WO2011153120A1 - Novel low molecular weight cationic lipids for oligonucleotide delivery - Google Patents

Novel low molecular weight cationic lipids for oligonucleotide delivery Download PDF

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Publication number
WO2011153120A1
WO2011153120A1 PCT/US2011/038490 US2011038490W WO2011153120A1 WO 2011153120 A1 WO2011153120 A1 WO 2011153120A1 US 2011038490 W US2011038490 W US 2011038490W WO 2011153120 A1 WO2011153120 A1 WO 2011153120A1
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Prior art keywords
compound
amine
yloxy
alkyl
propan
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PCT/US2011/038490
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French (fr)
Inventor
Brian W. Budzik
Steven L. Colletti
Jennifer R. Davis
Ivory D. Hills
Darla D. Seifried
Matthew G. Stanton
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Merck Sharp & Dohme Corp.
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Application filed by Merck Sharp & Dohme Corp. filed Critical Merck Sharp & Dohme Corp.
Priority to EP11790268.4A priority Critical patent/EP2575767B1/en
Priority to DK11790268.4T priority patent/DK2575767T3/en
Priority to JP2013513262A priority patent/JP5957646B2/en
Priority to US13/701,636 priority patent/US8748667B2/en
Publication of WO2011153120A1 publication Critical patent/WO2011153120A1/en
Priority to US14/265,029 priority patent/US9062021B2/en
Priority to US14/711,246 priority patent/US9315437B2/en
Priority to US15/077,447 priority patent/US9643916B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • 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
    • C07C217/46—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 unsaturated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • 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
    • C07C217/44—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 saturated and containing rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/02—Ethers
    • C07C43/03—Ethers having all ether-oxygen atoms bound to acyclic carbon atoms
    • C07C43/14—Unsaturated ethers
    • C07C43/15—Unsaturated ethers containing only non-aromatic carbon-to-carbon double bonds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D205/00—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom
    • C07D205/02—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
    • C07D205/04—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/10—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings
    • C07D317/14—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D317/28—Radicals substituted by nitrogen atoms
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the present invention relates to novel cationic lipids that can be used in combination with other lipid components such as cholesterol and PEG-lipids to form lipid nanoparticles with oligonucleotides, to facilitate the cellular uptake and endosomal escape, and to knockdown target mRNA both in vitro and in vivo.
  • Cationic lipids and the use of cationic lipids in lipid nanoparticles for the delivery of oligonucleotides, in particular siRNA and miRNA, have been previously disclosed.
  • Lipid nanoparticles and use of lipid nanoparticles for the delivery of oligonucleotides, in particular siRNA and miRNA has been previously disclosed.
  • Oligonucleotides (including siRNA and miRNA) and the synthesis of oligonucleotides has been previously disclosed. (See US patent applications: US 2006/0083780, US 2006/0240554, US 2008/0020058, US
  • cationic lipids such as CLinDMA and DLinDMA have been employed for siRNA delivery to liver but suffer from non-optimal delivery efficiency along with liver toxicity at higher doses. It is an object of the instant invention to provide a cationic lipid scaffold that demonstrates enhanced efficacy along with lower liver toxicity as a result of lower lipid levels in the liver.
  • the present invention employs low molecular weight cationic lipids with one short lipid chain to enhance the efficiency and tolerability of in vivo delivery of siRNA.
  • the instant invention provides for novel cationic lipids that can be used in combination with other lipid components such as cholesterol and PEG-lipids to form lipid nanoparticles with oligonucleotides. It is an object of the instant invention to provide a cationic lipid scaffold that demonstrates enhanced efficacy along with lower liver toxicity as a result of lower lipid levels in the liver.
  • the present invention employs low molecular weight cationic lipids with one short lipid chain to enhance the efficiency and tolerability of in vivo delivery of siRNA.
  • FIGURE 1 LNP (Compound 1) efficacy in mice.
  • FIGURE 2 LNP (Compound 1) efficacy in rat.
  • FIGURE 3 LNP (Compound 1) LFT elevations in rat.
  • FIGURE 4 Cationic lipid (Compound 1) liver levels in rat. DETAILED DESCRIPTION OF THE INVENTION
  • the various aspects and embodiments of the invention are directed to the utility of novel cationic lipids useful in lipid nanoparticles to deliver oligonucleotides, in particular, siRNA and miR A, to any target gene.
  • novel cationic lipids useful in lipid nanoparticles to deliver oligonucleotides, in particular, siRNA and miR A, to any target gene.
  • the cationic lipids of the instant invention are useful components in a lipid nanoparticle for the delivery of oligonucleotides, specifically siRNA and miRNA.
  • R 1 and R 2 are independently selected from H, (C 1 -C 6 )alkyl, heterocyclyl, and polyamine, wherein said alkyl, heterocyclyl and polyamine are optionally substituted with one to three substituents selected from R', or R 1 and R 2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one to three substituents selected from R';
  • R' is independently selected from halogen, R", OR", SR", CN, CO 2 R" or
  • R" is independently selected from H and (Ci-C6)alkyl, wherein said alkyl is optionally substituted with halogen and OH;
  • Li is selected from C4-C22 alkyl and C4-C22 alkenyl, said alkyl and alkenyl are optionally substituted with one or more substituents selected from R'; and
  • L2 is selected from C3-C13 alkyl and C3-C13 alkenyl, said alkyl and alkenyl are optionally substituted with one or more substituents selected from R';
  • the invention features a compound having Formula A, wherein:
  • R 1 and R 2 are each methyl
  • Li is selected from C4-C22 alkyl and C4-C22 alkenyl
  • L2 is selected from C3-C13 alkyl and C3-C13 alkenyl
  • Specific cationic lipids are:
  • the cationic lipids disclosed are useful in the preparation of lipid nanoparticles.
  • the cationic lipids disclosed are useful components in a lipid nanoparticle for the delivery of oligonucleotides.
  • the cationic lipids disclosed are useful components in a lipid nanoparticle for the delivery of siRNA and miR A.
  • the cationic lipids disclosed are useful components in a lipid nanoparticle for the delivery of siRNA.
  • the cationic lipids of the present invention may have asymmetric centers, chiral axes, and chiral planes (as described in: E.L. Eliel and S.H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190), and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers and mixtures thereof, including optical isomers, being included in the present invention.
  • the cationic lipids disclosed herein may exist as tautomers and both tautomeric forms are intended to be encompassed by the scope of the invention, even though only one tautomeric structure is depicted.
  • substituents and substitution patterns on the cationic lipids of the instant invention can be selected by one of ordinary skill in the art to provide cationic lipids that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.
  • Si atoms can be incorporated into the cationic lipids of the instant invention by one of ordinary skill in the art to provide cationic lipids that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials.
  • the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature.
  • the present invention is meant to include all suitable isotopic variations of the compounds of Formula A.
  • different isotopic forms of hydrogen (H) include protium (lH) and deuterium (3 ⁇ 4H).
  • Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples.
  • Isotopically-enriched compounds within Formula A can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the Scheme and Examples herein using appropriate isotopically-enriched reagents and/or intermediates.
  • alkyl means a straight chain, cyclic or branched saturated aliphatic hydrocarbon having the specified number of carbon atoms.
  • alkenyl means a straight chain, cyclic or branched
  • unsaturated aliphatic hydrocarbon having the specified number of carbon atoms including but not limited to diene, triene and tetraene unsaturated aliphatic hydrocarbons.
  • heterocyclyl or “heterocycle” means a 4- to 10-membered aromatic or nonaromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, and includes bicyclic groups.
  • Heterocyclyl therefore includes, the following: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyri
  • dihydrothiadiazolyl dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and N-oxides thereof all of which are optionally substituted with one to three substituents selected from R".
  • polyamine means compounds having two or more amino groups. Examples include putrescine, cadaverine, spermidine, and spermine.
  • halogen means Br, CI, F and I.
  • R 1 and R 2 are independently selected from H and (C ⁇ -C6)alkyl, wherein said alkyl is optionally substituted with one to three substituents selected from R', or R 1 and R 2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, 0 and S, said monocyclic heterocycle is optionally substituted with one to three substituents selected from R ⁇
  • R 1 and R 2 are independently selected from H, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl are optionally substituted with one to three substituents selected from R', or R 1 and R 2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one to three substituents selected from R'.
  • R 1 and R 2 are independently selected from H, methyl, ethyl and propyl.
  • R 1 and R 2 are each methyl.
  • R' is R".
  • R" is independently selected from H, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl are optionally substituted with one or more halogen and OH.
  • R" is independently selected from H, methyl, ethyl and propyl.
  • Li is selected from C4-C22 alkyl and C4-C22 alkenyl, which are optionally substituted with halogen and OH. In an embodiment of Formula A, Li is selected from C4-C22 alkyl and C4-C22 alkenyl.
  • Li is selected from C4-C22 alkenyl.
  • Li is selected from C12-C22 alkenyl.
  • Li is Cig alkenyl.
  • Li is:
  • L2 is selected from C3-C13 alkyl and C3-C13 alkenyl, which are optionally substituted with halogen and OH.
  • L2 is selected from C3-C9 alkyl and C3-C9 alkenyl, which are optionally substituted with halogen and OH.
  • L2 is selected from C3-C8 alkyl and C3-C8 alkenyl, which are optionally substituted with halogen and OH.
  • L2 is selected from C3-C13 alkyl and C3-C13 alkenyl.
  • L2 is selected from C3-C9 alkyl and C3-C9 alkenyl.
  • L2 is selected from C3-C8 alkyl and C3-C8 alkenyl.
  • L2 is C3-C13 alkyl.
  • L2 is C3-C9 alkyl.
  • L2 is C3-C8 alkyl.
  • L2 is C8 alkyl.
  • heterocyclyl is pyrolidine, piperidine, morpholine, imidazole or piperazine.
  • "monocyclic heterocyclyl” is pyrolidine, piperidine, morpholine, imidazole or piperazine.
  • polyamine is putrescine, cadaverine, spermidine or spermine.
  • alkyl is a straight chain saturated aliphatic hydrocarbon having the specified number of carbon atoms.
  • alkenyl is a straight chain unsaturated aliphatic hydrocarbon having the specified number of carbon atoms.
  • the free form of cationic lipids of Formula A is the free form of cationic lipids of Formula A, as well as the pharmaceutically acceptable salts and stereoisomers thereof.
  • Some of the isolated specific cationic lipids exemplified herein are the protonated salts of amine cationic lipids.
  • the term "free form" refers to the amine cationic lipids in non-salt form.
  • the encompassed pharmaceutically acceptable salts not only include the isolated salts exemplified for the specific cationic lipids described herein, but also all the typical pharmaceutically acceptable salts of the free form of cationic lipids of Formula A.
  • the free form of the specific salt cationic lipids described may be isolated using techniques known in the art. For example, the free form may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium
  • the free forms may differ from their respective salt forms somewhat in certain physical properties, such as solubility in polar solvents, but the acid and base salts are otherwise pharmaceutically equivalent to their respective free forms for purposes of the invention.
  • the pharmaceutically acceptable salts of the instant cationic lipids can be synthesized from the cationic lipids of this invention which contain a basic or acidic moiety by conventional chemical methods.
  • the salts of the basic cationic lipids are prepared either by ion exchange chromatography or by reacting the free base with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents.
  • the salts of the acidic compounds are formed by reactions with the appropriate inorganic or organic base.
  • pharmaceutically acceptable salts of the cationic lipids of this invention include the conventional non-toxic salts of the cationic lipids of this invention as formed by reacting a basic instant cationic lipids with an inorganic or organic acid.
  • conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like, as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic (
  • salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine caffeine, choline, ⁇ , ⁇ 1 - dibenzylethylenediamine, diethylamin, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine tripropylamine, tromethamine and the like.
  • basic ion exchange resins such as arginine,
  • the cationic lipids of the present invention are potentially internal salts or zwitterions, since under physiological conditions a deprotonated acidic moiety in the compound, such as a carboxyl group, may be anionic, and this electronic charge might then be balanced off internally against the cationic charge of a protonated or alkylated basic moiety, such as a quaternary nitrogen atom.
  • the product of this reaction was first assigned the 2R stereochemistry, which would be obtained via a SN2 mechanism at the carbon bearing the halide atom with no change at the asymmetric carbon centre.
  • Compound 2 is a novel cationic lipid and was prepared according to the General Scheme 1 and Scheme 1 above, starting with S-epichlorohydrin.
  • Compound 23 is DLinDMA as described in J. Controlled Release, 2005, 107, 276-287, US 2006/0083780 Al, and US 2006/0008910 Al.
  • Compound 24 is DLinKC2DMA as described in Nature Biotechnology, 2010, 28, 172-176, WO 2010/042877 Al, WO 2010/048536 A2, WO 2010/088537 A2, and WO 2009/127060 Al.
  • lipid nanoparticle compositions of the instant invention are useful for the delivery of oligonucleotides, specifically siR A and miRNA:
  • the Lipid Nano-Particles are prepared by an impinging jet process.
  • the particles are formed by mixing lipids dissolved in alcohol with siRNA dissolved in a citrate buffer.
  • the mixing ratio of lipids to siRNA are targeted at 45-55% lipid and 65-45% siRNA.
  • the lipid solution contains a novel cationic lipid of the instant invention, a helper lipid (cholesterol) , PEG (e.g. PEG-C-DMA, PEG-DMG) lipid, and DSPC at a concentration of 5- 15 mg/mL with a target of 9-12 mg/mL in an alcohol (for example ethanol).
  • the ratio of the lipids has a mole percent range of 25-98 for the cationic lipid with a target of 35-65
  • the helper lipid has a mole percent range from 0-75 with a target of 30-50
  • the PEG lipid has a mole percent range from 1-15 with a target of 1 -6
  • the DSPC has a mole precent range of 0-15 with a target of 0-12.
  • the siRNA solution contains one or more siRNA sequences at a concentration range from 0.3 to 1 .0 mg/mL with a target of 0.3 -0.9 mg/mL in a sodium citrate buffered salt solution with pH in the range of 3.5-5.
  • the two liquids are heated to a temperature in the range of 15-40°C, targeting 30-40°C, and then mixed in an impinging jet mixer instantly forming the LNP.
  • the teelD has a range from 0.25 to 1.0 mm and a total flow rate from 10 -600 mL/min.
  • the combination of flow rate and tubing ID has effect of controlling the particle size of the LNPs between 30 and 200 nm.
  • the solution is then mixed with a buffered solution at a higher pH with a mixing ratio in the range of 1 : 1 to 1 :3 vol: vol but targeting 1 :2 vol:vol. This buffered solution is at a temperature in the range of 15-40°C, targeting 30-40°C.
  • the mixed LNPs are held from 30 minutes to 2 hrs prior to an anion exchange filtration step.
  • the temperature during incubating is in the range of 15-40°C, targeting 30-40°C.
  • the solution is filtered through a 0.8 um filter containing an anion exchange separation step.
  • This process uses tubing IDs ranging from 1 mm ID to 5 mm ID and a flow rate from 10 to 2000 mL/min.
  • the LNPs are concentrated and diafiltered via an ultrafiltration process where the alcohol is removed and the citrate buffer is exchanged for the final buffer solution such as phosphate buffered saline.
  • the ultrafiltration process uses a tangential flow filtration format (TFF). This process uses a membrane nominal molecular weight cutoff range from 30 -500 KD.
  • the membrane format can be hollow fiber or flat sheet cassette.
  • the TFF processes with the proper molecular weight cutoff retains the LNP in the retentate and the filtrate or permeate contains the alcohol; citrate buffer; final buffer wastes.
  • the TFF process is a multiple step process with an initial concentration to a siRN A
  • the LNPs solution is diafiltered against the final buffer for 10 -20 volumes to remove the alcohol and perform buffer exchange. The material is then concentrated an additional 1-3 fold. The final steps of the LNP process are to sterile filter the concentrated LNP solution and vial the product.
  • siRNA duplex concentrations are determined by Strong Anion-Exchange High-Performance Liquid Chromatography (SAX-HPLC) using Waters 2695 Alliance system (Water Corporation, Milford MA) with a 2996 PDA detector.
  • the LNPs otherwise referred to as RNAi Delivery Vehicles (RDVs)
  • RDVs RNAi Delivery Vehicles
  • SAX separation using a Dionex BioLC DNAPac PA 200 (4 ⁇ 250 mm) column with UV detection at 254 nm.
  • Mobile phase is composed of A: 25 mM NaClC , 10 mM Tris, 20% EtOH, pH 7.0 and B: 250 mM NaC10 4 , 10 mM Tris, 20% EtOH, pH 7.0 with liner gradient from 0-15 min and flow rate of 1 ml/min.
  • the siRNA amount is determined by comparing to the siRNA standard curve.
  • Fluorescence reagent SYBR Gold is employed for RNA quantitation to monitor the encapsulation rate of RDVs.
  • RDVs with or without Triton X-100 are used to determine the free siRNA and total siRNA amount.
  • the assay is performed using a SpectraMax M5e microplate spectrophotometer from Molecular Devices (Sunnyvale, CA). Samples are excited at 485 nm and fluorescence emission was measured at 530 nm. The siRNA amount is determined by comparing to the siRNA standard curve.
  • Encapsulation rate (1- free siRNA/total siRNA) *100%
  • RDVs containing 1 ⁇ g siRNA are diluted to a final volume of 3 ml with 1 *
  • the particle size and polydispersity of the samples is measured by a dynamic light scattering method using ZetaPALS instrument (Brookhaven Instruments Corporation,
  • the scattered intensity is measured with He-Ne laser at 25°C with a scattering angle of 90°.
  • RDVs containing 1 ⁇ g siRNA are diluted to a final volume of 2 ml with 1 mM Tris buffer (pH 7.4). Electrophoretic mobility of samples is determined using ZetaPALS instrument (Brookhaven Instruments Corporation, Holtsville, NY) with electrode and He-Ne laser as a light source. The Smoluchowski limit is assumed in the calculation of zeta potentials.
  • lipid concentrations are determined by Reverse Phase High- Performance Liquid Chromatography (RP-HPLC) using Waters 2695 Alliance system (Water Corporation, Milford MA) with a Corona charged aerosol detector (CAD) (ES A Biosciences, Inc, Chelmsford, MA). Individual lipids in RDVs are analyzed using an Agilent Zorbax SB- Cl 8 (50 x 4.6 mm, 1.8 um particle size) column with CAD at 60 °C. The mobile phase is composed of A: 0.1% TFA in H 2 0 and B: 0.1% TFA in IPA.
  • the gradient changes from 60% mobile phase A and 40% mobile phase B from time 0 to 40% mobile phase A and 60% mobile phase B at 1.00 min; 40% mobile phase A and 60% mobile phase B from 1.00 to 5.00 min; 40% mobile phase A and 60% mobile phase B from 5.00 min to 25% mobile phase A and 75% mobile phase B at 10.00 min; 25% mobile phase A and 75% mobile phase B from 10.00 min to 5% mobile phase A and 95% mobile phase B at 15.00 min; and 5% mobile phase A and 95% mobile phase B from 15.00 to 60% mobile phase A and 40% mobile phase B at 20.00 min with flow rate of 1 ml/min.
  • the individual lipid concentration is determined by comparing to the standard curve with all the lipid components in the RDVs with a quadratic curve fit. The molar percentage of each lipid is calculated based on its molecular weight.
  • oligonucleotides in particular siRNA and miRNA
  • the siRNA targets the mRNA transcript for the firefly (Photinus pyralis) luciferase gene (Accession # Ml 5077).
  • the primary sequence and chemical modification pattern of the luciferase siRNA is displayed above.
  • the in vivo luciferase model employs a transgenic mouse in which the firefly luciferase coding sequence is present in all cells.
  • ROSA26- LoxP-Stop-LoxP-Luc (LSL-Luc) transgenic mice licensed from the Dana Farber Cancer Institute are induced to express the Luciferase gene by first removing the LSL sequence with a recombinant Ad-Cre virus (Vector Biolabs). Due to the organo-tropic nature of the virus, expression is limited to the liver when delivered via tail vein injection. Luciferase expression levels in liver are quantitated by measuring light output, using an IVIS imager (Xenogen) following administration of the luciferin substrate (Caliper Life Sciences). Pre-dose luminescence levels are measured prior to administration of the RDVs.
  • Luciferin in PBS 15mg/mL is intraperitoneally (IP) injected in a volume of 150 ⁇ L. After a four minute incubation period mice are anesthetized with isoflurane and placed in the IVIS imager.
  • the RDVs (containing siRNA) in PBS vehicle were tail vein injected n a volume of 0.2 mL. Final dose levels ranged from 0.3 to 3 mg/kg siRNA.
  • PBS vehicle alone was dosed as a control.
  • mice Three hours post dose, mice were bled retro-orbitally to obtain plasma for cytokine analysis. Mice were imaged 48 hours post dose using the method described above. Changes in luciferin light output directly correlate with luciferase mRNA levels and represent an indirect measure of luciferase siRNA activity. In vivo efficacy results are expressed as % inhibition of luminescence relative to pre-dose luminescence levels. Plasma cytokine levels were determined using the SearchLight multiplexed cytokine chemoluminescent array
  • LNPs utilizing Compound 1 in the nominal compositions described above were evaluated for in vivo efficacy and increases in alanine amino transferase and aspartate amino transferase in Sprague-Dawley (Crl:CD(SD) female rats (Charles River Labs).
  • the siRNA targets the mRNA transcript for the ApoB gene (Accession # NM 019287).
  • the primary sequence and chemical modification pattern of the ApoB siRNA is displayed above.
  • the RDVs (containing siRNA) in PBS vehicle were tail vein injected in a volume of 1 to 1.5 mL. Infusion rate is approximately 3 ml/min. Five rats were used in each dosing group. After LNP administration, rats are placed in cages with normal diet and water present.
  • liver tissue was homogenized and total RNA isolated using a Qiagen bead mill and the Qiagen miRNA-Easy RNA isolation kit following the manufacturer's instructions. Liver ApoB mRNA levels were determined by quantitative RT-PCR. Message was amplified from purified RNA utilizing a rat ApoB commercial probe set (Applied Biosystems Cat #
  • RNA samples were analyzed for RNA expression in Eukaryotic cells.
  • the PCR reaction was performed on an ABI 7500 instrument with a 96- well Fast Block.
  • the ApoB mRNA level is normalized to the housekeeping PPIB (NM 011149) mRNA.
  • PPIB mRNA levels were determined by RT-PCR using a commercial probe set (Applied Biosytems Cat. No. Mm00478295_ml). Results are expressed as a ratio of ApoB mRNA/ PPIB mRNA. All mRNA data is expressed relative to the PBS control dose.
  • Serum ALT and AST analysis were performed on the Siemens Advia 1800 Clinical Chemistry Analyzer utilizing the Siemens alanine aminotransferase (Cat# 03039631) and aspartate aminotransferase (Cat# 03039631 ) reagents. Greater efficacy was observed in rats dosed with Compound 1 containing RDV than with the RDV containing the octyl-CLinDMA cationic lipid ( Figure 2). Additionally, lower elevations in LFTs (ALT/AST) were observed in rats dosed with Compound 1 containing RDV than with the RDV containing the octyl-CLinDMA cationic lipid ( Figure 3).
  • Liver tissue was weighed into 20-ml vials and homogenized in 9 v/w of water using a GenoGrinder 2000 (OPS Diagnostics, 1600 strokes/min, 5min). A 50 ⁇ , aliquot of each tissue homogenate was mixed with 300 uL of extraction/protein precipitating solvent (50/50 acetonitrile/methanol containing 500 nM internal standard) and the plate was centrifuged to sediment precipitated protein. A volume of 200 ⁇ , of each supernatant was then transferred to separate wells of a 96-well plate and 10 ⁇ samples were directly analyzed by LC/MS-MS.
  • OPS Diagnostics 1600 strokes/min, 5min.
  • a 50 ⁇ , aliquot of each tissue homogenate was mixed with 300 uL of extraction/protein precipitating solvent (50/50 acetonitrile/methanol containing 500 nM internal standard) and the plate was centrifuged to sediment precipitated protein. A volume of 200 ⁇ , of each supernatant was then
  • Absolute quantification versus standards prepared and extracted from rat liver homogenate was performed using an Aria LX-2 HPLC system (Thermo Scientific) coupled to an API 4000 triple quadrupole mass spectrometer (Applied Biosystems). For each run, a total of 10 L sample was injected onto a BDS Hypersil C8 HPLC column (Thermo, 50 x 2mm, 3 ⁇ ) at ambient temperature ( Figure 4).

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Abstract

The instant invention provides for novel cationic lipids that can be used in combination with other lipid components such as cholesterol and PEG-lipids to form lipid nanoparticles with oligonucleotides. It is an object of the instant invention to provide a cationic lipid scaffold that demonstrates enhanced efficacy along with lower liver toxicity as a result of lower lipid levels in the liver. The present invention employs low molecular weight cationic lipids with one short lipid chain to enhance the efficiency and tolerability of in vivo delivery of siRNA.

Description

TITLE OF THE INVENTION
NOVEL LOW MOLECULAR WEIGHT CATIONIC LIPIDS FOR OLIGONUCLEOTIDE DELIVERY
BACKGROUND OF THE INVENTION
The present invention relates to novel cationic lipids that can be used in combination with other lipid components such as cholesterol and PEG-lipids to form lipid nanoparticles with oligonucleotides, to facilitate the cellular uptake and endosomal escape, and to knockdown target mRNA both in vitro and in vivo.
Cationic lipids and the use of cationic lipids in lipid nanoparticles for the delivery of oligonucleotides, in particular siRNA and miRNA, have been previously disclosed. Lipid nanoparticles and use of lipid nanoparticles for the delivery of oligonucleotides, in particular siRNA and miRNA, has been previously disclosed. Oligonucleotides (including siRNA and miRNA) and the synthesis of oligonucleotides has been previously disclosed. (See US patent applications: US 2006/0083780, US 2006/0240554, US 2008/0020058, US
2009/0263407 and US 2009/0285881 and PCT patent applications: WO 2009/086558, WO2009/127060, WO2009/1321 1, WO2010/042877, WO2010/054384, WO2010/054401, WO2010/054405 and WO2010/054406). See also Semple S. C. et al., Rational design of cationic lipids for siRNA delivery, Nature Biotechnology, published online 17 January 2010; doi:10.1038/nbt.l602.
Other cationic lipids are disclosed in US patent applications: US 2009/0263407, US 2009/0285881, US 2010/0055168, US 2010/0055169, US 2010/0063135, US
2010/0076055, US 2010/0099738 and US 2010/0104629.
Traditional cationic lipids such as CLinDMA and DLinDMA have been employed for siRNA delivery to liver but suffer from non-optimal delivery efficiency along with liver toxicity at higher doses. It is an object of the instant invention to provide a cationic lipid scaffold that demonstrates enhanced efficacy along with lower liver toxicity as a result of lower lipid levels in the liver. The present invention employs low molecular weight cationic lipids with one short lipid chain to enhance the efficiency and tolerability of in vivo delivery of siRNA.
SUMMARY OF THE INVENTION
The instant invention provides for novel cationic lipids that can be used in combination with other lipid components such as cholesterol and PEG-lipids to form lipid nanoparticles with oligonucleotides. It is an object of the instant invention to provide a cationic lipid scaffold that demonstrates enhanced efficacy along with lower liver toxicity as a result of lower lipid levels in the liver. The present invention employs low molecular weight cationic lipids with one short lipid chain to enhance the efficiency and tolerability of in vivo delivery of siRNA.
BRIEF DESCRIPTION OF THE FIGURES FIGURE 1 : LNP (Compound 1) efficacy in mice.
FIGURE 2: LNP (Compound 1) efficacy in rat.
FIGURE 3: LNP (Compound 1) LFT elevations in rat.
FIGURE 4: Cationic lipid (Compound 1) liver levels in rat. DETAILED DESCRIPTION OF THE INVENTION
The various aspects and embodiments of the invention are directed to the utility of novel cationic lipids useful in lipid nanoparticles to deliver oligonucleotides, in particular, siRNA and miR A, to any target gene. (See US patent applications: US 2006/0083780, US 2006/0240554, US 2008/0020058, US 2009/0263407 and US 2009/0285881 and PCT patent applications: WO 2009/086558, WO2009/127060, WO2009/132131, WO2010/042877,
WO2010/054384, WO2010/054401, WO2010/054405 and WO2010/054406). See also Semple S. C. et al., Rational design of cationic lipids for siRNA delivery, Nature Biotechnology, published online 17 January 2010; doi:10.1038/nbt.l602.
The cationic lipids of the instant invention are useful components in a lipid nanoparticle for the delivery of oligonucleotides, specifically siRNA and miRNA.
In a first embodiment of this invention, the cationic lipids are illustrated by the
Formula A:
Figure imgf000003_0001
wherein:
R1 and R2 are independently selected from H, (C1-C6)alkyl, heterocyclyl, and polyamine, wherein said alkyl, heterocyclyl and polyamine are optionally substituted with one to three substituents selected from R', or R1 and R2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one to three substituents selected from R';
R' is independently selected from halogen, R", OR", SR", CN, CO2R" or
CON(R")2; R" is independently selected from H and (Ci-C6)alkyl, wherein said alkyl is optionally substituted with halogen and OH;
Li is selected from C4-C22 alkyl and C4-C22 alkenyl, said alkyl and alkenyl are optionally substituted with one or more substituents selected from R'; and
L2 is selected from C3-C13 alkyl and C3-C13 alkenyl, said alkyl and alkenyl are optionally substituted with one or more substituents selected from R';
or any pharmaceutically acceptable salt or stereoisomer thereof.
In a second embodiment, the invention features a compound having Formula A, wherein:
R1 and R2 are each methyl;
Li is selected from C4-C22 alkyl and C4-C22 alkenyl; and
L2 is selected from C3-C13 alkyl and C3-C13 alkenyl;
or any pharmaceutically acceptable salt or stereoisomer thereof.
Specific cationic lipids are:
R -N,N^ime 1 -[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-(octyloxy)propan-2-amine (Compound 2);
S -N,N-<limemyl-l-[(9Z,12Z)-octadeca-9^^
(Compound 1);
l-{2-[(9Z,12Z octadeca-9,12^ien-l-yloxy^
(Compound 3);
(2S)-N,N^imemyl-l-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-[(5Z)-oct-5^-l- yloxy]propan-2-amine (Compound 4);
l-{2-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-H^
5);
(2S)-l-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine (Compound 6);
(2S)-l-(heptyloxy)-N,N-dim^
(Compound 7);
N,N-dimethyl- 1 -(nonyloxy)-3-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]propan-2-amine
(Compound 8);
N,N-dimethyl-l-[(9Z)-octadec-9-en-l-yloxy]-3-(octyloxy)propan-2-amine (Compoiind 9); (2S)-N,N-dimemyl-l-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-l-yloxy]-3-(octyloxy)prop^ amine (Compound 10);
(2S)- 1 -[(11 Z, 14Z)-icosa- 11,14-dien- 1 -yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine (Compound 11);
(2S)-l-(hexyloxy)-3-[(l lZ,14Z)-icosa-ll,14-dien-l-yloxy]-N,N-dimethylpropan-2-amine (Compound 12); 1- [(1 lZ 4Z)-icosa-l 1,14-dien-l-yto^
(Compound 13);
1 -[(13Z, 16Z)-docosa- 13,16-dien-l -yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine
(Compound 14);
(2S)-l-[(13Z,16Z)-docosa-13 6-dien-l-yloxy
(Compound 15);
(2S)-l-[(13Z)-docos-13-en-l-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine (Compound 16);
1 -[(13Z)-docos- 13 -en- 1 -yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine (Compound 17); l-[(9Z)-hexadec-9-en-l-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine (Compound 18); (2R)-N,N-dimetoyl-l-[(l-methyto^^
2- amine (Compound 19);
(2R)-l-[(3,7-dime loctyl)oxy]-N^^
yloxy]propan-2-amine (Compound 20);
N,N-dimethyl-l-(octyloxy)-3-({8-[(l S,2S)-2-{ [(lR,2R)-2- pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine (Compound 21); and N,N^memyl-l-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine (Compound 22);
or any pharmaceutically acceptable salt or stereoisomer thereof.
In another embodiment, the cationic lipids disclosed are useful in the preparation of lipid nanoparticles.
In another embodiment, the cationic lipids disclosed are useful components in a lipid nanoparticle for the delivery of oligonucleotides.
In another embodiment, the cationic lipids disclosed are useful components in a lipid nanoparticle for the delivery of siRNA and miR A.
In another embodiment, the cationic lipids disclosed are useful components in a lipid nanoparticle for the delivery of siRNA.
The cationic lipids of the present invention may have asymmetric centers, chiral axes, and chiral planes (as described in: E.L. Eliel and S.H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190), and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers and mixtures thereof, including optical isomers, being included in the present invention. In addition, the cationic lipids disclosed herein may exist as tautomers and both tautomeric forms are intended to be encompassed by the scope of the invention, even though only one tautomeric structure is depicted.
It is understood that substituents and substitution patterns on the cationic lipids of the instant invention can be selected by one of ordinary skill in the art to provide cationic lipids that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.
It is understood that one or more Si atoms can be incorporated into the cationic lipids of the instant invention by one of ordinary skill in the art to provide cationic lipids that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials.
In the compounds of Formula A, the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. The present invention is meant to include all suitable isotopic variations of the compounds of Formula A. For example, different isotopic forms of hydrogen (H) include protium (lH) and deuterium (¾H). Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples.
Isotopically-enriched compounds within Formula A can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the Scheme and Examples herein using appropriate isotopically-enriched reagents and/or intermediates.
As used herein, "alkyl" means a straight chain, cyclic or branched saturated aliphatic hydrocarbon having the specified number of carbon atoms.
As used herein, "alkenyl" means a straight chain, cyclic or branched
unsaturated aliphatic hydrocarbon having the specified number of carbon atoms including but not limited to diene, triene and tetraene unsaturated aliphatic hydrocarbons.
Examples of a cyclic "alkyl" or "alkenyl are:
Figure imgf000006_0001
As used herein, "heterocyclyl" or "heterocycle" means a 4- to 10-membered aromatic or nonaromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, and includes bicyclic groups. "Heterocyclyl" therefore includes, the following: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, 1,4-dioxanyl,
hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, tfaiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofiiranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl,
dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and N-oxides thereof all of which are optionally substituted with one to three substituents selected from R".
As used herein, "polyamine" means compounds having two or more amino groups. Examples include putrescine, cadaverine, spermidine, and spermine.
As used herein, "halogen" means Br, CI, F and I.
In an embodiment of Formula A, R1 and R2 are independently selected from H and (C\ -C6)alkyl, wherein said alkyl is optionally substituted with one to three substituents selected from R', or R1 and R2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, 0 and S, said monocyclic heterocycle is optionally substituted with one to three substituents selected from R\
In an embodiment of Formula A, R1 and R2 are independently selected from H, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl are optionally substituted with one to three substituents selected from R', or R1 and R2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one to three substituents selected from R'.
In an embodiment of Formula A, R1 and R2 are independently selected from H, methyl, ethyl and propyl.
In an embodiment of Formula A, R1 and R2 are each methyl.
In an embodiment of Formula A, R' is R".
In an embodiment of Formula A, R" is independently selected from H, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl are optionally substituted with one or more halogen and OH.
In an embodiment of Formula A, R" is independently selected from H, methyl, ethyl and propyl.
In an embodiment of Formula A, Li is selected from C4-C22 alkyl and C4-C22 alkenyl, which are optionally substituted with halogen and OH. In an embodiment of Formula A, Li is selected from C4-C22 alkyl and C4-C22 alkenyl.
In an embodiment of Formula A, Li is selected from C4-C22 alkenyl.
In an embodiment of Formula A, Li is selected from C12-C22 alkenyl.
In an embodiment of Formula A, Li is Cig alkenyl.
In an embodiment of Formula A, Li is:
Figure imgf000008_0001
In an embodiment of Formula A, L2 is selected from C3-C13 alkyl and C3-C13 alkenyl, which are optionally substituted with halogen and OH.
In an embodiment of Formula A, L2 is selected from C3-C9 alkyl and C3-C9 alkenyl, which are optionally substituted with halogen and OH.
In an embodiment of Formula A, L2 is selected from C3-C8 alkyl and C3-C8 alkenyl, which are optionally substituted with halogen and OH.
In an embodiment of Formula A, L2 is selected from C3-C13 alkyl and C3-C13 alkenyl.
In an embodiment of Formula A, L2 is selected from C3-C9 alkyl and C3-C9 alkenyl.
In an embodiment of Formula A, L2 is selected from C3-C8 alkyl and C3-C8 alkenyl.
In an embodiment of Formula A, L2 is C3-C13 alkyl.
In an embodiment of Formula A, L2 is C3-C9 alkyl.
In an embodiment of Formula A, L2 is C3-C8 alkyl.
In an embodiment of Formula A, L2 is C8 alkyl.
In an embodiment of Formula A, "heterocyclyl" is pyrolidine, piperidine, morpholine, imidazole or piperazine.
In an embodiment of Formula A, "monocyclic heterocyclyl" is pyrolidine, piperidine, morpholine, imidazole or piperazine.
In an embodiment of Formula A, "polyamine" is putrescine, cadaverine, spermidine or spermine. In an embodiment, "alkyl" is a straight chain saturated aliphatic hydrocarbon having the specified number of carbon atoms.
In an embodiment, "alkenyl" is a straight chain unsaturated aliphatic hydrocarbon having the specified number of carbon atoms.
Included in the instant invention is the free form of cationic lipids of Formula A, as well as the pharmaceutically acceptable salts and stereoisomers thereof. Some of the isolated specific cationic lipids exemplified herein are the protonated salts of amine cationic lipids. The term "free form" refers to the amine cationic lipids in non-salt form. The encompassed pharmaceutically acceptable salts not only include the isolated salts exemplified for the specific cationic lipids described herein, but also all the typical pharmaceutically acceptable salts of the free form of cationic lipids of Formula A. The free form of the specific salt cationic lipids described may be isolated using techniques known in the art. For example, the free form may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium
bicarbonate. The free forms may differ from their respective salt forms somewhat in certain physical properties, such as solubility in polar solvents, but the acid and base salts are otherwise pharmaceutically equivalent to their respective free forms for purposes of the invention.
The pharmaceutically acceptable salts of the instant cationic lipids can be synthesized from the cationic lipids of this invention which contain a basic or acidic moiety by conventional chemical methods. Generally, the salts of the basic cationic lipids are prepared either by ion exchange chromatography or by reacting the free base with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents. Similarly, the salts of the acidic compounds are formed by reactions with the appropriate inorganic or organic base.
Thus, pharmaceutically acceptable salts of the cationic lipids of this invention include the conventional non-toxic salts of the cationic lipids of this invention as formed by reacting a basic instant cationic lipids with an inorganic or organic acid. For example, conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like, as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic (TFA) and the like.
When the cationic lipids of the present invention are acidic, suitable
"pharmaceutically acceptable salts" refers to salts prepared form pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine caffeine, choline, Ν,Ν1- dibenzylethylenediamine, diethylamin, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine tripropylamine, tromethamine and the like.
The preparation of the pharmaceutically acceptable salts described above and other typical pharmaceutically acceptable salts is more fully described by Berg et al,
"Pharmaceutical Salts," J. Pharm. Set, 1977:66:1-19.
It will also be noted that the cationic lipids of the present invention are potentially internal salts or zwitterions, since under physiological conditions a deprotonated acidic moiety in the compound, such as a carboxyl group, may be anionic, and this electronic charge might then be balanced off internally against the cationic charge of a protonated or alkylated basic moiety, such as a quaternary nitrogen atom.
EXAMPLES
Examples provided are intended to assist in a further understanding of the invention. Particular materials employed, species and conditions are intended to be further illustrative of the invention and not limitative of the reasonable scope thereof. The reagents utilized in synthesizing the cationic lipids are either commercially available or are readily prepared by one of ordinary skill in the art.
Synthesis of the novel cationic lipids is a linear process starting epichlorohydrin (i). Reaction of epichlorohydrin with lipid alcohol gives intermediate epoxide ii. Lewis acid epoxide opening with the second lipid alcohol affords secondary alcohol iii. Triflate formation and amine substitution gives final products of type iv. GENERAL SCHEME 1
Figure imgf000010_0001
Synthesis of intermediate cyclopropyl analogs (vi) was conducted by Simmons- Smith cyclopropanation of ole ins of type v. GENERAL SCHEME 2
Figure imgf000011_0001
SCHEME 1
S -N,N-dimetoyl-l-[(9Z,12Z)-ocl^
(Compound 1)
Figure imgf000011_0002
A solution of octanol (1.5mol, 195g), tetrabutyl ammonium bromide (75mmol, 24. lg) and sodium hydroxide (2.4mol, 96g) was cooled to 3 C and R-epichlorohydrin was added via dropping funnel . The mixture was stirred at 3 C for 6 hours and then allowed to warm to ambient temperature for 16 hours. The reaction was partitioned between hexanes and aqueous saturated sodium bicarbonate. The organics were washed with water (2x 625mL) and brine (625mL) and evaporated in vacuo. The crude oil was purified by flash chromatography (silica, 0-20% ethyl acetate/heptane) to give 236g (85%) of one of the enantiomers of 2- [(octyloxy)methyl]oxirane as a clear oil. ¾ NMR (400MHz, CDC13): δ 3.68 (m, 1H), 3.45 (m, 2H), 3.38 (m, 1H), 3.16 (m, 1H), 2.78 (m, 1H), 2.59 (m, 1H), 1.58 (m, 2H), 1.30 (m, 10H), 0.83 (m, 3H).
The product of this reaction was first assigned the 2R stereochemistry, which would be obtained via a SN2 mechanism at the carbon bearing the halide atom with no change at the asymmetric carbon centre.
However, subsequent studies (experiments on the reaction of lineolyl alcohol with S-epichlorohydrin), under conditions described, Lewis acid conditions and in conjunction with vibrational circular dichroism (VCD), a valid spectroscopic method for determining absolute configuration of chiral molecules (R. K. Dukor and L. A. Nafie, in Encyclopedia of Analytical Chemistry: Instrumentation and Applications, Ed. R. A. Meyers (Wiley, Chichester, 2000) 662-676.) have revealed the reaction occured via a SN2' mechanism ie reaction at the terminal carbon of the epoxide leading to subsequent ring opening, followed by an in situ ring closing step that leads to inversion of stereochemistry at the asymmetric carbon.
As a result of these studies, the product from this reaction was reassigned the 2S stereochemistry: (2S)-[(octyloxy)methyl]oxirane.
Figure imgf000012_0003
A solution of epoxide (3.86g, 20.7mmol) and linoleyl alcohol (6.6g, 24.8mmol) in dichloromethane (lOOmL) was treated with tin tetrachloride (4.44g, 2.0mmol) at ambient temperature. After stirring for 3 hours, the reaction was quenched with brine and partitioned between brine and dichloromethane. The organics were dried over sodium sulfate, filtered and evaporated in vacuo. The crude oil was purified by flash chromatography (silica, 2-5% acetone/hexanes) to give 5.85g (62%) of R-l-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3- (octyloxy)propan-2-ol as a clear oil. !H NMR (400MHz, CDC13): δ 5.38 (m, 4H), 3.93 (m, 1H), 3.49 (m, 8H), 2.79 (m, 2H), 2.45 (m, 1H), 2.06 (m, 4H), 1.58 (m, 6H), 1.30 (m, 26H), 0.89 (m, 6H).
Iridic anhydride, DCM |
(CH,)2NH.2,e-lutidine
Figure imgf000012_0002
Figure imgf000012_0001
Compound iii (5.08 g, 11.2 mmol) was dissolved into dichloromethane (100 ml) and treated with 2,6-lutidine (1.44g, 13.5 mmol). Triflic anhydride (3.8g, 13.5 mmol) was added dropwise into the reaction system at 0 °C. After 2.5 hours, the reaction was cannulated into a cold solution of dimethylamine (39.3mmol) in dichloromethane (85mL). After 2 hours at 0 C, the reaction was quenched with saturated sodium bicarbonate solution and partitioned between water/dichloromethane. The organics were dried over magnesium sulfate, filtered and evaporated in vacuo. The crude oil was purified by flash chromatography (silica, 0-30% ethyl acetate hexanes) to give 3.66g (65%) of S-N,N-dimethyl-l-[(9Z,12Z)-octadeca-9,12-dien-l- yloxy]-3-(octyloxy)propan-2-amine as an amber oil. 1H NMR (400MHz, CDC13): δ 5.38 (m, 4H), 3.52 (m, 4H), 3.40 (m, 4H), 2.79 (m, 3H), 2.40 (s, 6H), 2.05 (m, 4H), 1.58 (m, 4H), 1.30 (m, 26H), 0.89 (m, 6H). HRMS: cal'd - 480.4775, found - 480.4769.
Compound 2 is a novel cationic lipid and was prepared according to the General Scheme 1 and Scheme 1 above, starting with S-epichlorohydrin.
Figure imgf000012_0004
Compound 2
R- Ν,Ν-dimethyl- 1 -[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]-3-(octyloxy)propan-2-amine
(Compound 2)
Ή NMR (400MHz, CDC13): δ 5.38 (m, 4H), 3.52 (m, 4H), 3.40 (m, 4H), 2.79 (m, 3H), 2.40 (s, 6H), 2.05 (m, 4H), 1.58 (m, 4H), 1.30 (m, 26H), 0.89 (m, 6H).
Compounds 3-20 were prepared in a manner analogous to that described for
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0002
SCHEME 2
N,N-dimethyl-l-(octyloxy)-3-((8-[(1S,2S)-2-{[(1R,2R)-2- pentylcyclopropyl]methyl}cyclopropyl]octyl oxy)propan-2-amine (Compound 21)
Figure imgf000015_0001
A solution of l-[(9Z,122 -octadeca-942^en-l-yloxy]-3-(octyloxy)propan-2-ol in DCM (166 ml) in a 1 L flask under nitrogen was cooled in an ice/IP A bath (-15 °C) and then 1 M diethylzinc in hexane (199 ml, 199 mmol) added, followed by diiodomethane (24.05 ml, 298 mmol). The mixture was allowed to stir in the cooling bath overnight to gradually come up to room temperature. Reaction was quenched by addition of excess saturated ammonium chloride. Washed reaction into 2 L separatory funnel with hexane and water and added excess hexane, shook vigorously. Discarded lower aqueous layer, dried organic layer with sodium sulfate and evaporated to l-(octyloxy)-3-[(8-{2-[(2- pentylcyclopropyl)methyl]cyclopropyl}octyl)oxy]propan-2-ol (15.5g, 32.2 mmol, 97 % yield) as a yellow oil. Material was used with no further purification. MS (ESI): 481 [M+H]+.
Figure imgf000016_0001
vl 21
Conversion of alcohol vi to final compound 21 was accomplished as described for Compound 1. C33H65N02 [M+H], calc 508.5, obs 508.5.
N,N-dimethyl- 1 -{ [8-(2-octylcyclopropyl)octyl]oxy } -3-(octyloxy)propan-2- amine (Compound 22) was prepared in a manner analogous to that described for compound 21. C32H65N02 [M+H], calc 496.5, obs 496.5.
Figure imgf000016_0002
Compound 23 is DLinDMA as described in J. Controlled Release, 2005, 107, 276-287, US 2006/0083780 Al, and US 2006/0008910 Al.
Figure imgf000016_0003
Compound 24 is DLinKC2DMA as described in Nature Biotechnology, 2010, 28, 172-176, WO 2010/042877 Al, WO 2010/048536 A2, WO 2010/088537 A2, and WO 2009/127060 Al.
Figure imgf000016_0004
LNP COMPOSITIONS
The following lipid nanoparticle compositions (LNPs) of the instant invention are useful for the delivery of oligonucleotides, specifically siR A and miRNA:
Cationic Lipid / Cholesterol / PEG-DMG 56.6/38/5.4;
Cationic Lipid / Cholesterol / PEG-DMG 60/38/2;
Cationic Lipid/ Cholesterol / PEG-DMG 67.3/29/3.7; Cationic Lipid / Cholesterol / PEG-DMG 49.3/47/3.7;
Cationic Lipid / Cholesterol / PEG-DMG 50.3/44.3/5.4;
Cationic Lipid / Cholesterol / PEG-C-DMA / DSPC 40/48/2/10;
Cationic Lipid / Cholesterol / PEG-DMG / DSPC 40/48/2/10; and
Cationic Lipid / Cholesterol / PEG-DMG / DSPC 58/30/2/10.
The synthesis and use of LNPs are known. (See US patent applications: US 2006/0083780, US 2006/0240554, US 2008/0020058, US 2009/0263407 and US
2009/0285881 and PCT patent applications: WO 2009/086558, WO2009/127060,
WO2009/132131, WO2010/042877, WO2010/054384, WO2010/054401, WO2010/054405 and WO2010/054406). See also Semple S. C. et al., Rational design of cationic lipids for siRNA delivery, Nature Biotechnology, published online 17 January 2010;
doi:10.1038/nbt.l602.
LNP process description:
The Lipid Nano-Particles (LNP) are prepared by an impinging jet process. The particles are formed by mixing lipids dissolved in alcohol with siRNA dissolved in a citrate buffer. The mixing ratio of lipids to siRNA are targeted at 45-55% lipid and 65-45% siRNA. The lipid solution contains a novel cationic lipid of the instant invention, a helper lipid (cholesterol) , PEG (e.g. PEG-C-DMA, PEG-DMG) lipid, and DSPC at a concentration of 5- 15 mg/mL with a target of 9-12 mg/mL in an alcohol (for example ethanol). The ratio of the lipids has a mole percent range of 25-98 for the cationic lipid with a target of 35-65, the helper lipid has a mole percent range from 0-75 with a target of 30-50, the PEG lipid has a mole percent range from 1-15 with a target of 1 -6, and the DSPC has a mole precent range of 0-15 with a target of 0-12. The siRNA solution contains one or more siRNA sequences at a concentration range from 0.3 to 1 .0 mg/mL with a target of 0.3 -0.9 mg/mL in a sodium citrate buffered salt solution with pH in the range of 3.5-5. The two liquids are heated to a temperature in the range of 15-40°C, targeting 30-40°C, and then mixed in an impinging jet mixer instantly forming the LNP. The teelD has a range from 0.25 to 1.0 mm and a total flow rate from 10 -600 mL/min. The combination of flow rate and tubing ID has effect of controlling the particle size of the LNPs between 30 and 200 nm. The solution is then mixed with a buffered solution at a higher pH with a mixing ratio in the range of 1 : 1 to 1 :3 vol: vol but targeting 1 :2 vol:vol. This buffered solution is at a temperature in the range of 15-40°C, targeting 30-40°C. The mixed LNPs are held from 30 minutes to 2 hrs prior to an anion exchange filtration step. The temperature during incubating is in the range of 15-40°C, targeting 30-40°C. After incubating the solution is filtered through a 0.8 um filter containing an anion exchange separation step. This process uses tubing IDs ranging from 1 mm ID to 5 mm ID and a flow rate from 10 to 2000 mL/min. The LNPs are concentrated and diafiltered via an ultrafiltration process where the alcohol is removed and the citrate buffer is exchanged for the final buffer solution such as phosphate buffered saline. The ultrafiltration process uses a tangential flow filtration format (TFF). This process uses a membrane nominal molecular weight cutoff range from 30 -500 KD. The membrane format can be hollow fiber or flat sheet cassette. The TFF processes with the proper molecular weight cutoff retains the LNP in the retentate and the filtrate or permeate contains the alcohol; citrate buffer; final buffer wastes. The TFF process is a multiple step process with an initial concentration to a siRN A
concentration of 1 -3 mg/mL. Following concentration, the LNPs solution is diafiltered against the final buffer for 10 -20 volumes to remove the alcohol and perform buffer exchange. The material is then concentrated an additional 1-3 fold. The final steps of the LNP process are to sterile filter the concentrated LNP solution and vial the product.
Analytical Procedure:
1) siR A concentration
The siRNA duplex concentrations are determined by Strong Anion-Exchange High-Performance Liquid Chromatography (SAX-HPLC) using Waters 2695 Alliance system (Water Corporation, Milford MA) with a 2996 PDA detector. The LNPs, otherwise referred to as RNAi Delivery Vehicles (RDVs), are treated with 0.5% Triton X-100 to free total siRNA and analyzed by SAX separation using a Dionex BioLC DNAPac PA 200 (4 χ 250 mm) column with UV detection at 254 nm. Mobile phase is composed of A: 25 mM NaClC , 10 mM Tris, 20% EtOH, pH 7.0 and B: 250 mM NaC104, 10 mM Tris, 20% EtOH, pH 7.0 with liner gradient from 0-15 min and flow rate of 1 ml/min. The siRNA amount is determined by comparing to the siRNA standard curve.
2) Encapsulation rate
Fluorescence reagent SYBR Gold is employed for RNA quantitation to monitor the encapsulation rate of RDVs. RDVs with or without Triton X-100 are used to determine the free siRNA and total siRNA amount. The assay is performed using a SpectraMax M5e microplate spectrophotometer from Molecular Devices (Sunnyvale, CA). Samples are excited at 485 nm and fluorescence emission was measured at 530 nm. The siRNA amount is determined by comparing to the siRNA standard curve.
Encapsulation rate = (1- free siRNA/total siRNA) *100%
3) Particle size and polvdispersitv
RDVs containing 1 μg siRNA are diluted to a final volume of 3 ml with 1 *
PBS. The particle size and polydispersity of the samples is measured by a dynamic light scattering method using ZetaPALS instrument (Brookhaven Instruments Corporation,
Holtsville, NY). The scattered intensity is measured with He-Ne laser at 25°C with a scattering angle of 90°.
4^ Zeta Potential analysis
RDVs containing 1 μg siRNA are diluted to a final volume of 2 ml with 1 mM Tris buffer (pH 7.4). Electrophoretic mobility of samples is determined using ZetaPALS instrument (Brookhaven Instruments Corporation, Holtsville, NY) with electrode and He-Ne laser as a light source. The Smoluchowski limit is assumed in the calculation of zeta potentials.
Lipid analysis
Individual lipid concentrations are determined by Reverse Phase High- Performance Liquid Chromatography (RP-HPLC) using Waters 2695 Alliance system (Water Corporation, Milford MA) with a Corona charged aerosol detector (CAD) (ES A Biosciences, Inc, Chelmsford, MA). Individual lipids in RDVs are analyzed using an Agilent Zorbax SB- Cl 8 (50 x 4.6 mm, 1.8 um particle size) column with CAD at 60 °C. The mobile phase is composed of A: 0.1% TFA in H20 and B: 0.1% TFA in IPA. The gradient changes from 60% mobile phase A and 40% mobile phase B from time 0 to 40% mobile phase A and 60% mobile phase B at 1.00 min; 40% mobile phase A and 60% mobile phase B from 1.00 to 5.00 min; 40% mobile phase A and 60% mobile phase B from 5.00 min to 25% mobile phase A and 75% mobile phase B at 10.00 min; 25% mobile phase A and 75% mobile phase B from 10.00 min to 5% mobile phase A and 95% mobile phase B at 15.00 min; and 5% mobile phase A and 95% mobile phase B from 15.00 to 60% mobile phase A and 40% mobile phase B at 20.00 min with flow rate of 1 ml/min. The individual lipid concentration is determined by comparing to the standard curve with all the lipid components in the RDVs with a quadratic curve fit. The molar percentage of each lipid is calculated based on its molecular weight.
Utilizing the above described LNP process, specific LNPs with the following ratios were identified:
Nominal composition:
Cationic Lipid / Cholesterol / PEG-DMG 60/38/2
Cationic Lipid / Cholesterol / PEG-DMG 67.3/29/3.7
Luc siRNA
5'-iB-AC/AAGGCi/AC/GAAGAGA£/ATT-iB 3' (SEQ.ID.NO.:l) y JOUAUUCCGAUACmCUCUM -y { EQ D^O. 2)
AUGQ-Ribose
iB - Inverted deoxy abasic
UC- 2' Fluoro
AGT - 2' Deoxy
AGU - 2' OCH3
Nominal composition
Cationic Lipid /Cholesterol/PEG-DMG 60/38/2
Cationic Lipid / Cholesterol / PEG-DMG / DSPC 40/48/2/10
Cationic Lipid / Cholesterol / PEG-DMG / DSPC 58/30/2/10
ApoB siRNA
S'-iB-CUUVAACAAUUCCUGAAAUTT-iB (SEQ ID NO.:3) -in GAAAUUGUUAAGGACUUUA-y (SEQ ID NO.:4)
AUGC -Ribose
iB - Inverted deoxy abasic
C/C- 2' Fluoro
AGT -2' Deoxy
AGU - 2' OCH3
Nominal composition
Cationic Lipid /Cholesterol PEG-DMG 60/38/2
Cationic Lipid / Cholesterol / PEG-DMG / DSPC 40/48/2/10
Cationic Lipid / Cholesterol / PEG-DMG / DSPC 58/30/2/10
ApoB siRNA
5,^B-CUUU^4Gt4UUCCUG^4-4UTsT-^B-3, (SEQ ID NO.:5) 3'AJsυGAAAυUGυυAAGGACυslMJs£-5, (SEQ ID NO.:6)
AUGC -Ribose
iB - Inverted deoxy abasic
C/C- 2' Fluoro
AGT - 2' Deoxy
AGU - 2' OCH3
UsA - phophorothioate linkage
The synthesis and use of oligonucleotides, in particular siRNA and miRNA, are known. (See US patent applications: US 2006/0083780, US 2006/0240554, US 2008/0020058, US 2009/0263407 and US 2009/0285881 and PCT patent applications: WO 2009/086558, WO2009/127060, WO2009/132131, WO2010/042877, WO2010/054384, WO2010/054401, WO2010/054405 and WO2010/054406). See also Semple S. C. et al., Rational design of cationic lipids for siRNA delivery, Nature Biotechnology, published online 17 January 2010; doi:10.1038/nbt.l602.
EXAMPLE 1
Mouse In Vivo Evaluation of Efficacy
LNPs utilizing Compounds 1-2, in the nominal compositions described immediately above, were evaluated for in vivo efficacy and induction of inflammatory cytokines in a luciferase mouse model. The siRNA targets the mRNA transcript for the firefly (Photinus pyralis) luciferase gene (Accession # Ml 5077). The primary sequence and chemical modification pattern of the luciferase siRNA is displayed above. The in vivo luciferase model employs a transgenic mouse in which the firefly luciferase coding sequence is present in all cells. ROSA26- LoxP-Stop-LoxP-Luc (LSL-Luc) transgenic mice licensed from the Dana Farber Cancer Institute are induced to express the Luciferase gene by first removing the LSL sequence with a recombinant Ad-Cre virus (Vector Biolabs). Due to the organo-tropic nature of the virus, expression is limited to the liver when delivered via tail vein injection. Luciferase expression levels in liver are quantitated by measuring light output, using an IVIS imager (Xenogen) following administration of the luciferin substrate (Caliper Life Sciences). Pre-dose luminescence levels are measured prior to administration of the RDVs. Luciferin in PBS (15mg/mL) is intraperitoneally (IP) injected in a volume of 150 μL. After a four minute incubation period mice are anesthetized with isoflurane and placed in the IVIS imager. The RDVs (containing siRNA) in PBS vehicle were tail vein injected n a volume of 0.2 mL. Final dose levels ranged from 0.3 to 3 mg/kg siRNA. PBS vehicle alone was dosed as a control.
Three hours post dose, mice were bled retro-orbitally to obtain plasma for cytokine analysis. Mice were imaged 48 hours post dose using the method described above. Changes in luciferin light output directly correlate with luciferase mRNA levels and represent an indirect measure of luciferase siRNA activity. In vivo efficacy results are expressed as % inhibition of luminescence relative to pre-dose luminescence levels. Plasma cytokine levels were determined using the SearchLight multiplexed cytokine chemoluminescent array
(Pierce/Thermo). Systemic administration of the luciferase siRNA RDVs decreased luciferase expression in a dose dependant manner. Greater efficacy was observed in mice dosed with Compound 1 containing RDVs than with the RDV containing the octyl-CLinDMA (OCD) cationic lipid (Figure 1 ). OCD is known and described in WO2010/021865.
Rat In Vivo Evaluation of Efficacy and Toxicity
LNPs utilizing Compound 1 in the nominal compositions described above, were evaluated for in vivo efficacy and increases in alanine amino transferase and aspartate amino transferase in Sprague-Dawley (Crl:CD(SD) female rats (Charles River Labs). The siRNA targets the mRNA transcript for the ApoB gene (Accession # NM 019287). The primary sequence and chemical modification pattern of the ApoB siRNA is displayed above. The RDVs (containing siRNA) in PBS vehicle were tail vein injected in a volume of 1 to 1.5 mL. Infusion rate is approximately 3 ml/min. Five rats were used in each dosing group. After LNP administration, rats are placed in cages with normal diet and water present. Six hours post dose, food is removed from the cages. Animal necropsy is performed 24 hours after LNP dosing. Rats are anesthetized under isoflurane for 5 minutes, then maintained under anesthesia by placing them in nose cones continuing the delivery of isoflurane until ex-sanguination is completed. Blood is collected from the vena cava using a 23 guage butterfly venipuncture set and aliquoted to serum separator vacutainers for serum chemistry analysis. Punches of the excised caudate liver lobe are taken and placed in RNALater (Ambion) for mRNA analysis. Preserved liver tissue was homogenized and total RNA isolated using a Qiagen bead mill and the Qiagen miRNA-Easy RNA isolation kit following the manufacturer's instructions. Liver ApoB mRNA levels were determined by quantitative RT-PCR. Message was amplified from purified RNA utilizing a rat ApoB commercial probe set (Applied Biosystems Cat #
RN01499054_ml). The PCR reaction was performed on an ABI 7500 instrument with a 96- well Fast Block. The ApoB mRNA level is normalized to the housekeeping PPIB (NM 011149) mRNA. PPIB mRNA levels were determined by RT-PCR using a commercial probe set (Applied Biosytems Cat. No. Mm00478295_ml). Results are expressed as a ratio of ApoB mRNA/ PPIB mRNA. All mRNA data is expressed relative to the PBS control dose. Serum ALT and AST analysis were performed on the Siemens Advia 1800 Clinical Chemistry Analyzer utilizing the Siemens alanine aminotransferase (Cat# 03039631) and aspartate aminotransferase (Cat# 03039631 ) reagents. Greater efficacy was observed in rats dosed with Compound 1 containing RDV than with the RDV containing the octyl-CLinDMA cationic lipid (Figure 2). Additionally, lower elevations in LFTs (ALT/AST) were observed in rats dosed with Compound 1 containing RDV than with the RDV containing the octyl-CLinDMA cationic lipid (Figure 3).
Determination of Cationic Lipid Levels in Rat Liver
Liver tissue was weighed into 20-ml vials and homogenized in 9 v/w of water using a GenoGrinder 2000 (OPS Diagnostics, 1600 strokes/min, 5min). A 50 μΐ, aliquot of each tissue homogenate was mixed with 300 uL of extraction/protein precipitating solvent (50/50 acetonitrile/methanol containing 500 nM internal standard) and the plate was centrifuged to sediment precipitated protein. A volume of 200 μΐ, of each supernatant was then transferred to separate wells of a 96-well plate and 10 μΐ samples were directly analyzed by LC/MS-MS.
Standards were prepared by spiking known amounts of a methanol stock solution of Compound 1 or OCD into untreated rat liver homogenate (9 vol water/weight liver). Aliquots (50 μί) each standard/liver homogenate was mixed with 300 μΐ, of extraction/protein precipitating solvent (50/50 acetonitrile/methanol containing 500 nM internal standard) and the plate was centrifuged to sediment precipitated protein. A volume of 200 uL of each supernatant was transferred to separate wells of a 96-well plate and 10 μΐ of each standard was directly analyzed by LC/MS-MS.
Absolute quantification versus standards prepared and extracted from rat liver homogenate was performed using an Aria LX-2 HPLC system (Thermo Scientific) coupled to an API 4000 triple quadrupole mass spectrometer (Applied Biosystems). For each run, a total of 10 L sample was injected onto a BDS Hypersil C8 HPLC column (Thermo, 50 x 2mm, 3 μηι) at ambient temperature (Figure 4).
Mobile Phase A: 95% H20/5% methanol/10 raM ammonium
formate/0. l%formic acid Mobile Phase B: 40% methanol/60% n-propanol/10 mM ammonium formate/0. l%formic acid The flow rate was 0.5 mL/min and gradient elution profile was as follows: hold at 80% A for 0.25 min, linear ramp to 100% B over 1.6 min, hold at 100% B for 2.5 min, then return and hold at 80% A for 1.75 min. Total run time was 5.8 min. API 4000 source parameters were CAD: 4, CUR: 15, GS1: 65, GS2: 35, IS: 4000, TEM: 550, CXP: 15, DP: 60, EP: 10.

Claims

WHAT IS CLAIMED IS:
1. A cationic lipid of Formula A :
Figure imgf000024_0001
wherein: R1 and R2 are independently selected from H, (Ci-C6)alkyl, heterocyclyl, and polyamine, wherein said alkyl, heterocyclyl and polyamine are optionally substituted with one to three substituents selected from R', or R1 and R2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one to three substituents selected from
R'; R' is independently selected from halogen, R", OR", SR", CN, C02R" or CON(R")2;
R" is independently selected from H and (Ci-C6)alkyl, wherein said alkyl is optionally substituted with halogen and OH; Li is selected from C4-C22 alkyl and C4-C22 alkenyl, said alkyl and alkenyl are optionally substituted with one or more substituents selected from R'; and
L2 is selected from C3-C13 alkyl and C3-C13 alkenyl, said alkyl and alkenyl are optionally substituted with one or more substituents selected from R'; or any pharmaceutically acceptable salt or stereoisomer thereof.
2. A cationic lipid of Formula A according to Claim 1 ,
wherein: R1 and R2 are each methyl;
Li is selected from C4-C22 alkyl and C4-C22 alkenyl; and L2 is selected from C3-C13 alkyl and C3-C13 alkenyl; or any pharmaceutically acceptable salt or stereoisomer thereof.
3. A cationic lipid which is selected from:
R -N,N-dimemyl-l-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-(octyloxy)propan-2-amine (Compound 2);
S -N,N-dimetoyl-l-[(9Z,12Z)-octadeca-9,12-d^
(Compound 1);
l-{2-[(9Z.12Z)-octadeca-9,12-dien-l-yto
(Compound 3);
(2S>N,N-dime l-l-[(9Z,12Z octad^
yloxy]propan-2-amine (Compound 4);
l-{2-[(9Z912Z)-octadeca-9,12-dien-l-yloxyH^
5);
(2S)-l-(hexyloxy)-N,N-fflme l-3-[(9Z^
(Compound 6);
(2S l-(heptyloxy)-N,N-dimemyl-3-[(9Z ^
(Compound 7);
Ν,Ν-dimethyl- 1 -(nonyloxy)-3-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]propan-2-amine (Compound 8);
Ν,Ν-dimethyl-l -[(9Z)-octadec-9-en-l -yloxy]-3-(octyloxy)propan-2-amine (Compound 9); (2S)-N,N-dimetoyl-l-[(6Z,9Z 2Z)-oc^
amine (Compound 10);
(2S)-l-[(l lZ,14Z)-icosa-ll,14-dien-l-yloxy]^
(Compound 11);
(2S)- 1 -(hexyloxy)-3 -[(11 Z, 14Z)-icosa- 11,14-dien- 1 -yloxy]-N,N-dimethylpropan-2-amine (Compound 12);
1 - [( 11 Z, 14Z)-icosa- 11 , 14-dien- 1 -y loxy] -Ν,Ν-dimethy 1-3 -(octyloxy)propan-2-amine
(Compound 13);
1 -[(13Z, 16Z)-docosa-l 3,16-dien-l -yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine (Compound 14);
(2S)- 1 -[( 13Z, 16Z)-docosa- 13,16-dien- 1 -yloxy]-3 -(hexyloxy)-N,N-dimethy lpropan-2-amine (Compound 15);
(2S)-l-[(13Z)-docos-13-en-l-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine (Compound 16); 1- [(13Z)-docos-13-en-l-yloxy]-N,N-dime^ (Compound 17); 1 -[(9Z)-hexadec-9-en- 1 -yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine (Compound 18); (2R)-N,N-dimetoyl-H(l -metoyto
2- amine (Compound 19);
(2R)-l-[(3,7-dimemyloctyl)oxy]-N^
yloxy]propan-2-amine (Compound 20);
N,N-dimethyl-l-(octyloxy)-3-({8-[(lS,2S)-2-{[(lR,2R)-2- pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine (Compound 21); and N,N-dimemyl-l-{[8-(2-oc1ylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine (Compound 22);
or any pharmaceutically acceptable salt or stereoisomer thereof.
4. The use of a cationic lipid according to Claim 1 for the preparation of lipid nanoparticles.
5. The use of a cationic lipid according to Claim 1 as a component in a lipid nanoparticle for the delivery of oligonucleotides.
6. The use according to Claim 5 wherein the oligonucleotides are siR A or miRNA.
7, The use according to Claim 5 wherein the oligonucleotides are siRNA.
PCT/US2011/038490 2010-06-04 2011-05-31 Novel low molecular weight cationic lipids for oligonucleotide delivery WO2011153120A1 (en)

Priority Applications (7)

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EP11790268.4A EP2575767B1 (en) 2010-06-04 2011-05-31 Novel low molecular weight cationic lipids for oligonucleotide delivery
DK11790268.4T DK2575767T3 (en) 2010-06-04 2011-05-31 HOWEVER UNKNOWN LOW MOLECULAR CATIONIC LIPIDS TO PROCESS OIGONUCLEOTIDES
JP2013513262A JP5957646B2 (en) 2010-06-04 2011-05-31 Novel low molecular weight cationic lipids for oligonucleotide delivery
US13/701,636 US8748667B2 (en) 2010-06-04 2011-05-31 Low molecular weight cationic lipids for oligonucleotide delivery
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Cited By (100)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013148541A1 (en) * 2012-03-27 2013-10-03 Merck Sharp & Dohme Corp. DIETHER BASED BIODEGRADABLE CATIONIC LIPIDS FOR siRNA DELIVERY
WO2013151666A2 (en) 2012-04-02 2013-10-10 modeRNA Therapeutics Modified polynucleotides for the production of biologics and proteins associated with human disease
WO2013151736A2 (en) 2012-04-02 2013-10-10 modeRNA Therapeutics In vivo production of proteins
WO2014152540A1 (en) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
WO2014152211A1 (en) 2013-03-14 2014-09-25 Moderna Therapeutics, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
WO2015006747A2 (en) 2013-07-11 2015-01-15 Moderna Therapeutics, Inc. Compositions comprising synthetic polynucleotides encoding crispr related proteins and synthetic sgrnas and methods of use.
WO2015034925A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Circular polynucleotides
JP2015509505A (en) * 2012-02-24 2015-03-30 プロティバ バイオセラピューティクス インコーポレイテッド Trialkylcationic lipids and methods of use thereof
WO2015051214A1 (en) 2013-10-03 2015-04-09 Moderna Therapeutics, Inc. Polynucleotides encoding low density lipoprotein receptor
WO2015075557A2 (en) 2013-11-22 2015-05-28 Mina Alpha Limited C/ebp alpha compositions and methods of use
JP2015520733A (en) * 2012-04-19 2015-07-23 サーナ・セラピューティクス・インコーポレイテッドＳｉｒｎａ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． Novel diester and triester-based low molecular weight, biodegradable cationic lipids for oligonucleotide delivery
WO2015110957A2 (en) 2014-01-21 2015-07-30 De Beer Joel Hybridosomes, compositions comprising the same, processes for their production and uses thereof
WO2016014846A1 (en) 2014-07-23 2016-01-28 Moderna Therapeutics, Inc. Modified polynucleotides for the production of intrabodies
WO2016065349A2 (en) 2014-10-24 2016-04-28 University Of Maryland, Baltimore Short non-coding protein regulatory rnas (sprrnas) and methods of use
WO2017070613A1 (en) 2015-10-22 2017-04-27 Modernatx, Inc. Human cytomegalovirus vaccine
WO2017112943A1 (en) 2015-12-23 2017-06-29 Modernatx, Inc. Methods of using ox40 ligand encoding polynucleotides
WO2017120612A1 (en) 2016-01-10 2017-07-13 Modernatx, Inc. Therapeutic mrnas encoding anti ctla-4 antibodies
US10106490B2 (en) 2014-06-25 2018-10-23 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2018213731A1 (en) 2017-05-18 2018-11-22 Modernatx, Inc. Polynucleotides encoding tethered interleukin-12 (il12) polypeptides and uses thereof
WO2018232006A1 (en) 2017-06-14 2018-12-20 Modernatx, Inc. Polynucleotides encoding coagulation factor viii
US10221127B2 (en) 2015-06-29 2019-03-05 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2019048631A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Hnf4a sarna compositions and methods of use
WO2019048645A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Stabilized cebpa sarna compositions and methods of use
WO2019197845A1 (en) 2018-04-12 2019-10-17 Mina Therapeutics Limited Sirt1-sarna compositions and methods of use
WO2019217964A1 (en) 2018-05-11 2019-11-14 Lupagen, Inc. Systems and methods for closed loop, real-time modifications of patient cells
WO2020033791A1 (en) 2018-08-09 2020-02-13 Verseau Therapeutics, Inc. Oligonucleotide compositions for targeting ccr2 and csf1r and uses thereof
WO2020061295A1 (en) 2018-09-19 2020-03-26 Modernatx, Inc. High-purity peg lipids and uses thereof
WO2020061284A1 (en) 2018-09-19 2020-03-26 Modernatx, Inc. Peg lipids and uses thereof
WO2020128031A2 (en) 2018-12-21 2020-06-25 Curevac Ag Rna for malaria vaccines
WO2020161342A1 (en) 2019-02-08 2020-08-13 Curevac Ag Coding rna administered into the suprachoroidal space in the treatment of ophtalmic diseases
WO2020208361A1 (en) 2019-04-12 2020-10-15 Mina Therapeutics Limited Sirt1-sarna compositions and methods of use
WO2020254535A1 (en) 2019-06-18 2020-12-24 Curevac Ag Rotavirus mrna vaccine
WO2021026310A1 (en) 2019-08-06 2021-02-11 L.E.A.F. Holdings Group Llc Processes of preparing polyglutamated antifolates and uses of their compositions
WO2021028439A1 (en) 2019-08-14 2021-02-18 Curevac Ag Rna combinations and compositions with decreased immunostimulatory properties
WO2021061815A1 (en) 2019-09-23 2021-04-01 Omega Therapeutics, Inc. COMPOSITIONS AND METHODS FOR MODULATING HEPATOCYTE NUCLEAR FACTOR 4-ALPHA (HNF4α) GENE EXPRESSION
WO2021061707A1 (en) 2019-09-23 2021-04-01 Omega Therapeutics, Inc. Compositions and methods for modulating apolipoprotein b (apob) gene expression
WO2021156267A1 (en) 2020-02-04 2021-08-12 Curevac Ag Coronavirus vaccine
WO2021183720A1 (en) 2020-03-11 2021-09-16 Omega Therapeutics, Inc. Compositions and methods for modulating forkhead box p3 (foxp3) gene expression
WO2021195214A1 (en) 2020-03-24 2021-09-30 Generation Bio Co. Non-viral dna vectors and uses thereof for expressing factor ix therapeutics
WO2021195218A1 (en) 2020-03-24 2021-09-30 Generation Bio Co. Non-viral dna vectors and uses thereof for expressing gaucher therapeutics
WO2021239880A1 (en) 2020-05-29 2021-12-02 Curevac Ag Nucleic acid based combination vaccines
WO2022023559A1 (en) 2020-07-31 2022-02-03 Curevac Ag Nucleic acid encoded antibody mixtures
WO2022023284A1 (en) 2020-07-27 2022-02-03 Anjarium Biosciences Ag Compositions of dna molecules, methods of making therefor, and methods of use thereof
WO2022032154A2 (en) 2020-08-06 2022-02-10 Modernatx, Inc. Compositions for the delivery of payload molecules to airway epithelium
WO2022043551A2 (en) 2020-08-31 2022-03-03 Curevac Ag Multivalent nucleic acid based coronavirus vaccines
WO2022122872A1 (en) 2020-12-09 2022-06-16 Ucl Business Ltd Therapeutics for the treatment of neurodegenerative disorders
WO2022137133A1 (en) 2020-12-22 2022-06-30 Curevac Ag Rna vaccine against sars-cov-2 variants
WO2022135993A2 (en) 2020-12-22 2022-06-30 Curevac Ag Pharmaceutical composition comprising lipid-based carriers encapsulating rna for multidose administration
WO2022162027A2 (en) 2021-01-27 2022-08-04 Curevac Ag Method of reducing the immunostimulatory properties of in vitro transcribed rna
US11453639B2 (en) 2019-01-11 2022-09-27 Acuitas Therapeutics, Inc. Lipids for lipid nanoparticle delivery of active agents
WO2022200575A1 (en) 2021-03-26 2022-09-29 Glaxosmithkline Biologicals Sa Immunogenic compositions
WO2022200810A1 (en) 2021-03-26 2022-09-29 Mina Therapeutics Limited Tmem173 sarna compositions and methods of use
WO2022207862A2 (en) 2021-03-31 2022-10-06 Curevac Ag Syringes containing pharmaceutical compositions comprising rna
EP4074834A1 (en) 2012-11-26 2022-10-19 ModernaTX, Inc. Terminally modified rna
WO2022223556A1 (en) 2021-04-20 2022-10-27 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
WO2022232289A1 (en) 2021-04-27 2022-11-03 Generation Bio Co. 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
WO2022261394A1 (en) 2021-06-11 2022-12-15 LifeEDIT Therapeutics, Inc. Rna polymerase iii promoters and methods of use
WO2023283359A2 (en) 2021-07-07 2023-01-12 Omega Therapeutics, Inc. Compositions and methods for modulating secreted frizzled receptor protein 1 (sfrp1) gene expression
WO2023014974A1 (en) 2021-08-06 2023-02-09 University Of Iowa Research Foundation Double stranded mrna vaccines
US11590229B2 (en) 2011-12-07 2023-02-28 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
EP4144378A1 (en) 2011-12-16 2023-03-08 ModernaTX, Inc. Modified nucleoside, nucleotide, and nucleic acid compositions
WO2023031394A1 (en) 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids
WO2023031392A2 (en) 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids comprising phosphatidylserine
EP4159741A1 (en) 2014-07-16 2023-04-05 ModernaTX, Inc. Method for producing a chimeric polynucleotide encoding a polypeptide having a triazole-containing internucleotide linkage
WO2023073228A1 (en) 2021-10-29 2023-05-04 CureVac SE Improved circular rna for expressing therapeutic proteins
WO2023081526A1 (en) 2021-11-08 2023-05-11 Orna Therapeutics, Inc. Lipid nanoparticle compositions for delivering circular polynucleotides
US11648324B2 (en) 2015-10-28 2023-05-16 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2023086465A1 (en) 2021-11-12 2023-05-19 Modernatx, Inc. Compositions for the delivery of payload molecules to airway epithelium
WO2023099884A1 (en) 2021-12-01 2023-06-08 Mina Therapeutics Limited Pax6 sarna compositions and methods of use
WO2023104964A1 (en) 2021-12-09 2023-06-15 Ucl Business Ltd Therapeutics for the treatment of neurodegenerative disorders
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
WO2023144330A1 (en) 2022-01-28 2023-08-03 CureVac SE Nucleic acid encoded transcription factor inhibitors
WO2023154818A1 (en) 2022-02-09 2023-08-17 Modernatx, Inc. Mucosal administration methods and formulations
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
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
WO2024033901A1 (en) 2022-08-12 2024-02-15 LifeEDIT Therapeutics, Inc. Rna-guided nucleases and active fragments and variants thereof and methods of use
WO2024040222A1 (en) 2022-08-19 2024-02-22 Generation Bio Co. Cleavable closed-ended dna (cedna) and methods of use thereof
DE202023106198U1 (en) 2022-10-28 2024-03-21 CureVac SE Nucleic acid-based vaccine
EP4342460A1 (en) 2022-09-21 2024-03-27 NovoArc GmbH Lipid nanoparticle with nucleic acid cargo
WO2024068545A1 (en) 2022-09-26 2024-04-04 Glaxosmithkline Biologicals Sa Influenza virus vaccines
US11976019B2 (en) 2020-07-16 2024-05-07 Acuitas Therapeutics, Inc. Cationic lipids for use in lipid nanoparticles
GB202404607D0 (en) 2024-03-29 2024-05-15 Glaxosmithkline Biologicals Sa RNA formulation
WO2024102730A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Lipids and nanoparticle compositions for delivering polynucleotides
WO2024102762A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Lipids and lipid nanoparticle compositions for delivering polynucleotides
WO2024102677A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Circular rna compositions
WO2024119051A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Novel polyglycerol-conjugated lipids and lipid nanoparticle compositions comprising the same
WO2024119074A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Stealth lipid nanoparticle compositions for cell targeting
WO2024119103A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Lipid nanoparticles comprising nucleic acids and lipid-anchored polymers
WO2024119039A2 (en) 2022-12-01 2024-06-06 Generation Bio Co. Stealth lipid nanoparticles and uses thereof
WO2024134199A1 (en) 2022-12-22 2024-06-27 Mina Therapeutics Limited Chemically modified sarna compositions and methods of use
WO2024160936A1 (en) 2023-02-03 2024-08-08 Glaxosmithkline Biologicals Sa Rna formulation
WO2024171052A1 (en) 2023-02-14 2024-08-22 Glaxosmithkline Biologicals Sa Analytical method
WO2024184500A1 (en) 2023-03-08 2024-09-12 CureVac SE Novel lipid nanoparticle formulations for delivery of nucleic acids
WO2024205657A2 (en) 2023-03-29 2024-10-03 Orna Therapeutics, Inc. Lipids and lipid nanoparticle compositions for delivering polynucleotides
US12129223B2 (en) 2022-12-15 2024-10-29 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations

Families Citing this family (108)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5957646B2 (en) 2010-06-04 2016-07-27 サーナ・セラピューティクス・インコーポレイテッドＳｉｒｎａ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． Novel low molecular weight cationic lipids for oligonucleotide delivery
WO2012019168A2 (en) 2010-08-06 2012-02-09 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
CA2821992A1 (en) 2010-10-01 2012-04-05 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
DE12722942T1 (en) 2011-03-31 2021-09-30 Modernatx, Inc. RELEASE AND FORMULATION OF MANIPULATED NUCLEIC ACIDS
WO2013066438A2 (en) 2011-07-22 2013-05-10 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
EP3492109B1 (en) 2011-10-03 2020-03-04 ModernaTX, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9878056B2 (en) 2012-04-02 2018-01-30 Modernatx, Inc. Modified polynucleotides for the production of cosmetic proteins and peptides
US20150044192A1 (en) 2013-08-09 2015-02-12 President And Fellows Of Harvard College Methods for identifying a target site of a cas9 nuclease
US9359599B2 (en) 2013-08-22 2016-06-07 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
EP3041934A1 (en) 2013-09-03 2016-07-13 Moderna Therapeutics, Inc. Chimeric polynucleotides
WO2016070129A1 (en) 2014-10-30 2016-05-06 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US9340799B2 (en) 2013-09-06 2016-05-17 President And Fellows Of Harvard College MRNA-sensing switchable gRNAs
US9322037B2 (en) 2013-09-06 2016-04-26 President And Fellows Of Harvard College Cas9-FokI fusion proteins and uses thereof
US9526784B2 (en) 2013-09-06 2016-12-27 President And Fellows Of Harvard College Delivery system for functional nucleases
WO2015048744A2 (en) 2013-09-30 2015-04-02 Moderna Therapeutics, Inc. Polynucleotides encoding immune modulating polypeptides
JP2016536021A (en) 2013-11-07 2016-11-24 エディタス・メディシン，インコーポレイテッド CRISPR-related methods and compositions with governing gRNA
US9068179B1 (en) 2013-12-12 2015-06-30 President And Fellows Of Harvard College Methods for correcting presenilin point mutations
SG10201912038TA (en) 2014-04-23 2020-02-27 Modernatx Inc Nucleic acid vaccines
WO2015178343A1 (en) * 2014-05-20 2015-11-26 国立大学法人北海道大学 LIPID MEMBRANE STRUCTURE FOR siRNA INTRACELLULAR DELIVERY
WO2016022363A2 (en) 2014-07-30 2016-02-11 President And Fellows Of Harvard College Cas9 proteins including ligand-dependent inteins
US9816080B2 (en) 2014-10-31 2017-11-14 President And Fellows Of Harvard College Delivery of CAS9 via ARRDC1-mediated microvesicles (ARMMs)
EP4088741A1 (en) 2014-12-08 2022-11-16 The Board of Regents of the University of Texas System Lipocationic polymers and uses thereof
WO2017031232A1 (en) 2015-08-17 2017-02-23 Modernatx, Inc. Methods for preparing particles and related compositions
HUE057613T2 (en) 2015-09-17 2022-05-28 Modernatx Inc Compounds and compositions for intracellular delivery of therapeutic agents
HUE059127T2 (en) 2015-10-22 2022-10-28 Modernatx Inc Respiratory virus vaccines
IL294014B2 (en) 2015-10-23 2024-07-01 Harvard College Nucleobase editors and uses thereof
PL3386484T3 (en) 2015-12-10 2022-07-25 Modernatx, Inc. Compositions and methods for delivery of therapeutic agents
DK3394030T3 (en) 2015-12-22 2022-03-28 Modernatx Inc COMPOUNDS AND COMPOSITIONS FOR INTRACELLULAR RELEASE OF FUNDS
EP3442590A2 (en) 2016-04-13 2019-02-20 Modernatx, Inc. Lipid compositions and their uses for intratumoral polynucleotide delivery
CA3024500A1 (en) 2016-05-18 2017-11-23 Modernatx, Inc. Polynucleotides encoding relaxin
CA3032699A1 (en) 2016-08-03 2018-02-08 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
AU2017308889B2 (en) 2016-08-09 2023-11-09 President And Fellows Of Harvard College Programmable Cas9-recombinase fusion proteins and uses thereof
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
TW201815736A (en) * 2016-09-27 2018-05-01 日商協和醱酵麒麟有限公司 Cationic lipid compound
WO2018071868A1 (en) 2016-10-14 2018-04-19 President And Fellows Of Harvard College Aav delivery of nucleobase editors
EP3538067A1 (en) 2016-11-08 2019-09-18 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
EP3592853A1 (en) 2017-03-09 2020-01-15 President and Fellows of Harvard College Suppression of pain by gene editing
JP2020510439A (en) 2017-03-10 2020-04-09 プレジデント アンド フェローズ オブ ハーバード カレッジ Base-editing factor from cytosine to guanine
CA3056133A1 (en) 2017-03-15 2018-09-20 Modernatx, Inc. Crystal forms of amino lipids
US11969506B2 (en) 2017-03-15 2024-04-30 Modernatx, Inc. Lipid nanoparticle formulation
FI3596041T3 (en) 2017-03-15 2023-01-31 Compound and compositions for intracellular delivery of therapeutic agents
IL269458B2 (en) 2017-03-23 2024-02-01 Harvard College Nucleobase editors comprising nucleic acid programmable dna binding proteins
WO2018209320A1 (en) 2017-05-12 2018-11-15 President And Fellows Of Harvard College Aptazyme-embedded guide rnas for use with crispr-cas9 in genome editing and transcriptional activation
WO2018232120A1 (en) 2017-06-14 2018-12-20 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
US11786607B2 (en) 2017-06-15 2023-10-17 Modernatx, Inc. RNA formulations
JP2020534795A (en) 2017-07-28 2020-12-03 プレジデント アンド フェローズ オブ ハーバード カレッジ Methods and Compositions for Evolving Base Editing Factors Using Phage-Supported Continuous Evolution (PACE)
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
WO2019046809A1 (en) 2017-08-31 2019-03-07 Modernatx, Inc. Methods of making lipid nanoparticles
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
WO2020056161A1 (en) 2018-09-12 2020-03-19 University Of Florida Research Foundation, Inc. Slow-cycling cell-rna based nanoparticle vaccine to treat cancer
EP3852728B1 (en) 2018-09-20 2024-09-18 ModernaTX, Inc. Preparation of lipid nanoparticles and methods of administration thereof
SG11202102164PA (en) 2018-09-28 2021-04-29 Nutcracker Therapeutics Inc Tertiary amino lipidated cationic peptides for nucleic acid delivery
CA3113472A1 (en) 2018-09-28 2020-04-02 President And Fellows Of Harvard College Cellular reprogramming to reverse aging and promote organ and tissue regeneration
DE112020001342T5 (en) 2019-03-19 2022-01-13 President and Fellows of Harvard College Methods and compositions for editing nucleotide sequences
JP7156154B2 (en) * 2019-04-18 2022-10-19 株式会社島津製作所 Medium processing system and medium processing method
US20220218808A1 (en) 2019-05-02 2022-07-14 University Of Florida Research Foundation, Inc. Compositions for treatment of diffuse intrinsic pontine glioma
CA3147875A1 (en) 2019-07-19 2021-01-28 Flagship Pioneering Innovations Vi, Llc Recombinase compositions and methods of use
US11066355B2 (en) 2019-09-19 2021-07-20 Modernatx, Inc. Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents
EP4126030A1 (en) 2020-04-01 2023-02-08 University Of Florida Research Foundation, Incorporated Multilamellar rna nanoparticle vaccine against sars-cov-2
DE112021002672T5 (en) 2020-05-08 2023-04-13 President And Fellows Of Harvard College METHODS AND COMPOSITIONS FOR EDIT BOTH STRANDS SIMULTANEOUSLY OF A DOUBLE STRANDED NUCLEOTIDE TARGET SEQUENCE
IL298363A (en) 2020-05-20 2023-01-01 Flagship Pioneering Innovations Vi Llc Immunogenic compositions and uses thereof
IL298362A (en) 2020-05-20 2023-01-01 Flagship Pioneering Innovations Vi Llc Coronavirus antigen compositions and their uses
EP4158032A2 (en) 2020-05-29 2023-04-05 Flagship Pioneering Innovations VI, LLC Trem compositions and methods relating thereto
CA3180101A1 (en) 2020-05-29 2021-12-02 Flagship Pioneering Innovations Vi, Llc Trem compositions and methods relating thereto
TW202218669A (en) 2020-09-03 2022-05-16 美商旗艦先鋒創新有限責任公司 Immunogenic compositions and uses thereof
US20240175020A1 (en) 2020-12-23 2024-05-30 Flagship Pioneering Innovations Vi, Llc Compositions of modified trems and uses thereof
US11524023B2 (en) 2021-02-19 2022-12-13 Modernatx, Inc. Lipid nanoparticle compositions and methods of formulating the same
WO2022212784A1 (en) 2021-03-31 2022-10-06 Flagship Pioneering Innovations V, Inc. Thanotransmission polypeptides and their use in treating cancer
CA3211623A1 (en) 2021-05-24 2022-12-01 Bo YING Lipid compounds and lipid nanoparticle compositions
EP4377457A1 (en) 2021-07-26 2024-06-05 Flagship Pioneering Innovations VI, LLC Trem compositions and uses thereof
EP4396355A1 (en) 2021-09-03 2024-07-10 GlaxoSmithKline Biologicals S.A. Substitution of nucleotide bases in self-amplifying messenger ribonucleic acids
CA3232635A1 (en) 2021-09-17 2023-03-23 Flagship Pioneering Innovations Vi, Llc Compositions and methods for producing circular polyribonucleotides
AU2022370530A1 (en) 2021-10-18 2024-05-02 Flagship Pioneering Innovations Vi, Llc Compositions and methods for purifying polyribonucleotides
EP4436984A1 (en) 2021-11-24 2024-10-02 Flagship Pioneering Innovations VI, LLC Coronavirus immunogen compositions and their uses
CN118555966A (en) 2021-11-24 2024-08-27 旗舰创业创新六公司 Immunogenic compositions and uses thereof
AU2022397292A1 (en) 2021-11-24 2024-05-30 Flagship Pioneering Innovations Vi, Llc Varicella-zoster virus immunogen compositions and their uses
AR128002A1 (en) 2021-12-17 2024-03-20 Flagship Pioneering Innovations Vi Llc CIRCULAR RNA ENRICHMENT METHODS UNDER DENATURALING CONDITIONS
TW202340461A (en) 2021-12-22 2023-10-16 美商旗艦先鋒創新有限責任公司 Compositions and methods for purifying polyribonucleotides
WO2023122789A1 (en) 2021-12-23 2023-06-29 Flagship Pioneering Innovations Vi, Llc Circular polyribonucleotides encoding antifusogenic polypeptides
AU2023239151A1 (en) 2022-03-25 2024-10-03 Sail Biomedicines, Inc. Novel ionizable lipids and lipid nanoparticles and methods of using the same
WO2023196851A1 (en) 2022-04-06 2023-10-12 President And Fellows Of Harvard College Reversing aging of the central nervous system
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
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
WO2024030856A2 (en) 2022-08-01 2024-02-08 Flagship Pioneering Innovations Vii, Llc Immunomodulatory proteins and related methods
WO2024035952A1 (en) 2022-08-12 2024-02-15 Remix Therapeutics Inc. Methods and compositions for modulating splicing at alternative splice sites
WO2024049979A2 (en) 2022-08-31 2024-03-07 Senda Biosciences, Inc. Novel ionizable lipids and lipid nanoparticles and methods of using the same
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
WO2024097664A1 (en) 2022-10-31 2024-05-10 Flagship Pioneering Innovations Vi, Llc Compositions and methods for purifying polyribonucleotides
WO2024102799A1 (en) 2022-11-08 2024-05-16 Flagship Pioneering Innovations Vi, Llc Compositions and methods for producing circular polyribonucleotides
WO2024129988A1 (en) 2022-12-14 2024-06-20 Flagship Pioneering Innovations Vii, Llc Compositions and methods for delivery of therapeutic agents to bone
WO2024133160A1 (en) 2022-12-19 2024-06-27 Glaxosmithkline Biologicals Sa Hepatitis b compositions
US20240238473A1 (en) 2023-01-09 2024-07-18 Beth Israel Deaconess Medical Center, Inc. Recombinant nucleic acid molecules and their use in wound healing
US20240252520A1 (en) 2023-01-09 2024-08-01 Beth Israel Deaconess Medical Center, Inc. Therapeutic agents and their use for treating chronic wounds
WO2024151687A1 (en) 2023-01-09 2024-07-18 Flagship Pioneering Innovations V, Inc. Genetic switches and their use in treating cancer
WO2024151583A2 (en) 2023-01-09 2024-07-18 Flagship Pioneering Innovations Vii, Llc Vaccines and related methods
WO2024167885A1 (en) 2023-02-06 2024-08-15 Flagship Pioneering Innovations Vii, Llc Immunomodulatory compositions and related methods
US20240293318A1 (en) 2023-02-13 2024-09-05 Flagship Pioneering Innovations Vii, Llc Cleavable linker-containing ionizable lipids and lipid carriers for therapeutic compositions
US20240293582A1 (en) 2023-02-17 2024-09-05 Flagship Pioneering Innovations Vii, Llc Dna compositions comprising modified cytosine
WO2024173828A1 (en) 2023-02-17 2024-08-22 Flagship Pioneering Innovations Vii, Llc Dna compositions comprising modified uracil
WO2024192422A1 (en) 2023-03-15 2024-09-19 Flagship Pioneering Innovations Vi, Llc Immunogenic compositions and uses thereof
WO2024192420A1 (en) 2023-03-15 2024-09-19 Flagship Pioneering Innovations Vi, Llc Compositions comprising polyribonucleotides and uses thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6060455A (en) * 1995-06-14 2000-05-09 Boehringer Mannheim Gmbh Cationic and polycationic amphiphiles, reactives containing the same and their use
US20090285881A1 (en) * 2008-04-16 2009-11-19 Abbott Laboratories Cationic lipids and uses thereof

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE537263T1 (en) * 2004-06-07 2011-12-15 Protiva Biotherapeutics Inc CATIONIC LIPIDS AND METHODS OF USE
US20100055168A1 (en) * 2008-04-16 2010-03-04 Abbott Laboratories Cationic lipids and uses thereof
WO2009129385A1 (en) * 2008-04-16 2009-10-22 Abbott Laboratories Cationic lipids and uses thereof
TW201019963A (en) * 2008-09-10 2010-06-01 Abbott Lab Polyethylene glycol lipid conjugates and uses thereof
WO2010030730A1 (en) * 2008-09-10 2010-03-18 Abbott Laboratories Polyethylene glycol lipid conjugates and uses thereof
JP5957646B2 (en) * 2010-06-04 2016-07-27 サーナ・セラピューティクス・インコーポレイテッドＳｉｒｎａ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． Novel low molecular weight cationic lipids for oligonucleotide delivery

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6060455A (en) * 1995-06-14 2000-05-09 Boehringer Mannheim Gmbh Cationic and polycationic amphiphiles, reactives containing the same and their use
US20090285881A1 (en) * 2008-04-16 2009-11-19 Abbott Laboratories Cationic lipids and uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP2575767A4 *
SEMPLE ET AL.: "Rational design of cationic lipids for siRNA delivery.", NAT BIOTECHNOL., vol. 28, no. 2, February 2010 (2010-02-01), pages 172 - 176, XP002633693 *

Cited By (129)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11679158B2 (en) 2011-12-07 2023-06-20 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
US11633479B2 (en) 2011-12-07 2023-04-25 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
US11633480B2 (en) 2011-12-07 2023-04-25 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
US11612657B2 (en) 2011-12-07 2023-03-28 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
US11590229B2 (en) 2011-12-07 2023-02-28 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
EP4144378A1 (en) 2011-12-16 2023-03-08 ModernaTX, Inc. Modified nucleoside, nucleotide, and nucleic acid compositions
US11395854B2 (en) 2012-02-24 2022-07-26 Arbutus Biopharma Corporation Trialkyl cationic lipids and methods of use thereof
US10561732B2 (en) 2012-02-24 2020-02-18 Arbutus Biopharma Corporation Trialkyl cationic lipids and methods of use thereof
JP2018039844A (en) * 2012-02-24 2018-03-15 プロティバ バイオセラピューティクス インコーポレイテッド Trialkyl cationic lipids and methods of use thereof
JP2015509505A (en) * 2012-02-24 2015-03-30 プロティバ バイオセラピューティクス インコーポレイテッド Trialkylcationic lipids and methods of use thereof
AU2013240051B2 (en) * 2012-03-27 2017-11-30 Sirna Therapeutics, Inc. Diether based biodegradable cationic lipids for siRNA delivery
WO2013148541A1 (en) * 2012-03-27 2013-10-03 Merck Sharp & Dohme Corp. DIETHER BASED BIODEGRADABLE CATIONIC LIPIDS FOR siRNA DELIVERY
EP2830594A4 (en) * 2012-03-27 2015-11-11 Sirna Therapeutics Inc DIETHER BASED BIODEGRADABLE CATIONIC LIPIDS FOR siRNA DELIVERY
US9446132B2 (en) 2012-03-27 2016-09-20 Sima Therapeutics, Inc. Diether based biodegradable cationic lipids for siRNA delivery
US9604908B2 (en) 2012-03-27 2017-03-28 Sima Therapeutics, Inc. Diether based biodegradable cationic lipids for siRNA delivery
WO2013151666A2 (en) 2012-04-02 2013-10-10 modeRNA Therapeutics Modified polynucleotides for the production of biologics and proteins associated with human disease
WO2013151736A2 (en) 2012-04-02 2013-10-10 modeRNA Therapeutics In vivo production of proteins
JP2015520733A (en) * 2012-04-19 2015-07-23 サーナ・セラピューティクス・インコーポレイテッドＳｉｒｎａ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． Novel diester and triester-based low molecular weight, biodegradable cationic lipids for oligonucleotide delivery
EP4074834A1 (en) 2012-11-26 2022-10-19 ModernaTX, Inc. Terminally modified rna
WO2014152211A1 (en) 2013-03-14 2014-09-25 Moderna Therapeutics, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
WO2014152540A1 (en) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
WO2015006747A2 (en) 2013-07-11 2015-01-15 Moderna Therapeutics, Inc. Compositions comprising synthetic polynucleotides encoding crispr related proteins and synthetic sgrnas and methods of use.
EP3971287A1 (en) 2013-07-11 2022-03-23 ModernaTX, Inc. Compositions comprising synthetic polynucleotides encoding crispr related proteins and synthetic sgrnas and methods of use
WO2015034925A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Circular polynucleotides
WO2015051214A1 (en) 2013-10-03 2015-04-09 Moderna Therapeutics, Inc. Polynucleotides encoding low density lipoprotein receptor
EP3985118A1 (en) 2013-11-22 2022-04-20 MiNA Therapeutics Limited C/ebp alpha short activating rna compositions and methods of use
EP3594348A1 (en) 2013-11-22 2020-01-15 Mina Therapeutics Limited C/ebp alpha short activating rna compositions and methods of use
WO2015075557A2 (en) 2013-11-22 2015-05-28 Mina Alpha Limited C/ebp alpha compositions and methods of use
WO2015110957A2 (en) 2014-01-21 2015-07-30 De Beer Joel Hybridosomes, compositions comprising the same, processes for their production and uses thereof
US11944706B2 (en) 2014-01-21 2024-04-02 Anjarium Biosciences Ag Hybridosomes, compositions comprising the same, processes for their production and uses thereof
EP3791863A1 (en) 2014-01-21 2021-03-17 Anjarium Biosciences AG Process for the production of hybridosomes
US10106490B2 (en) 2014-06-25 2018-10-23 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
EP4159741A1 (en) 2014-07-16 2023-04-05 ModernaTX, Inc. Method for producing a chimeric polynucleotide encoding a polypeptide having a triazole-containing internucleotide linkage
WO2016014846A1 (en) 2014-07-23 2016-01-28 Moderna Therapeutics, Inc. Modified polynucleotides for the production of intrabodies
WO2016065349A2 (en) 2014-10-24 2016-04-28 University Of Maryland, Baltimore Short non-coding protein regulatory rnas (sprrnas) and methods of use
US10221127B2 (en) 2015-06-29 2019-03-05 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US11168051B2 (en) 2015-06-29 2021-11-09 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2017070613A1 (en) 2015-10-22 2017-04-27 Modernatx, Inc. Human cytomegalovirus vaccine
US11648324B2 (en) 2015-10-28 2023-05-16 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
EP4039699A1 (en) 2015-12-23 2022-08-10 ModernaTX, Inc. Methods of using ox40 ligand encoding polynucleotides
WO2017112943A1 (en) 2015-12-23 2017-06-29 Modernatx, Inc. Methods of using ox40 ligand encoding polynucleotides
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
WO2018232006A1 (en) 2017-06-14 2018-12-20 Modernatx, Inc. Polynucleotides encoding coagulation factor viii
EP4219715A2 (en) 2017-09-08 2023-08-02 MiNA Therapeutics Limited Stabilized cebpa sarna compositions and methods of use
WO2019048645A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Stabilized cebpa sarna compositions and methods of use
WO2019048632A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Stabilized hnf4a sarna compositions and methods of use
EP4233880A2 (en) 2017-09-08 2023-08-30 MiNA Therapeutics Limited Hnf4a sarna compositions and methods of use
EP4183882A1 (en) 2017-09-08 2023-05-24 MiNA Therapeutics Limited Stabilized hnf4a sarna compositions and methods of use
WO2019048631A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Hnf4a sarna compositions and methods of use
WO2019197845A1 (en) 2018-04-12 2019-10-17 Mina Therapeutics Limited Sirt1-sarna compositions and methods of use
EP4242307A2 (en) 2018-04-12 2023-09-13 MiNA Therapeutics Limited Sirt1-sarna compositions and methods of use
WO2019217964A1 (en) 2018-05-11 2019-11-14 Lupagen, Inc. Systems and methods for closed loop, real-time modifications of patient cells
WO2020033791A1 (en) 2018-08-09 2020-02-13 Verseau Therapeutics, Inc. Oligonucleotide compositions for targeting ccr2 and csf1r and uses thereof
WO2020061295A1 (en) 2018-09-19 2020-03-26 Modernatx, Inc. High-purity peg lipids and uses thereof
WO2020061284A1 (en) 2018-09-19 2020-03-26 Modernatx, Inc. Peg lipids and uses thereof
WO2020128031A2 (en) 2018-12-21 2020-06-25 Curevac Ag Rna for malaria vaccines
US11453639B2 (en) 2019-01-11 2022-09-27 Acuitas Therapeutics, Inc. Lipids for lipid nanoparticle delivery of active agents
WO2020161342A1 (en) 2019-02-08 2020-08-13 Curevac Ag Coding rna administered into the suprachoroidal space in the treatment of ophtalmic diseases
WO2020208361A1 (en) 2019-04-12 2020-10-15 Mina Therapeutics Limited Sirt1-sarna compositions and methods of use
WO2020254535A1 (en) 2019-06-18 2020-12-24 Curevac Ag Rotavirus mrna vaccine
WO2021026310A1 (en) 2019-08-06 2021-02-11 L.E.A.F. Holdings Group Llc Processes of preparing polyglutamated antifolates and uses of their compositions
WO2021028439A1 (en) 2019-08-14 2021-02-18 Curevac Ag Rna combinations and compositions with decreased immunostimulatory properties
WO2021061815A1 (en) 2019-09-23 2021-04-01 Omega Therapeutics, Inc. COMPOSITIONS AND METHODS FOR MODULATING HEPATOCYTE NUCLEAR FACTOR 4-ALPHA (HNF4α) GENE EXPRESSION
WO2021061707A1 (en) 2019-09-23 2021-04-01 Omega Therapeutics, Inc. Compositions and methods for modulating apolipoprotein b (apob) gene expression
DE202021003575U1 (en) 2020-02-04 2022-01-17 Curevac Ag Coronavirus Vaccine
WO2021156267A1 (en) 2020-02-04 2021-08-12 Curevac Ag Coronavirus vaccine
EP4147717A1 (en) 2020-02-04 2023-03-15 CureVac SE Coronavirus vaccine
DE202021004130U1 (en) 2020-02-04 2022-10-26 Curevac Ag Coronavirus Vaccine
DE202021004123U1 (en) 2020-02-04 2022-10-26 Curevac Ag Coronavirus Vaccine
DE112021000012T5 (en) 2020-02-04 2021-11-18 Curevac Ag Coronavirus vaccine
WO2021183720A1 (en) 2020-03-11 2021-09-16 Omega Therapeutics, Inc. Compositions and methods for modulating forkhead box p3 (foxp3) gene expression
WO2021195218A1 (en) 2020-03-24 2021-09-30 Generation Bio Co. Non-viral dna vectors and uses thereof for expressing gaucher therapeutics
WO2021195214A1 (en) 2020-03-24 2021-09-30 Generation Bio Co. Non-viral dna vectors and uses thereof for expressing factor ix therapeutics
WO2021239880A1 (en) 2020-05-29 2021-12-02 Curevac Ag Nucleic acid based combination vaccines
US11976019B2 (en) 2020-07-16 2024-05-07 Acuitas Therapeutics, Inc. Cationic lipids for use in lipid nanoparticles
WO2022023284A1 (en) 2020-07-27 2022-02-03 Anjarium Biosciences Ag Compositions of dna molecules, methods of making therefor, and methods of use thereof
WO2022023559A1 (en) 2020-07-31 2022-02-03 Curevac Ag Nucleic acid encoded antibody mixtures
WO2022032154A2 (en) 2020-08-06 2022-02-10 Modernatx, Inc. Compositions for the delivery of payload molecules to airway epithelium
WO2022043551A2 (en) 2020-08-31 2022-03-03 Curevac Ag Multivalent nucleic acid based coronavirus vaccines
WO2022122872A1 (en) 2020-12-09 2022-06-16 Ucl Business Ltd Therapeutics for the treatment of neurodegenerative disorders
WO2022135993A2 (en) 2020-12-22 2022-06-30 Curevac Ag Pharmaceutical composition comprising lipid-based carriers encapsulating rna for multidose administration
WO2022137133A1 (en) 2020-12-22 2022-06-30 Curevac Ag Rna vaccine against sars-cov-2 variants
WO2022162027A2 (en) 2021-01-27 2022-08-04 Curevac Ag Method of reducing the immunostimulatory properties of in vitro transcribed rna
WO2022200575A1 (en) 2021-03-26 2022-09-29 Glaxosmithkline Biologicals Sa Immunogenic compositions
WO2022200810A1 (en) 2021-03-26 2022-09-29 Mina Therapeutics Limited Tmem173 sarna compositions and methods of use
WO2022207862A2 (en) 2021-03-31 2022-10-06 Curevac Ag Syringes containing pharmaceutical compositions comprising rna
WO2022223556A1 (en) 2021-04-20 2022-10-27 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
WO2022232289A1 (en) 2021-04-27 2022-11-03 Generation Bio Co. 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
WO2022261394A1 (en) 2021-06-11 2022-12-15 LifeEDIT Therapeutics, Inc. Rna polymerase iii promoters and methods of use
WO2023283359A2 (en) 2021-07-07 2023-01-12 Omega Therapeutics, Inc. Compositions and methods for modulating secreted frizzled receptor protein 1 (sfrp1) gene expression
WO2023014974A1 (en) 2021-08-06 2023-02-09 University Of Iowa Research Foundation Double stranded mrna vaccines
WO2023031392A2 (en) 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids comprising phosphatidylserine
WO2023031394A1 (en) 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids
WO2023073228A1 (en) 2021-10-29 2023-05-04 CureVac SE Improved circular rna for expressing therapeutic proteins
WO2023081526A1 (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
WO2023099884A1 (en) 2021-12-01 2023-06-08 Mina Therapeutics Limited Pax6 sarna compositions and methods of use
WO2023104964A1 (en) 2021-12-09 2023-06-15 Ucl Business Ltd Therapeutics for the treatment of neurodegenerative disorders
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
WO2023144330A1 (en) 2022-01-28 2023-08-03 CureVac SE Nucleic acid encoded transcription factor inhibitors
WO2023154818A1 (en) 2022-02-09 2023-08-17 Modernatx, Inc. Mucosal administration methods and formulations
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
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
WO2024033901A1 (en) 2022-08-12 2024-02-15 LifeEDIT Therapeutics, Inc. Rna-guided nucleases and active fragments and variants thereof and methods of use
WO2024040222A1 (en) 2022-08-19 2024-02-22 Generation Bio Co. Cleavable closed-ended dna (cedna) and methods of use thereof
EP4342460A1 (en) 2022-09-21 2024-03-27 NovoArc GmbH Lipid nanoparticle with nucleic acid cargo
WO2024062001A1 (en) 2022-09-21 2024-03-28 Novoarc Gmbh Lipid nanoparticle with nucleic acid cargo
WO2024068545A1 (en) 2022-09-26 2024-04-04 Glaxosmithkline Biologicals Sa Influenza virus vaccines
DE202023106198U1 (en) 2022-10-28 2024-03-21 CureVac SE Nucleic acid-based vaccine
WO2024102677A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Circular rna compositions
WO2024102730A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Lipids and nanoparticle compositions for delivering polynucleotides
WO2024102762A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Lipids and lipid nanoparticle compositions for delivering polynucleotides
WO2024119039A2 (en) 2022-12-01 2024-06-06 Generation Bio Co. Stealth lipid nanoparticles and uses thereof
WO2024119051A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Novel polyglycerol-conjugated lipids and lipid nanoparticle compositions comprising the same
WO2024119074A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Stealth lipid nanoparticle compositions for cell targeting
WO2024119103A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Lipid nanoparticles comprising nucleic acids and lipid-anchored polymers
US12129223B2 (en) 2022-12-15 2024-10-29 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
WO2024134199A1 (en) 2022-12-22 2024-06-27 Mina Therapeutics Limited Chemically modified sarna compositions and methods of use
WO2024160936A1 (en) 2023-02-03 2024-08-08 Glaxosmithkline Biologicals Sa Rna formulation
WO2024171052A1 (en) 2023-02-14 2024-08-22 Glaxosmithkline Biologicals Sa Analytical method
WO2024184500A1 (en) 2023-03-08 2024-09-12 CureVac SE Novel lipid nanoparticle formulations for delivery of nucleic acids
WO2024205657A2 (en) 2023-03-29 2024-10-03 Orna Therapeutics, Inc. Lipids and lipid nanoparticle compositions for delivering polynucleotides
GB202404607D0 (en) 2024-03-29 2024-05-15 Glaxosmithkline Biologicals Sa RNA formulation

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US20130090372A1 (en) 2013-04-11
JP5957646B2 (en) 2016-07-27
EP2575767A1 (en) 2013-04-10
US20150315112A1 (en) 2015-11-05
JP2013533223A (en) 2013-08-22
US8748667B2 (en) 2014-06-10
US20170050917A1 (en) 2017-02-23
US9643916B2 (en) 2017-05-09
US20140235872A1 (en) 2014-08-21
EP2575767B1 (en) 2017-01-04
US9315437B2 (en) 2016-04-19
US9062021B2 (en) 2015-06-23

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