hybridization assays were developed (see below) to measure the tissue concentrations of the ApoC3/ApoB ASO heterodimer linked with four diester bases (cleavable; SEQ ID NO: 21), the ApoC3/ApoB ASO heterodimer linked with four phosphothioate bases (stable; SEQ ID NO: 59), and the ApoB monomer ASO (SEQ ID NO: 13) in plasma and homogenates of liver and kidney. Samples from the experiment described in Example 16 were measured.
• the detection probes contain Spacer- 18s at the 3 '-end of the specific probe sequence and were biotin labeled at the 3 '-end (Elfer et al., 2005).
• the specific sequences of capture and detection probes used in the assays are showed in table below.
• the oligonucleotide was cleaved from solid support using a solution of ammonium hydroxide and ethanol (3: 1) at 55 * C for 17 hours.
• the crude oligonucleotides were purified in a two-step IEX-purification procedure using a Source 30Q column and buffer system containing sodium hydroxide.
• the mass spectrometer analysis was done using ESTMS and the purity was established using HPLC and generic method.
• the endotoxin levels were measured using LAL-test procedure.
• the oligonucleotide was cleaved from solid support using a solution of ammonium hydroxide and ethanol (3: 1) at 55 * C for 17 hours.
• the crude oligonucleotides were purified in a two-step RP- / IEX - purification procedure.
• the RP-purification was by applying a TEAA-containing buffer system, the IEX purification was carried out at physiological conditions.
• the mass spectrometer analysis was done using ESTMS and the purity was established using HPLC and generic method.
• Liver and kidney homogenate was prepared from animals treated with heterodimeric or monomeric ASOs. Tissue samples collected at specified time points were minced and weighed in ready-to-use Lysing Matrix D tubes containing 1.4 mm ceramic spheres beads (Catalogue* 6913-100, MP Biomedicals). DNase/RNAse free water (Catalogue # SH30538.02, Thermo) was added to the tube with ratio of 5 or 10 mL per g of tissue. Each tissue sample was mixed and homogenized using a MP Biomedicals Fast Prep-24 at 4 °C for 20 seconds twice. The tissue homogenate was stored in freezer or kept on ice before analyzed with the hybridization assay.
• DNA-Bind plates (96-well) (Catalogue #2505, Costar) were coated overnight at 4 °C with 100 of 50 nM capture probes in HEPES/lmM Na 2 EDTA buffer. The plates were then washed three times with wash buffer (Tris Buffer/0.1% Tween 20) and incubated in blocking buffer (PBS/3% BSA) for 1-2 hrs.
• ApoC3/ApoB ASO heterodimer linked with four diester bases SEQ ID NO: 21
• ApoC3/ApoB ASO heterodimer linked with four phosphothioate bases stable; SEQ ID NO: 59).
• heterodimer ASOs and the ApoB monomer were measured using the methods above in 2 pools of 3 individuals each after treatment with the ApoC3/ApoB ASO heterodimer linked with four diester bases (cleavable; SEQ ID NO: 21), the ApoC3/ApoB ASO heterodimer linked with four phosphothioate bases (stable; SEQ ID NO: 59), the ApoB monomer ASO (SEQ ID NO: 13) or the physical combination of the ApoC3 and ApoB monomer ASOs (SEQ ID NO: 30 plus SEQ ID NO: 13). As shown in Figure 10, both heterodimer ASOs were detected in plasma 3 days post-treatment.
• ApoB monomer was also detected 3 days after treatment with the ApoB monomer ASO alone or in physical combination with the ApoC3 monomer.
• ApoB monomer ASO was detected as a metabolite of the ApoC3/ApoB ASO heterodimer linked with four diester bases (cleavable; SEQ ID NO: 21) 3 days after treatment, but not after administration of the ApoC3/ApoB ASO heterodimer linked with four phosphothioate bases (stable; SEQ ID NO: 59), demonstrating that the endonuclease sensitive linker resulted in enhanced metabolism to active ASO monomers. None of the analytes were detected in plasma pools taken 14 days after treatment.
• the concentration of ApoB monomer present in the liver as a metabolite after heterodimer administration was significantly higher after administration of the ApoC3/ApoB ASO heterodimer linked with four phosphothioate bases (stable; SEQ ID NO: 59) than the ApoC3/ApoB ASO heterodimer linked with four diester bases (cleavable; SEQ ID NO: 21). Since the phosphothioate linked heterodimer also degrades in tissue, albeit at a slower rate, relatively more monomer is present at this time point.
• the levels of ApoB monomer, after its administration or as a metabolite of administered ASO heterodimers is related to target mRNA knockdown, e.g., the highest levels of ApoB monomer are present after administration of the ApoC3/ApoB ASO heterodimer linked with four diester bases (cleavable; SEQ ID NO: 21) and the highest level of target mRNA knockdown is also observed in this treatment group (compare Figures 12A and 12B to Figures 8A and 8B)
• Oligo sequences [00299] 15-mer gapmer oligos were designed as single monomers or as homodimers (30-mers) linked by an oligo-dT linker (4 bases) via cleavable phosphodiester bonds.
• the oligos were designed to target either miR-122 or MALAT-1 and consisted of three LNA-modified bases at each end of the monomer with 9 unmodified DNA bases in the center or gap region.
• the gapmer design facilitated cleavage of the bound target mRNA by RNAseH resulting in a decrease in target mRNA (either miR-122 or MALAT-1).
• the sequences of the following table correspond, from top to bottom, to SEQ ID NOS: 128 to 135.
• mice Female C57BL6/J mice were obtained from the Jackson
• mice Female Balb/C mice obtained from the Charles River Laboratories. Oligonucleotides were dissolved in phosphate buffered saline (PBS) and administered to mice based on body weight by subcutaneous injection. Mice were injected once per week (MALAT-1) at 50mg/kg or twice per week (MIR-122) at lOmg/kg or 50mg/kg. Mice were sacrificed after one week and at study termination (four weeks) and liver, kidney and plasma harvested for further analysis.
• PBS phosphate buffered saline
• Triglycerides HDL and total cholesterol measurements.
• Plasma concentrations of Triglycerides, total cholesterol and LDL cholesterol were determined by enzymatic assay (Bioo Scientific) on a Molecular Devices SpectraMax M5 plate reader according to manufacturer's recommendations.
• RNA extraction, reverse transcription and mRNA qPCR RNA extraction, reverse transcription and mRNA qPCR.
• MPBio FastPrep-24 tissue homogenizer
• Trizol Invitrogen
• miRNEasy columns Qiagen
• RNA concentration was assessed using RIboGreen plates (Molecular Probes) and a Molecular Dynamics M5 multimodal plate reader. 250 ng of total RNA was reverse transcribed with random hexamers
• oligonucleotides targeting miR-122 decreased target miRNA in vivo by 75-90% compared to PBS treated controls. Monomers exhibited 75% knockdown of miR-122; whereas dimers caused 90% knockdown of miR-122.

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