WO2017027814A1 - Agents oligomères una pour la stimulation du régulateur de la conductance transmembranaire impliqué dans la fibrose kystique et leurs utilisations - Google Patents

Agents oligomères una pour la stimulation du régulateur de la conductance transmembranaire impliqué dans la fibrose kystique et leurs utilisations Download PDF

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WO2017027814A1
WO2017027814A1 PCT/US2016/046805 US2016046805W WO2017027814A1 WO 2017027814 A1 WO2017027814 A1 WO 2017027814A1 US 2016046805 W US2016046805 W US 2016046805W WO 2017027814 A1 WO2017027814 A1 WO 2017027814A1
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monomers
una
strand
compound
monomer
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PCT/US2016/046805
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Padmanabh Chivukula
Kiyoshi Tachikawa
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Arcturus Therapeutics, Inc.
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Publication of WO2017027814A1 publication Critical patent/WO2017027814A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • 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
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • This invention relates to the fields of biopharmaceuticals and therapeutics composed of oligomers for gene silencing. More particularly, this invention relates to structures, compositions and methods for therapeutic oligomers directed to stimulating cystic fibrosis transmembrane conductance regulator.
  • Cystic fibrosis is a genetic disorder that substantially affects the respiratory system, causing abnormally thick mucus linings in the lungs.
  • the disease can lead to fatal lung infections, and may also result in various obstructions of the pancreas, hindering digestion.
  • Symptoms of CF include persistent coughing, wheezing or shortness of breath, and an excessive appetite but poor weight gain. Deterioration is inevitable, leading to debility and eventually death. In the United States, the incidence of CF is reported to be 1 in every 3500 births.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the defective CFTR gene produces the defective protein cystic fibrosis transmembrane conductance regulator, which does not properly regulate the movement of salt and water in and out of cells. The result is thick, sticky mucus in the respiratory, digestive and reproductive systems, as well as increased salt in sweat. There are more than one thousand possible mutations of the CFTR gene.
  • the CFTR gene may be regulated by an associated long, non-coding RNA identified as BGAS (Homo sapiens, EST sequence BG213071, intron 11 of CFTR). Consequently, suppressing BGAS RNA or its promoter may result in increased CFTR gene expression.
  • BG213071 is embedded in the transcribed region, between exons 11 and 12, and in the antisense orientation of CFTR.
  • This invention provides novel molecules to be used as therapeutic agents against cystic fibrosis.
  • the molecules of this invention can be used as active
  • compositions for ameliorating, preventing or treating cystic fibrosis are provided.
  • this invention can provide methods for modulating or increasing the cellular level of cystic fibrosis transmembrane conductance regulator (CFTR) in a cell by contacting the cell with a UNA oligomer that specifically modulates an antisense long non-coding RNA (IncRNA) which is associated with CFTR.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the cell may be an epithelial cell.
  • An epithelial cell may be present in lung, liver, pancreas, or intestine.
  • compositions and methods of this invention can be used for enhancing the function of CFTR, and to provide increased CFTR in a subject.
  • compositions and methods of this invention can be used for:
  • Molecules of this invention may act to reduce expression of long, non- coding RNA (IncRNA) BGAS, or of the gene for BG213071. Inhibition of the IncRNA BGAS can result in a surprising increase in the level of CFTR mRNA and/or a CFTR protein. [0013] Molecules of this invention can be used for ameliorating and/or treating cystic fibrosis.
  • IncRNA non- coding RNA
  • Embodiments of this invention can provide molecules having one or more properties that advantageously provide inhibition of the BGAS, as well as compositions or formulations for therapeutic agents in cystic fibrosis, which can provide clinical agents.
  • the properties of the molecules of this invention arise according to their structure, and the molecular structure in its entirety, as a whole, can provide significant benefits and properties.
  • linker groups can be attached in a chain in the molecule.
  • Each linker group can also be attached to a nucleobase.
  • a linker group can be a monomer. Monomers can be attached to form a chain molecule. In a chain molecule of this invention, a linker group monomer can be attached at any point in the chain.
  • linker group monomers can be attached in a chain molecule of this invention so that the linker group monomers reside near the ends of the chain.
  • the ends of the chain molecule can be formed by linker group monomers.
  • the linker groups of a chain molecule can each be attached to a nucleobase.
  • the presence of nucleobases in the chain molecule can provide a sequence of nucleobases.
  • this invention provides oligomer molecules having chain structures that incorporate novel combinations of the linker group monomers, along with certain natural nucleotides, or non-natural nucleotides, or modified nucleotides, or chemically-modified nucleotides.
  • the oligomer molecules of this invention can display a sequence of nucleobases that is targeted to at least a portion of a BGAS IncRNA.
  • this invention provides therapeutics for preventing, ameliorating, or treating cystic fibrosis.
  • An active compound or molecule of this invention may be used in the prevention or treatment of cystic fibrosis.
  • This invention provides structures, methods and compositions for oligomeric agents that incorporate the linker group monomers. The oligomeric molecules of this invention can be used as active agents in formulations for antisense and gene silencing therapeutics targeted to BGAS.
  • Embodiments of this invention include the following:
  • a compound comprising a first strand and a second strand, each of the strands being 19-29 monomers in length, the monomers comprising UNA monomers and nucleic acid monomers, wherein the compound has a duplex region of from 14 to 29 contiguous monomers in length, wherein the first strand is a passenger strand for RNA interference and the second strand is a guide strand for RNA interference, and wherein the compound comprises a sequence of bases targeted to inhibit expression of a BGAS transcript or BGAS promoter.
  • a compound may contain one to seven UNA monomers.
  • a compound may contain a UNA monomer at the 5' end (1-end for UNA) of the first strand, a UNA monomer at the second position from the 3 '-end of the first strand, and a UNA monomer at the second position from the 3' end of the second strand.
  • the second strand may comprise a contiguous sequence that is complementary to a contiguous sequence of 19 nucleotides of BGAS IncRNA or BGAS promoter.
  • a compound may have a 3' overhang comprising one or more UNA monomers, natural nucleotides, non-natural nucleotides, modified nucleotides, or chemically-modified nucleotides, and combinations thereof.
  • a 3' overhang can comprise one or more deoxythymidine nucleotides, 2'-0-methyl nucleotides, inverted abasic monomers, inverted thymidine monomers, L-thymidine monomers, or glyceryl nucleotides.
  • a compound may have one or more nucleic acid monomers being a non-natural nucleotide, a modified nucleotide, or a chemically-modified nucleotide.
  • one or more of three monomers at each end of each strand may be connected by a phosphorothioate, a chiral phosphorothioate, or a phosphorodithioate linkage.
  • each strand can be from 19 to 23 monomers in length.
  • a compound may contain one or more nucleic acid monomers modified with a 2'-0-methyl group.
  • Embodiments of this invention further contemplate a lipid nanoparticle- oligomer compound, which can have one or more compounds of this invention attached to the lipid nanoparticle.
  • compositions containing one or more compounds and a pharmaceutically acceptable carrier include compositions containing one or more compounds and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may include lipid nanoparticles or liposomes.
  • This invention also contemplates methods for preventing, ameliorating or treating a disease associated with CFTR function in a subject in need, by administering to the subject an effective amount of a composition above.
  • Certain embodiments include methods for inhibiting expression of a
  • BGAS polynucleotide in a subject in need, by administering to the subject a composition above.
  • this invention includes the use of a composition above for preventing, ameliorating or treating a disease or condition associated with CFTR function in a subject in need.
  • a composition of this invention can be used in medical therapy, or in the treatment of the human or animal body.
  • compositions of this disclosure can be used for preparing or
  • FIG. 1 shows a schematic representation of the CFTR gene (NM_000492.3) and a schematic representation of BGAS gene BG213071, which is located between introns 11 and 12 of the CFTR gene, shown in the complementary strand in the diagram expansion.
  • BG213071 transcribes a long, non-coding RNA (IncRNA), which is antisense to a CFTR trancript, and may be involved in regulating CFTR.
  • BG213071 has an associated Promoter.
  • UNA oligomeric agents were used to increase CFTR expression.
  • CFTR expression can be increased by suppressing BG213071 IncRNA or its promoter.
  • FIG. 2 shows the results of a method for increasing CFTR expression in cells by administering UNA oligomeric agents of this invention.
  • UNA oligomeric agent BG4 was used to increase the transport function of the ziF508-CFTR chloride channel in Cystic Fibrosis Human Bronchial Epithelial (CFhBE) Primary Cells.
  • CFhBE Cystic Fibrosis Human Bronchial Epithelial
  • One of the most common cystic fibrosis-associated mutations is the deletion of phenylalanine 508 (F508del), which results in channels with poor membrane expression and impaired function. As shown in FIG.
  • the UNA oligomeric agent BG4 activated CFTR-mediated chloride channel transport function in the CFhBE homozygous AF508-CFTR primary cells in a dose-dependent manner. These results showed that the UNA oligomer increased expression of functionally active CFTR by inhibiting BGAS IncRNA.
  • the UNA oligomer was transfected into AF508-CFTR primary cells, and an epithelial voltage clamp assay was performed 16 hours after transfection.
  • This invention provides a range of novel agents and compositions to be used as therapeutics for cystic fibrosis.
  • Molecules of this invention can be used as active pharmaceutical ingredients in compositions for ameliorating, preventing or treating cystic fibrosis and related diseases associated with CFTR function.
  • Molecules of this invention can prevent BGAS IncRNA from carrying out one or more of its processes or cellular functions.
  • Molecules of this invention can be used for ameliorating or treating cystic fibrosis, and may act against any of the transcription, translation, splicing, maturation, or function of BGAS IncRNA.
  • CFTR may be regulated in part by the presence of IncRNA BG213071.
  • compositions for upregulating CFTR expression may be used as agents for treating cystic fibrosis.
  • the agents of this invention can be UNA oligomers, which promote CFTR expression by targeting any site in IncRNA BG213071.
  • a UNA oligomer may be targeted to any portion, locus or sequence of BG213071.
  • a UNA oligomer may be used for inhibiting expression of a BGAS-associated protein.
  • compositions or formulations for therapeutic agents for cystic fibrosis which can provide clinical agents.
  • linker groups can be attached in a chain in the molecule.
  • linker group can also be attached to a nucleobase.
  • a linker group can be a monomer. Monomers can be attached to form a chain molecule. In a chain molecule of this invention, a linker group monomer can be attached at any point in the chain.
  • linker group monomers can be attached in a chain molecule of this invention so that the linker group monomers reside near the ends of the chain.
  • the ends of the chain molecule can be formed by linker group monomers.
  • a chain molecule can also be referred to as an oligomer.
  • the linker groups of a chain molecule can each be attached to a nucleobase.
  • the presence of nucleobases in the chain molecule can provide a sequence of nucleobases.
  • this invention provides oligomer molecules having chain structures that incorporate novel combinations of the linker group monomers, along with certain natural nucleotides, or non-natural nucleotides, or modified nucleotides, or chemically-modified nucleotides.
  • the oligomer molecules of this invention can display a sequence of nucleobases that is targeted to at least a portion of BGAS IncRNA.
  • an oligomer can be targeted to at least a portion of BGAS IncRNA that is conserved, or highly conserved, among a number of variants.
  • this invention provides active oligomer molecules that correspond to, or are complementary to at least a fragment of a BGAS nucleic acid molecule, and that decrease expression of at least such a fragment present in a cell.
  • This invention provides structures, methods and compositions for oligomeric agents that incorporate the linker group monomers.
  • the oligomeric molecules of this invention can be used as active agents in formulations for gene silencing and antisense therapeutics targeted to BGAS.
  • This invention provides a range of molecules that are useful for providing therapeutic effects because of their activity in regulating expression of a gene or gene product.
  • the molecules of this invention are structured to provide gene regulating or silencing activity in vitro and in vivo.
  • Embodiments of this invention can provide molecules for use as therapeutic agents for cystic fibrosis.
  • the molecules can be used as active
  • compositions for ameliorating, preventing or treating cystic fibrosis are provided.
  • an active molecule can be structured as an oligomer composed of monomers.
  • the oligomeric structures of this invention may contain one or more linker group monomers, along with certain nucleotides.
  • Embodiments of this invention can provide an active molecule, which can increase CFTR in a cell, thereby increasing CFTR-associated cAMP-dependent chloride ion transport in a cell membrane.
  • BG213071 IncRNA has its own promoter, which transcribes an antisense
  • RNA to the pre-mRNA of CFTR RNA to the pre-mRNA of CFTR.
  • Embodiments of this invention provide UNA oligomers targeted to BGAS
  • IncRNA itself, as well as sites including BGAS promoter, and introns and exons of BG213071.
  • the second strand (antisense) of a UNA oligomeric agent can comprise a contiguous sequence that is complementary to a contiguous sequence of 19 nucleotides of BGAS IncRNA.
  • the second strand (antisense) of a UNA oligomeric agent can comprise a contiguous sequence that is complementary to a contiguous sequence of 19 nucleotides of a putative BGAS promoter transcript.
  • the BGAS promoter does not have to be transcribed for this activity of the agent.
  • BG213071 Various sites in BG213071 were targeted for suppression of gene expression using UNA oligomeric agents of this invention, including the BG213071 Promoter, as well as BG213071 exons and introns.
  • UNA oligomeric agents BG2 and BG3 were targeted to BG213071 Exon 1 (arrows indicating 2 and 3).
  • UNA oligomeric agents BG1 and BG4 were targeted to the BGAS Promoter (arrows indicating 1 and 4).
  • An active oligomer molecule of this invention can be double-stranded or single-stranded.
  • an active oligomer molecule of this invention can be double-stranded.
  • a double-stranded oligomer molecule of this invention which is an RNA interference molecule targeted to the BG213071 IncRNA, can be used to suppress the BG213071 IncRNA by post-transcriptional gene silencing.
  • An active oligomer of this invention that can suppress BG213071 IncRNA can be used to increase CFTR in a cell, thereby increasing CFTR-associated cAMP-dependent chloride ion transport in a cell membrane.
  • any portion of BG213071 can be targeted with a double-stranded oligomer molecule of this invention.
  • a cellular pathway may use active oligomers of this invention to be sequence-specific regulators in an RNA interference pathway.
  • the active oligomers may bind to the RNA-induced silencing complex (RISC complex), where a sense strand, also referred to as the passenger strand, and an anti sense strand, also referred to as the guide strand, can be unwound, and the antisense strand complexed in the RISC complex.
  • RISC complex RNA-induced silencing complex
  • the guide strand can bind to a complementary sequence to which it was targeted, for example, a target sequence in an mRNA, which can be subsequently cleaved, resulting in inactivation of the nucleic acid molecule containing the target sequence.
  • a target sequence in an mRNA which can be subsequently cleaved, resulting in inactivation of the nucleic acid molecule containing the target sequence.
  • an oligomeric molecule may be attached to a delivery moiety.
  • delivery moieties include glycoprotein receptors, galactoses, galactosamines, N-acetylgalactosamines, GalNAc groups, cholesterols, sterols, phytosterols, steroids, zoosterols, lanosterols, stigmastanols, dihydrolanosterols, zymosterols, zymostenols, desmosterols, and 7-dehydrocholesterols.
  • an active oligomer molecule of this invention can be double-stranded.
  • a double stranded oligomer of this invention may operate by transcriptional gene silencing when targeted directly to the BG213071 Promoter, and suppress BG213071 IncRNA.
  • a cellular pathway may use active oligomers of this invention to be sequence-specific regulators in a transcription pathway.
  • the active oligomers may be targeted to a promoter region, and may bind to an Argonaute or transcription complex, causing inactivation of transcription of a nucleic acid molecule related to the target sequence.
  • a single-stranded oligomer molecule of this invention which is targeted to the BG213071 IncRNA as an antisense molecule, can be used to suppress the IncRNA by post-transcriptional gene silencing.
  • An active oligomer of this invention that can suppress BG213071 IncRNA can increase CFTR in a cell, thereby increasing CFTR-associated cAMP-dependent chloride ion transport in a cell membrane.
  • any portion of BG213071 can be targeted with a single- stranded oligomer molecule of this invention.
  • a cellular pathway may use active oligomers of this invention to be sequence-specific regulators in an antisense pathway.
  • the active oligomers may bind to a target RNA, such as a IncRNA.
  • An active oligomer can bind to a complementary sequence to which it was targeted, for example, a target sequence in an IncRNA. This may recruit or activate an RNase H, which can subsequently cleave and inactivate the nucleic acid molecule containing the target sequence. As a result, the expression of IncRNA containing the target sequence can be reduced.
  • the oligomeric compounds of this invention can be used as an active pharmaceutical ingredient for regulating or modulating gene expression, and in RNA interference methods, as well as antisense, RNA blocking, and micro-RNA strategies.
  • linker group monomers can be unlocked nucleomonomers (UNA monomers), which are small organic molecules based on a propane- 1,2,3 -tri-yl- trisoxy structure as shown below:
  • UNA monomers unlocked nucleomonomers
  • R 1 and R 2 are H, and R 1 and R 2 can be phosphodiester linkages
  • Base can be a nucleobase
  • R 3 is a functional group described below.
  • UNA monomer main atoms can be drawn in IUPAC notation as follows:
  • nucleobase examples include uracil, thymine, cytosine, 5- methylcytosine, adenine, guanine, inosine, and natural and non-natural nucleobase analogues.
  • a UNA monomer can be an internal monomer in an oligomer, where the UNA monomer is flanked by other monomers on both sides.
  • the UNA monomer can participate in base pairing when the oligomer is a duplex, for example, and there are other monomers with nucleobases in the duplex.
  • a UNA monomer can be a monomer in an overhang of an oligomer duplex, where the UNA monomer is flanked by other monomers on both sides. In this form, the UNA monomer does not participate in base pairing. Because the UNA monomers are flexible organic structures, unlike nucleotides, the overhang containing a UNA monomer will be a flexible terminator for the oligomer.
  • a UNA monomer can be a terminal monomer in an overhang of an oligomer, where the UNA monomer is attached to only one monomer at either the propane-l-yl position or the propane-3-yl position. In this form, the UNA monomer does not participate in base pairing. Because the UNA monomers are flexible organic structures, unlike nucleotides, the overhang containing a UNA monomer can be a flexible terminator for the oligomer.
  • a UNA monomer can be a flexible molecule
  • a UNA monomer as a terminal monomer can assume widely differing conformations.
  • An example of an energy minimized UNA monomer conformation as a terminal monomer attached at the propane-3-yl position is shown below.
  • UNA oligomers having a terminal UNA monomer are significantly different in structure from conventional nucleic acid agents, such as siRNAs.
  • siRNAs may require that terminal monomers or overhangs in a duplex be stabilized.
  • the conformability of a terminal UNA monomer can provide UNA oligomers with different properties.
  • a UNA oligomer can be a chain composed of UNA monomers, as well as various nucleotides that may be based on naturally- occurring nucleosides.
  • the functional group R 3 of a UNA monomer can be any suitable functional group R 3 of a UNA monomer.
  • R 4 is the same or different for each occurrence, and can be H, alkyl, a cholesterol, a lipid molecule, a polyamine, an amino acid, or a polypeptide.
  • the UNA monomers are organic molecules. UNA monomers are not nucleic acid monomers or nucleotides, nor are they naturally-occurring nucleosides or modified naturally-occurring nucleosides.
  • a UNA oligomer of this invention is a synthetic chain molecule.
  • a UNA oligomer of this invention is not a nucleic acid, nor an oligonucleotide.
  • a UNA monomer can be UNA-A (designated A), UNA-U (designated U), UNA-C (designated C), and UNA-G (designated G).
  • Designations that may be used herein include mA, mG, mC, and mU, which refer to the 2'-0-Methyl modified ribonucleotides.
  • Designations that may be used herein include lower case c and u, which refer to the 2'-0-methyl modified ribonucleotides.
  • Designations that may be used herein include dT, which refers to a 2'- deoxy T nucleotide.
  • N represents any natural nucleotide monomer, or a modified nucleotide monomer.
  • the symbol Q represents a non-natural, modified, or chemically-modified nucleotide monomer.
  • the monomer can have any base attached.
  • the Q monomer can have any base attached that would be complementary to the monomer in the corresponding paired position in the other strand.
  • nucleic acid monomers include non-natural, modified, and chemically-modified nucleotides, including any such nucleotides known in the art.
  • non-natural, modified, and chemically-modified nucleotide monomers include any such nucleotides known in the art, for example, 2'-0-methyl ribonucleotides, 2'-0-methyl purine nucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'- deoxy-2'-fluoro pyrimidine nucleotides, 2'-deoxy ribonucleotides, 2'-deoxy purine nucleotides, universal base nucleotides, 5-C-methyl-nucleotides, and inverted
  • Examples of non-natural, modified, and chemically-modified nucleotide monomers include 3 '-end stabilized nucleotides, 3 '-glyceryl nucleotides, 3 '-inverted abasic nucleotides, and 3 '-inverted thymidine, and L-thymidine.
  • Non-natural, modified, and chemically-modified nucleotide monomers include locked nucleic acid nucleotides (LNA), 2'-0,4'-C-methylene-(D- ribofuranosyl) nucleotides, 2'-methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotides, and 2'-0-methyl nucleotides.
  • LNA locked nucleic acid nucleotides
  • MOE methoxyethoxy
  • Examples of non-natural, modified, and chemically-modified nucleotide monomers include 2',4'-Constrained 2'-0-Methoxyethyl (cMOE) and 2'-0-Ethyl (cEt) Modified DNAs.
  • cMOE 2',4'-Constrained 2'-0-Methoxyethyl
  • cEt 2'-0-Ethyl
  • Examples of non-natural, modified, and chemically-modified nucleotide monomers include 2'-amino nucleotides, 2'-0-amino nucleotides, 2'-C-allyl nucleotides, and 2'-0-allyl nucleotides.
  • non-natural, modified, and chemically-modified nucleotide monomers include N 6 -methyladenosine nucleotides.
  • nucleotide monomers examples include nucleotide monomers with modified bases 5-(3-amino)propyluridine, 5-(2-mercapto)ethyluridine, 5-bromouridine; 8-bromoguanosine, or 7-deazaadenosine.
  • Examples of non-natural, modified, and chemically-modified nucleotide monomers include 2'-0-aminopropyl substituted nucleotides.
  • non-natural, modified, and chemically-modified nucleotide monomers include 2'-0-guanidinopropyl substituted nucleotides.
  • Examples of non-natural, modified, and chemically-modified nucleotide monomers include Pseudouridines.
  • Examples of non-natural, modified, and chemically-modified nucleotide monomers include replacing the 2'-OH group of a nucleotide with a 2'-R, a 2'-OR, a 2'- halogen, a 2'-SR, or a 2'-amino, 2'-azido, where R can be H, alkyl, fluorine-substituted alkyl, alkenyl, or alkynyl.
  • Examples of non-natural, modified, and chemically-modified nucleotide monomers include replacing the 2'-OH group of a nucleotide with a 2'-R or 2'-OR, where R can be CN, CF 3 , alkylamino, or aralkyl.
  • non-natural, modified, and chemically-modified nucleotide monomers include nucleotides with a modified sugar such as an F-HNA, an FINA, a CeNA, a bicyclic sugar, or an LNA.
  • a modified sugar such as an F-HNA, an FINA, a CeNA, a bicyclic sugar, or an LNA.
  • Examples of non-natural, modified, and chemically-modified nucleotide monomers include 2'-oxa-3'-aza-4'a-carbanucleoside monomers, 3-hydroxymethyl-5- (lH-l,2,3-triazol)-isoxazolidine monomers, and 5'-triazolyl-2'-oxa-3'-aza-4'a- carbanucleoside monomers.
  • aspects of this invention can provide structures and compositions for UNA-containing oligomeric compounds.
  • the oligomeric agents may incorporate one or more UNA monomers.
  • Oligomeric molecules of this invention can be used as active agents in formulations for gene regulating or gene silencing therapeutics.
  • An oligomer can be single stranded, or double stranded, or may have additional strands or non-strand structures.
  • this invention provides oligomeric compounds having a structure that incorporates novel combinations of UNA monomers with certain natural nucleotides, non-natural nucleotides, modified nucleotides, or chemically- modified nucleotides.
  • the oligomeric compounds can be pharmacologically active molecules.
  • a UNA oligomer of this invention can be used as an active pharmaceutical ingredient for regulating or modulating gene expression, and in RNA interference methods, as well as antisense, RNA blocking, and micro-RNA strategies.
  • a UNA oligomer of this invention can be a short chain molecule.
  • a UNA oligomer can be a duplex pair.
  • a UNA oligomer can have a first strand of the duplex and a second strand of the duplex, which is complementary to the first strand with respect to the nucleobases, although up to three mismatches can occur.
  • a UNA oligomer duplex can have overhangs.
  • the target of a UNA oligomer can be a target nucleic acid.
  • the target can be any mRNA of a subject.
  • a UNA oligomer can be active for gene silencing in RNA interference.
  • a UNA oligomer may comprise two strands that together provide a duplex.
  • the duplex may be composed of a first strand, which may also be referred to as a passenger strand or sense strand, and a second strand, which may also be referred to as a guide strand or antisense strand.
  • a UNA oligomer of this invention can have any number of phosphorothioate intermonomer linkages in any position in any strand, or in both strands of a duplex structure.
  • any one or more of the intermonomer linkages of a UNA oligomer can be a phosphodiester, a phosphorothioate including dithioates, a chiral phosphorothioate, and other chemically modified forms.
  • Examples of UNA oligomers of this invention include duplex pairs, which are in general complementary.
  • SEQ ID NO: 1 can represent a first strand of a duplex and SEQ ID NO:2 can represent a second strand of the duplex, which is complementary to the first strand.
  • N in the first strand can represent any nucleotide that is complementary to the monomer in the corresponding position in the second strand.
  • Example UNA oligomers of this disclosure are shown with 2-monomer length overhangs, although overhangs of from 1 to 8 monomers, or longer, can be used.
  • the symbol "X" in a strand or oligomer represents a UNA monomer.
  • the monomer can have any base attached.
  • the UNA monomer can have any base attached that would be complementary to the monomer in the corresponding paired position in the other strand.
  • the terminal position has a 1-end, according to the positional numbering shown above, instead of a 5'- end as for a nucleotide, or the terminal position has a 3 -end, according to the positional numbering shown above, instead of a 3 '-end as for a nucleotide.
  • the UNA oligomer terminates in a UNA monomer
  • the terminal position has a 1-end, according to the positional numbering shown above, instead of a 5'- end as for a nucleotide, or the terminal position has a 3 -end, according to the positional numbering shown above, instead of a 3 '-end as for a nucleotide.
  • the UNA oligomer terminates in a UNA monomer
  • SEQIDNO:2 has a UNA monomer 1-end on the first strand, a UNA monomer 3 -end on the first strand, a UNA monomer 3 -end on the second strand, and a nucleotide 5 '-end on the second strand.
  • Complementarity of strands can involve mismatches.
  • complementarity of strands can include one to three, or more, mismatches.
  • a UNA oligomer of this invention can have one or more UNA monomers at the 1-end of the first strand, and one or more UNA monomers at the 3 -end of the first strand.
  • a UNA oligomer of this invention can have one or more UNA monomers at the 3 -end of the second strand.
  • a duplex UNA oligomer of this invention can have one or more UNA monomers at the 1 -end of the first strand, one or more UNA monomers at the 3 -end of the first strand, and one or more UNA monomers at the 3 -end of the second strand.
  • a UNA oligomer of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length.
  • a UNA oligomer of this invention may have a first strand that is 19-23 monomers in length.
  • a UNA oligomer of this invention may have a duplex region that is 19-21 monomers in length.
  • a UNA oligomer of this invention may have a second strand that is 19-23 monomers in length.
  • a UNA oligomer of this invention may have a first strand that is 19 monomers in length, and a second strand that is 21 monomers in length.
  • a UNA oligomer of this invention may have a first strand that is 20 monomers in length, and a second strand that is 21 monomers in length.
  • a UNA oligomer of this invention may have a first strand that is 21 monomers in length, and a second strand that is 21 monomers in length.
  • a UNA oligomer of this invention may have a first strand that is 22 monomers in length, and a second strand that is 21 monomers in length.
  • a UNA oligomer of this invention for inhibiting gene expression can have a first strand and a second strand, each of the strands being 19-29 monomers in length.
  • the monomers can be UNA monomers and nucleic acid monomers.
  • the oligomer can have a duplex structure of from 14 to 29 monomers in length.
  • the UNA oligomer can be targeted to a target gene and can exhibit reduced off-target effects as compared to a conventional siRNA.
  • a UNA oligomer of this invention can have a first strand and a second strand, each of the strands being 19-23 monomers in length.
  • the UNA oligomer may have a blunt end, or may have one or more overhangs.
  • the first and second strands may be connected with a connecting oligomer in between the strands, and form a duplex region with a connecting loop at one end.
  • an overhang can be one or two monomers in length.
  • Examples of an overhang can contain one or more UNA monomers, natural nucleotides, non-natural nucleotides, modified nucleotides, or chemically- modified nucleotides, and combinations thereof.
  • Examples of an overhang can contain one or more deoxythymidine nucleotides, 2'-0-methyl nucleotides, inverted abasic monomers, inverted thymidine monomers, L-thymidine monomers, or glyceryl nucleotides.
  • a UNA oligomer can mediate cleavage of a target nucleic acid in a cell.
  • the second strand of the UNA oligomer at least a portion of which can be complementary to the target nucleic acid, can act as a guide strand that can hybridize to the target nucleic acid.
  • the second strand can be incorporated into an RNA Induced Silencing Complex (RISC).
  • RISC RNA Induced Silencing Complex
  • a UNA oligomer of this disclosure may comprise naturally-occurring nucleic acid nucleotides, and modifications thereof that are compatible with gene silencing activity.
  • a UNA oligomer is a double stranded construct molecule that is able to inhibit gene expression.
  • strand refers to a single, contiguous chain of monomers, the chain having any number of internal monomers and two end monomers, where each end monomer is attached to one internal monomer on one side, and is not attached to a monomer on the other side, so that it ends the chain.
  • the monomers of a UNA oligomer may be attached via phosphodiester linkages, phosphorothioate linkages, gapped linkages, and other variations.
  • a UNA oligomer can include mismatches in complementarity between the first and second strands.
  • a UNA oligomer may have 1, or 2, or 3 mismatches. The mismatches may occur at any position in the duplex region.
  • the target of a UNA oligomer can be a target nucleic acid of a target gene.
  • a UNA oligomer may have one or two overhangs outside the duplex region.
  • the overhangs can be an unpaired portion at the end of the first strand or second strand.
  • the lengths of the overhang portions of the first and second strands can be the same or different.
  • a UNA oligomer may have at least one blunt end.
  • a blunt end does not have an overhang portion, and the duplex region at a blunt end terminates at the same position for both the first and second strands.
  • a UNA oligomer can be RISC length, which means that it has a duplex length of less than 25 base pairs.
  • a UNA oligomer can be a single strand that folds upon itself and hybridizes to itself to form a double stranded region having a connecting loop at the end of the double stranded region.
  • an oligomeric compound of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a Q monomer.
  • an oligomeric compound of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a Q monomer, and where the number of Q monomers is less than twenty.
  • an oligomeric compound of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a Q monomer, and where the number of Q monomers is less than twelve.
  • an oligomeric compound of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a Q monomer, and where the number of Q monomers is less than ten.
  • an oligomeric compound of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a Q monomer, and where the number of Q monomers is less than eight.
  • an oligomeric compound of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a Q monomer, and where the number of Q monomers is from 1 to 20.
  • an oligomeric compound of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a Q monomer, and where the number of Q monomers is from 1 to 15.
  • an oligomeric compound of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a Q monomer, and where the number of Q monomers is from 1 to 9.
  • an oligomeric compound of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a 2'-0- Methyl modified ribonucleotide.
  • an oligomeric compound of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a 2'-0- Methyl modified ribonucleotide, and where the number of 2'-0-Methyl modified ribonucleotides is less than twenty.
  • an oligomeric compound of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a 2'-0- Methyl modified ribonucleotide, and where the number of 2'-0-Methyl modified ribonucleotides is less than twelve.
  • an oligomeric compound of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a 2'-0- Methyl modified ribonucleotide, and where the number of 2'-0-Methyl modified ribonucleotides is less than ten.
  • an oligomeric compound of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a 2'-0- Methyl modified ribonucleotide, and where the number of 2'-0-Methyl modified ribonucleotides is less than eight.
  • an oligomeric compound of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a 2'-0- Methyl modified ribonucleotide, and where the number of 2'-0-Methyl modified ribonucleotides is from 1 to 20.
  • an oligomeric compound of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a 2'-0- Methyl modified ribonucleotide, and where the number of 2'-0-Methyl modified ribonucleotides is from 1 to 15.
  • an oligomeric compound of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a 2'-0- Methyl modified ribonucleotide, and where the number of 2'-0-Methyl modified ribonucleotides is from 1 to 9.
  • An oligomeric compound of this invention may be a single stranded molecule, wherein the single strand is any one of the strands shown in Table 1.
  • an oligomeric compound of this invention may have a single strand being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a Q monomer.
  • an oligomeric compound of this invention may have a single strand being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a Q monomer, and where the number of Q monomers is less than twenty.
  • an oligomeric compound of this invention may have a single strand being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a Q monomer, and where the number of Q monomers is less than twelve.
  • an oligomeric compound of this invention may have a single strand being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a Q monomer, and where the number of Q monomers is less than ten.
  • an oligomeric compound of this invention may have a single strand being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a Q monomer, and where the number of Q monomers is less than eight.
  • an oligomeric compound of this invention may have a single strand being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a Q monomer, and where the number of Q monomers is from 1 to 20.
  • an oligomeric compound of this invention may have a single strand being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a Q monomer, and where the number of Q monomers is from 1 to 15.
  • an oligomeric compound of this invention may have a single strand being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a Q monomer, and where the number of Q monomers is from 1 to 9.
  • an oligomeric compound of this invention may have a single strand being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a 2'-0-Methyl modified ribonucleotide.
  • an oligomeric compound of this invention may have a single strand being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a 2'-0-Methyl modified ribonucleotide, and where the number of 2'-0-Methyl modified ribonucleotides is less than twenty.
  • an oligomeric compound of this invention may have a single strand being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a 2'-0-Methyl modified ribonucleotide, and where the number of 2'-0-Methyl modified ribonucleotides is less than twelve.
  • an oligomeric compound of this invention may have a single strand being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a 2'-0-Methyl modified ribonucleotide, and where the number of 2'-0-Methyl modified ribonucleotides is less than ten.
  • an oligomeric compound of this invention may have a single strand being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a 2'-0-Methyl modified ribonucleotide, and where the number of 2'-0-Methyl modified ribonucleotides is less than eight.
  • an oligomeric compound of this invention may have a single strand being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a 2'-0-Methyl modified ribonucleotide, and where the number of 2'-0-Methyl modified ribonucleotides is from 1 to 20.
  • an oligomeric compound of this invention may have a single strand being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a 2'-0-Methyl modified ribonucleotide, and where the number of 2'-0-Methyl modified ribonucleotides is from 1 to 15.
  • an oligomeric compound of this invention may have a single strand being 19-23 monomers in length, where any monomer that is not a UNA monomer can be a 2'-0-Methyl modified ribonucleotide, and where the number of 2'-0-Methyl modified ribonucleotides is from 1 to 9.
  • Methods of this invention include the treatment and/or prevention of cystic fibrosis disease in a subject.
  • a subject can be a mammalian subject, including a human subject.
  • An oligomeric compound of this invention can be formed having a first strand and a second strand each being 21 monomers in length.
  • the first strand can have 19 contiguous monomers with a sequence of attached bases shown in Table 1 (sense), and two additional overhang monomers on the 3' end.
  • the second strand can have 19 contiguous monomers with a sequence of attached bases shown in Table 1 (antisense), and two additional overhang monomers on the 3' end.
  • the overhang monomers can be any of NN, QQ, XX, NX, NQ, XN, XQ, QN, and QX.
  • XQ can be UNA- U/mU, or UNA-U/*/dT.
  • An oligomeric compound of this invention can be composed of monomers.
  • the monomers can have attached bases.
  • An oligomeric compound of this invention can have a sequence of attached bases.
  • the sequences of bases shown in Table 1 do not indicate to which monomer each of the bases in the sequence is attached. Thus, each sequence shown in Table 1 refers to a large number of small molecules, each of which is composed of UNA monomers, as well as nucleic acid monomers.
  • an oligomeric compound of this invention can be described by a sequence of attached bases, for example as shown in Table 1, and being substituted forms thereof.
  • substituted forms include differently substituted UNA monomers, as well as differently substituted or modified nucleic acid monomers, as are further described herein.
  • one or more of three monomers at each end of each strand can be connected by a phosphorothioate, a chiral phosphorothioate, or a phosphorodithioate linkage.
  • a compound may have a deoxythymidine nucleotide at the 3' end of the first strand, at the 3' end of the second strand, or at both the 3' end of the first strand and the 3' end of the second strand.
  • a compound may contain one to five UNA monomers.
  • a compound may contain three UNA monomers.
  • a compound may contain a UNA monomer at the 5' end (1-end for UNA) of the first strand, a UNA monomer at the second position from the 3 '-end of the first strand, and a UNA monomer at the second position from the 3' end of the second strand.
  • a compound may contain a UNA monomer at any one or more of positions 2 to 8 from the 5' end of the second strand (seed region).
  • Ref Pos refers to reference position, which is the numerical position of a reference nucleotide in the CFTR genome. Referring to FIG. 1, the reference positions of the sequences in Table 1 were from the region 1 to 5240. This 5240 nt length region encompassed Exon 2, Intron 1, and Exon 1 of the BGAS gene, as well as the BGAS Promoter, taken as a 1250 nt length region as shown in FIG. 1.
  • any of the 19-mer sequences represented in Table 1, in corresponding "sense” and “antisense” pairs on the left and right hand sides, respectively, can correspond to the "sense” and “antisense” sequences of the double stranded region of a UNA oligomer of this invention.
  • any of the 19-mer sequences represented in Table 1, in corresponding "sense” and “antisense” pairs on the left and right hand sides, respectively, can correspond to the "sense” and "antisense” sequences of the monomers in the double stranded region of a UNA oligomer, for which the tail UNA-U/mU is added at the 3' end of the sense strand, and the tail UNA-U/mU is added at the 3' end of the antisense strand.
  • a monomer in an overhang can have any nucleobase.
  • Embodiments of this invention can provide oligomeric molecules that are active agents targeted to BGAS.
  • Embodiments of this invention can provide oligomeric molecules that are active agents targeted to BGAS.
  • rN refers to N, which is a ribonucleotide
  • mN refers to a chemically-modified 2'-OMe ribonucleotide
  • an asterisk * between characters refers to a phosphorothioate linkage
  • dN refers to a deoxyribonucleotide.
  • Methods of this invention include the treatment and prevention of various diseases in mammalian subjects.
  • a subject can be a human or mammal.
  • a subject in need of treatment or prevention can be administered an effective amount of an oligomeric compound of this invention.
  • An effective amount of an oligomeric compound of this invention can be a dose ranging from 0.001 mg/kg to 100 mg/kg.
  • target RNA expression can be reduced in a subject for at least 5 days. In certain embodiments, target RNA expression can be reduced in a subject for at least 10 days, or 15 days.
  • an oligomeric compound may not result in an inflammatory response in a subject.
  • this invention includes methods for inhibiting expression of a target gene in a cell, by treating the cell with an oligomeric compound of this invention.
  • this invention includes methods for inhibiting expression of a target gene in a mammal, by administering to the mammal a composition containing an oligomeric compound of this invention.
  • this invention provides pharmaceutical compositions containing an oligomeric compound and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition can be capable of local or systemic administration.
  • a pharmaceutical composition can be capable of any modality of administration.
  • the administration can be intravenous, subcutaneous, pulmonary, intramuscular, intraperitoneal, dermal, oral, or nasal administration.
  • Embodiments of this invention include pharmaceutical compositions containing an oligomeric compound in a lipid formulation.
  • a pharmaceutical composition may comprise one or more lipids selected from cationic lipids, anionic lipids, sterols, pegylated lipids, and any combination of the foregoing.
  • a pharmaceutical composition can be any suitable pharmaceutical composition.
  • a pharmaceutical composition can include liposomes or nanoparticles.
  • lipids and lipid compositions for delivery of an active molecule of this invention are given in WO/2015/074085, which is hereby incorporated by reference in its entirety.
  • a pharmaceutical composition can contain an oligomeric compound within a viral or bacterial vector.
  • a pharmaceutical composition of this disclosure may include carriers, diluents or excipients as are known in the art. Examples of pharmaceutical compositions are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro ed. 1985), as well as in Remington, The Science and Practice of Pharmacy, Mack Publishing Co. (2000), and J.R. Robinson, Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker (1978).
  • excipients for a pharmaceutical composition include antioxidants, suspending agents, dispersing agents, preservatives, buffering agents, tonicity agents, and surfactants.
  • Example 1 Silencing of CFTR-associated long non-coding RNA with a double-stranded UNA Oligomer.
  • a double-stranded UNA oligomer BG4 was made to target a 21-nt sequence in the BGAS promoter region of the CFTR gene. Referring to FIG. 1, BG4 was targeted to the BG213071 promoter. BG4 is shown in Table 4.
  • UNA oligomer BG4 was transfected into CF patient-derived human Bronchial Epithelial (CFhBE) homozygous AF508-CFTR primary cells, and an epithelial voltage clamp assay was performed 16 hours after transfection.
  • CFhBE CF patient-derived human Bronchial Epithelial
  • FIG. 2 demonstrates the results of a method for increasing CFTR expression in cells by administering a UNA oligomeric agent.
  • UNA oligomeric agent BG4 was used to increase the transport function of the ziF508-CFTR chloride channel in Cystic Fibrosis Human Bronchial Epithelial (CFhBE) Primary Cells.
  • CFhBE Cystic Fibrosis Human Bronchial Epithelial
  • One of the most common cystic fibrosis-associated mutations is the deletion of phenylalanine 508 (F508del), which results in channels with poor membrane expression and impaired function.
  • the potentiator ivacaftor (VX-770), a clinically approved drug for treatment of CF patients carrying the G551D mutation, was introduced to increase channel transport function.
  • the UNA oligomeric agent BG4 was introduced along with the VX-770, and the results in FIG. 2 show a significant dose-dependent increase due to UNA oligomeric agent BG4.
  • the UNA oligomeric agent BG4 activated CFTR- mediated chloride channel transport function in the CFhBE homozygous AF508-CFTR primary cells in a dose-dependent manner.
  • Example 2 Silencing of CFTR-associated long non-coding RNA with a UNA Oligomer in CFPAC-1 cells increased expression of CFTR.
  • the double-stranded UNA oligomer BG4 was used to inhibit CFTR- associated long non-coding RNA in CFPAC-1 cells. As shown in Table 5, the suppression of CFTR-associated long non-coding RNA in CFPAC-1 cells caused a dose- dependent increase in CFTR expression. A nearly three-fold increase in CFTR expression was achieved.
  • the human immortalized pancreatic adenocarcinoma cell line CFPACl (American Type Culture Collection) was maintained in DMEM supplemented with supplemented with 0.1 mM nonessential amino acids and 10% FBS (Life Technologies, Carlsbad, CA). A total of 10,000 CFPACl cells were plated onto a well of 96-well plate one day before the transfection. The culture medium was changed to 90 ⁇ of fresh medium just before the transfection. Cells were transfected with UNA oligomer using LIPOFECTAMINE RNAiMAX (Life Technologies, Carlsbad, CA, USA) in accordance with the manufacturer's instructions.
  • cell lysate was prepared with CELL-TO-CT lysis buffer (Life Technologies) according to manufacture's instruction. Quantitative PCR analysis of the CFTR mRNA expression level was carried out by using TaqMan chemistry on QuantStudio 6 Flex (Applied Biosystems).
  • Example 3 Silencing of CFTR-associated long non-coding RNA with an siRNA in CFPAC-1 cells increased expression of CFTR.
  • siRNA targeted to inhibit CFTR-associated long non-coding RNA was tested in CFPAC-1 cells.
  • the siRNA suppressed CFTR-associated long non-coding RNA in CFPAC-1 cells, and caused a 90% increase in CFTR expression at a concentration of 50 nM.
  • This preliminary example shows that suppression of CFTR-associated long non- coding RNA enhances CFTR expression.
  • Example 4 Silencing of CFTR-associated long non-coding RNA with an siRNA in CFPAC-1 cells increased expression of CFTR.
  • siRNA targeted to inhibit CFTR-associated long non-coding RNA was tested in CFPAC-1 cells.
  • the siRNA suppressed CFTR-associated long non-coding RNA in CFPAC-1 cells, and caused a 98% increase in CFTR expression at a concentration of 50 nM. This preliminary example shows that suppression of CFTR-associated long non-coding RNA enhances CFTR expression.

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Abstract

La présente invention concerne des composés, des structures et des compositions utiles dans des méthodes pour thérapie médicale en général et, plus précisément, pour stimuler le régulateur de la conductance transmembranaire impliqué dans la fibrose kystique (CFTR) chez un sujet. Les composés peuvent comprendre des monomères UNA et des monomères d'acide nucléique, et ils sont destinés à inhiber l'ARN non codant long BGAS. Les molécules de cette invention peuvent être utilisées comme ingrédients pharmaceutiques actifs dans des compositions visant à améliorer ou traiter une maladie associée au fonctionnement du CFTR, y compris la fibrose kystique.
PCT/US2016/046805 2015-08-13 2016-08-12 Agents oligomères una pour la stimulation du régulateur de la conductance transmembranaire impliqué dans la fibrose kystique et leurs utilisations WO2017027814A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014066915A2 (fr) * 2012-10-26 2014-05-01 Smith Larry J Procédés et compositions pour produire une activité ss-arni ayant une puissance accrue
WO2015179656A2 (fr) * 2014-05-23 2015-11-26 The Scripps Research Institute Activation ciblée spécifique de régulateur de la conductance transmembranaire de la fibrose kystique (cftr)

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014066915A2 (fr) * 2012-10-26 2014-05-01 Smith Larry J Procédés et compositions pour produire une activité ss-arni ayant une puissance accrue
WO2015179656A2 (fr) * 2014-05-23 2015-11-26 The Scripps Research Institute Activation ciblée spécifique de régulateur de la conductance transmembranaire de la fibrose kystique (cftr)

Non-Patent Citations (2)

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Title
OTT ET AL.: "Genomic approaches for the discovery of CFTR regulatory elements.", TRANSCRIPTION., vol. 2, no. 1, January 2011 (2011-01-01), pages 23 - 27, XP055364427 *
SAAYMAN ET AL.: "Long Non-coding RNA BGas Regulates the Cystic Fibrosis Transmembrance Conductance Regulator.", MOLECULAR THERAPY, vol. 24, no. 8, August 2016 (2016-08-01), pages 1351 - 1357, XP055364431 *

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