US20130116419A1 - Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry - Google Patents

Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry Download PDF

Info

Publication number
US20130116419A1
US20130116419A1 US13/574,136 US201113574136A US2013116419A1 US 20130116419 A1 US20130116419 A1 US 20130116419A1 US 201113574136 A US201113574136 A US 201113574136A US 2013116419 A1 US2013116419 A1 US 2013116419A1
Authority
US
United States
Prior art keywords
rna
functional group
ribose rings
alkyne functional
provides
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US13/574,136
Other languages
English (en)
Inventor
Daniel Zewge
Francis Gosselin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sirna Therapeutics Inc
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to US13/574,136 priority Critical patent/US20130116419A1/en
Priority claimed from PCT/US2011/021629 external-priority patent/WO2011090968A1/en
Publication of US20130116419A1 publication Critical patent/US20130116419A1/en
Assigned to MERCK SHARP & DOHME CORP. reassignment MERCK SHARP & DOHME CORP. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GOSSELIN, FRANCIS, ZEWGE, DANIEL
Assigned to SIRNA THERAPEUTICS, INC. reassignment SIRNA THERAPEUTICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MERCK SHARP & DOHME CORP.
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/28—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having one amino group and at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the carbon skeleton, e.g. ethers of polyhydroxy amines
    • C07C217/40—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having one amino group and at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the carbon skeleton, e.g. ethers of polyhydroxy amines having at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the same carbon atom of the carbon skeleton, e.g. amino-ketals, ortho esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/52—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups or amino groups bound to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/08—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon radicals, substituted by hetero atoms, attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/12—Oxygen or sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/44—Iso-indoles; Hydrogenated iso-indoles
    • C07D209/48—Iso-indoles; Hydrogenated iso-indoles with oxygen atoms in positions 1 and 3, e.g. phthalimide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D211/20—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms
    • C07D211/22—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/40—Oxygen atoms
    • C07D211/44—Oxygen atoms attached in position 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/40—Oxygen atoms
    • C07D211/44—Oxygen atoms attached in position 4
    • C07D211/46—Oxygen atoms attached in position 4 having a hydrogen atom as the second substituent in position 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
    • C07D295/084—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/088—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • 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
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/02—Systems containing only non-condensed rings with a three-membered ring
    • 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.
    • 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.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • 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
    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised

Definitions

  • RNA interference is an evolutionarily conserved cellular mechanism of post-transcriptional gene silencing found in fungi, plants and animals that uses small RNA molecules to inhibit gene expression in a sequence-specific manner.
  • the RNAi machinery can be harnessed to destruct any mRNA of a known sequence. This allows for suppression (knock-down) of any gene from which it was generated and consequently preventing the synthesis of the target protein.
  • Smaller siRNA duplexes introduced exogenously were found to be equally effective triggers of RNAi (Zamore, P. D., Tuschl, T., Sharp, P. A., Bartel, D. P. Cell 2000, 101, 25-33).
  • Synthetic RNA duplexes can be used to modulate therapeutically relevant biochemical pathways, including ones which are not accessible through traditional small molecule control.
  • RNA modification of RNA leads to improved physical and biological properties such as nuclease stability (Damha et al Drug Discovery Today 2008, 13(19/20), 842-855), reduced immune stimulation (Sioud TRENDS in Molecular Medicine 2006, 12(4), 167-176), enhanced binding (Koller, E. et al Nucl. Acids Res. 2006, 34, 4467-4476), enhanced lipophilic character to improve cellular uptake and delivery to the cytoplasm.
  • RNA modifications of RNA have relied heavily on work-intensive, cumbersome, multi-step syntheses of structurally novel nucleoside analogues and their corresponding phosphoramidites prior to RNA assembly.
  • a major emphasis has been placed on chemical modification of the 2′-position of nucleosides.
  • a rigorous approach to structure-activity-relationship (SAR) studies of chemical modifications will obviously require synthesis and evaluation of all four canonical ribonucleosides [adenosine (A), cytidine (C), uridine (U), guanosine (G)].
  • RNA Post-synthetic chemical modifications of RNA have centered for the most part on simple conjugation chemistry. Conjugation has largely been performed on either the 3′ or the 5′-end of the RNA via alkylamine and disulfide linkers. These modifications have allowed conjugation of RNA to various compounds such as cholesterol, fatty acids, poly(ethylene)glycols, various delivery vehicles and targeting agents such as poly(amines), peptides, peptidomimetics, and carbohydrates.
  • This invention relates to the post-synthetic chemical modification of RNA at the 2′-position on the ribose ring via a copper catalyzed Huisgen cycloaddition (“click” chemistry: Kolb, Sharpless Drug Discovery Today 2003, 8, 1128).
  • the invention 1) avoids complex, tedious multi-step syntheses of each desired modified ribonucleoside; 2) allows diverse chemical modifications using high-fidelity chemistry that is completely orthogonal to commonly used alkylamino, carboxylate and disulfide linker reactivities; 3) allows introduction of functional groups that are incompatible with modern automated solid-phase synthesis of RNA and subsequent cleavage-deprotection steps; 4) allows introduction of functional groups useful as targeting ligands; and 5) enables high-throughput structure-activity relationship studies on chemically modified RNA in 96-well format.
  • FIG. 1 Systematic evaluation of the impact on knockdown of the 2′-O-benzyl-triazole inosine chemical modification along positions 1 through 19 of the guide strand of a SSB(291) siRNA.
  • FIG. 2 Systematic evaluation of the impact on knockdown of the 2′-O-phenylthiomethyl-triazole inosine chemical modification along positions 1 through 19 of the guide strand of a SSB(291) siRNA.
  • FIG. 3 Systematic evaluation of the impact on knockdown of the 2′-O-benzyl-triazole inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of a Luc(80) siRNA.
  • FIG. 4 Systematic evaluation of the impact on knockdown of the 2′-O-phenylthiomethyl-triazole inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of a Luc(80) siRNA.
  • FIG. 5 Duration of knockdown activity of the 2′-O-benzyl-triazole inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of a Luc(80) siRNA.
  • FIG. 6 Duration of knockdown activity of the 2′-O-phenylthiornethyl inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of a Luc(80) siRNA.
  • FIG. 7 Introduction of N-acetyl-galactosamine as chemical modification.
  • FIG. 8 Introduction of poly(ethylene)glycol amine in SSB(291) RNA.
  • FIG. 9 Multi-click for introduction of multiple N-acetylgalactosamine chemical modifications in one synthetic operation.
  • This invention relates to the post-synthetic chemical modification of RNA at the 2′-position on the ribose ring via a copper catalyzed Huisgen cycloaddition (“click” chemistry: Kolb, Sharpless Drug Discovery Today 2003, 8, 1128).
  • the invention 1) avoids complex, tedious multi-step syntheses of each desired modified ribonucleoside; 2) allows diverse chemical modifications using high-fidelity chemistry that is completely orthogonal to commonly used alkylamino, carboxylate and disulfide linker reactivities; 3) allows introduction of functional groups that are incompatible with modern automated solid-phase synthesis of RNA and subsequent cleavage-deprotection steps; 4) allows introduction of functional groups useful as targeting ligands; and 5) enables high-throughput structure-activity relationship studies on chemically modified RNA in 96-well format.
  • the prior art discloses the use of “click chemistry” to generate modified oligonucleotides wherein the alkyne functional group is on the phosphate backbone or the base in DNA and RNA molecules or the alkyne functional group is on the ribose of DNA molecules.
  • the modification is for labeling purposes.
  • RNA with alkyne functional group at the 2′-position is not known.
  • click chemistry to generate 2′-modified RNA wherein the alkyne functional group is on the ribose is not known.
  • RNA can undergo auto-catalytic cleavage via intramolecular cyclization of the 2′-position onto the 3′-phosphodiester. Modification of the 2′-position is critical for RNA stability and therapeutic applicability.
  • RNA with alkyne functional group at the 2′-position is critical for RNA stability and therapeutic applicability.
  • the current invention relates to chemical modification of RNA at the 2′-position of the ribose ring based on the 1,3-dipolar cycloaddition (Huisgen reaction) between alkynes and azides.
  • the 1,3-dipolar cycloaddition (Huisgen reaction) between alkynes and azides is known. (Torn ⁇ e, Christensen, Meldal J. Org. Chem. 2002, 67, 3057; Rostovstev, Green, Fokin, Sharpless Angew. Chem. Int. Ed. 2002, 41, 2596).
  • the invention provides a process for introducing 2′-modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2′-position on at least one ribose ring; b) creating a solution of RNA in a solvent; and c) adding an organic azide and a metal catalyst to the solution to form a reaction and creating a 2′-modified RNA.
  • the process is conducted in high-throughput format.
  • the step (a) RNA may be purchased or synthesized.
  • the step (b) solvent is selected from aqueous buffer solutions (including phosphate buffers), aqueous DMSO, CH 3 CN, DMF, DMAc, NMP and a suitable ionic liquid.
  • the step (b) solvent is aqueous DMSO.
  • the step (c) metal catalyst is selected from copper and ruthenium.
  • the step (c) metal catalyst is copper.
  • the step (c) metal catalyst is copper with a suitable ligand to stabilize the Cu(I) oxidation state.
  • the step (c) reaction is performed at temperatures between ⁇ 20-300° C. for 0 to 18 h.
  • step (c) reaction is performed at temperatures between 5-120° C. for 0.5 to 18 h.
  • the step (c) reaction is performed at temperatures between 20-100° C. for 0.5 to 18 h.
  • the step (c) reaction is performed at temperatures between 60-90° C. for 0.5 to 18 h.
  • step (c) reaction is performed at temperatures between 65-80° C. for 0.5 to 18 h.
  • the invention provides a process for introducing 2′-modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2′-position on at least one ribose ring of an internal nucleotide; b) creating a solution of RNA in a solvent; and c) adding an organic azide and a metal catalyst to the solution to form a reaction and creating a 2′-modified RNA.
  • the process is conducted in high-throughput format.
  • the step (a) RNA may be purchased or synthesized.
  • the step (b) solvent is selected from aqueous buffer solutions (including phosphate buffers), aqueous DMSO, CH 3 CN, DMF, DMAc, NMP and a suitable ionic liquid.
  • the step (b) solvent is aqueous DMSO.
  • the step (c) metal catalyst is selected from copper and ruthenium.
  • the step (c) metal catalyst is copper.
  • the step (c) metal catalyst is copper with a suitable ligand to stabilize the Cu(I) oxidation state.
  • the step (e) reaction is performed at temperatures between ⁇ 20-300° C. for 0 to 18 h.
  • step (c) reaction is performed at temperatures between 5-120° C. for 0.5 to 18 h.
  • the step (c) reaction is performed at temperatures between 20-100° C. for 0.5 to 18 h. In an embodiment, the step (c) reaction is performed at temperatures between 60-90° C. for 0.5 to 18 h.
  • step (c) reaction is performed at temperatures between 65-80° C. for 0.5 to 18 h.
  • the invention provides a process for introducing 2′-modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2′-position on at least one ribose ring of an internal nucleotide; b) creating a solution of RNA in a solvent; c) adding an organic azide and a metal catalyst to the solution to form a reaction and creating a 2′-modified RNA; and d) purifying the 2 1 -modified RNA.
  • the step (a) RNA may be purchased or synthesized.
  • the step (c) solvent is selected from aqueous buffer solutions (including phosphate buffers), aqueous DMSO, CH 3 CN, DMF, DMAc, NMP and a suitable ionic liquid.
  • the step (c) solvent is aqueous DMSO.
  • the step (c) metal catalyst is selected from copper and ruthenium.
  • the step (c) metal catalyst is copper.
  • the step (c) metal catalyst is copper with a suitable ligand to stabilize Cu(I) oxidation state.
  • the step (c) reaction is performed at temperatures between ⁇ 20-300° C. for 0 to 18 h.
  • step (c) reaction is performed at temperatures between 5-120° C. for 0.5 to 18 h.
  • the step (c) reaction is performed at temperatures between 20-100° C. for 0.5 to 18 h.
  • the step (c) reaction is performed at temperatures between 60-90° C. for 0.5 to 18 h.
  • step (c) reaction is performed at temperatures between 65-80° C. for 0.5 to 18 h.
  • the step (d) purification is performed in high-throughput format on 96-well C18 cartridges (solid-phase extraction) or strong-anion-exchange-HPLC or reverse-phase HPLC or poly(acrylamide) gel electrophoresis (PAGE) or size-exclusion chromatography.
  • the invention provides a process for introducing 2′-modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2′-position on at least one ribose ring of an internal nucleotide; b) creating a solution of RNA in a solvent; c) adding an organic azide and a metal catalyst to the solution to form a reaction and creating a 2′-modified RNA; d) cooling the solution and adding a fluoride source; e) heating the solution; f) cooling the solution and adding a diluent; and g) purifying the 2′-modified RNA.
  • the step (a) RNA may be purchased or synthesized.
  • the step (c) solvent is selected from aqueous buffer solutions (including phosphate buffers), aqueous DMSO, CH 3 CN, DMF, DMAc, NMP and a suitable ionic liquid.
  • the step (c) solvent is aqueous DMSO.
  • the step (e) metal catalyst is selected from copper and ruthenium.
  • the step (c) metal catalyst is copper.
  • the step (c) metal catalyst is copper with a suitable ligand to stabilize Cu(I) oxidation state.
  • the step (c) reaction is performed at temperatures between ⁇ 20-300° C. for 0 to 18 h.
  • step (c) reaction is performed at temperatures between 5-120° C. for 0.5 to 18 h.
  • the step (c) reaction is performed at temperatures between 20-100° C. for 0.5 to 18 h.
  • the step (c) reaction is performed at temperatures between 60-90° C. for 0.5 to 18 h. In an embodiment, the step (c) reaction is performed at temperatures between 65-80° C. for 0.5 to 18 h.
  • the step (e) fluoride source is Et 3 N.3HF, tetrabutylammonium fluoride, potassium fluoride and ammonium fluoride,
  • the step (e) fluoride source is ammonium fluoride.
  • the step (f) diluent is NaCl.
  • the step (g) purification is performed in high-throughput format on 96-well C18 cartridges (solid-phase extraction) or strong-anion-exchange-HPLC or reverse-phase HPLC or poly(acrylamide) gel electrophoresis (PAGE) or size-exclusion chromatography.
  • the instant invention also discloses a method for attaching targeting ligands to RNA utilizing the process described herein.
  • the instant invention further discloses a method for attaching targeting ligands to internal nucleotides in RNA utilizing the process described herein.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on one or more ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on two or more ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on three or more ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on four or more ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on five or more ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on six or more ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on seven or more ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on eight or more ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on nine or more ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on ten or more ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on one or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on two or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on three or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on four or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on five or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on six or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on seven or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on eight or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on nine or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a RNA with an alkyne functional group at the 2′-position on ten or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on one or more ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on two or more ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on three or more ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on four or more ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on five or more ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on six or more ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on seven or more ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on eight or more ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on nine or more ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on ten or more ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on one or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on two or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on three or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on four or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on five or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on six or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on seven or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on eight or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on nine or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a miRNA with an alkyne functional group at the 2′-position on ten or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on one or more ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on two or more ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on three or more ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on four or more ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on five or more ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on six or more ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on seven or more ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on eight or more ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on nine or more ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on ten or more ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on one or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on two or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on three or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on four or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on five or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on six or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on seven or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on eight or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on nine or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on ten or more ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on one ribose ring.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on two ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on three ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2 1 -position on four ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on five ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on six ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on seven ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on eight ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on nine ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on ten ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on one ribose ring excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on two ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on three ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on four ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on five ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on six ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on seven ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on eight ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on nine ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides a siRNA with an alkyne functional group at the 2′-position on ten ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on one ribose ring.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on two ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on three ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on four ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on five ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on six ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on seven ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on eight ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on nine ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on ten ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2 1 -position on one ribose ring excluding the external 5′ and 3′ ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on two ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on three ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on four ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on five ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on six ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on seven ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on eight ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on nine ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on ten ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on one ribose ring.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on two ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on three ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on four ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on five ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on six ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on seven ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on eight ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on nine ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on ten ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on one ribose ring excluding the external 5′ and 3′ ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on two ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on three ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on four ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on five ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on six ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on seven ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on eight ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on nine ribose rings excluding the external 5′ and 3′ ribose rings.
  • the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on ten ribose rings excluding the external 5′ and 3′ ribose rings.
  • 2′-modified RNA means a RNA wherein at least one ribose ring is modified at the 2′-position.
  • Alkyne functional group means any chemical compound containing an alkyne functional group.
  • the preferred “Alkyne functional group” is the propargyl moiety shown throughout this disclosure.
  • High-throughput format means that several operations are run in parallel fashion such as for example in 96-well plate chemical synthesis, 96-well plate purification, 96-well plate chromatographic analysis and 96-well plate mass spectrometric analysis.
  • Internal nucleotide means a nucleotide in an RNA molecule that is not at the 3′- or 5′-end. For example, the internal nucleotides in a 21 mer siRNA occur at positions 2-20.
  • RNA means a chemically modified or unmodified ribonucleic acid molecule (single stranded or double stranded) comprising at least 3 nucleotides, including but not limited to miRNA and siRNA. In another embodiment, “RNA” means miRNA. In another embodiment, “RNA” means siRNA. Chemical modifications include, for example, modifications to the base, ribose ring (excluding modifications to the 2′-position), and phosphate backbone. The base can be a canonical base (A, G, T and U) or a modified or universal base (including but not limited to inosine and nitroindole).
  • Organic azide means any chemical compound containing the azide functional group.
  • Metal catalyst means any chemical form of copper and ruthenium, including solid-supported variants.
  • metal catalyst include CuBr, CuBrMe 2 S, CuI, CuSO 4 or CuOAc and a suitable reducing agent such as sodium ascorbate, Cu(CH 3 CN) 4 PF 6 , CpRuCl(PPh 3 ) 2 , and Cp*RuCl(PPh 3 ) 2 .
  • Ribose ring means the ribose moiety in a ribonucleotide.
  • Targeting ligand means a conjugate delivery moiety capable of delivering an oligonucleotide to a target cell of interest.
  • Targeting ligands include, but are not limited to, lipids (cholesterol), sugars (NAG), proteins (transferrin), peptides, poly(ethylene)glycols and antibodies. See Juliano et al., Nucleic Acids Research, 2008, 1-14, doi:10.1093/narigkn342.
  • the present invention provides a process for introducing chemical modifications into RNA at the 2′-position on the ribose ring. It is well known in the art that RNA are useful for therapeutic and research purposes.
  • RNA The synthesis of RNA is well known in the art.
  • a suitable 2′-O-propargyl nucleoside phosphoramidite is incorporated into RNA using modern techniques based on the phosphoramidite approach.
  • the crude, solid-support bound protected oligonucleotide is then treated with aqueous methylamine to remove nucleobase and phosphate protecting groups.
  • the crude product is then lyophilized to remove volatiles.
  • the crude product is dissolved in DMSO:H 2 O, treated with a suitable organic azide and a copper catalyst. After aging an appropriate amount of time, the reaction mixture is treated with fluoride to remove the 2′-O-tert-butyldimethylsilyl protecting groups.
  • the crude product is then purified to obtain the chemically modified RNA.
  • RNA Lyophilized crude RNA ( ⁇ 50 nmol) containing at least one alkyne functional group (shown below) in 96-well format was dissolved in DMSO:water (75:25, 40 jut). Benzyl azide (1M in DMSO, 40 pi) was added, followed by a freshly prepared solution of CuBr.Me 2 S in DMSO (12 mM, 40 ⁇ L). The reaction block was sealed and heated at 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 ⁇ L, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 ⁇ L). The crude product was purified on a C18 cartridge to afford the desired chemically modified benzyl-triazole-linked RNA as determined by HPLC and LC-MS analyses.
  • RNA ( ⁇ 50 nmol) containing at least one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 ⁇ L). Azidomethyl phenyl sulfide (1M in DMSO, 40 ⁇ L) was added, followed by a freshly prepared solution of CuBr.Me 2 S in DMSO (12 mM, 40 ⁇ L). The reaction block was sealed and heated to 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 ⁇ L, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 ⁇ L). The crude product was purified on a C18 cartridge to afford the desired chemically modified phenylthiomethyl-triazole-linked RNA as determined by HPLC and LC-MS analyses.
  • RNA ( ⁇ 50 nmol) containing at least one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 ⁇ L). Ethyl azidoacetate (1M in DMSO, 40 ⁇ L) was added, followed by a freshly prepared solution of CuBr.Me 2 S in DMSO (12 mM, 40 ⁇ L). The reaction block was sealed and heated to 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 ⁇ L, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 ⁇ L). The crude product was purified on a C18 cartridge to afford the desired chemically modified ethyl-carboxymethyl-1,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.
  • RNA ( ⁇ 50 nmol) containing at least one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 ⁇ L). Modified N-acetyl galactosamine azide (1M in DMSO, 40 ⁇ L) was added, followed by a freshly prepared solution of CuBr.Me 2 S in DMSO (12 mM, 40 ⁇ L). The reaction block was sealed and heated to 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 ⁇ L, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 ⁇ L). The crude product was purified on a C18 cartridge to afford the desired chemically modified N-acetylgalactosamine-1,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.
  • RNA ( ⁇ 50 nmol) containing more than one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 ⁇ L). Modified N-acetylgalactosamine azide (1M in DMSO, 40 ⁇ L) was added, followed by a freshly prepared solution of CuBr.Me 2 S in DMSO (12 mM, 40 ⁇ L). The reaction block was sealed and heated to 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 ⁇ L, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 ⁇ L). The crude product was purified on a C18 cartridge to afford the desired chemically modified N-acetylgalactosamine-1,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.
  • RNA 50 nmol containing at least one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 uL). Benzyl azide (1M in DMSO, 40 uL) was added, followed by a freshly prepared solution of CuBr.Me 2 S in DMSO (12 mM, 40 uL). The reaction block was sealed and heated at 65-SO ° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 ⁇ L, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 uL). The crude product was purified on a C18 cartridge to afford the desired chemically modified benzyl-1,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.
  • RNA 50 nmol containing at least one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 uL). 11-Azido-3,6,9-trioxaundecan-1-amine (1M in DMSO, 40 uL) was added, followed by a freshly prepared solution of CuBr.Me 2 S in DMSO (12 mM, 40 uL). The reaction block was sealed and heated at 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 ⁇ L, 5.4M in water) was added. The solution was heated at 65° C.
  • RNA oligomers with the first nucleotide, uridine (U), replaced with 2′-O-propargyl-inosine. Then, a second sequence, in which the second nucleoside (U) was replaced with 2′-O-propargyl-inosine was synthesized, keeping all other nucleotides unchanged.
  • Hepa1-6 cells were transfected with 10 nM of either the unmodified, modified, or negative control siRNA using a commercial lipid transfection reagent.
  • the target mRNA was assessed for degradation using standard Taqman procedures.
  • Multiplex luciferase assay for in vitro duration study is modified from the manufacturer's instruction using HeLa-luc cell line. Briefly, the cell viability and the luciferease expression at the same well are determined by CellTiter-FluorTM (Promega, Cat #G6082) and Bright-GloTM (Promega Cat #E2620) sequentially.
  • HeLa-luc cell line is a stable firefly luciferase reporter expression cell line.
  • Bright-GloTM luciferase assay system contains the stable substrate—luciferin and assay buffer.
  • the luminescent reaction of luciferease and luciferin has high quantum yield and can be detected as luminescence intensity, which represents the luciferase expression level.
  • Target siRNAs containing luciferase coding region is designed to be transfected into the HeLa-luc cells. Once the target is effected, the luciferase expression is reduced accordingly, Therefore, the siRNA silencing efficacy can be determined by the relative luminecence intensity of treated cells.
  • CellTiter-fluor kit measures the conserved and constitutive protease activity within live cells and therefore serves as a marker of cell viability, using a fluorogenic, cell-permeable peptide substrate (glycyl-phenylalanyl-aminofluorocoumarin; GF-AFC).
  • Luciferase stable expressed HeLa-luc cell cells are plated in 96-well plates at density of 4,500 cells per well in 100 ⁇ L DMEM media without antibiotics 24 hours prior to transfection.
  • siRNA transfection is performed using the RNAiMAXTM (Invitrogen). Briefly, 0.05 ⁇ M siRNA are mixed with Opti-MEMmedia and RNAiMAX and incubated at room temperature for 15 min. The mix is then added to the cells. The final siRNA concentration is 1 nM. Cell plates for all time points are transfected at same time with a medium change at 6 hours post-transfection into 100 ⁇ L of fresh completed DMEM (DMEM+10% FBS+Pen/strep).
  • In vitro duration is determined by the luciferase expression post-transfection at four time points: day 1, day 2, day 5 and day 7. Addition medium changes are performed at day 2 and day 5 into 100 ⁇ L of fresh completed DMEM (DMEM+10% FBS+Penn/strep). Luciferase levels are determined using the Bright-Glo Luminescence Assay (Promega) and measuring the wells on an Envison instrument (Perkin Elmer) according to manufacturer's instructions.
  • the cell viability of the same treatment wells is measured using CellTiter-fluor kit (Promega) according to manufacturer's instructions.
  • This assay measures the conserved and constitutive protease activity within live cells and therefore servers as a marker of cell viability, using a fluorogenic, cell-permeable peptide substrate (glycyl-phenylalanyl-aminofluorocoumarin; GF-AFC).
  • the fluorescence was measured on the Envision using exciton filter at 405 nm and emission filter at 510 nm.
  • the luciferase expression was normalized to cell viability. The log of this number was calculated to determine the luciferase protein that was degraded (knockdown). A non-targeting siRNA was subtracted from this value to account for non-specific background.
  • RNAs made by the process of the invention are useful in high-throughput structure-activity relationship studies on chemically modified RNA in 96-well format.
  • FIG. 1 the impact on knockdown of the 2′-O-benzyl-triazole inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of an siRNA targeting mRNA SSB(291).
  • the liver targeting compound N-acetyl-galactosamine can be introduced as a chemical modification that may help with specific cell targeting, cellular uptake and delivery of RNA.
  • poly(ethylene)glycol amines can be introduced to improve solubility properties, cellular uptake, immune stealth, reduce metabolic clearance and delivery of RNA.
  • the “click” reaction can be utilized to introduce multiple chemical modifications in one synthetic operation.
  • the click reaction was performed to introduce three units of protected N-acetylgalactosamine on RNA. This may lead to improved physical properties towards solubility, cellular uptake, and delivery of siRNA.
US13/574,136 2010-01-22 2011-01-19 Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry Abandoned US20130116419A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US13/574,136 US20130116419A1 (en) 2010-01-22 2011-01-19 Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US29737710P 2010-01-22 2010-01-22
US32590810P 2010-04-20 2010-04-20
US13/574,136 US20130116419A1 (en) 2010-01-22 2011-01-19 Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry
PCT/US2011/021629 WO2011090968A1 (en) 2010-01-22 2011-01-19 Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/021629 A-371-Of-International WO2011090968A1 (en) 2010-01-22 2011-01-19 Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14/609,118 Continuation US9441228B2 (en) 2010-01-22 2015-01-29 Post-synthetic chemical modification of RNA at the 2′-position of the ribose ring via “click” chemistry

Publications (1)

Publication Number Publication Date
US20130116419A1 true US20130116419A1 (en) 2013-05-09

Family

ID=44307168

Family Applications (4)

Application Number Title Priority Date Filing Date
US13/574,315 Active 2031-04-14 US9670487B2 (en) 2010-01-22 2011-01-19 Cationic lipids for oligonucleotide delivery
US13/574,136 Abandoned US20130116419A1 (en) 2010-01-22 2011-01-19 Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry
US14/609,118 Active US9441228B2 (en) 2010-01-22 2015-01-29 Post-synthetic chemical modification of RNA at the 2′-position of the ribose ring via “click” chemistry
US15/234,736 Abandoned US20160348109A1 (en) 2010-01-22 2016-08-11 Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US13/574,315 Active 2031-04-14 US9670487B2 (en) 2010-01-22 2011-01-19 Cationic lipids for oligonucleotide delivery

Family Applications After (2)

Application Number Title Priority Date Filing Date
US14/609,118 Active US9441228B2 (en) 2010-01-22 2015-01-29 Post-synthetic chemical modification of RNA at the 2′-position of the ribose ring via “click” chemistry
US15/234,736 Abandoned US20160348109A1 (en) 2010-01-22 2016-08-11 Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry

Country Status (3)

Country Link
US (4) US9670487B2 (de)
EP (2) EP2525781A1 (de)
WO (1) WO2011090965A1 (de)

Families Citing this family (130)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9670487B2 (en) * 2010-01-22 2017-06-06 Sirna Therapeutics, Inc. Cationic lipids for oligonucleotide delivery
EP3960726A1 (de) 2011-10-18 2022-03-02 Dicerna Pharmaceuticals, Inc. Kationische aminlipide und verwendungen davon
JP6133883B2 (ja) * 2011-11-04 2017-05-24 日東電工株式会社 薬物送達用の脂質ナノ粒子の生成方法
US9579338B2 (en) 2011-11-04 2017-02-28 Nitto Denko Corporation Method of producing lipid nanoparticles for drug delivery
CA2856742A1 (en) 2011-12-07 2013-06-13 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
EP2791160B1 (de) 2011-12-16 2022-03-02 ModernaTX, Inc. Modifizierte mrna zusammensetzungen
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US10501513B2 (en) 2012-04-02 2019-12-10 Modernatx, Inc. Modified polynucleotides for the production of oncology-related proteins and peptides
US9303079B2 (en) 2012-04-02 2016-04-05 Moderna Therapeutics, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
AU2013243949A1 (en) * 2012-04-02 2014-10-30 Moderna Therapeutics, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
AU2013243948A1 (en) 2012-04-02 2014-10-30 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins associated with human disease
EP4074834A1 (de) 2012-11-26 2022-10-19 ModernaTX, Inc. Am kettenende modifizierte rna
AU2013361605A1 (en) * 2012-12-20 2015-06-04 Sirna Therapeutics, Inc. Post-synthetic orthogonal amidation plus metal catalyzed azide-alkyne cycloaddition click chemistry on siRNA
US10258698B2 (en) 2013-03-14 2019-04-16 Modernatx, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
HRP20211563T1 (hr) 2013-07-11 2022-01-07 Modernatx, Inc. Pripravci koji sadrže sintetske polinukleotide koji kodiraju proteine srodne crispr-u i sintetske sgrna, te postupci njihove uporabe
US20160194625A1 (en) 2013-09-03 2016-07-07 Moderna Therapeutics, Inc. Chimeric polynucleotides
WO2015034925A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Circular polynucleotides
CA2926218A1 (en) 2013-10-03 2015-04-09 Moderna Therapeutics, Inc. Polynucleotides encoding low density lipoprotein receptor
JP6621409B2 (ja) 2013-11-22 2019-12-18 ミナ セラピューティクス リミテッド Ｃ／ＥＢＰα小分子活性化ＲＮＡ組成物
KR102396026B1 (ko) 2014-01-21 2022-05-09 안자리움 바이오사이언시스 아게 하이브리도좀, 이를 포함하는 조성물, 이의 제조 방법 및 이의 용도
US9738593B2 (en) 2014-06-25 2017-08-22 Acuitas Therapeutics Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
CA2955250A1 (en) 2014-07-16 2016-01-21 Moderna Therapeutics, Inc. Chimeric polynucleotides
EP3171895A1 (de) 2014-07-23 2017-05-31 Modernatx, Inc. Modifizierte polynukleotide zur herstellung von intrabodys
EP3209780A4 (de) 2014-10-24 2018-09-19 University of Maryland, Baltimore Kurze nichtcodierende proteinregulatorische rnas (sprrnas) und verfahren zur verwendung
IL283545B2 (en) 2015-06-29 2023-09-01 Acuitas Therapeutics Inc Lipids and nanoparticulate lipid formulations for delivery of nucleic acids
EP3364981A4 (de) 2015-10-22 2019-08-07 ModernaTX, Inc. Impfstoff gegen das humane cytomegalovirus
PL3368507T3 (pl) 2015-10-28 2023-03-27 Acuitas Therapeutics Inc. Nowe preparaty lipidów i nanocząstek lipidowych do dostarczania kwasów nukleinowych
HRP20220525T1 (hr) 2015-12-23 2022-05-27 Modernatx, Inc. Postupci uporabe polinukleotida koji kodiraju ligand ox40
TW201726614A (zh) * 2015-12-25 2017-08-01 Kyowa Hakko Kirin Co Ltd 作為陽離子性脂質之化合物
WO2017120612A1 (en) 2016-01-10 2017-07-13 Modernatx, Inc. Therapeutic mrnas encoding anti ctla-4 antibodies
WO2018213731A1 (en) 2017-05-18 2018-11-22 Modernatx, Inc. Polynucleotides encoding tethered interleukin-12 (il12) polypeptides and uses thereof
US20200268666A1 (en) 2017-06-14 2020-08-27 Modernatx, Inc. Polynucleotides encoding coagulation factor viii
EP4219715A3 (de) 2017-09-08 2023-09-06 MiNA Therapeutics Limited Stabilisierte cebpa-sarna-zusammensetzungen und verfahren zur verwendung
AU2018330495A1 (en) 2017-09-08 2020-03-26 Mina Therapeutics Limited Stabilized hnf4a sarna compositions and methods of use
EP3775211B1 (de) 2018-04-12 2023-04-05 MiNA Therapeutics Limited Sirt1-sarna kompositionen und methoden zu deren verwendung
WO2019217964A1 (en) 2018-05-11 2019-11-14 Lupagen, Inc. Systems and methods for closed loop, real-time modifications of patient cells
EP3833762A4 (de) 2018-08-09 2022-09-28 Verseau Therapeutics, Inc. Oligonukleotidzusammensetzungen zum targeting von ccr2 und csf1r und verwendungen davon
EP3852732A1 (de) 2018-09-19 2021-07-28 ModernaTX, Inc. Peg-lipide und deren verwendung
CA3113353A1 (en) 2018-09-19 2020-03-26 Modernatx, Inc. High-purity peg lipids and uses thereof
EP3897702A2 (de) 2018-12-21 2021-10-27 CureVac AG Rna für malaria-impfstoffe
US11453639B2 (en) 2019-01-11 2022-09-27 Acuitas Therapeutics, Inc. Lipids for lipid nanoparticle delivery of active agents
EP3920950A1 (de) 2019-02-08 2021-12-15 CureVac AG In den suprachoroidalen raum verabreichte codierende rna zur behandlung von augenerkrankungen
US20220211740A1 (en) 2019-04-12 2022-07-07 Mina Therapeutics Limited Sirt1-sarna compositions and methods of use
US20220313813A1 (en) 2019-06-18 2022-10-06 Curevac Ag Rotavirus mrna vaccine
WO2021016075A1 (en) 2019-07-19 2021-01-28 Flagship Pioneering Innovations Vi, Llc Recombinase compositions and methods of use
EP4010031A1 (de) 2019-08-06 2022-06-15 L.E.A.F Holdings Group LLC Verfahren zur herstellung von polyglutamat-antifolaten und ihre verwendungen
MX2022001870A (es) 2019-08-14 2022-05-30 Curevac Ag Combinaciones y composiciones de arn con propiedades inmunoestimuladoras disminuidas.
EP4048807A1 (de) 2019-09-23 2022-08-31 Omega Therapeutics, Inc. Zusammensetzungen und verfahren zur modulation von apolipoprotein-b(apob)-genexpression
AU2020352552A1 (en) 2019-09-23 2022-03-17 Omega Therapeutics, Inc. Compositions and methods for modulating hepatocyte nuclear factor 4-alpha (HNF4α) gene expression
EP3886897A1 (de) 2020-02-04 2021-10-06 CureVac AG Coronavirusimpfstoff
CA3173528A1 (en) 2020-03-11 2021-09-16 Omega Therapeutics, Inc. Compositions and methods for modulating forkhead box p3 (foxp3) gene expression
CA3172591A1 (en) 2020-03-24 2021-09-30 Douglas Anthony KERR Non-viral dna vectors and uses thereof for expressing gaucher therapeutics
US20230138409A1 (en) 2020-03-24 2023-05-04 Generation Bio Co. Non-viral dna vectors and uses thereof for expressing factor ix therapeutics
CA3179420A1 (en) 2020-05-20 2021-11-25 Avak Kahvejian Coronavirus antigen compositions and their uses
WO2021236930A1 (en) 2020-05-20 2021-11-25 Flagship Pioneering Innovations Vi, Llc Immunogenic compositions and uses thereof
EP3993828A1 (de) 2020-05-29 2022-05-11 CureVac AG Kombinationsimpfstoffe auf nukleinsäurebasis
EP4158032A2 (de) 2020-05-29 2023-04-05 Flagship Pioneering Innovations VI, LLC Trem-zusammensetzungen und damit in zusammenhang stehende verfahren
EP4158031A1 (de) 2020-05-29 2023-04-05 Flagship Pioneering Innovations VI, LLC Trem-zusammensetzungen und damit in zusammenhang stehende verfahren
JP2023535365A (ja) 2020-07-16 2023-08-17 アクイタス セラピューティクス インコーポレイテッド 脂質ナノ粒子に使用するためのカチオン性脂質
JP2023535632A (ja) 2020-07-27 2023-08-18 アンジャリウム バイオサイエンシズ エージー Ｄｎａ分子の組成物、その作製方法、及びその使用方法
EP4172194A1 (de) 2020-07-31 2023-05-03 CureVac SE Nukleinsäurecodierte antikörpermischungen
BR112023001955A2 (pt) 2020-08-06 2023-04-11 Modernatx Inc Composições para a distribuição de moléculas de carga útil ao epitélio das vias aéreas
EP4157344A2 (de) 2020-08-31 2023-04-05 CureVac SE Coronavirus-impfstoffe auf basis multivalenter nukleinsäuren
AU2021336976A1 (en) 2020-09-03 2023-03-23 Flagship Pioneering Innovations Vi, Llc Immunogenic compositions and uses thereof
GB2603454A (en) 2020-12-09 2022-08-10 Ucl Business Ltd Novel therapeutics for the treatment of neurodegenerative disorders
WO2022137133A1 (en) 2020-12-22 2022-06-30 Curevac Ag Rna vaccine against sars-cov-2 variants
CA3171051A1 (en) 2020-12-22 2022-06-30 Curevac Ag Pharmaceutical composition comprising lipid-based carriers encapsulating rna for multidose administration
WO2022140702A1 (en) 2020-12-23 2022-06-30 Flagship Pioneering, Inc. Compositions of modified trems and uses thereof
WO2022162027A2 (en) 2021-01-27 2022-08-04 Curevac Ag Method of reducing the immunostimulatory properties of in vitro transcribed rna
WO2022200574A1 (en) 2021-03-26 2022-09-29 Glaxosmithkline Biologicals Sa Immunogenic compositions
EP4314292A1 (de) 2021-03-26 2024-02-07 MiNA Therapeutics Limited Tmem173-sarna-zusammensetzungen und verfahren zur verwendung
CA3171429A1 (en) 2021-03-31 2022-09-30 Alexander SCHWENGER Syringes containing pharmaceutical compositions comprising rna
CA3214085A1 (en) 2021-03-31 2022-10-06 Darby Rye Schmidt Thanotransmission polypeptides and their use in treating cancer
AU2022260111A1 (en) 2021-04-20 2023-11-30 Anjarium Biosciences Ag Compositions of dna molecules encoding amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase, methods of making thereof, and methods of use thereof
CA3216585A1 (en) 2021-04-27 2022-11-03 Nathaniel SILVER Non-viral dna vectors expressing therapeutic antibodies and uses thereof
WO2022232286A1 (en) 2021-04-27 2022-11-03 Generation Bio Co. Non-viral dna vectors expressing anti-coronavirus antibodies and uses thereof
WO2022233880A1 (en) 2021-05-03 2022-11-10 Curevac Ag Improved nucleic acid sequence for cell type specific expression
CA3173953A1 (en) 2021-06-11 2023-12-10 Tyson D. BOWEN Rna polymerase iii promoters and methods of use
EP4367242A2 (de) 2021-07-07 2024-05-15 Omega Therapeutics, Inc. Zusammensetzungen und verfahren zur modulation der genexpression des sekretierten frizzled-rezeptorproteins 1 (sfrp1)
WO2023009547A1 (en) 2021-07-26 2023-02-02 Flagship Pioneering Innovations Vi, Llc Trem compositions and uses thereof
WO2023014974A1 (en) 2021-08-06 2023-02-09 University Of Iowa Research Foundation Double stranded mrna vaccines
WO2023023055A1 (en) 2021-08-16 2023-02-23 Renagade Therapeutics Management Inc. Compositions and methods for optimizing tropism of delivery systems for rna
AU2022336664A1 (en) 2021-09-03 2024-01-18 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids comprising phosphatidylserine
WO2023031855A1 (en) 2021-09-03 2023-03-09 Glaxosmithkline Biologicals Sa Substitution of nucleotide bases in self-amplifying messenger ribonucleic acids
CA3230031A1 (en) 2021-09-03 2023-03-09 Patrick Baumhof Novel lipid nanoparticles for delivery of nucleic acids
WO2023044343A1 (en) 2021-09-14 2023-03-23 Renagade Therapeutics Management Inc. Acyclic lipids and methods of use thereof
CA3232386A1 (en) 2021-09-14 2023-03-23 Renagade Therapeutics Management Inc. Cyclic lipids and methods of use thereof
EP4271818A1 (de) 2021-09-17 2023-11-08 Flagship Pioneering Innovations VI, LLC Zusammensetzungen und verfahren zur herstellung von zirkulären polyribonukleotiden
TW202322826A (zh) 2021-10-18 2023-06-16 美商旗艦先鋒創新有限責任公司 用於純化多核糖核苷酸之組成物及方法
WO2023073228A1 (en) 2021-10-29 2023-05-04 CureVac SE Improved circular rna for expressing therapeutic proteins
WO2023081756A1 (en) 2021-11-03 2023-05-11 The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone Precise genome editing using retrons
CA3236235A1 (en) 2021-11-08 2023-05-11 Orna Therapeutics, Inc. Lipid nanoparticle compositions for delivering circular polynucleotides
WO2023086465A1 (en) 2021-11-12 2023-05-19 Modernatx, Inc. Compositions for the delivery of payload molecules to airway epithelium
WO2023097003A2 (en) 2021-11-24 2023-06-01 Flagship Pioneering Innovations Vi, Llc Immunogenic compositions and their uses
WO2023096990A1 (en) 2021-11-24 2023-06-01 Flagship Pioneering Innovation Vi, Llc Coronavirus immunogen compositions and their uses
WO2023096963A1 (en) 2021-11-24 2023-06-01 Flagship Pioneering Innovations Vi, Llc Varicella-zoster virus immunogen compositions and their uses
WO2023099884A1 (en) 2021-12-01 2023-06-08 Mina Therapeutics Limited Pax6 sarna compositions and methods of use
GB202117758D0 (en) 2021-12-09 2022-01-26 Ucl Business Ltd Therapeutics for the treatment of neurodegenerative disorders
AR128002A1 (es) 2021-12-17 2024-03-20 Flagship Pioneering Innovations Vi Llc Métodos de enriquecimiento de rna circular en condiciones desnaturalizantes
TW202340461A (zh) 2021-12-22 2023-10-16 美商旗艦先鋒創新有限責任公司 用於純化多核糖核苷酸之組成物和方法
WO2023122752A1 (en) 2021-12-23 2023-06-29 Renagade Therapeutics Management Inc. Constrained lipids and methods of use thereof
WO2023122789A1 (en) 2021-12-23 2023-06-29 Flagship Pioneering Innovations Vi, Llc Circular polyribonucleotides encoding antifusogenic polypeptides
WO2023135273A2 (en) 2022-01-14 2023-07-20 Anjarium Biosciences Ag Compositions of dna molecules encoding factor viii, methods of making thereof, and methods of use thereof
WO2023141602A2 (en) 2022-01-21 2023-07-27 Renagade Therapeutics Management Inc. Engineered retrons and methods of use
WO2023144330A1 (en) 2022-01-28 2023-08-03 CureVac SE Nucleic acid encoded transcription factor inhibitors
TW202345863A (zh) 2022-02-09 2023-12-01 美商現代公司 黏膜投與方法及調配物
WO2023161350A1 (en) 2022-02-24 2023-08-31 Io Biotech Aps Nucleotide delivery of cancer therapy
WO2023170435A1 (en) 2022-03-07 2023-09-14 Mina Therapeutics Limited Il10 sarna compositions and methods of use
WO2023177655A1 (en) 2022-03-14 2023-09-21 Generation Bio Co. Heterologous prime boost vaccine compositions and methods of use
WO2023183616A1 (en) 2022-03-25 2023-09-28 Senda Biosciences, Inc. Novel ionizable lipids and lipid nanoparticles and methods of using the same
WO2023196931A1 (en) 2022-04-07 2023-10-12 Renagade Therapeutics Management Inc. Cyclic lipids and lipid nanoparticles (lnp) for the delivery of nucleic acids or peptides for use in vaccinating against infectious agents
WO2023196634A2 (en) 2022-04-08 2023-10-12 Flagship Pioneering Innovations Vii, Llc Vaccines and related methods
WO2023220083A1 (en) 2022-05-09 2023-11-16 Flagship Pioneering Innovations Vi, Llc Trem compositions and methods of use for treating proliferative disorders
WO2023220729A2 (en) 2022-05-13 2023-11-16 Flagship Pioneering Innovations Vii, Llc Double stranded dna compositions and related methods
WO2023227608A1 (en) 2022-05-25 2023-11-30 Glaxosmithkline Biologicals Sa Nucleic acid based vaccine encoding an escherichia coli fimh antigenic polypeptide
WO2023239756A1 (en) 2022-06-07 2023-12-14 Generation Bio Co. Lipid nanoparticle compositions and uses thereof
WO2023242817A2 (en) 2022-06-18 2023-12-21 Glaxosmithkline Biologicals Sa Recombinant rna molecules comprising untranslated regions or segments encoding spike protein from the omicron strain of severe acute respiratory coronavirus-2
WO2023250112A1 (en) 2022-06-22 2023-12-28 Flagship Pioneering Innovations Vi, Llc Compositions of modified trems and uses thereof
WO2024020346A2 (en) 2022-07-18 2024-01-25 Renagade Therapeutics Management Inc. Gene editing components, systems, and methods of use
WO2024030856A2 (en) 2022-08-01 2024-02-08 Flagship Pioneering Innovations Vii, Llc Immunomodulatory proteins and related methods
WO2024033901A1 (en) 2022-08-12 2024-02-15 LifeEDIT Therapeutics, Inc. Rna-guided nucleases and active fragments and variants thereof and methods of use
WO2024035952A1 (en) 2022-08-12 2024-02-15 Remix Therapeutics Inc. Methods and compositions for modulating splicing at alternative splice sites
WO2024040222A1 (en) 2022-08-19 2024-02-22 Generation Bio Co. Cleavable closed-ended dna (cedna) and methods of use thereof
WO2024044723A1 (en) 2022-08-25 2024-02-29 Renagade Therapeutics Management Inc. Engineered retrons and methods of use
WO2024049979A2 (en) 2022-08-31 2024-03-07 Senda Biosciences, Inc. Novel ionizable lipids and lipid nanoparticles and methods of using the same
EP4342460A1 (de) 2022-09-21 2024-03-27 NovoArc GmbH Lipidnanopartikel mit nukleinsäurefracht
WO2024068545A1 (en) 2022-09-26 2024-04-04 Glaxosmithkline Biologicals Sa Influenza virus vaccines
WO2024077191A1 (en) 2022-10-05 2024-04-11 Flagship Pioneering Innovations V, Inc. Nucleic acid molecules encoding trif and additionalpolypeptides and their use in treating cancer
WO2024089638A1 (en) 2022-10-28 2024-05-02 Glaxosmithkline Biologicals Sa Nucleic acid based vaccine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7919591B2 (en) * 2006-09-08 2011-04-05 Ambrx, Inc. Modified human plasma polypeptide or Fc scaffolds and their uses

Family Cites Families (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1075718A (en) * 1975-12-12 1980-04-15 Gerhard W. H. Scherf Acetals as enervatable surface-active organic compounds
US7799565B2 (en) 2004-06-07 2010-09-21 Protiva Biotherapeutics, Inc. Lipid encapsulated interfering RNA
US7745651B2 (en) 2004-06-07 2010-06-29 Protiva Biotherapeutics, Inc. Cationic lipids and methods of use
CA2597724A1 (en) 2005-02-14 2007-08-02 Sirna Therapeutics, Inc. Cationic lipids and formulated molecular compositions containing them
US7404969B2 (en) 2005-02-14 2008-07-29 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
US8114636B2 (en) * 2006-02-10 2012-02-14 Life Technologies Corporation Labeling and detection of nucleic acids
MX2009003548A (es) * 2006-10-03 2009-04-15 Alnylam Pharmaceuticals Inc Formulaciones que contienen lipidos.
CA2689042A1 (en) * 2007-02-16 2008-08-28 Merck & Co., Inc. Compositions and methods for potentiated activity of biologicaly active molecules
WO2009086558A1 (en) 2008-01-02 2009-07-09 Tekmira Pharmaceuticals Corporation Improved compositions and methods for the delivery of nucleic acids
US20090263407A1 (en) 2008-04-16 2009-10-22 Abbott Laboratories Cationic Lipids and Uses Thereof
WO2010039548A2 (en) 2008-09-23 2010-04-08 Alnylam Pharmaceuticals, Inc. Chemical modifications of monomers and oligonucleotides with cycloaddition
US9139554B2 (en) 2008-10-09 2015-09-22 Tekmira Pharmaceuticals Corporation Amino lipids and methods for the delivery of nucleic acids
WO2010048536A2 (en) 2008-10-23 2010-04-29 Alnylam Pharmaceuticals, Inc. Processes for preparing lipids
CA2751342C (en) 2009-01-29 2019-05-07 Alnylam Pharmaceuticals, Inc. Lipid formulations comprising cationic lipid and a targeting lipid comprising n-acetyl galactosamine for delivery of nucleic acid
US8569256B2 (en) * 2009-07-01 2013-10-29 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
US9670487B2 (en) * 2010-01-22 2017-06-06 Sirna Therapeutics, Inc. Cationic lipids for oligonucleotide delivery
US20110250641A1 (en) * 2010-04-08 2011-10-13 Matthew Jacob Powell Zwitterionic acid-labile surfactants and methods of use

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7919591B2 (en) * 2006-09-08 2011-04-05 Ambrx, Inc. Modified human plasma polypeptide or Fc scaffolds and their uses
US8022186B2 (en) * 2006-09-08 2011-09-20 Ambrx, Inc. Modified human plasma polypeptide or Fc scaffolds and their uses
US8053560B2 (en) * 2006-09-08 2011-11-08 Ambrx, Inc. Modified human plasma polypeptide or Fc scaffolds and their uses
US8618257B2 (en) * 2006-09-08 2013-12-31 Ambrx, Inc. Modified human plasma polypeptide or Fc scaffolds and their uses

Also Published As

Publication number Publication date
WO2011090965A1 (en) 2011-07-28
US20150211008A1 (en) 2015-07-30
US9670487B2 (en) 2017-06-06
EP2525781A1 (de) 2012-11-28
EP2526113A4 (de) 2013-11-27
EP2526113A1 (de) 2012-11-28
EP2526113B1 (de) 2016-08-10
US20130053572A1 (en) 2013-02-28
US20160348109A1 (en) 2016-12-01
US9441228B2 (en) 2016-09-13

Similar Documents

Publication Publication Date Title
US9441228B2 (en) Post-synthetic chemical modification of RNA at the 2′-position of the ribose ring via “click” chemistry
US9914745B2 (en) Morpholino-based antisense agent
CN102639552A (zh) 含有O-缩醛乙酰丙基酯基团(O-acetal levulinyl ester)的RNA单体及其在RNA微阵列中的应用
US10385090B2 (en) Nucleotide derivative or salt thereof, nucleotide-derived 5′-phosphate ester or salt thereof, nucleotide-derived 3′-phosphoramidite compound or salt thereof, and polynucleotide
US20210277047A1 (en) Synthesis and Biological Activity of Phosphoramidimidate and Phosphoramidate DNA
AU2018223027A1 (en) Post-synthetic orthogonal amidation plus metal catalyzed azide-alkyne cycloaddition click chemistry on siRNA
US10844376B2 (en) Structurally-enhanced miRNA inhibitor S-TuD
Gebhard et al. Hybridization-Sensitive Fluorescent Probes for DNA and RNA by a Modular “Click” Approach
Abdelhady et al. Rapid alkene–alkene photo-cross-linking on the base-flipping-out field in duplex DNA
Sato et al. Synthesis and characterization of novel (S)-5′-C-aminopropyl-2′-fluorouridine modified oligonucleotides as therapeutic siRNAs
WO2011090968A1 (en) Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry
Winkler et al. 2′‐O‐Lysylaminohexyl Oligonucleotides: Modifications for Antisense and siRNA
Kajino et al. (S)-5′-C-Aminopropyl-2′-O-methyl nucleosides enhance antisense activity in cultured cells and binding affinity to complementary single-stranded RNA
Zubin et al. Oligonucleotides containing 2′-O-[2-(2, 3-dihydroxypropyl) amino-2-oxoethyl] uridine as suitable precursors of 2′-aldehyde oligonucleotides for chemoselective ligation
US20210032282A1 (en) Nucleic acid complex, method for forming nucleic acid hybridization, pharmaceutical composition, nucleic acid probe, and complementary-strand nucleic acid complex
AU2012254580B2 (en) Process for preparing phosphate compound bearing isotope
WO2012036972A1 (en) Chemical modification of rna at the 2'-position of the ribose ring via aaa coupling
Taniguchi et al. Enhancement of TFO triplex formation by conjugation with pyrene via click chemistry
Patrushev et al. New Zwitter-Ionic Oligonucleotides: Preparation and Complementary Binding
Hirano et al. Synthesis and Application of Interstrand Cross‐Linked Duplexes by Covalently Linking a Pair of Abasic Sites
WO2014016202A1 (en) Methods for catalytic alkylation of nucleic acids
Matsubara et al. Synthesis of siRNAs incorporated with cationic peptides R8G7 and R8A7 and the effect of the modifications on siRNA properties
JP5569768B2 (ja) ヌクレオシド三リン酸誘導体、核酸プローブ、マルチラベル化核酸プローブおよび標的核酸の検出方法
JP5424236B2 (ja) オリゴヌクレオチド誘導体、オリゴヌクレオチド誘導体を用いたオリゴヌクレオチド構築物、オリゴヌクレオチド誘導体を合成するための化合物及びオリゴヌクレオチド誘導体の製造方法
Wu et al. Synthesis of Site‐Specifically Phosphate‐Caged siRNAs

Legal Events

Date Code Title Description
AS Assignment

Owner name: MERCK SHARP & DOHME CORP., NEW JERSEY

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:GOSSELIN, FRANCIS;ZEWGE, DANIEL;SIGNING DATES FROM 20100614 TO 20100617;REEL/FRAME:031594/0575

AS Assignment

Owner name: SIRNA THERAPEUTICS, INC., MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:MERCK SHARP & DOHME CORP.;REEL/FRAME:032584/0215

Effective date: 20140324

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO PAY ISSUE FEE