WO2014201413A1 - Composés et procédés permettant de moduler l'arn non codant - Google Patents

Composés et procédés permettant de moduler l'arn non codant Download PDF

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Publication number
WO2014201413A1
WO2014201413A1 PCT/US2014/042401 US2014042401W WO2014201413A1 WO 2014201413 A1 WO2014201413 A1 WO 2014201413A1 US 2014042401 W US2014042401 W US 2014042401W WO 2014201413 A1 WO2014201413 A1 WO 2014201413A1
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Prior art keywords
antisense compound
modified
certain embodiments
simtron
nucleosides
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PCT/US2014/042401
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English (en)
Inventor
Mallory A. HAVENS
Frank Rigo
Michelle L. HASTINGS
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Isis Pharmaceuticals, Inc.
Rosalind Franklin University Of Medicine And Science
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Publication of WO2014201413A1 publication Critical patent/WO2014201413A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs

Definitions

  • Sequence Listing is provided as a file entitled CORE0117WOSEQ_ST25.txt, created June 13, 2014, which is 204 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
  • Antisense compounds have been used to modulate target nucleic acids. Antisense compounds comprising a variety of chemical modifications and motifs have been reported. In certain instances, such compounds are useful as research tools, diagnostic reagents, and as therapeutic agents. In certain instances antisense compounds have been shown to modulate protein expression by binding to a target messenger RNA (mRNA) encoding the protein. In certain instances, such binding of an antisense compound to its target mRNA results in cleavage of the mRNA. Antisense compounds that modulate processing of a pre -mRNA have also been reported. Such antisense compounds alter splicing, interfere with polyadenlyation or prevent formation of the 5 '-cap of a pre -mRNA. SUMMARY OF THE INVENTION
  • the present disclosure provides compounds and methods for modulating a non-coding RNA.
  • Embodiment 1 A method of modulating the amount or activity of a simtron in a cell comprising contacting the cell with an antisense compound complementary to a simtron precursor and thereby modulating the amount or activity of the simtron in the cell.
  • Embodiment 2 The method of embodiment 1, wherein the amount or activity of the simtron is increased.
  • Embodiment 3 The method of embodiment 1, wherein the amount or activity of the simtron is decreased.
  • Embodiment 4 The method of any of embodiments 1-3, wherein the antisense compound is complementary to a portion of a host transcript encoding the simtron.
  • Embodiment 5 The method of embodiment 4, wherein the antisense compound is complementary to a portion of an intron encoding the simtron.
  • Embodiment 6 The embodiment of claim 4 or 5, wherein the antisense compound is complementary to an SRSF5 binding site.
  • Embodiment 7 The method of any of embodiments 1 -6, wherein splicing of the host transcript is not affected by contacting the cell with the antisense compound.
  • Embodiment 8 The method of any of embodiments 1 -7, wherein expression of the host protein is not altered by contacting the cell with the antisense compound.
  • Embodiment 9. The method of any of embodiments 1-6, wherein splicing of the synthetic intron is not affected by contacting the cell with the antisense compound.
  • Embodiment 10 The method of any of embodiments 1-9, wherein the simtron precursor comprises at least one mutation.
  • Embodiment 11 The method of embodiment 10, wherein the simtron precursor comprises at least one point mutation.
  • Embodiment 12 The method of embodiment 11, wherein the antisense compound is complementary to a region of the simtron precursor that comprises at least one point mutation.
  • Embodiment 13 The method of any of embodiments 1-12, wherein hybridization of the antisense compound to the simtron precursor results in reduced SRSF5 activity at the simtron precursor.
  • Embodiment 14 The method of any of embodiments 1-12, wherein hybridization of the antisense compound to the simtron precursor results in increased SRSF5 activity at the simtron precursor.
  • Embodiment 15 The method of any of embodiments 1-14, wherein hybridization of the antisense compound to the simtron precursor results in recruitment of SRSF5 to the simtron precursor.
  • Embodiment 16 The method of any of embodiments 1-15, wherein the simtron is miR-1225.
  • Embodiment 17 The method of any of embodiments 1-16, wherein the host transcript encodes PKDl .
  • Embodiment 18 The method of any of embodiments 1-17, wherein the antisense compound comprises an antisense oligonucleotide comprising at least one modified nucleoside.
  • Embodiment 19 The method of embodiment 15, wherein the antisense oligonucleotide comprises at least one modified nucleoside comprising a 2'-MOE modification.
  • Embodiment 20 The method of embodiment 19, wherein each nucleoside of the antisense oligonucleotide comprises a 2'-MOE modification.
  • Embodiment 21 The method of any of embodiments 1-20, wherein the cell is in vitro.
  • Embodiment 22 The method of any of embodiments 1-21, wherein the cell is in an animal.
  • Embodiment 23 The method of embodiment 22 wherein the animal is a human.
  • Embodiment 24 The method of embodiment 23, wherein the human has kidney disease.
  • Embodiment 25 The method of embodiment 24, wherein the human has autosomal dominant polycystic kidney disease.
  • Embodiment 26 A method of identifying one or more simtron comprising isolating nucleic acids associated with SRSF5 from a cell or cell extract and identifying the nucleic acids.
  • Figure 1 Quantification of PKDl and miR-1225 RNA expression in various human tissues
  • Figure 2 Quantification of PKDl and miR-1225 RNA expression over time in mouse kidney
  • Figure 3 Effect of altering SRSF5 expression on PKDl splicing products
  • miR-1225 Figure 4: Quantification of PKD1 and miR-1225 RNA expression in cells treated with ADPKD patient minigenes containing exons 43 through 46 and intervening introns
  • Figure 5 Quantification of pri-miRNA and pre-miRNA from a cell-free in vitro processing assay using RNA transcribed from a PKD1 WT or IVS45-14T>C DNA template
  • Figure 6 Targeting human PKD1 intron 45/miR-1225 using ASOs
  • Figure 7 Targeting mouse PKD1 intron 45/miR-1225 using ASOs.
  • Figure 8 Effect of in vivo treatment with antisense oligonucleotides targeting PKD1 intron 45 on miR-1225 abundance
  • Figure 9 Effect of in vivo treatment with antisense oligonucleotides targeting PKD1 intron 45 on miR-1225 cleavage sites
  • FIG. 10 A summary of the regulation of miR-1225 biogenesis.
  • nucleoside means a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.
  • chemical modification means a chemical difference in a compound when compared to a naturally occurring counterpart.
  • chemical modification does not include differences only in nucleobase sequence.
  • Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications.
  • furanosyl means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.
  • naturally occurring sugar moiety means a ribofuranosyl as found in naturally occurring RNA or a deoxyribofuranosyl as found in naturally occurring DNA.
  • sugar moiety means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.
  • modified sugar moiety means a substituted sugar moiety or a sugar surrogate.
  • substituted sugar moiety means a furanosyl that is not a naturally occurring sugar moiety.
  • Substituted sugar moieties include, but are not limited to furanosyls comprising modifications at the 2'-position, the 5'-position and/or the 4'-position. Certain substituted sugar moieties are bicyclic sugar moieties.
  • MOE means -OCH 2 CH 2 OCH 3 .
  • sucrose surrogate means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside is capable of (1) incorporation into an oligonucleotide and (2) hybridization to a complementary nucleoside.
  • Such structures include relatively simple changes to the furanosyl, such as rings comprising a different number of atoms (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of the furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen.
  • Such structures may also comprise substitutions corresponding with those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents).
  • Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid).
  • Sugar surrogates include without limitation morpholinos, cyclohexenyls and cyclohexitols.
  • bicyclic sugar moiety means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure.
  • the 4 to 7 membered ring is a sugar ring.
  • the 4 to 7 membered ring is a furanosyl.
  • the bridge connects the 2'-carbon and the 4'-carbon of the furanosyl.
  • nucleotide means a nucleoside further comprising a phosphate linking group.
  • linked nucleosides may or may not be linked by phosphate linkages and thus includes, but is not limited to “linked nucleotides.”
  • nucleobase means group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of nucleobase atoms are capable of bonding with a complementary nucleobase of another oligonucleotide or nucleic acid. Nucleobases may be naturally occurring or may be modified.
  • heterocyclic base or “heterocyclic nucleobase” means a nucleobase comprising a heterocyclic structure.
  • unmodified nucleobase or “naturally occurring nucleobase” means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).
  • modified nucleobase means any nucleobase that is not an unmodified nucleobase.
  • modified nucleoside means a nucleoside comprising at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides comprise a modified sugar moiety and/or a modified nucleobase.
  • bicyclic nucleoside or "BNA” means a nucleoside comprising a bicyclic sugar moiety.
  • oligonucleotide means a compound comprising a plurality of linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified
  • RNA ribonucleosides
  • DNA deoxyribonucleosides
  • oligonucleoside means an oligonucleotide in which none of the internucleoside linkages contains a phosphorus atom.
  • oligonucleotides include oligonucleosides.
  • modified oligonucleotide means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.
  • nucleoside linkage means a covalent linkage between adjacent nucleosides in an oligonucleotide.
  • naturally occurring internucleoside linkage means a 3' to 5' phosphodiester linkage.
  • modified internucleoside linkage means any internucleoside linkage other than a naturally occurring internucleoside linkage.
  • oligomeric compound means a polymeric structure comprising two or more sub-structures.
  • an oligomeric compound comprises an oligonucleotide.
  • an oligomeric compound comprises one or more conjugate groups and/or terminal groups.
  • an oligomeric compound consists of an oligonucleotide.
  • conjugate means an atom or group of atoms bound to an oligonucleotide or oligomeric compound.
  • conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and clearance.
  • antisense compound means a compound comprising or consisting of an oligonucleotide at least a portion of which is at least partially complementary to a target nucleic acid to which it is capable of hybridizing, resulting in at least one antisense activity.
  • antisense activity means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid.
  • detecting or “measuring” means that a test or assay for detecting or measuring is performed. Such detection and/or measuring may result in a value of zero. Thus, if a test for detection or measuring results in a finding of no activity (activity of zero), the step of detecting or measuring the activity has nevertheless been performed.
  • detecttable and/or measureable activity means an activity that is not zero.
  • essentially unchanged means little or no change in a particular parameter, particularly relative to another parameter which changes much more.
  • a parameter is essentially unchanged when it changes less than 5%.
  • a parameter is essentially unchanged if it changes less than two-fold while another parameter changes at least ten-fold.
  • an antisense activity is a change in the amount of a target nucleic acid.
  • the amount of a non-target nucleic acid is essentially unchanged if it changes much less than the target nucleic acid does, but need not be zero.
  • expression means the process by which a gene ultimately results in a protein.
  • Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenlyation, addition of 5 '-cap), and translation.
  • an oligonucleotide that recruits a protein to a target region of a target nucleic acid causes the protein to become in association with the target region of the target nucleic acid where the protein would not otherwise be in association with that region of that nucleic acid.
  • An oligonucleotide capable of recruiting a protein does not include an oligonucleotide to which a protein has been covalently bound.
  • “recruited protein” means a protein that becomes associated with an antisense compound/target nucleic acid duplex. Typically, recruited proteins are endogenous cellular proteins.
  • recruiting nucleoside means a modified nucleoside which, when incorporated into an oligonucleotide is capable of recruiting a protein.
  • a contiguous region of 4 recruiting nucleosides is required for recruitment.
  • a contiguous region of 5 recruiting nucleosides is required for recruitment.
  • a contiguous region of 6 recruiting nucleosides is required for recruitment.
  • a contiguous region of 7 recruiting nucleosides is required for recruitment.
  • a contiguous region of 8 recruiting nucleosides is required for recruitment.
  • high affinity nucleoside means a nucleoside which, when incorporated into an oligonucleotide, increases the affinity of the oligonucleotide for a nucleic acid target, compared to an unmodified nucleoside.
  • stabilizing nucleoside means a nucleoside that is resistant to degradation, including, but not limited to nuclease degradation, compared to unmodified nucleases.
  • duplex stabilizing nucleoside means a nucleoside that is either a high affinity nucleoside or stabilizing nucleoside or both a high affinity nucleoside and stabilizing nucleoside.
  • non-cleaving nucleic acid binding protein means a protein capable of being recruited to a target nucleic acid/oligonucleotide duplex that does not cleave either strand of that duplex.
  • RNase H and Ago2 are not non-cleaving nucleic acid binding proteins.
  • target nucleic acid means a nucleic acid molecule to which an antisense compound hybridizes.
  • mRNA means an RNA molecule that encodes a protein.
  • pre-mRNA means an RNA transcript that has not been fully processed into mRNA. Pre -RNA includes one or more intron.
  • exon means a portion of a pre-mRNA which, after splicing, is typically included in the mature mRNA.
  • intron means a portion of a pre-mRNA which, after splicing, is typically excluded in the mature mRNA or non-coding RNA.
  • microRNA means a naturally occurring, small, non-coding RNA that represses gene expression of at least one mRNA.
  • a microRNA represses gene expression by binding to a target site within a 3 ' untranslated region of an mRNA.
  • a microRNA has a nucleobase sequence as set forth in miRBase, a database of published microRNA sequences found at http://microrna.sanger.ac.uk/sequences/.
  • a microRNA has a nucleobase sequence as set forth in miRBase version 12.0 released September 2008, which is herein incorporated by reference in its entirety.
  • non-canonical microRNA means a microRNA that is processed by a non- canonical pathway not dependent on microprocessor, as described in Havens et. Al., Nucleic Acid Research, 2012, Vol. 40, No. 10 4626-4640.
  • mirtron means a non-canonical microRNA that is defined by the entire length of the intron in which it is located, and require pre-mRNA splicing rather than the microprocessor for the first step in biogenesis.
  • Mirtrons are splicing-dependent microRNAs where spliceosome-excised introns direct Dicer substrates.
  • Simtron means a non-canonical microRNA that does not require splicing for genesis. Simtron biogenesis does not depend on DGCR98, Dicer, Exportin-5, or Argonaute 2.
  • precursor-microRNA means a transcript or portion thereof comprising a microRNA.
  • RNA transcript means an RNA transcript comprising a precursor RNA and a coding or non-coding RNA.
  • host protein means a protein encoded by a host transcript.
  • a human PKDl nucleic acid means a nucleic acid encoding human PKDl .
  • Examples of a human PKDl nucleic acid include without limitation nucleotides 2077700 to 2126900 of GENBANK Accession No: NT 010393.16 (herein SEQ ID NO: 1), GENBANK Accession No:
  • NM 001009944.2 (herein SEQ ID NO: 2), GENBANK Accession No: NM 000296.3 (herein SEQ ID NO: 3), and GENBANK Accession No: L33243.1 (herein SEQ ID NO: 4).
  • a mouse PKDl nucleic acid means a nucleic acid encoding human PKDl .
  • Examples of a mouse PKDl nucleic acid include without limitation nucleotides 10894338 to 10942902 of GENABANK Accession No: NT 039649.8 (herein SEQ ID NO: 5) and GENBANK Accession no: NM 013630.2 (herein SEQ ID NO: 6).
  • targeting means the association of an antisense compound to a particular target nucleic acid molecule or a particular region of nucleotides within a target nucleic acid molecule.
  • An antisense compound targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to allow hybridization under physiological conditions.
  • nucleobase complementarity or “complementarity” when in reference to nucleobases means a nucleobase that is capable of base pairing with another nucleobase.
  • adenine (A) is complementary to thymine (T).
  • T thymine
  • adenine (A) is
  • complementary nucleobase means a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair. Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.
  • non-complementary in reference to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.
  • oligomeric compounds e.g., linked nucleosides, oligonucleotides, or nucleic acids
  • complementary means the capacity of such oligomeric compounds or regions thereof to hybridize to another oligomeric compound or region thereof through nucleobase complementarity.
  • hybridization means the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
  • oligomeric compound specifically hybridizes to more than one target site.
  • oligonucleotide or portion thereof means that each nucleobase of the oligonucleotide or portion thereof is capable of pairing with a nucleobase of a complementary nucleic acid or contiguous portion thereof.
  • fully complementary region comprises no mismatches or unhybridized nucleobases in either strand.
  • percent complementarity means the percentage of nucleobases of an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound.
  • percent identity means the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.
  • modulation means a perturbation of amount or quality of a function or activity when compared to the function or activity prior to modulation.
  • modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression.
  • modulation of expression can include perturbing splice site selection of pre-mRNA processing, resulting in a change in the amount of a particular splice-variant present compared to conditions that were not perturbed.
  • modulation includes perturbing translation of a protein.
  • motif means a pattern of chemical modifications in an oligomeric compound or a region thereof. Motifs may be defined by modifications at certain nucleosides and/or at certain linking groups of an oligomeric compound.
  • nucleoside motif means a pattern of nucleoside modifications in an oligomeric compound or a region thereof.
  • the linkages of such an oligomeric compound may be modified or unmodified.
  • motifs herein describing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited.
  • linkage motif means a pattern of linkage modifications in an oligomeric compound or region thereof.
  • the nucleosides of such an oligomeric compound may be modified or unmodified.
  • motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited.
  • nucleobase modification motif means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, a nucleobase modification motif is independent of the nucleobase sequence.
  • sequence motif means a pattern of nucleobases arranged along an
  • oligonucleotide or portion thereof Unless otherwise indicated, a sequence motif is independent of chemical modifications.
  • nucleoside having a modification of a first type may be an unmodified nucleoside.
  • telomeres As used herein, “differently modified” mean chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are “differently modified,” even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are “differently modified,” even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified, unless otherwise indicated.
  • nucleoside comprising a 2'-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2'-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.
  • the same type of modifications refers to modifications that are the same as one another, including absence of modifications.
  • two unmodified DNA nucleoside have “the same type of modification,” even though the DNA nucleoside is unmodified.
  • Such nucleosides having the same type modification may comprise different nucleobases.
  • separate regions means a portion of an oligonucleotide wherein the chemical modifications or the motif of chemical modifications within the portion are the same and the chemical modifications or motif of chemical modifications of any neighboring portions include at least one difference to allow the separate regions to be distinguished from one another.
  • pharmaceutically acceptable carrier or diluent means any substance suitable for use in administering to an animal.
  • a pharmaceutically acceptable carrier or diluent is sterile saline.
  • such sterile saline is pharmaceutical grade saline.
  • substituted nucleoside and “substituent group,” means an atom or group that replaces the atom or group of a named parent compound.
  • a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e.g., a modified 2'-substuent is any atom or group at the 2'-position of a nucleoside other than H or OH).
  • Substituent groups can be protected or unprotected.
  • compounds of the present invention have substituents at one or at more than one position of the parent compound.
  • Substituents may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound.
  • substituted in reference to a chemical functional group means an atom or group of atoms differs from the atom or a group of atoms normally present in the named functional group.
  • a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen which replaces one of the hydrogen atoms of an unsubstituted methyl group).
  • each R aa , R bb and R cc is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree.
  • recursive substituent means that a substituent may recite another instance of itself. Because of the recursive nature of such substituents, theoretically, a large number may be present in any given claim.
  • One of ordinary skill in the art of medicinal chemistry and organic chemistry understands that the total number of such substituents is reasonably limited by the desired properties of the compound intended. Such properties include, by way of example and not limitation, physical properties such as molecular weight, solubility or log P, application properties such as activity against the intended target and practical properties such as ease of synthesis.
  • Recursive substituents are an intended aspect of the invention.
  • One of ordinary skill in the art of medicinal and organic chemistry understands the versatility of such substituents.
  • stable compound and “stable structure” mean a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an therapeutic agent.
  • alkyl means a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms.
  • alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.
  • Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C 1-C12 alkyl) with from 1 to about 6 carbon atoms being more preferred.
  • the term "lower alkyl” as used herein includes from 1 to about 6 carbon atoms.
  • Alkyl groups as used herein may optionally include one or more further substituent groups.
  • alkenyl means a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond.
  • alkenyl groups include without limitation, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl, dienes such as 1,3- butadiene and the like.
  • Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred.
  • Alkenyl groups as used herein may optionally include one or more further substituent groups.
  • alkynyl means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond.
  • alkynyl groups include, without limitation, ethynyl, 1 -propynyl, 1 -butynyl, and the like.
  • Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred.
  • Alkynyl groups as used herein may optionally include one or more further substituent groups.
  • acyl means a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula -C(0)-X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.
  • alicyclic means a cyclic ring system wherein the ring is aliphatic.
  • the ring system can comprise one or more rings wherein at least one ring is aliphatic.
  • Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring.
  • Alicyclic as used herein may optionally include further substituent groups.
  • aliphatic means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond.
  • An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred.
  • the straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus.
  • Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups.
  • alkoxy means a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule.
  • alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert- butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like.
  • Alkoxy groups as used herein may optionally include further substituent groups.
  • aminoalkyl means an amino substituted C1-C12 alkyl radical.
  • the alkyl portion of the radical forms a covalent bond with a parent molecule.
  • the amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.
  • aralkyl and arylalkyl mean an aromatic group that is covalently linked to a C1-C12 alkyl radical.
  • the alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like.
  • Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.
  • aryl and aromatic mean a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings.
  • aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like.
  • Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings.
  • Aryl groups as used herein may optionally include further substituent groups.
  • halo and halogen mean an atom selected from fluorine, chlorine, bromine and iodine.
  • heteroaryl and “heteroaromatic,” mean a radical comprising a mono- or poly- cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen.
  • heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like.
  • Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom.
  • Heteroaryl groups as used herein may optionally include further substituent groups.
  • the present invention provides antisense compounds.
  • such antisense compounds comprise an antisense oligonucleotide.
  • such antisense compounds comprise an antisense oligonucleotide and one or more conjugates.
  • antisense oligonucleotides comprise one or more chemical modification. Such chemical modifications include modifications one or more nucleoside (including modifications to the sugar moiety and/or the nucleobase) and/or modifications to one or more internucleoside linkage.
  • antisense oligonucleotides of the invention comprise one or more modifed nucleoside comprising a modifed sugar moiety.
  • Such antisense oligonucleotide comprising one or more sugar-modified nucleoside may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to oligomeric compounds comprising only nucleosides comprising naturally occurring sugar moieties.
  • modified sugar moieties are substitued sugar moieties.
  • modified sugar moieties are sugar surrogates. Such sugar surogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.
  • modified sugar moieties are substituted sugar moieties comprising one or more non-bridging sugar substituent, including but not limited to substituents at the 2' and/or 5' positions.
  • sugar substituents suitable for the 2'-position include, but are not limited to: 2'- F, 2'-OCH 3 ("OMe” or "O-methyl"), and 2'-0(CH 2 ) 2 OCH 3 (“MOE").
  • sugar substituents at the 5'-position include, but are not limited to:, 5'-methyl (R or S); 5'-vinyl, and 5'- methoxy.
  • substituted sugars comprise more than one non-bridging sugar substituent, for example, 2'-F-5'-methyl sugar moieties (see,Q.g., PCT International Application WO 2008/101157, for additional 5', 2'-bis substituted sugar moieties and nucleosides).
  • Nucleosides comprising 2'-modifed sugar moieties are referred to as 2'-modifed nucleosides.
  • These 2'-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (N0 2 ), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
  • a 2'-modified nucleoside comprises a sugar moiety comprising a 2'- substituent group selected from F, 0-CH 3 , and OCH 2 CH 2 OCH 3 .
  • Certain substituted sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety.
  • the bicyclic sugar moiety comprises a bridge between the 4' and the 2' furanose ring atoms.
  • Examples of such 4' to 2' sugar substituents include, but are not limited to: -[C(R a )(R b )] n -, -[C(R a )(R b )] n -0-, -C(R a R b )-N(R)-0- or, - C(R a R b )-0-N(R)-; 4'-CH 2 -2', 4'-(CH 2 ) 2 -2', 4'-(CH 2 ) 3 -2',.
  • Patent 7,427,672 issued on September 23, 2008
  • 4'-CH 2 -C(H)(CH 3 )-2' see, e.g., Chattopadhyaya, et al, J. Org. Chem.,2009, 74, 118-134
  • x 0, 1, or 2;
  • n 1, 2, 3, or 4;
  • Bicyclic nucleosides include, but are not limited to, (A) a-L-Methyleneoxy (4'-CH 2 -0-2') BNA , (B) ⁇ - D-Methyleneoxy (4'-CH 2 -0-2') BNA , (C) Ethyleneoxy (4'-(CH 2 ) 2 -0-2') BNA , (D) Aminooxy (4'-CH 2 - 0-N(R)-2') BNA, (E) Oxyamino (4'-CH 2 -N(R)-0-2') BNA, (F) Methyl(methyleneoxy) (4'-CH(CH 3 )-0- 2') BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4'-CH 2 -S-2') BNA, (H) methylene-thio (4'-CH 2 -S-2') BNA, (H) methylene-thio (4'-CH
  • Bx is a nucleobase moiety and R is, independently, H, a protecting group, or alkyl.
  • modified sugar moieties are sugar surrogates.
  • the oxygen atom of the naturally occuring sugar is substituted, e.g., with a sulfer, carbon or nitrogen atom.
  • such modified sugar moiety also comprises bridging and/or non-bridging substituents as described above.
  • certain sugar surogates comprise a 4 '-sulfer atom and a substitution at the 2'-position (see,c.g., published U.S. Patent Application US2005/0130923, published on June 16, 2005) and/or the 5' position.
  • carbocyclic bicyclic nucleosides having a 4'-2' bridge have been described (see, e.g., Freier et ah, Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al, J. Org. Chem., 2006, 71, 7731-7740).
  • sugar surrogates comprise rings having other than 5-atoms.
  • a sugar surrogate comprises a six-membered tetrahydropyran.
  • Such tetrahydropyrans may be further modified or substituted.
  • Nucleosides comprising such modifed tetrahydropyrans include, but are not limited to, hexitol nucleic acid (UNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, CJ. Bioorg. & Med. Chem. (2002) 10:841 -854), fluoro HNA (F-HNA), and Formula VII:
  • Bx is a nucleobase moiety
  • T 3 and T 4 are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T 3 and T 4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T 3 and T is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or 3'-terminal group;
  • qi, q2, q 3 , q 4 , qs, q6 and q 7 are each, independently, H, Ci-Ce alkyl, substituted Ci-Ce alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; and
  • the modified THP nucleosides of Formula VII are provided wherein q 3 ⁇ 42, q3, q4, qs, q6 and q 7 are each H. In certain embodiments, at least one of q q 2 , q 3 , q 4 , qs, q6 and q 7 is other than H. In certain embodiments, at least one of q b q 2 , q 3 , q 4 , qs, q6 and q 7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of Ri and R 2 is F. In certain embodiments, Ri is fluoro and R 2 is H, R t is methoxy and R 2 is H, and Ri is methoxyethoxy and R 2 is H.
  • Patent Application US2005-0130923, published on June 16, 2005) or alternatively 5'-substitution of a bicyclic nucleic acid see PCT International Application WO 2007/134181 , published on 1 1/22/07 wherein a 4'-CH 2 -0-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or a 5'-vinyl group).
  • PCT International Application WO 2007/134181 published on 1 1/22/07 wherein a 4'-CH 2 -0-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or a 5'-vinyl group.
  • carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et ah, J. Am. Chem. Soc. 2007, 129(26), 8362-8379).
  • the present invention provides oligomeric compounds comprising recruiting nucleosides.
  • recruiting nucleosides comprise a modified sugar moiety.
  • any of the above described sugar modifications may be incorporated into a recruiting nucleoside.
  • the present invention provides oligomeric compounds comprising duplex stabilizing nucleosides.
  • duplex stabilizing nucleosides comprise a modified sugar moiety.
  • any of the above described sugar modifications may be incorporated into a stabiliazing nucleoside.
  • nucleosides of the present invention comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present invention comprise one or more modifed nucleobases.
  • modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein.
  • nucleobases include tricyclic pyrimidines such as phenoxazine cytidine( [5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.
  • nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
  • nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J.I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al. , Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, Crooke, S.T. and Lebleu, B., Eds., CRC Press, 1993, 273-288.
  • the present invention provides oligomeric compounds comprising linked nucleosides.
  • nucleosides may be linked together using any internucleoside linkage.
  • the two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom.
  • Non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH 2 -N(CH 3 )-0-CH 2 -), thiodiester (-O-C(O)-S-), thionocarbamate (-0-C(0)(NH)-S-); siloxane (-0-Si(H) 2 -0-); and ⁇ , ⁇ '-dimethylhydrazine (-CH 2 -
  • Modified linkages compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligomeric compound.
  • Modified linkages compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligomeric compound.
  • internucleoside linkages having a chiral atom can be prepared a racemic mixture, as separate enantomers.
  • Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.
  • oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), a or ⁇ such as for sugar anomers, or as (D) or (L) such as for amino acids et al. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
  • Neutral internucleoside linkages include without limitation, phosphotriesters,
  • Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y.S.
  • Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH 2 component parts.
  • the present invention provides oligomeric compounds comprising oligonucleotides.
  • such oligonucleotides comprise one or more chemical modification.
  • chemically modified oligonucleotides comprise one or more modified sugar.
  • chemically modified oligonucleotides comprise one or more modified nucleobase.
  • chemically modified oligonucleotides comprise one or more modified internucleoside linkage.
  • the chemically modifications (sugar modifications, nucleobase modifications, and/or linkage modifications) define a pattern or motif.
  • the patterns of chemically modifications of sugar moieties, internucleoside linkages, and nucleobases are each independent of one another.
  • an oligonucleotide may be described by its sugar modification motif, internucleoside linkage motif and/or nucleobase modification motif (as used herein, nucleobase modification motif describes the chemically modifications to the nucleobases independent of the sequence of nucleobases).
  • oligonucleotides comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif.
  • the oligonucleotides of the present invention comprise or consist of a region that is uniformly sugar modified.
  • the uniform sugar region comprises at least 5 contiguous nucleosides.
  • the uniform sugar region comprises at least 7 contiguous nucleosides.
  • the uniform sugar region comprises at least 9 contiguous nucleosides.
  • the uniform sugar region comprises at least 10 contiguous nucleosides.
  • the uniform sugar region comprises at least 11 contiguous nucleosides. In certain embodiments, the uniform sugar region comprises at least 12 contiguous nucleosides. In certain embodiments, the uniform sugar region comprises at least 13 contiguous nucleosides. In certain embodiments, the uniform sugar region comprises at least 14 contiguous nucleosides. In certain embodiments, the uniform sugar region comprises at least 15 contiguous nucleosides. In certain embodiments, the uniform sugar region comprises at least 16 contiguous nucleosides. In certain embodiments, the uniform sugar region comprises at least 18 contiguous nucleosides. In certain embodiments, the uniform sugar region comprises at least 20 contiguous nucleosides.
  • the nucleosides of a uniform sugar region are recruiting nucleosides.
  • the sugar moieties of the nucleosides of the uniform sugar region comprise a 2'-MOE.
  • the sugar moieties of the nucleosides of the uniform sugar region comprise a 2'-F.
  • oligonucleotides consist of a uniform sugar region region (i.e., comprise no nucleosides with different sugar modifications). In certain embodiments, oligonucleotides comprise one or more uniform sugar regions and one or more differently modified nucleosides.
  • the oligonucleotides comprise or consist of a region having a gapmer sugar modification motif, which comprises two external regions or "wings" and an internal region or "gap."
  • the three regions of a gapmer motif (the 5 '-wing, the gap, and the 3 '-wing) form a contiguous sequence of nucleosides wherein the sugar moieties of the nucleosides of each of the wings are different from the sugar moieties of the nucleosides of the gap.
  • the sugar moieties within each of the two wings are the same as one another and the sugar moieties within the gap are the same as one another.
  • the sugar moieties of the two wings are the same as one another (symmetric gapmer).
  • the sugar moieties in the 5'-wing are different from the sugar moieties in the 3 '-wing (asymmetric gapmer).
  • the nucleosides of the 5 '-wing and the nucleosides of the 3 '-wing are sugar modified nucleosides.
  • the nucleosides of the gap comprise 2'-F sugar moieties.
  • the wings are each from 1 to 10 nucleosides in length. In certain embodiments, the wings are each from 1 to 5 nucleosides in length. In certain embodiments, the gap is from 5 to 25 nucleosides in length. In certain embodiments, the gap is from 8 to 18 nucleosides in length. In certain embodiments, gapmers may be described using the following formula:
  • Nui, ⁇ 3 ⁇ 4, and ⁇ 3 ⁇ 4 are nucleosides, wherein the sugar moieties of the Nu 2 nucleosides are different from the sugar moieties of the Nui nucleosides and from the sugar moieties of the ⁇ 3 ⁇ 4 nucleosides and wherein the sugar moieties of the Nui nucleosides and the ⁇ 3 ⁇ 4 nucleosides may be the same or different from one another.
  • each Nui is a duplex stabilizing nucleoside; each Nu 3 is a duplex stabilizing nucleoside; and each Nu 2 is a recruiting nucleoside.
  • each Nui is a 2'-MOE modified nucleoside; each Nu 3 is a 2'-MOE modified nucleoside; and each Nu 2 is a 2'-F modified nucleoside.
  • the oligonucleotides of the present invention comprise a region that is fully sugar modified, meaning that each nucleoside is a sugar modified nucleoside.
  • the modifications of the nucleosides of a fully modified oligomeric compound may all be the same or one or more may be different from one another.
  • oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif.
  • internucleoside linkages are arranged in a gapped motif, as described above for sugar modification motif.
  • the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region.
  • internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate.
  • the sugar modification motif is independently selected, so such oligonucleotides having a gapped internucleotide linkage motif may or may not have a gapped sugar modification motif and if it does have a gapped sugar motif, the wing and gap lengths may or may not be the same.
  • oligonucleotides of the present invention comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides of the present invention comprise a region of fully modified internucleoside linkages. In certain embodiments, oligonucleotides of the present invention comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate.
  • the oligonucleotide comprises at least 6 phosphorothioate
  • the oligonucleotide comprises at least 8
  • the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least 10 consecutive phosphorothioate internucleoside linkages.
  • the oligonucleotide comprises at least block of at least 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3 ' end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3' end of the oligonucleotide.
  • oligonucleotides comprise chemical modifications to nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or nucleobases modification motif.
  • nucleobase modifications are arranged in a gapped motif.
  • nucleobase modifications are arranged in an alternating motif.
  • each nucleobase is modified (fully modified nucleobase motif).
  • nucleobase modifications are uniform throughout an oligonucleotide. In certain embodiments, none of the nucleobases is chemically modified.
  • oligonucleotides comprise a block of modified nucleobases.
  • the block is at the 3 '-end of the oligonucleotide.
  • the block is within 3 nucleotides of the 3 '-end of the oligonucleotide.
  • the block is at the 5 '-end of the oligonucleotide.
  • the block is within 3 nucleotides of the 5 '-end of the oligonucleotide.
  • nucleobase modifications are a function of the natural base at a particular position of an oligonucleotide.
  • each purine or each pyrimidine in an oligonucleotide is modified.
  • each adenine is modified.
  • each guanine is modified.
  • each thymine is modified.
  • each cytosine is modified.
  • each uracil is modified.
  • some, all, or none of the cytosine moieties in an oligonucleotide are 5- methyl cytosine moieties.
  • 5-methyl cytosine is not a "modified nucleobase.”
  • unmodified nucleobases include both cytosine residues having a 5-methyl and those lacking a 5 methyl.
  • the methyl state of all or some cytosine nucleobases is specified.
  • the present invention provides oligomeric compounds including oligonucleotides of any of a variety of ranges of lengths.
  • the invention provides oligomeric compounds or oligonucleotides consisting of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number of nucleosides in the range.
  • X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X ⁇ Y.
  • the invention provides oligomeric compounds which comprise oligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23,
  • an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotides having 31 nucleosides, but, unless otherwise indicated, such an oligonucleotide may further comprise, for example one or more conjugates, terminal groups, or other substituents.
  • oligonucleotides of the present invention are characterized by their sugar motif, internucleoside linkage motif, nucleobase modification motif and overall length. In certain embodiments, such parameters are each independent of one another. Thus, each internucleoside linkage of an oligonucleotide having a gaped sugar motif may be modified or unmodified and may or may not follow the modification pattern of the sugar modifications. Thus, the internucleoside linkages within the wing regions of a sugar-gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region. Likewise, such sugar-gapmer
  • oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications.
  • modified nucleobase independent of the gapmer pattern of the sugar modifications.
  • One of skill in the art will appreciate that such motifs may be combined to create or to describe a variety of oligonucleotides. Unless otherwise indicated, all chemical modifications are independent of nucleobase sequence.
  • the present invention provides oligomeric compounds comprising recruiting nucleosides.
  • recruiting nucleosides comprise a modified sugar moiety.
  • each modified sugar moiety is selected from: a 2'-F sugar moiety; tetrahydropyran sugar moiety (including, but not limited to, a F-substituted tetrahydropyran); a BNA (including, but not limited to, LNA, ENA, and cEt).
  • antisense compounds of the present invention comprise a region of contiguous recruiting nucleosides.
  • a region of recruiting nucleosides comprises at least 4 contiguous recruiting nucleosides.
  • a region of recruiting nucleosides comprises at least 6 contiguous recruiting nucleosides. In certain embodiments, a region of recruiting nucleosides comprises at least 8 contiguous recruiting nucleosides. In certain embodiments, a region of recruiting nucleosides comprises at least 10 contiguous recruiting nucleosides. In certain embodiments, a region of recruiting nucleosides comprises at least 12 contiguous recruiting nucleosides. In certain embodiments, a region of recruiting nucleosides comprises at least 14 contiguous recruiting nucleosides. In certain embodiments, a region of recruiting nucleosides comprises at least 16 contiguous recruiting nucleosides.
  • a region of recruiting nucleosides comprises at least 18 contiguous recruiting nucleosides. In certain embodiments, a region of recruiting nucleosides comprises at least 20 contiguous recruiting nucleosides. In certain embodiments, a region of recruiting nucleosides comprises at least 22 contiguous recruiting nucleosides. In certain embodiments, the recruiting nucleosides within a region all comprise the same modified sugar moiety.
  • oligonucleotides of the present invention comprise one or more duplex stabilizing nucleosides, which comprises a modified sugar moiety making them more stable than naturally occurring nucleosides.
  • duplex stabilizing nucleosides comprise a substituted sugar moiety.
  • duplex stabilizing nucleosides comprise a sugar surrogate.
  • duplex stabilizing nucleosides comprise 2'-MOE sugar moieties.
  • the invention provides a region of 1 -5 duplex stabilizing nucleosides at one or both ends of an oligonucleotide.
  • antisense compounds of the present invention comprise a gapmer sugar motif, wherein the nucleosides of the gap are recruiting nucleosides and the nucleosides of the wings are duplex stabilizing nucleosides.
  • antisense compounds comprise the sugar motif: 2'-MOE:2'-F:2'-MOE.
  • antisense compounds comprise the sugar motif: 2'-MOE:BNA:2'-MOE.
  • antisense compounds comprise the sugar motif: 2'-MOE:cEt:2'-MOE.
  • oligomeric compounds are modified by attachment of one or more conjugate groups.
  • conjugate groups modify one or more properties of the attached oligomeric compound including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance.
  • Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligomeric compound, such as an oligonucleotide.
  • Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.
  • Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci.
  • Acids Res., 1992, 20, 533- 538 an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett, 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl.
  • a phospholipid e.g., di-hexadecyl-rac-glyce
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
  • conjugate groups are directly attached to oligonucleotides in oligomeric compounds.
  • conjugate groups are attached to oligonucleotides by a conjugate linking group.
  • conjugate linking groups including, but not limited to, bifunctional linking moieties such as those known in the art are amenable to the compounds provided herein.
  • Conjugate linking groups are useful for attachment of conjugate groups, such as chemical stabilizing groups, functional groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligomeric compound.
  • a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups.
  • One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group.
  • the conjugate linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units.
  • functional groups that are routinely used in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups.
  • bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like.
  • the present invention provides antisense compounds comprising oligonucleotides that are fully complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, oligonucleotides are 99% complementary to the target nucleic acid. In certain embodiments, oligonucleotides are 95% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 90% complementary to the target nucleic acid.
  • such oligonucleotides are 85% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In certain embodiments, an antisense compound comprises a region that is fully complementary to a target nucleic acid and is at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain such embodiments, the region of full complementarity is from 6 to 14 nucleobases in length.
  • target nucleic acids are precursors to microRNAs.
  • the microRNA is a non-canonical microRNA.
  • target nucleic acids are mirtrons.
  • MicroRNAs are small non-coding RNAs that regulate gene expression by direct base-pairing with target mRNAs (Bartel, Cell, 2004, 116, 281-297).
  • Canonical microRNAs in animals are transcribed as primary microRNAs (pri-microRNAs) and subsequently cleaved by the Microprocessor complex, comprised of the RNase III enzyme Drosha and the double-stranded RNA (dsRNA)-binding protein, DGCR8/Pasha to yield a pre-microRNA that is then exported to the cytoplasm by Exportin-5 (XP05)(Han et al., Genes Dev., 2004, 18, 3016-3027, Bohnsack et al., RNA, 2004, 10, 185-191), .
  • dsRNA double-stranded RNA
  • pre- microRNA is processed in to a -21-23 nucleotide mature microRNA duplex by the RNAse III enzyme, Dicer (Bernstein E., et al, Nature, 2001, 409, 363-366).
  • Dicer RNAse III enzyme
  • One strand of the mature microRNA duplex is preferentially loaded into the RNA-induced silencing complex (RISC) with members of the Argonaute family of proteins, producing a functional complex for targeting mRNA via direct base pairing (Du and Zamore, Development, 2005, 132, 4645-4652).
  • RISC RNA-induced silencing complex
  • microRNA/mRNA hybrids can alter protein expression of the targeted mRNA by different mechanisms, such as translational repression and mRNA degradation (Chekulaeva and Filipowicz, Curr. Opin, Cell Biol., 2009, 21, 452-460).
  • Mirtrons are a type of microRNA that are processed by a non-canonical microRNA pathway. Mirtrons have a pre-microRNA that is defined by the entire length of the intron in which it is located, and require pre-mRNA splicing rather than the Microprocessor for the first step in their biogenesis.
  • the biogenesis pathway for a number of mirtrons has been characterized in Drosophila melanogaster and Caenorhabditis elegans (Okamura et al, Cell, 2007, 130, 89-100).
  • the pre -microRNA excised by splicing is initially in the form of an intron lariat which is subsequently linearized by the debranching enzyme, Ldbr (DBR1 in humans), allowing the intron to form a structure that is exported to the cytoplasm by XP05 and recognized and cleaved by the Dicer complex to from a mature microRNA (Ruby et al, Nature, 2007, 448, 83-86).
  • mirtronic microRNAs contain all or a portion of either the 5; splice site, in the case of 5' microRNAs, or the 3' splice site, if a 3' microRNA is formed.
  • Mirtrons have been documented in mammals, avians and plants by deep-sequencing approaches (Glazov, et al, Genome Res., 2008, 18, 957-964). For humans, 13 mirtrons were predicted bases on their structure, conservation, location within small introns and cloning evidence (Berezikov, et al, Mol. Cell, 2007, 28, 328-336). Two mirtrons, miR-877 and miR-1224, were shown to be insensitive to changes in cellular DGCR8 or Drosha levels, as expected for a mirtron (Babiarz, et al, Genes Dev., 2008, 22, 2773- 2785).
  • a target nucleic acid is a simtron.
  • Simtrons have been described previously. See Havens et. AL, Nucleic Acid Research, 2012, Vol. 40, No. 10 4626-4640, which is hereby incorporated by reference in its entirety. Simtrons are microRNAs that are processed by a non- canonical pathway that does not require DGCR8 or splicing of the host introns.
  • an antisense compound hybridizes to an intronic microRNA precursor (an intronic region comprising a microRNA).
  • the microRNA is a mirtron.
  • the microRNA is a simtron.
  • hybridization of the antisense compound to the intron results in an increase in processing of the microRNA and thus an increase in the amount of microRNA.
  • hybridization of the antisense compound to the intron results in a decrease in processing of the microRNA and thus a decrease in the amount of the microRNA.
  • hybridization of the antisense compound to the microRNA precursor prevents or inhibits interaction with one or more protein involved in processing the microRNA.
  • hybridization of the antisense compound to the microRNA precursor recruits or increases interaction with one or more protein involved in processing the microRNA.
  • the protein involved in processing microRNA is associated with splicing.
  • the microRNA is a mirtron.
  • the protein involved in processing microRNA is SRSF5.
  • the microRNA is a simtron.
  • ADPKD Autosomal Dominant Polycystic Kidney Disease
  • the gene PKDl is associated with 85% of ADPKD cases and contains the microRNA miR-1225 within highly conserved intron 45.
  • miR-1225 expression is regulated in a tissue-specific manner and is inversely related to PKDl expression during mouse kidney development.
  • the SR protein SRSF5 is a regulator of miR-1225 biogenesis.
  • human pathogenic mutations in PKDl intron 45 effect miR-1225 abundance.
  • antisense oligonucleotides alter miR-1225 abundance without affecting PKDl pre-mRNA splicing.
  • the present invention provides pharmaceutical compositions comprising one or more antisense compound.
  • such pharmaceutical composition comprises a a suitable pharmaceutically acceptable diluent or carrier.
  • a pharmaceutical composition comprises a sterile saline solution and one or more antisense compound.
  • such pharmaceutical composition consists of a sterile saline solution and one or more antisense compound.
  • the sterile saline is pharmaceutical grade saline.
  • a pharmaceutical composition comprises one or more antisense compound and sterile water.
  • a pharmaceutical composition consists of one or more antisense compound and sterile water.
  • the sterile saline is pharmaceutical grade water.
  • a pharmaceutical composition comprises one or more antisense compound and phosphate-buffered saline (PBS).
  • a pharmaceutical composition consists of one or more antisense compound and sterile phosphate -buffered saline (PBS).
  • the sterile saline is pharmaceutical grade PBS.
  • antisense compounds may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations.
  • compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
  • compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters.
  • pharmaceutical compositions comprising antisense compounds comprise one or more oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
  • the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
  • Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
  • a prodrug can include the incorporation of additional nucleosides at one or both ends of an oligomeric compound which are cleaved by endogenous nucleases within the body, to form the active antisense oligomeric compound.
  • Lipid moieties have been used in nucleic acid therapies in a variety of methods.
  • the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids.
  • DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.
  • RNA nucleoside comprising a 2' -OH sugar moiety and a thymine base
  • RNA having a modified sugar 2'-OH for the natural 2'-H of DNA
  • RNA having a modified base thymine (methylated uracil) for natural uracil of RNA
  • nucleic acid sequences provided herein are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified
  • an oligomeric compound having the nucleobase sequence "ATCGATCG” encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence "AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and oligomeric compounds having other modified bases, such as "AT me CGAUCG,” wherein me C indicates a cytosine base comprising a methyl group at the 5-position.
  • an antisense oligomeric compound having two non-hybridizing 3 '-terminal 2'-MOE modified nucleosides, but otherwise fully complementary to a target nucleic acid may be described as an oligonucleotide comprising a region of 2'-MOE-modified nucleosides, wherein the oligonucleotide is less than 100% complementary to its target.
  • oligomeric compound comprising: (1) an oligonucleotide that is 100%> complementary to its nucleic acid target and (2) a terminal group wherein the terminal group comprises two 2'-MOE modified terminal-group nucleosides.
  • Such descriptions are not intended to be exclusive of one another or to exclude overlapping subject matter.
  • Example 1 Quantification of PKD1 and miR-1225 RNA expression in various human tissues
  • snoRNA65 and GAPDH were quantified across 20 different human tissues.
  • GAPDH and PKD1 radiolabeled RT-PCR reactions were separated by PAGE.
  • miR-1225-5p and snoRNA65 radiolabeled stemloop-RT-PCR reactions were separated on 15% denaturing polyacrylamide gels. Bands were quantitated on a typhoon phosphorimager.
  • the top graph depicts the zmol of miR-1225 and PKD1 in 1 ⁇ g of total RNA.
  • expression of PKD1 and miR-1225 RNA are differentially expressed in various human tissues.
  • PKD1 and miR-1225 RNA expression in mice from embryonic day 13 to 13 months old was measured in kidney.
  • Radiolabeled, stemloop RT-PCR and RT-PCR analysis were used to quantify miR- 1225 and PKD1 RNA expression.
  • snoRNA65 and GAPDH were used as loading controls.
  • the gels show 3 representative animals from each age group.
  • the top graph represents miR-1225/snoRNA65
  • the middle graph represents PKD1 /GAPDH
  • the bottom graph shows the correlation between the two RNA species.
  • PCR products were separated on 12% (miR-1225 and snoRNA65) or 6%> (PKD1 and GAPDH) native PAGE gels.
  • Example 3 Effect of altering SRSF5 expression on PKD1 splicing products and miR-1225 SRSF5 interacts with intron 45 of PKDl to promote biogenesis of miR-1225 and PKDl splicing.
  • the top panel shows putative SRSF5 binding sites in intron 45 of PKDl and the predicted RNA structure (mfold).
  • PKDl splicing products and miR-1225 RNA expression were quantified after treatment with siRNA targeting SRSF5 or treatment with a plasmid overexpressing SRSF5.
  • Provided in the middle and bottom panel are the results of a radiolabeled stemloop RT-PCR analysis of miR-1225 and let-7a (control) and radiolabeled RT-PCR analysis of PKDl, SRSF5, and GAPDH (control) from HeLa cells transfected with a PKDl expression minigene and siRNA targeting SRSF5 (middle panel) or a plasmid overexpression SRSF5 (bottom panel). PCR products were separated on 12% (miRNAs) or 6% (mRNAs) native PAGE gels.
  • PKDl splicing products are represented on the right of the gels where boxes represent exons and lines represent introns. Bands were quantitated on a typhoon phosphorimager. Graphs represent relative RNA abundance. Bars represent SEM. Statistics were performed using Student's T-tests. Values of p ⁇ 0.05 (*) were considered significant. As shown in Figure 3, proper levels of SRSF5 are necessary for miR-1225 biogenesis and PKDl splicing.
  • Example 4 Quantification of PKDl and miR-1225 RNA expression in cells expressing ADPKD patient minigenes containing exons 43 through 46 and intervening introns
  • the top panel shows the location of documented, pathogenic, patient mutations in intron 45 of PKDl . Boxed letters represent flanking exons, underlined nucleotides represent mature miR-1225 species. Mutations are indicated. Minigenes of exons 43 through 46 and intervening introns with the indicated mutations were transfected into HEK-293T cells. Splicing of PKDl and miRNA abundance was analyzed using Radiolabeled RT-PCR and stemloop RT-PCR, respectively. Bands were quantitated on a typhoon phosphorimager. miR-16 and snoRNA65 are loading controls for miR-1225.
  • Example 5 Quantification of pri-miRNA and pre -miRNA from a cell-free in vitro processing assay using RNA transcribed from a PKDl WT or IVS45-14T>C DNA template
  • RNA transcribed from a PKDl WT or rVS45-14T>C DNA template was incubated with the FLAG-immunoprecipitates from HEK-293T cells.
  • FLAG-immunoprecipitates were derived from cells transfected pFLAG-GFP (GFP) or pFLAG- Drosha and pFLAG-DGCR8 (Drosha/DGCR8).
  • Template RNA was included as a control (RNA). Reaction products were separated by 8% denaturing PAGE. The pri- and pre-miRNAs are indicated. As shown in Figure 5, the IVS45-14T>C mutation increases pre -miR-1225.
  • Example 6 Targeting human PKD1 intron 45/miR-1225 using ASOs
  • a diagram of antisense oligonucleotides (ASOs) targeting intron 45 of human PKD1 is provided in Figure 6. Double underline indicates exons, Bold indicates intron 45, underline indicates mature miR- 1225 sequences. ASOs numbered 5, 14, and 16 were the most effective at altering miR-1225 abundance.
  • Table 1 provides the sequence of the antisense oligonucleotides targeting a human PKD1 nucleic acid.
  • the antisense oligonucleotides provided in Table 1 are uniformly modified, wherein each nucleoside comprises a 2'-MOE modification.
  • HEK-293T cells were transfected with ASOs and minigene expressing human PKDl and miR- 1225. Abundance and splicing were analyzed by radiolabeled RT-PCR and stemloop RT-PCR. PCR products were separated on 6% and 12% native PAGE gels respectively. GAPDH is the loading control of PKDl snoRNA65 is a loading control for miR-1225. Graphs indicate intron 45 retention/spliced (top), overall PKDl abundance (middle) and miR-1225 abundance (bottom).
  • Example 7 Targeting mouse PKDl intron 45/miR-1225 using ASOs.
  • a diagram of antisense oligonucleotides (ASOs) targeting intron 45 of mouse PKDl is provided in Figure 7. Double underline indicates exons, Black indicates intron 45, underline indicates mature miR-1225 sequences. ASOs depicted in blue were the most effective at altering miR-1225 abundance.
  • Table 2 provides the sequence of the antisense oligonucleotides targeting a mouse PKDl nucleic acid.
  • the antisense oligonucleotides provided in Table 1 are uniformly modified, wherein each nucleoside comprises a 2'-MOE modification. Each internucleoside linkage throughout the
  • HEK-293T cells were transfected with ASOs and minigene expressing mouse PKDl and miR- 1225. Abundance and splicing were analyzed by radiolabeled RT-PCR and stemloop RT-PCR. PCR products were separated on 6% and 12% native PAGE gels respectively. GAPDH is the loading control of PKDl snoRNA65 is a loading control for miR-1225. Graphs indicate intron 45 retention/spliced (top), overall PKDl abundance (middle) and miR-1225 abundance (bottom).
  • Example 8 Effect of in vivo treatment with antisense oligonucleotides targeting PKDl intron 45 on miR-1225 abundance
  • Example 9 Effect of in vivo treatment with antisense oligonucleotides targeting PKDl intron 45 on miR-1225 cleavage sites
  • Figure 10 A summary of the regulation of miR-1225 biogenesis Shown in the top panel of Figure 10 (physiological conditions) is the promotion of miR-1225 biogenesis by SRSF5.
  • the second panel shows the reduction of miR-1225 expression in the absence of SRSF5 or when intron 45 contained the point mutation IVS45-14T>C found in some patients.
  • the third panel shows intron retention in PKDl caused by overexpression of SRSF5.
  • the bottom panel shows the increased expression of miR-1225 in the presence ASO 16.

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Abstract

La présente invention concerne des composés antisens et des procédés de mobilisation d'une ou plusieurs protéines non clivantes en direction d'un acide nucléique cible dans une cellule. Dans certains cas, ladite mobilisation d'une protéine non clivante modifie le fonctionnement ou l'activité de l'acide nucléique cible. Dans certains de ces cas, l'acide nucléique cible est un pré-ARNm et la mobilisation de la protéine non clivante entraîne une modification de l'épissage dudit pré-ARNm.
PCT/US2014/042401 2013-06-14 2014-06-13 Composés et procédés permettant de moduler l'arn non codant WO2014201413A1 (fr)

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US10696969B2 (en) 2014-10-03 2020-06-30 Cold Spring Harbor Laboratory Targeted augmentation of nuclear gene output
US10941405B2 (en) 2015-10-09 2021-03-09 University Of Southampton Modulation of gene expression and screening for deregulated protein expression
US11083745B2 (en) 2015-12-14 2021-08-10 Cold Spring Harbor Laboratory Antisense oligomers for treatment of autosomal dominant mental retardation-5 and Dravet Syndrome
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Cited By (13)

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Publication number Priority date Publication date Assignee Title
US11891605B2 (en) 2014-06-16 2024-02-06 University Of Southampton Reducing intron retention
US10538764B2 (en) 2014-06-16 2020-01-21 University Of Southampton Reducing intron retention
US10696969B2 (en) 2014-10-03 2020-06-30 Cold Spring Harbor Laboratory Targeted augmentation of nuclear gene output
US11702660B2 (en) 2015-10-09 2023-07-18 University Of Southampton Modulation of gene expression and screening for deregulated protein expression
US10941405B2 (en) 2015-10-09 2021-03-09 University Of Southampton Modulation of gene expression and screening for deregulated protein expression
US11083745B2 (en) 2015-12-14 2021-08-10 Cold Spring Harbor Laboratory Antisense oligomers for treatment of autosomal dominant mental retardation-5 and Dravet Syndrome
US11096956B2 (en) 2015-12-14 2021-08-24 Stoke Therapeutics, Inc. Antisense oligomers and uses thereof
CN107014998A (zh) * 2017-04-01 2017-08-04 中国人民解放军军事医学科学院放射与辐射医学研究所 一种肺癌调节因子srsf5及其抑制剂与应用
CN107014998B (zh) * 2017-04-01 2019-04-02 中国人民解放军军事医学科学院放射与辐射医学研究所 一种肺癌调节因子srsf5及其抑制剂与应用
US10683503B2 (en) 2017-08-25 2020-06-16 Stoke Therapeutics, Inc. Antisense oligomers for treatment of conditions and diseases
US10913947B2 (en) 2017-08-25 2021-02-09 Stoke Therapeutics, Inc. Antisense oligomers for treatment of conditions and diseases
US11873490B2 (en) 2017-08-25 2024-01-16 Stoke Therapeutics, Inc. Antisense oligomers for treatment of conditions and diseases
US11814622B2 (en) 2020-05-11 2023-11-14 Stoke Therapeutics, Inc. OPA1 antisense oligomers for treatment of conditions and diseases

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