US20120136073A1 - Amine-Containing Transfection Reagents and methods for making and using same - Google Patents

Amine-Containing Transfection Reagents and methods for making and using same Download PDF

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US20120136073A1
US20120136073A1 US13/297,231 US201113297231A US2012136073A1 US 20120136073 A1 US20120136073 A1 US 20120136073A1 US 201113297231 A US201113297231 A US 201113297231A US 2012136073 A1 US2012136073 A1 US 2012136073A1
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transfection
substituted
unsubstituted
pharmaceutically acceptable
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Zhiwei Yang
Parul Angrish
Xavier de Mollerat du Jeu
Kristin Wiederholt
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Life Technologies Corp
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Life Technologies Corp
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Assigned to Life Technologies Corporation reassignment Life Technologies Corporation ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DE MOLLERAT DU JEU, XAVIER, WEIDERHOLT, KRISTEN, ANGRISH, PARUL, YANG, ZHIWEI
Publication of US20120136073A1 publication Critical patent/US20120136073A1/en
Assigned to Life Technologies Corporation reassignment Life Technologies Corporation ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WU, TAO
Priority to US14/340,395 priority patent/US9901642B2/en
Priority to US15/844,380 priority patent/US10406237B2/en
Priority to US16/557,967 priority patent/US11464863B2/en
Priority to US17/821,122 priority patent/US20230181742A1/en
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    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/04—Formation of amino groups in compounds containing carboxyl groups
    • C07C227/06—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/08—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to hydrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D211/26—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by nitrogen atoms
    • C07D211/28—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by nitrogen atoms to which a second hetero atom is attached
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/56—Nitrogen atoms
    • C07D211/58—Nitrogen atoms attached in position 4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • This invention relates generally to the field of transfection reagents for the in vitro and in vivo delivery of biologically active agents. More specifically, this invention relates to biodegradable and biocompatible lipids, and transfection complexes made using same, that may be used to introduce nucleic acids or other biologically active agents into cells in vitro or in vivo.
  • Gene therapy such as the treatment of diseases through the application of nucleotide based drugs has become an important medical field.
  • modified viruses as gene transfer vectors have been used in recent years.
  • concerns over possible undesirable side effects, such as unsolicited immune responses, when viral vectors are used have resulted in efforts to develop non-viral alternatives (e.g., polymeric delivery systems, liposomal formulations and “naked” DNA injections). While these alternative approaches have not yet achieved the clinical effectiveness of viral vectors, the potential safety, processing, and economic benefits offered by these methods are promising.
  • lipids are needed that is easily and economically efficiently prepared to be used to transfect nucleic acids.
  • Such lipids would have several uses, including the delivery of nucleic acids in gene therapy as well as in the packaging and/or delivery of diagnostic, therapeutic, and prophylactic agents.
  • the instant specification describes such new transfection reagents and methods for synthesizing thereof.
  • the present invention is directed towards amine-containing transfection reagents and methods for synthesizing the same. Additional embodiments of the present invention relate to the use of the amine-containing transfection reagents to make transfection complexes suitable for use in the intracellular delivery of one or more biologically active agents to a cell in vitro or a tissue in a human or an animal in vivo.
  • amine-containing transfection compounds having the general structure I, or pharmaceutically acceptable salts or derivatives thereof are provided:
  • each of X 1 and X 2 is a moiety independently selected from the group consisting of O, S, N-A and C-A, wherein A is selected from the group consisting of hydrogen and a C 1 -C 20 hydrocarbon chain; each of Y and Z is a moiety independently selected from the group consisting of CH—OH, C ⁇ O, C ⁇ S, S ⁇ O and SO 2 ; each of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 is a moiety independently selected from the group consisting of hydrogen, a cyclic or an acyclic, substituted or unsubstituted, branched or unbranched aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic group, a substituted or unsubstituted, branched or unbranched acyl group, a substituted or unsubstituted, branched or unbranched acy
  • each of g, e and f is an integer independently having the value between 1 and 6, inclusively, “HCC” symbolizes a hydrocarbon chain, and each * indicates the nitrogen atom in structure I.
  • amine-containing transfection compounds having the general structure II or pharmaceutically acceptable salts thereof:
  • R 2 is H.
  • amine-containing transfection compounds having the structure I, or pharmaceutically acceptable salts thereof, the method comprising reacting one or more equivalents of an unsaturated component comprising at least two compounds selected from the group consisting of the first intermediate having the structure R 1 —X 1 —Y—(CR 4 R 5 ) n —Br and the second intermediate having the structure R 2 —X 2 —Z—(CR 6 R 7 ) m —Br, wherein in (CR 4 R 5 ) n and (CR 6 R 2 ) m portions of the structures, each R 4 is the same or different, each R 5 is the same or different, each R 6 is the same or different, and each R 7 is the same or different, wherein the first and the second intermediates are the same or different, with one equivalent of an amino component comprising a primary amine NH 2 —R 3 , a diamine, a polyamine or a combination thereof.
  • the amine-containing transfection compounds of the present invention may exist in neutral form or as a cation.
  • a pH at or near physiologically neutral e.g. pH from about 5 to about 8
  • the predominant form of an amine-containing transfection compound according to the presently described embodiments is a cation.
  • a pH at or near physiologically neutral e.g., pH from about 5 to about 8
  • the predominant form of the amine-containing transfection compound according to the presently described embodiments is neutral.
  • transfection complexes suitable for the delivery of one or more biologically active agents to a cell or a tissue in vitro or in vivo are provided for herein.
  • the transfection complexes may include one or more of the amine-containing transfection compounds described herein.
  • the transfection complexes may optionally be made in combination with one or more helper lipids, optionally in combination with one or more pegylated lipids, optionally in combination with one or more cationic lipids, and optionally in combination with one or more targeting moieties.
  • transfection may be made with peptide or non-peptide transfection enhancers, fusogenic peptide or non-peptide agents, peptide or non-peptide endosomal release agents, or nuclear targeting agents (such as, e.g., a peptide containing one or more nuclear localization sequences, such as will be readily apparent to one skilled in the art without undue experimentation.
  • peptide or non-peptide transfection enhancers such as, e.g., a peptide containing one or more nuclear localization sequences, such as will be readily apparent to one skilled in the art without undue experimentation.
  • Helper lipids suitable for use in the preparation and formation of transfection complexes disclosed herein may include, though are not limited to a cholesterol, a cholesterol derivative, one or more sterols, including phytosterols, zoosterols and hopanoids, or any of the neutral or cationic lipids that are known to allow or to facilitate the introduction of exogenous bioactive molecules to the interior of a cell or of a tissue.
  • more than one helper lipid may be used in the formulation of the transfection complexes described herein.
  • Illustrative though non-limiting neutral or cationic lipids suitable for use as helper lipids in accordance with the embodiments set forth herein may include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides or derivatives thereof.
  • Straight-chain and branched alkyl and alkene groups of cationic lipids can contain from 1 to about 25 carbon atoms. In some embodiments, straight-chain or branched alkyl or alkene groups have six or more carbon atoms. In some embodiments, straight-chain or branched alkyl or alkene groups have eight to about twenty carbon atoms.
  • Alicyclic groups can contain from about 6 to 30 carbon atoms, or, in some embodiments eight to twenty carbon atoms. In some embodiments, the alicyclic groups include cholesterol and other steroid groups. Cationic lipids can be prepared with a variety of counter ions (anions) including among others: Cl-, Br-, I-, F-, acetate, trifluoroacetate, sulfate, nitrite, triflate, and nitrate
  • Exemplary though non-limiting neutral or cationic lipids contemplated for use in the preparation of the presently disclosed transfection complexes may include one or lipids selected from the following: BMOP (N-(2-bromoethyl)-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-propana minimun bromide), DDPES (Dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide), DSPC, CTAB:DOPE (formulation of cetyltrimethylammonium bromide (CATB) and DOPE), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPE (dioleoylphosphatidylethanolamine), DMG, DMAP (4-dimethylaminopyridine), DMPE (Dimyristoylphospatidylethanolamine), DOMG, DMA, DOPC (Dioleoy
  • Neutral lipids also include cholesterol and other 3 ⁇ OH-sterols as well as derivatives thereof phosphatidyl choline or commercially available cationic lipid mixtures such as, for example, LIPOFECTIN® CELLFECTIN® (1:1.5 (M/M) formulation of N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmitylspermine (TMTPS) and dioleoyl phosphatidylethanolamine (DOPE), LIPOFECTACE®, GS 2888 CYTOFECTIN®, FUGENE 6®, EFFECTENE®, and LIPOFECTAMINE®, LIPOFECTAMINE 2000®, LIPOFECTAMINE PLUS®, LIPOTAXI®, POLYECT®, SUPERFECT®, TFXNTM, TRANSFASTTM, TRANSFECTAM®, TRANSMESSENGER®, vectamidine (3-tetradecylamino-N-tert-butyl-N
  • Pegylated lipids suitable for use in the preparation and formation of transfection complexes disclosed herein can be any lipid or mixture of lipids that are compatible with the formation of transfection complexes described herein, and with the administration thereof to an animal or to a human in vivo, or to tissues or cells in vitro.
  • the pegylated lipids used in the present invention include a PEG polymer having a molecular weight between about 250 daltons and about 12,000, or in some embodiments, about 350 daltons and about 6,000 daltons, or, in some embodiments, between about 500 daltons and about 1,000 daltons, or, in some embodiments, between about 1,000 daltons and about 2,000 daltons, or, in some embodiments, between about 2,000 daltons and 5,000 daltons.
  • the transfection complexes may include one or more biologically active agents to be delivered to a cell or to a target tissue in vitro or in vivo.
  • suitable biologically active agents may include any molecule that is capable of forming a transfection complex with the presently described amine-containing transfection reagents and that elicits a biological response when delivered to the interior of a cell or cells or to a tissue in vivo or in vitro.
  • Biologically active agents contemplated for use in the presently described embodiments may be cationic, neutral or anionic agents.
  • exemplary biologically active agents suitable for formulation in a transfection complex may include, though are not limited to; nucleic acids (including but not limited to single or double stranded linear or circular DNA molecules including cDNA molecules, single or double stranded RNA molecules, small interfereing RNA (siRNA) molecules, small hairpin RNA (shRNA) molecules, microRNA (miRNA) molecules, oligonucleotides, anti-sense oligonucleotides, sense oligonucleotides), polypeptides, antibodies, oligopeptides, therapeutic peptides or protein molecules, peptide nucleic acids (PNAs), cationic, anionic or neutral organic molecules or drugs, in addition to pharmaceutically acceptable salts thereof.
  • nucleic acids including but not limited to single or double stranded linear or circular DNA molecules including cDNA molecules, single or double stranded RNA molecules, small interfereing RNA (siRNA) molecules, small hairpin RNA (shRNA) molecules, microRNA (m
  • transfection complexes and methods that use the compounds of the present invention to deliver nucleic acid molecules into cells or tissues in vitro or in vivo, including the delivery of RNA interference molecules (RNAi) or small interfering RNA molecules (siRNA, shRNA or miRNA) into cells for inhibition of gene expression.
  • RNAi RNA interference molecules
  • shRNA small interfering RNA molecules
  • transfection complexes and methods that use the compounds of the present invention to deliver mRNA molecules into a cell or a tissue in vivo or in vitro to promote the expression of a specific protein or proteins are also provided.
  • transfection complexes and methods that use the compounds of the present invention to deliver DNA molecules (including cDNA molecules) into a cell or a tissue in vivo or in vitro to promote the expression of a specific protein or proteins or to synthesize specific RNA molecules, including but not limited to mRNA molecules or RNAi or miRNA or shRNA molecules are also provided.
  • the transfection complexes described herein may optionally include one or more fusogenic or cell-penetrating peptides.
  • a fusogenic or cell-penetrating peptide is any peptide molecule that is capable of promoting the fusion of a lipid-containing complex to a cell membrane (either a plasma membrane or an endosomal membrane).
  • a variety of fusogenic or cell-penetrating peptides are known in the art and it is well within the skill level of a practitioner to identify suitable fusogenic or cell-penetrating peptides and condition for the use thereof in the present invention without undue experimentation.
  • the transfection complexes described herein may optionally include one or more transfection helpers or targeting moieties.
  • a targeting moiety may be a peptide, a modified peptide, an antibody, a modified antibody, a receptor molecule, a modified receptor molecule, a single or a double stranded nucleic acid molecule, a modified single or double stranded nucleic acid molecule, a peptide or nucleic acid aptamer, a modified peptide or nucleic acid aptamer, an organic molecule, a polysaccharide, or any other molecule that is capable of targeting a transfection complex to specific tissue or cell type for targeted delivery of a biologically agent thereto, such as will be readily apparent to have having ordinary skill level in the art.
  • modification of a peptide, an antibody, a nucleic acid, an aptamer, and the like may include conjugating the peptide, antibody, nucleic acid, aptamer, and the like to a PEG moiety.
  • modification of a peptide, an antibody, a nucleic acid, an aptamer, and the like may include conjugating the peptide, antibody, nucleic acid, aptamer, and the like to a PEG-lipid moiety
  • targeting moieties are widely known to those skilled in the art, and all are contemplated for use with the presently described embodiments, without limitation.
  • the transfection complexes provided for herein may be stable for up to 1 year and may either be contacted with the cells or tissues to be transfected, or be administered to an animal or to a human immediately or shortly after being formed, or optionally may stored for a period of time prior to being contacted with the cells or tissues, or being administered to an animal or a human.
  • the transfection complexes are stable and may be stored for a time period of at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 5 days, at least 7 days, at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months or at least 1 year at room temperature, or at a temperature greater than freezing, up to about room temperature.
  • the formulation described herein may include one or more stabilizing agents, preservatives, buffers, etc, that aid in the long-term stabilization and storage of bioactive formulation, such as will be readily understood by the skilled practitioner of the biological and pharmaceutical arts, and without requiring undue experimentation to achieve. It is also understood, that the storage period can be between any of these time periods, for example between 31 minutes and 1 hour or between 1 hour and 24 hours.
  • methods for the preparation of functional transfection complexes generally include forming a lipid-aggregate by encapsulating a biologically active agent in a composition containing one or more of the amine-containing transfection compounds described herein, optionally in combination with one or more helper lipids, stabilizing lipids, transfection helpers, pegylated lipids or targeting moieties.
  • Such methods may include a1) mixing one or more amine-containing transfection compounds, at least one helper lipid, optionally more than one helper lipid and one or more pegylated lipids, or a salt thereof, in an alcohol/aqueous solution wherein the alcohol concentration is ⁇ 50%; a2) mixing one or more amine-containing transfection compounds, at least one helper, optionally more than one helper lipid and one or more pegylated lipids, or a salt thereof, in a molar percentage such that the one or more amine-containing transfection compounds are present at 15%-50%; a3) mixing one or more amine-containing transfection compounds, at least one helper lipid, optionally more tha one helper lipid and one or more pegylated lipids, or a salt thereof, in a molar percentage such that the Pegylated lipids are present at ⁇ 50%; and a4) mixing one or more amine-containing transfection compounds, at least one helper lipid, optional
  • the method includes a1) and a2), a2) and a3), a1) and a3), a2) and a4), a3) and a4), a1) and a4), or a1)-a)4, for example.
  • the alcohol is a C1-C4 alcohol.
  • the alcohol is ethanol.
  • the alcohol is a pharmaceutically acceptable alcohol such as an alcohol that is liquid at about room temperature, for example, ethanol, propylene glycol, 2-(2-ethoxyethoxy)ethanol (TranscutolTM), benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 or a mixture thereof.
  • the alcohol for mixing is different than the alcohol for complexing.
  • Such methods may include preparing a plurality of transfection complexes containing a compound that readily facilitates the detection of a marker in combination with a test transfection compound, delivering each of the plurality of transfection complexes to a test animal, and detecting the marker.
  • a method for screening tissue-biased delivery of a transfection complex may include preparing a plurality of transfection complexes, each transfection complex having at least one test transfection compound in combination with at least one nucleic acid that facilitates detection of delivery to a tissue.
  • the nucleic acid may be an RNA molecule or a DNA molecule that encodes a protein that can be directly detected (such as, e.g., Green Fluorescent Protein (GFP), red Fluorescent Protein, Luciferase, or the like), or encode a protein that effects expression of a protein that can be directly detected.
  • GFP Green Fluorescent Protein
  • Luciferase or the like
  • a method for screening tissue-biased delivery of a transfection complex may include preparing a plurality of unique transfection complexes, each transfection complex having at least one test transfection compound in combination with an mRNA or a cDNA that encodes Green Fluorescent Protein.
  • Each unique transfection complex may be delivered either intravenously, subcutaneously, or to a tissue to a test animal, such as a mouse. After a predetermined amount of time, tissues from the mouse may be harvested and the expression of GFP in various tissues may be detected by gross examination, histological examination or by molecular detection (PCR, Western blotting, or the like) to determine which to tissue or tissues transfection complexes containing specific transfection compounds are delivered to.
  • a method for screening tissue-biased delivery of a transfection complex may include preparing a plurality of unique transfection complexes, each transfection complex having at least one test transfection compound in combination with an mRNA or a cDNA that encodes Luciferase.
  • Each unique transfection complex may be delivered either intravenously, subcutaneously, or to a tissue to a test animal, such as a mouse. After a predetermined amount of time, tissues from the mouse may be harvested and the expression of Luciferase in various tissues may be detected by gross examination, histological examination or by molecular detection (PCR, Western blotting, or the like), or imaged in-vivo using the IVIS® Imaging System (Caliper).
  • a method for screening tissue-biased delivery of a transfection complex may include preparing a plurality of unique transfection complexes, each transfection complex having at least one test transfection compound in combination with an mRNA or a cDNA that encodes a specific transcription factor.
  • Each unique transfection complex may be delivered either intravenously, subcutaneously, or to a tissue to a transgenic animal that expresses a reporter gene (such as, e.g., luciferase) under the control of the specific transcription factor.
  • a reporter gene such as, e.g., luciferase
  • tissues from the transgenic animal may be harvested and the expression of reporter gene in various tissues may be detected by gross examination, histological examination or by molecular detection (PCR, Western blotting, or the like). If the reporter gene is luciferase, detection may be accomplished in-vivo using the IVIS® Imaging System (Caliper).
  • FIG. 1 shows a graph depicting some properties of a lipid composition prepared using compounds according to some embodiments of the present invention
  • FIG. 2 shows a graph depicting some properties of a lipid composition prepared using compounds according to other embodiments of the present invention
  • FIG. 3 shows a graph depicting some properties of a lipid composition prepared using compounds according to other embodiments of the present invention.
  • FIG. 4 shows a graph depicting some properties of a lipid composition prepared using compounds according to other embodiments of the present invention.
  • FIG. 5 shows a graph depicting some properties of a lipid composition prepared using compounds according to other embodiments of the present invention.
  • FIGS. 6 A and 6 B shows whole animal and whole tissue mount images depicting some properties of a lipid composition prepared using compounds according to other embodiments of the present invention.
  • FIG. 7A-7F show graphs depicting some properties of a lipid composition prepared using compounds according to other embodiments of the present invention.
  • Compounds of the present invention may exist in particular geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis- and toms-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention.
  • Additional asymmetric carbon atoms is present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.
  • Isomeric mixtures containing any of a variety of isomer ratios is utilized in accordance with the present invention. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios are all contemplated by the present invention. Those of ordinary skill in the art will readily appreciate that analogous ratios are contemplated for more complex isomer mixtures.
  • a particular enantiomer of a compound of the present invention is prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers.
  • the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
  • protecting group refers to a group that temporarily blocks a particular functional moiety, e.g., O, S, or N, is so that a reaction is carried out selectively at another reactive site in a multifunctional compound.
  • a protecting group reacts selectively in good yield to give a protected substrate that is stable to the projected reactions, and the protecting group is selectively removable in good yield by readily available reagents that do not attack the other functional groups; the protecting group forms an easily separable derivative; and the protecting group has a minimum of additional functionality to avoid further sites of reaction.
  • Non-limiting examples of exemplary hydroxyl protecting groups include methyl, methoxy]methyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl,
  • non-limiting examples of exemplary protecting groups include methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1-methoxyethylid
  • Non-limiting examples of exemplary amino-protecting groups include methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-f-butyl-[9-(10,10-dioxo-10, 10, 10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,
  • substituted refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure is substituted with more than one substituent selected from a specified group, the substituent is either the same or different at every position.
  • substituted is inclusive of all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Heteroatoms may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. Furthermore, this invention is not intended to be limited in any manner by the permissible substituents of organic compounds. Combinations of substituents and variables are those that result in the formation of stable compounds.
  • stable refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be detected and preferably for a sufficient period of time to be useful for the purposes detailed herein.
  • aliphatic includes both saturated and unsaturated, straight chain (i.e., unbranched), branched, acyclic, cyclic, or polycyclic aliphatic hydrocarbons, which are optionally substituted with one or more functional groups.
  • aliphatic is inclusive of, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties.
  • alkyl includes straight, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as “alkenyl” or “alkynyl.”
  • alkyl encompass both substituted and unsubstituted groups.
  • the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-20 aliphatic carbon atoms. “Lower alkyl” is used to indicate those alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1-6 carbon atoms.
  • Exemplary aliphatic groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, —CH 2 -cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, —CH 2 -cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, —CH 2 -cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, and —CH 2 -cyclohexyl moieties which isar one or more substituents.
  • Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl and 1-methyl-2-buten-1-yl.
  • Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl) and 1-propynyl.
  • alkyl refers to saturated, straight- or branched-chain hydrocarbon radicals derived from a hydrocarbon moiety containing between one and twenty carbon atoms by removal of a single hydrogen atom.
  • alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl.
  • alkenyl refers to a monovalent group derived from a hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom.
  • alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl and 1-methyl-2-buten-1-yl.
  • alkynyl refers to a monovalent group derived from a hydrocarbon having at least one carbon-carbon triple bond by the removal of a single hydrogen atom.
  • exemplary alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl) and 1-propynyl, and the like.
  • alkoxy and thioalkyl refer to an alkyl group, as previously defined, attached to the parent molecule through an oxygen atom or through a sulfur atom, respectively.
  • the alkyl, alkenyl, and alkynyl groups contain 1-20 alipahtic carbon atoms.
  • Exemplary alkoxy groups include but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy and n-hexoxy.
  • Exemplary thioalkyl groups include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio and n-butylthio.
  • alkylamino refers to a group having the structure —NHR′, wherein R′ is aliphatic, as defined above, containing 1-20 aliphatic carbon atoms.
  • exemplary alkylamino groups include, but are not limited to, methylamino, ethylamino, n-propylamino, iso-propylamino, cyclopropylamino, n-butylamino, tert-butylamino, neopentylamino, n-pentylamino, hexylamino and cyclohexylamino
  • dialkylamino refers to a group having the structure —NRR 1 , wherein R and R 1 are each an aliphatic group, as defined herein, containing 1-20 aliphatic carbon atoms. R and R 1 is the same or different or is linked to form an aromatic or non-aromatic cyclic structure.
  • Exemplary dialkylamino groups include, but are not limited to, dimethylamino, methyl ethylamino, diethylamino, methylpropylamino, di(n-propyl)amino, di(iso-propyl)amino, di(cyclopropyl)amino, di(n-butyl)amino, di(tert-butyl)amino, di(neopentyl)amino-5 di(n-pentyl)amino, di(hexyl)amino and di(cyclohexyl)amino
  • Exemplary cyclic diaminoalkyl groups include, but are not limited to, aziridinyl, pyrrolidinyl, piperidinyl, morpholinyl, pyrrolyl, imidazolyl, 1,3,4-trianolyl and tetrazolyl.
  • carboxylic acid refers to a compound comprising a group of formula —COOH.
  • substituents of the above-described aliphatic and other moieties of compounds of the invention include, but are not limited to to aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxy, aryloxy, heteroalkoxy, heteroaryloxy, alkylthio, arylthio, heteroalkylthio, heteroarylthio, —F, —Cl, —Br, —I, —OH, —NO 2 , —CN, —CF 3 , —CH 2 CF 3 , —CHCl 2 , —CH 2 OH, —CH 2 CH 2 OH, —CH 2 NH 2 , —CH 2 SO 2 CH 3 , —C(O)R x , —CO 2 (R x ), —CON(R X ) 2 , —OC(O)R x , —OCO 2 R x , —CO
  • aryl and heteroaryl refer to stable mono- or polycyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated moieties having 3-14 carbon atoms, each of which is substituted or unsubstituted.
  • Substituents include, but are not limited to, any of the substituents recited above for aliphatic moieties.
  • aryl is inclusive of mono- or bicyclic carbocyclic ring systems having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl and
  • heteroaryl is inclusive of cyclic aromatic radicals having from five to ten ring atoms, of which 1-3 ring atoms is selected from S, O, and N, and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl or isoquinolinyl.
  • Aryl and heteroaryl groups is unsubstituted or substituted, wherein substitution includes replacement of one, two, three, or more of the hydrogen atoms thereon independently with any one or more of the following moieties including, but not limited to aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxy, aryloxy, heteroalkoxy, heteroaryloxy, alkylthio, arylthio, heteroalkylthio, heteroarylthio, —F, —Cl, —Br, —I, —OH, —NO 2 , —CN, —CF 3 , —CH 2 CF 3 , —CHCl 2 , —CH 2 OH, —CH 2 CH 2 OH, —CH 2 NH 2 , —CH 2 SO 2 CH 3 , —C(O)R x , —CO 2 (R x ), —CON(R X )
  • cycloalkyl refers specifically to groups having three to seven, preferably three to ten carbon atoms. Suitable cycloalkyls include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, which may optionally be substituted with substituents including, but not limited to aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxy, aryloxy, heteroalkoxy, heteroaryloxy, alkylthio, arylthio, heteroalkylthio, heteroarylthio, —F, —Cl, —Br, —I, —OH, —NO 2 , —CN, —CF 3 , —CH 2 CF 3 , —CHCl 2 , —CH 2 OH, —CH 2 CH 2 OH, —CH
  • heteroaliphatic refers to aliphatic moieties that contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moieties is branched, unbranched, cyclic or acyclic and include saturated and unsaturated heterocycles such as morpholino, pyrrolidinyl, etc.
  • Heteroaliphatic moieties are substituted by independent replacement of one or more of the hydrogen atoms thereon with one or more moieties including, but not limited to aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxy, aryloxy, heteroalkoxy, heteroaryloxy, alkylthio, arylthio, heteroalkylthio, heteroarylthio, —F, —Cl, —Br, —I, —OH, —NO 2 , —CN, —CF 3 , —CH 2 CF 3 , —CHCl 2 , —CH 2 OH, —CH 2 CH 2 OH, —CH 2 NH 2 , —CH 2 SO 2 CH 3 , —C(O)R x , —CO 2 (R x ), —CON(R x ) 2 , —OC(O)R x , —OCO
  • halo and “halogen,” as used herein refer to an atom selected from fluorine, chlorine, bromine, and iodine.
  • haloalkyl denotes an alkyl group, as defined above, having one, two, or three halogen atoms attached thereto and is exemplified by such groups as chloromethyl, bromoethyl and trifluoromethyl.
  • heterocycloalkyl refers to a non-aromatic 5-, 6-, or 7-membered ring or a polycyclic group, including, but not limited to a bi- or tri-cyclic group comprising fused six-membered rings having between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein (i) each 5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms is optionally be oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings is fused to a benzene ring.
  • heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl and tetrahydrofuryl.
  • a “substituted” heterocycloalkyl or heterocycle group refers to a heterocycloalkyl or heterocycle group, as defined above, substituted by the independent replacement of one, two or three of the hydrogen atoms thereon with but are not to aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxy, aryloxy, heteroalkoxy, heteroaryloxy, alkylthio, arylthio, heteroalkylthio, heteroarylthio, —F, —Cl, —Br, —I, —OH, —NO 2 , —CN, —CF 3 , —CH 2 CF 3 , —CHCl 2 , —CH 2 OH, —CH 2 CH 2 OH, —CH 2 NH 2 , —CH 2 SO 2 CH 3 , —C(O)R x , —CO 2
  • Exemplary non-limiting heterocyclic and aromatic heterocyclic groups that is included in the compounds of the invention include 3-methyl-4-(3-methylphenyl)piperazine, 3 methylpiperidine, 4-(bis-(4-fluorophenyl)methyl)piperazine, 4-(diphenylmethyl)piperazine, 4-(ethoxycarbonyl)piperazine, 4-(ethoxycarbonylmethyl)piperazine, A-(phenylmethyl)piperazine, 4-(1-phenylethyl)piperazine, 4-(1,1-dimethylethoxycarbonyl)piperazine, 4-(2-(bis-(2-propenyl)amino)ethyl)piperazine, A-(2-(diethylamino)ethyl)piperazine, 4-(2-chlorophenyl)piperazine, 4-(2-cyanophenyl)piperazine, 4-(2-ethoxyphenyl)piperazine, 4-(2-ethylphenyl)piperazine
  • carrier refers to an aromatic or non-aromatic ring in which each atom of the ring is a carbon atom.
  • labeled is intended to mean that a compound has at least one element, isotope, or chemical compound attached to enable the detection of the compound by using a radioactive or heavy isotope label, or an immune label such as an antibody or antigen or a label derived from a colored, luminescent, phosphorescent, or fluorescent dye.
  • Photoaffinity labeling employing, for example, o-, m- and p-azidobenzoyls, substituted with one or more halogen moieties, including, but not limited to 4-azido-2,3,5,6-tetrafluorobenzoic acid, is utilized for the direct elucidation of intermolecular interactions in biological systems.
  • animal may refer to humans as well as non-human animals, including, for example, mammals (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig), birds, reptiles, amphibians, and fish.
  • mammals e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig
  • birds reptiles, amphibians, and fish.
  • cell generally refers to eukaryotic cells of any type and from any source.
  • Types of eukaryotic cells include epithelial, fibroblastic, neuronal, hematopoietic cells and the like from primary cells, tumor cells or immortalized cell lines.
  • Sources of such cells include any animal such as human, canine, mouse, hamster, cat, bovine, porcine, monkey, ape, sheep, fish, insect, fungus, and any plant including crop plants, algae, ornamentals and trees.
  • Delivery is used to denote a process by which a desired compound is transferred to a target cell such that the desired compound is ultimately located inside the target cell or in, or on, the target cell membrane.
  • the desired compound is not readily taken up by the target cell and delivery via lipid aggregates or transfection complexes a means for delivering the desired compound to the appropriate cellular compartment within a cell.
  • delivery to a specific target cell type is preferable and can be facilitated by compounds of the invention.
  • Drug refers to any therapeutic or prophylactic agent other than food which is used in the prevention, diagnosis, alleviation, treatment, or cure of disease in man or animal.
  • Kit refers to transfection or protein expression kits which include one or more of the compounds of the present invention or mixtures thereof. Such kits may comprise a carrying means being compartmentalized to receive in close confinement one or more container means such as vials, test tubes and the like. Each of such container means comprises components or a mixture of components needed to perform transfection. Such kits may include one or more components selected from nucleic acids (preferably one or more vectors), cells, one or more compounds of the present invention, lipid-aggregate forming compounds, transfection enhancers, biologically active substances, etc.
  • association with refers to two entities linked by a direct or indirect covalent or non-covalent interaction, such as hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, etc.
  • biocompatible refers to compounds that are not toxic to cells. Compounds are biocompatible if their addition to cells in vitro results in less than or equal to 20% cell death, and their administration in vivo does not induce inflammation or other such adverse effects.
  • biodegradable refers to compounds that, when introduced into cells, are broken down into components that the cells can either reuse or dispose of without significant toxic effect on the cells (i.e., fewer than about 20% of the cells are killed when the components are added to cells in vitro). The components do not induce inflammation or other adverse effects in vivo. The chemical reactions relied upon to break down the biodegradable compounds are typically uncatalyzed.
  • effective amount refers to the amount necessary to elicit the desired biological response. The effective amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the encapsulating matrix, the target tissue, etc.
  • an “effective amount of a molecule” is the delivery of the molecule into a cell in sufficient amounts so that the molecule elicits a biological response, for example, modulating the expression of one or more genes in the cell.
  • an effective amount of a molecule is delivered to a cell such that an amelioration or improvement in a disease, condition, or disorder related to the cell can be obtained.
  • Delivery of an “effective amount of siRNA” or an “effective amount or RNAi” is the delivery of siRNA or other RNAi into a cell in sufficient amounts to cause a reduction in expression of the target gene in the cell.
  • biologically active agent generally refers to a composition, complex, compound or molecule which has a biological effect or that modifies, causes, promotes, enhances, blocks or reduces a biological effect, or that enhances or limits the production or activity of, reacts with and/or binds to a second molecules which has a biological effect.
  • the second molecule can, but need not be, an endogenous molecule (e.g., a molecule, such as a protein or nucleic acid, normally present in the target cell).
  • a biological effect may be, but is not limited to, one that stimulates or causes an immunoreactive response; one that impacts a biological process in a cell, tissue or organism (e.g., in an animal); one that imparts a biological process in a pathogen or parasite; one that generated or causes to be generated a detectable signal; one that regulates the expression of a protein or polypeptide; one that stops or inhibits the expression of a protein or polypeptide; or one that causes or enhances the expression of a protein or polypeptide.
  • Biologically active compositions, complexes, compounds or molecules may be used in investigative, therapeutic, prophylactic and diagnostic methods and compositions and generally act to cause.
  • cationic lipid refers to any cationic lipids which may be used for transfection and which under physiological conditions possess at least one positive charge. While it is to be understood that certain of the amine-containing transfection agents that form the basis of the present disclosure also exist as cations under physiological conditions, the term is also extended without limitation to any cationic helper lipids that may be used to co-formulate transfection complexes as described herein. Additional cationic lipids other than the novel amine-containing transfection agents described herein may include, but not limited to, e.g., DOSPA, DOTMA, DMRIE, DOT AP, DOGS and TMTPS, as well as any of the cationic lipids described herein as “helper lipid”.
  • Target cell or “target tissue” refers to any cell or tissue to which a desired compound is delivered, using a lipid aggregate or transfection complex as carrier for the desired compound.
  • Transfection is used herein to mean the delivery of any nucleic acid, protein or other macromolecule to a target cell or tissue in vitro or in vivo (i.e., in an animal, a plant or a human), such that the nucleic acid, protein or other macromolecule is expressed in, confers a phenotype to, or has a biological function in the cell.
  • expressible nucleic acid includes both DNA and RNA without regard to molecular weight, and the term “expression” means any manifestation of the functional presence of the nucleic acid within the cell including, without limitation, both transient expression and stable expression.
  • transfection complex generally refers to a composition formulated for the delivery of a biologically active agent, such as a nucleic acid, a protein, a macromolecule, or the like, to a cell or to a tissue in vivo or in vitro.
  • Transfection complexes as used herein may include at least one or more of the amine-containing transfection compounds in combination with the biologically active compound to be delivered, optionally in combination with one or more helper lipids, one or more pegylated lipids, one or more targeting moieties, in addition to the bioactive agent that is to be delivered.
  • transfection complex may be thought of as a lipoplex or a lipid aggregate contacted with a bioactive agent.
  • terms such as lipoplex, lipid aggregate and the like may be used to make reference a transfection complex that lacks the one or more bioactive agents or “payloads”.
  • helper lipid generally refers to a lipid that is suitable for use in the preparation and formation of transfection complexes disclosed herein.
  • Suitable helper lipids may include, though are not limited to cholesterols, cholesterol derivatives, sterols, including phytosterols, zoosterols and hopanoids, or any of the neutral or cationic lipids that are known to allow or to facilitate the introduction of exogenous bioactive molecules to the interior of a cell or of a tissue.
  • more than one helper lipid may be used in the formulation of the transfection complxes described herein.
  • Exemplary though non-limiting neutral or cationic lipids contemplated for use in the preparation of the presently disclosed transfection complexes may include one or lipids selected from the following: BMOP (N-(2-bromoethyl)-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-propana minimun bromide), DDPES (Dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide), DSPC, CTAB:DOPE (formulation of cetyltrimethylammonium bromide (CATB) and DOPE), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPE (dioleoylphosphatidylethanolamine), DMG, DMAP (4-dimethylaminopyridine), DMPE (Dimyristoylphospatidylethanolamine), DOMG, DMA, DOPC (Dioleoy
  • Neutral lipids also include cholesterol and other 3 ⁇ OH-sterols as well as derivatives thereof phosphatidyl choline or commercially available cationic lipid mixtures such as, for example, LIPOFECTIN® CELLFECTIN® (1:1.5 (M/M) formulation of N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmitylspermine (TMTPS) and dioleoyl phosphatidylethanolamine (DOPE), LIPOFECTACE®, GS 2888 CYTOFECTIN®, FUGENE 6®, EFFECTENE®, and LIPOFECTAMINE®, LIPOFECTAMINE 2000®, LIPOFECTAMINE PLUS®, LIPOTAXI®, POLYECT®, SUPERFECT®, TFXNTTM, TRANSFASTTM, TRANSFECTAM®, TRANSMESSENGER®, vectamidine (3-tetradecylamino-N-tert-butyl-N
  • pegylated lipid generally refers to a lipid that is covalently conjugated to one or more polyethylene glycol moieties.
  • Pegylated lipids for lipoplex embodiments herein include phosphatidylethanolamine (PE) based pegylated lipids such as, for example, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-MW] where MW refers to average MW of the polyethylene glycol moiety.
  • PE phosphatidylethanolamine
  • MW dimyristoyl-PEG-PE lipids are commonly designated 14:0 PEG (MW) PE.
  • the average MW of the polyethylene glycol moiety can be 25, 350, 550, 750, 1000, 2000, 3000, 5000, 6000, 8000 or 12000, for example.
  • the fatty acid chains of the phosphatidylethanolamine based pegylated lipids may include, for example, a 1,2-dioleoyl group such as for 18:1 PEG (MW) PE, a 1,2-dipalmitoyl group such as for 16:0 PEG (MW) PE, or a 1,2-distearoyl-group such as for 18:0 PEG (MW) PE.
  • PE phosphatidylethanolamine
  • pegylated lipids include, for example, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[MOD(polyethylene glycol)-MW], also referred to as DSPE-MOD PEG(MW) wherein MOD refers to a functional moiety such as an amine, biotin, carboxylic acid, folate, maleimide, PDP, or carboxyfluorescein moiety.
  • the MW may be 2000 or 5000, for example.
  • Pegylated lipids for the embodiments described herein also include ceramide based pegylated lipids such as, for example, N-octanoyl-sphingosine-1- ⁇ succinyl[methoxy(polyethylene glycol)MW] ⁇ , designated C8 PEG (MW) ceramide, where MW is 750, 2000, or 5000, for example.
  • the fatty acid moiety may have an N-palmitoyl (C16) group such as for C16 PEG (MW) ceramide.
  • a “liposomal composition” generally is a formulation that includes one or more liposomes.
  • the term “liposomal composition” may be used interchangeably with the term “transfection complex”. These formulations are typically colloids, but can be dried formulations as well.
  • a liposome is a vesicular colloidal particle composed of self assembled amphiphilic molecules.
  • Liposomal compositions of the present invention typically include at least one or more cationic lipids either alone or optionally in combination with one or more helper lipids (i.e., a neutral lipid, a cholesterol or cholesterol derivative, a cationic lipid) that are processed using standard methods to form a liposome-containing colloid suspension.
  • Liposomal compositions of the present invention are those containing one or more amine-containing transfection lipids, one or more helper lipids, one or more pegylated lipids, optionally, in combination with one or more neutral and/or helper lipids or targeting moieties which are treated by any of the standard methods known in the art without limitation to form liposomes.
  • Liposomal compositions can be distinguished one from another by particle size measurements. Different compositions will exhibit differences in particle size and uniformity of particle size, e.g., average particle size, and polydispersity. Different compositions will exhibit differences in the extent of the composition that is in the form of liposomes.
  • liposomal compositions will exhibit particle size in the range 120 nm and 800 nm and will exhibit generally lower polydispersity.
  • Lipoplex particle size (with siRNA or other cargo) may range from about 40 nm to 135 nm. In some embodiments, lipoplex particle size is 50 nm to 120 nm, 50 nm to 100 nm, 60 nm to 90 nm, 70 nm to 90 nm, or about 85 nm.
  • Lipid aggregate or “lipoplex” is a generic term that includes liposomes of all types, both unilamellar and multilamellar, as well as vesicles, micelles and more amorphous aggregates.
  • a cationic lipid aggregate is a lipid aggregate comprising a combination of one or more cationic compounds, optionally in combination with non-cationic lipids (including neutral lipids), such that the lipid aggregate has a net positive charge.
  • Amine-containing transfection compounds of the present invention can form a lipid aggregate, optionally with a helper lipid and further optionally with one or more pegylated lipids and/or one or more targeting moieties, which can then form a lipid-bioactive agent complex when contacted with a suitable bioactive agent.
  • lipid aggregate or “lipoplex” are generally used herein to refer to a “naked” transfection complex, i.e., a transfection complex that generally lacks a payload of bioactive agent to be delivered to a cell or to a tissue in vitro or in vivo.
  • lipid-bioactive agent generally refers to the noncovalent association between a lipid or lipid aggregate and a bioactive agent, such as a nucleic acid, a polypeptide, and the like.
  • nucleic acid and its grammatical equivalents will include the full range of polymers of single or double stranded nucleotides and includes nucleic acids (including DNA, RNA, and DNA-RNA hybrid molecules) that are isolated from a natural source; that are prepared in vitro, using techniques such as PCR amplification or chemical synthesis; that are prepared in vivo, e.g., via recombinant DNA technology; or that are prepared or obtained by any known method.
  • nucleic acids including DNA, RNA, and DNA-RNA hybrid molecules
  • a nucleic acid typically refers to a polynucleotide molecule comprised of a linear strand of two or more nucleotides (deoxyribonucleotides and/or ribonucleotides) or variants, derivatives and/or analogs thereof. The exact size will depend on many factors, which in turn depends on the ultimate conditions of use, as is well known in the art.
  • the nucleic acids of the present invention include without limitation primers, probes, oligonucleotides, vectors, constructs, plasmids, genes, transgenes, genomic DNA, cDNA, RNA, RNAi, siRNA, shRNA, stRNA, PCR products, restriction fragments, oligonucleotides and the like.
  • nucleotide includes any monomeric unit of DNA or RNA containing a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base and may also include mono-, di- and triphosphate forms of such nucleotides.
  • the base is usually linked to the sugar moiety via the glycosidic carbon (at the 1′ carbon of pentose) and that combination of base and sugar is called a “nucleoside.”
  • the base characterizes the nucleotide with the four customary bases of DNA being adenine (A), guanine (G), cytosine (C) and thymine (T).
  • Inosine (I) is an example of a synthetic base that can be used to substitute for any of the four, naturally occurring bases (A, C, G, or T).
  • the four RNA bases are A, G, C, and uracil (U).
  • a nucleic acid may be a nucleotide sequence comprising a linear array of nucleotides connected by phosphodiester bonds between the 3′ and 5′ carbons of adjacent pentoses. Other modified nucleotides are known and may be sued in the practice of the invention.
  • nucleotide includes ribonucleoside triphosphates ATP, UTP, ITP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof.
  • Such derivatives include, for example, [ ⁇ S]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them.
  • nucleotide as used herein also refers to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.
  • ddATP dideoxyribonucleoside triphosphates
  • ddCTP dideoxyribonucleoside triphosphates
  • ddGTP dideoxyribonucleoside triphosphates
  • ddTTP dideoxyribonucleoside triphosphates
  • Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.
  • Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.
  • Various labeling methods known in the art can be employed in the practice of this invention.
  • RNA refers to any RNA molecule or functional portion thereof, of any size and having any sequence, from any source, including RNA from viral, prokaryotic, and eukaryotic organisms.
  • the RNA molecule may be chemically modified and in any form, including, but not limited to, linear or circular, and single or double stranded.
  • Non-limiting examples of RNA molecules include rRNA, tRNA, mRNA, mtRNA, tmRNA, RNAi, siRNA, shRNA, and stRNA.
  • siRNA molecules useful in the practice of the invention include, for example, those described in U.S. patent application Ser. No. 10/357,529 published as U.S. 2004/0014956, Ser. No.
  • siRNA molecules useful in the practice of the invention include, for example, those described in PCT patent application PCT/US2008/076675 published as WO 2009/039173 on Mar. 26, 2009; which application is incorporated by reference herein.
  • peptide refers to a string of at least three amino acids linked together by peptide bonds.
  • protein and “peptide” may be used interchangeably, though it is generally understood that a “polypeptide” or “protein” is larger than a peptide.
  • eptide may refer to an individual peptide or a collection of peptides.
  • polynucleotide or “oligonucleotide,” as used herein, refer to a polymer of nucleotides. Typically, a polynucleotide comprises at least three nucleotides.
  • the polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaaden
  • lipid refers to hydrophobic or amphiphilic organic compounds inclusive of fats, oils and triglyderides.
  • the present invention describes various amine-containing compounds useful as transfection reagents and methods of synthesizing thereof. More particularly, according to some embodiments of the invention, there are provided compounds having the general structure I, or pharmaceutically acceptable salts thereof:
  • each of X 1 and X 2 is a moiety independently selected from the group consisting of O, S, N-A and C-A, wherein A is selected from the group consisting of hydrogen and a C 1 -C 20 hydrocarbon chain; each of Y and Z is a moiety independently selected from the group consisting of CH—OH, C ⁇ O, C ⁇ S, S ⁇ O and SO 2 ; each of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 is a moiety independently selected from the group consisting of hydrogen, a cyclic or an acyclic, substituted or unsubstituted, branched or unbranched aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic group, a substituted or unsubstituted, branched or unbranched acyl group, a substituted or unsubstituted, branched or unbranched acy
  • each of g, e and f is an integer independently having the value between 1 and 6, inclusively, “HCC” symbolizes a hydrocarbon chain, and each * indicates the nitrogen atom in structure I.
  • R 3 is a polyamine. In other embodiments, R 3 is a ketal. In some embodiments, each of R 1 and R 2 in the general structure I is independently any of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, such as between 8 and about 18 carbon atoms, and between 0 and 4 double bonds, such as between 0 and 2 double bonds.
  • R 3 is any of the following moieties:
  • each “HCC” symbolizes a hydrocarbon chain, and each * shows a potential point of attachment of R 3 to the nitrogen atom in structure I, where each H on any * position can be replaced to achieve the attachment to the nitrogen atom in structure I.
  • compounds the general structure I may have each of R 4 , R 5 , R 6 and R 7 being hydrogen, each of Y and Z being C ⁇ O, each of R 1 and R 2 being the same and each of X 1 and X 2 also being the same.
  • Such compounds are represented by the general structure II (which is a sub-genus of the compound the general structure I):
  • R 2 is H.
  • each R 1 is independently any of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, e.g., between 8 and about 18 carbon atoms, and between 0 and 4 double bonds, e.g., between 0 and 2 double bonds.
  • R 3 is any of the following moieties:
  • each “HCC” symbolizes a hydrocarbon chain, and each * shows a potential point of attachment of R 3 to the nitrogen atom in structure II, where each H on any * position can be replaced to achieve the attachment to the nitrogen atom in structure II.
  • R 3 is either of the following moieties:
  • each of g, e and f is an integer independently having the value between 1 and 6, inclusively, each “HCC” symbolizes a hydrocarbon chain, and each * shows a point of attachment of R 3 to the nitrogen atom in structure II.
  • Non-limiting examples of such specific compounds are any of the following lipids 1-87, or any isomer of each of compounds 1-87, or any combination of isomers for each of compounds 1-87:
  • the above-described compounds of the invention may be synthesized by reacting an amino component with an unsaturated component, e.g., by the addition of the primary amino group of the amino component to a double bond of the unsaturated component where the double is conjugated with an electrophlic group such as, e.g., carbonyl.
  • the synthetic method includes reacting one equivalent of the amino component with one or more equivalents of the unsaturated component.
  • the amino component comprises a primary amine NH 2 —R 3 , a diamine, a polyamine or a combination thereof.
  • the unsaturated component comprises of at least one first intermediate having the structure R 1 —X 1 —Y—(CR 4 R 5 ) n —Br and the second intermediate having the structure R 2 —X 2 —Z—(CR 6 R 2 ) m —Br, wherein in (CR 4 R 5 ) n and (CR 6 R 2 ) m portions of the structures, each R 4 is the same or different, each R 5 is the same or different, each R 6 is the same or different, and each R 7 is the same or different, wherein the first and the second intermediates are the same or different.
  • the first and/or the second intermediate(s) of the unsaturated component can be an acrylate or acrylamide.
  • all the amino groups of the amine NH 2 —R 3 , a diamine or a polyamine are fully reacted with the unsaturated component to form tertiary amines. In other embodiments, not all the amino groups are so reacted to form tertiary amines thereby resulting in primary or secondary amines in the lipid molecule.
  • the synthesis of the compounds of the invention may be performed with or without solvent, and the synthesis may be performed at temperatures ranging between room temperature and about 100° C., for example, at about 95° C.
  • the reaction may be optionally catalyzed by adding an acid, a base or a metal.
  • Those having ordinary skill in the art can select the optimal conditions under which the synthesis is carried out, to choose an appropriate catalyst, if necessary, and to select the molar ratio between the amino component and the unsaturated component.
  • the amino component is the primary amine NH 2 —R 3
  • the molar ratio between the unsaturated component and the primary amine NH 2 —R 3 can be between about 1:1 and about 6:1.
  • the prepared lipids may be optionally purified.
  • the lipids may also be alkylated using an alkyl halide (e.g., methyl iodide) or other alkylating agent.
  • each of X 1 and X 2 is a moiety independently selected from the group consisting of O, S, N-A and C-A, wherein A is selected from the group consisting of hydrogen and a C 1 -C 20 hydrocarbon chain; each of Y and Z is a moiety independently selected from the group consisting of CH—OH, C ⁇ O, C ⁇ S, S ⁇ O and SO 2 ; each of R 1 , R 2 , R 4 , R 5 , R 6 and R 7 is a moiety independently selected from the group consisting of hydrogen, a cyclic or an acyclic, substituted or unsubstituted, branched or unbranched aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic group, a substituted or unsubstituted, branched or unbranched acyl group, a substituted or unsubstituted, branched or unbranched acyl group,
  • R 3 in the above-provided structure of the primary amine is either of the following moieties:
  • each of g, e and f is an integer independently having the value between 1 and 6, inclusively, each “HCC” symbolizes a hydrocarbon chain, and each * shows a point of attachment of R 3 to the amino group in the primary amine NH 2 —R 3 .
  • each of R 1 and R 2 in the above-provided structures of the first and the second intermediates of the unsaturated component is independently any of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, such as between 8 and about 18 carbon atoms, and between 0 and 4 double bonds, such as between 0 and 2 double bonds.
  • R 3 in the above-provided structure of the primary amine can be any of the following moieties:
  • each “HCC” symbolizes a hydrocarbon chain, and each * shows a potential point of attachment of R 3 to the amino group in the primary amine NH 2 —R 3 , where each H on any * position can be replaced to achieve the attachment to the nitrogen atom in the primary amine NH 2 —R 3 .
  • the above described compounds of the invention may be used in the delivery of biologically active or therapeutic agents to a subject, to an animal, or to a cell or a tissue in vitro or in vivo.
  • the compounds may be particularly suited to delivering negatively charged bioactive agents.
  • the amin-containing transfection compounds of the present invention may be used to delivery DNA, RNA, other polynucleotides, other anions or polyanions to a subject or to a cell.
  • the inventive lipids are combined with an agent to form transfection complexes, such as microparticles, liposomes or micelles.
  • the bioactive agent to be delivered e.g., a polynucleotide, a protein, a peptide or a small molecule
  • the inventive lipids may be combined with other lipids, polymers, surfactants, cholesterol, carbohydrates, proteins, etc. to form the particles. These particles may be combined with a pharmaceutically excipient to form pharmaceutical compositions.
  • the lipid synthesized as described above may be further purified by any known technique, such as by crystallization, chromatography, precipitation (e.g. repeated precipitations in diethyl ether, hexane or another organic solvent) or distillation.
  • the lipid may be also isolated as a salt that can be formed when the lipid is reacted with an organic acid or inorganic acid.
  • the lipid if the lipid comprises the tertiary amine moiety, it can be alkylated with any alkylating agent, for example, alkyl halides such as methyl iodide to form a quaternary ammonium salt of the lipid.
  • the anion associated with the quaternary amine may be any pharmaceutically acceptable organic or inorganic anion.
  • the synthetic process results in a mixture of isomers having acrylic tails, with varying numbers and positions of the acrylate tails. Such mixtures can be used with or without further purification, as desired.
  • an amine is not exhaustively alkylated, the resulting products may be further reacted with another electrophile, such as an acrylate or acrylamide optionally followed by further purification.
  • a library of different lipids can be prepared in parallel.
  • a different amine and/or unsaturated component can be added to each vial in a set of vials or to each well of a multi-well plate.
  • the array of reaction mixtures is incubated at a temperature and length of time sufficient to allow formation of the lipids to occur.
  • the lipids may then be isolated and purified using known techniques followed by screening using high-throughput techniques to identify lipids with a desired characteristic (e.g., solubility, ability to bind polynucleotides, ability to bind heparin, ability to bind small molecules, ability to form microparticles, ability to increase transfection efficiency and the like).
  • the delivery of a biologically active agent to cells or tissue as contemplated herein may be for the provision of a therapeutic modality for the treatment of a disorder, or may alternatively be provided during the course of conducting scientific research (e.g., as a research tool).
  • a transfection complex as provided for herein may include one or more amine-containing transfection agents formulated as a lipid aggregate such that the biologically active agent can be delivered to a cell or a tissue to affect a desired biological response.
  • a transfection complex may optionally include one or more helper lipids.
  • a transfection complex may optionally include one or more pegylated lipids.
  • a transfection complex may optionally include one or more targeting moieties or transfection enhancers.
  • an a transfection complex may include an amine-containing transfection compound having the general structure I, or pharmaceutically acceptable salts thereof:
  • each of X 1 and X 2 is a moiety independently selected from the group consisting of O, S, N-A and C-A, wherein A is selected from the group consisting of hydrogen and a C 1 -C 20 hydrocarbon chain; each of Y and Z is a moiety independently selected from the group consisting of CH—OH, C ⁇ O, C ⁇ S, S ⁇ O and SO 2 ; each of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 is a moiety independently selected from the group consisting of hydrogen, a cyclic or an acyclic, substituted or unsubstituted, branched or unbranched aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic group, a substituted or unsubstituted, branched or unbranched acyl group, a substituted or unsubstituted, branched or unbranched acy
  • each of g, e and f is an integer independently having the value between 1 and 6, inclusively, “HCC” symbolizes a hydrocarbon chain, and each * indicates the nitrogen atom in structure I.
  • R 3 is a polyamine. In other embodiments, R 3 is a ketal. In some embodiments, each of R 1 and R 2 in the general structure I is independently any of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, such as between 8 and about 18 carbon atoms, and between 0 and 4 double bonds, such as between 0 and 2 double bonds.
  • R 3 is any of the following moieties:
  • each “HCC” symbolizes a hydrocarbon chain, and each * shows a potential point of attachment of R 3 to the nitrogen atom in structure I, where each H on any * position can be replaced to achieve the attachment to the nitrogen atom in structure I.
  • transfection complexes containing amine-containing transfection compounds having the general structure I may have each of R 4 , R 5 , R 6 and R 7 being hydrogen, each of Y and Z being C ⁇ O, each of R 1 and R 2 being the same and each of X 1 and X 2 also being the same.
  • Such compounds are represented by the general structure II (which is a sub-genus of the compound the general structure I):
  • R 2 is H.
  • each R 1 is independently any of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, e.g., between 8 and about 18 carbon atoms, and between 0 and 4 double bonds, e.g., between 0 and 2 double bonds.
  • R 3 is any of the following moieties:
  • each “HCC” symbolizes a hydrocarbon chain, and each * shows a potential point of attachment of R 3 to the nitrogen atom in structure II, where each H on any * position can be replaced to achieve the attachment to the nitrogen atom in structure II.
  • R 3 is either of the following moieties:
  • each of g, e and f is an integer independently having the value between 1 and 6, inclusively, each “HCC” symbolizes a hydrocarbon chain, and each * shows a point of attachment of R 3 to the nitrogen atom in structure II.
  • transfection complexes may include one or more amine-containing transfection compounds that are species within either the general structure I or the general structure II, or both.
  • Non-limiting examples of such specific compounds are any of the following lipids 1-87 indicated above, or any isomer of each of compounds 1-87, or any combination of isomers for each of compounds 1-87:
  • the molar percentage of the amine-containing transfection compound is from about 15% to about 50% of the lipid aggregate; in other embodiments, the molar percentage of the cationic lipid is from about 20% to about 40% of the transfection complex; in some embodiments, the molar percentage of the amine-containing transfection compound is from about 25% to about 35% of the lipid aggregate; or, in some embodiments, the molar percentage of the amine-containing transfection compound is about 33% of the lipid aggregate.
  • the molar percentage of the amine-containing transfection compound is between about 15% and about 35% of the lipid aggregate; in other embodiments, the molar percentage of the amine-containing transfection compound is between about 20% and about 30% of the lipid aggregate; or in some embodiments, the molar percentage of the amine-containing transfection compound is approximately 25% of the lipid aggregate.
  • a transfection complex may optionally include one or more helper lipids.
  • helper lipids suitable for use in the formulation of the presently described transfection complexes include cholesterols, cholesterol derivatives, sterols, including phytosterols, zoosterols and hopanoids, or any of the neutral or cationic lipids that are known to allow or to facilitate the introduction of exogenous bioactive molecules to the interior of a cell or of a tissue.
  • more than one helper lipid may be used in the formulation of the transfection complxes described herein.
  • Exemplary though non-limiting neutral or cationic lipids contemplated for use in the preparation of the presently disclosed transfection complexes may include one or lipids selected from the following: BMOP(N-(2-bromoethyl)-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-propana minimun bromide), DDPES (Dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide), DSPC, CTAB:DOPE (formulation of cetyltrimethylammonium bromide (CATB) and DOPE), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPE (dioleoylphosphatidylethanolamine), DMG, DMAP (4-dimethylaminopyridine), DMPE (Dimyristoylphospatidylethanolamine), DOMG, DMA, DOPC (Dioleoy
  • Neutral lipids also include cholesterol and other 3 ⁇ OH-sterols as well as derivatives thereof phosphatidyl choline or commercially available cationic lipid mixtures such as, for example, LIPOFECTIN® CELLFECTIN® (1:1.5 (M/M) formulation of N, NI,NII,NIII-tetramethyl-N, NI, NII, NIII-tetrapalmitylspermine (TMTPS) and dioleoyl phosphatidylethanolamine (DOPE), LIPOFECTACE®, GS 2888 CYTOFECTIN®, FUGENE 6®, EFFECTENE®, and LIPOFECTAMINE®, LIPOFECTAMINE 2000®, LIPOFECTAMINE PLUS®, LIPOTAXI®, POLYECT®, SUPERFECT®, TFXNTM, TRANSFASTTM, TRANSFECTAM®, TRANSMESSENGER®, vectamidine (3-tetradecylamino-N-tert-buty
  • the molar percentage of the helper or neutral lipid is between about 60% and about 85% of the lipid aggregate; in some embodiments, the molar percentage of the helper or neutral lipid is between about 70% and about 80% of the lipid aggregate; or, in some embodiments, the molar percentage of the helper or neutral lipid is between about 70% and about 75% of the lipid aggregate.
  • a transfection complex may include one or more Pegylated lipids.
  • Pegylated lipids suitable for use in the preparation and formation of transfection complexes disclosed herein can be any lipid or mixture of lipids that are compatible with the formation of transfection complexes described herein, and with the administration thereof to an animal or to a human in vivo, or to tissues or cells in vitro.
  • the pegylated lipids used in the present invention include a PEG polymer having a molecular weight between about 250 daltons and about 12,000, or in some embodiments, about 350 daltons and about 6,000 daltons, or, in some embodiments, between about 500 daltons and about 1,000 daltons, or, in some embodiments, between about 1,000 daltons and about 2,000 daltons, or, in some embodiments, between about 2,000 daltons and 5,000 daltons.
  • suitable Pegylated lipids include phosphatidylethanolamine (PE) based pegylated lipids such as, for example, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-MW] where MW refers to average MW of the polyethylene glycol moiety.
  • PE phosphatidylethanolamine
  • Such dimyristoyl-PEG-PE lipids are commonly designated 14:0 PEG (MW) PE.
  • the average MW of the polyethylene glycol moiety can be 25, 350, 550, 750, 1000, 2000, 3000, 5000, 6000, 8000 or 12000, for example.
  • the fatty acid chains of the phosphatidylethanolamine based pegylated lipids may include, for example, a 1,2-dioleoyl group such as for 18:1 PEG (MW) PE, a 1,2-dipalmitoyl group such as for 16:0 PEG (MW) PE, or a 1,2-distearoyl-group such as for 18:0 PEG (MW) PE.
  • a 1,2-dioleoyl group such as for 18:1 PEG (MW) PE
  • a 1,2-dipalmitoyl group such as for 16:0 PEG (MW) PE
  • a 1,2-distearoyl-group such as for 18:0 PEG (MW) PE.
  • PE phosphatidylethanolamine
  • pegylated lipids include, for example, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[MOD(polyethylene glycol)-MW], also referred to as DSPE-MOD PEG(MW) wherein MOD refers to a functional moiety such as an amine, biotin, carboxylic acid, folate, maleimide, PDP, or carboxyfluorescein moiety.
  • the MW may be 2000 or 5000, for example.
  • Pegylated lipids for the embodiments described herein also include ceramide based pegylated lipids such as, for example, N-octanoyl-sphingosine-1- ⁇ succinyl[methoxy(polyethylene glycol)MW] ⁇ , designated C8 PEG (MW) ceramide, where MW is 750, 2000, or 5000, for example.
  • the fatty acid moiety may have an N-palmitoyl (C16) group such as for C16 PEG (MW) ceramide.
  • the molar percentage of the pegylated lipid is between about 0.5% and 15% of the transfection complex; in some embodiments, the molar percentage of the pegylated lipid is between about 1% and about 10% of the transfection complex; or in some embodiments, the molar percentage of the pegylated lipid is approximately 1% or 5% of the transfection complex.
  • the molar percentage of amine-containing transfection compound: helper lipid:pegylated lipid of the transfection complex ranges from 15:84:1 to 15:75:10, from 20:79:1 to 20:70:10, from 25:74:1 to 25:65:10, from 30:69:1 to 30:60:10, from 40:59:1 to 40:50:10, or from 50:49:1 to 50:40:10.
  • the molar percentage of amine-containing transfection compound: helper lipid:pegylated lipid of the transfection complex ranges from 10-90:7-35:5-70, from 15-85:5-35:8-50, from 30-85:5-35:8-50, from 35-70; 10-30:15-45, from 40-65:15-25:20-40, from 50-60:18-22:25-35, from 50-55:19-21:27-30, or from 51-53:20-20.5:28-29.
  • alternative ratios may be employed, and optimizing the ratios of such formulations is well within the skill level of such a person, without requiring undue experimentation.
  • a bioactive agent such as, e.g., a polyanion, a polynucleotide or a polypeptide into a cell or cells, or into a tissue
  • the method includes forming a lipid aggregate, such as a liposome, comprising one or more of the amine-containing transfection compounds described above, optionally with one or more helper lipids and/or one or more pegylated lipids, and contacting the lipid aggregate with the bioactive agent to form a neutral or positively charged bioactive agent-lipid aggregate complex, and incubating the complex with a cell or a tissue in vitro, or administering the resulting transfection complex to an animal or to a human, optionally as a therapeutic composition.
  • a bioactive agent such as, e.g., a polyanion, a polynucleotide or a polypeptide into a cell or cells, or into a tissue
  • a lipid aggregate such as a liposome
  • the method includes forming
  • the transfection complexes may include one or more biologically active agents to be delivered to a cell or to a target tissue in vitro or in vivo.
  • suitable biologically active agents may include any molecule that is capable of forming a transfection complex with the presently described amine-containing transfection reagents and that elicits a biological response when delivered to the interior of a cell or cells or to a tissue in vivo or in vitro.
  • Biologically active agents contemplated for use in the presently described embodiments may be cationic, neutral or anionic agents.
  • exemplary biologically active agents suitable for formulation in a transfection complex may include, though are not limited to; nucleic acids (including but not limited to single or double stranded linear or circular DNA molecules including cDNA molecules, single or double stranded RNA molecules, small interfereing RNA (siRNA) molecules, small hairpin RNA (shRNA) molecules, microRNA (miRNA) molecules, oligonucleotides, anti-sense oligonucleotides, sense oligonucleotides), polypeptides, antibodies, oligopeptides, therapeutic peptides or protein molecules, peptide nucleic acids (PNAs), cationic, anionic or neutral organic molecules or drugs, in addition to pharmaceutically acceptable salts thereof.
  • nucleic acids including but not limited to single or double stranded linear or circular DNA molecules including cDNA molecules, single or double stranded RNA molecules, small interfereing RNA (siRNA) molecules, small hairpin RNA (shRNA) molecules, microRNA (m
  • transfection complexes and methods that use the compounds of the present invention to deliver nucleic acid molecules into cells or tissues in vitro or in vivo, including the delivery of RNA interference molecules (RNAi) or small interfering RNA molecules (siRNA, shRNA or miRNA) into cells for inhibition of gene expression.
  • RNAi RNA interference molecules
  • shRNA small interfering RNA molecules
  • transfection complexes and methods that use the compounds of the present invention to deliver mRNA molecules into a cell or a tissue in vivo or in vitro to promote the expression of a specific protein or proteins are also provided.
  • transfection complexes and methods that use the compounds of the present invention to deliver DNA molecules (including cDNA molecules) into a cell or a tissue in vivo or in vitro to promote the expression of a specific protein or proteins or to synthesize specific RNA molecules, including but not limited to mRNA molecules or RNAi or miRNA or shRNA molecules are also provided.
  • the transfection complexes described herein may optionally include one or more fusogenic or cell-penetrating peptides.
  • a fusogenic or cell-penetrating peptide is any peptide molecule that is capable of promoting the fusion of a lipid-containing complex to a cell membrane (either a plasma membrane or an endosomal membrane).
  • a variety of fusogenic or cell-penetrating peptides are known in the art and it is well within the skill level of a practitioner to identify suitable fusogenic or cell-penetrating peptides and condition for the use thereof in the present invention without undue experimentation.
  • the transfection complexes described herein may optionally include one or more transfection helpers or targeting moieties.
  • a targeting moiety may be a peptide, a modified peptide, an antibody, a modified antibody, a receptor molecule, a modified receptor molecule, a single or a double stranded nucleic acid molecule, a modified single or double stranded nucleic acid molecule, a peptide or nucleic acid aptamer, a modified peptide or nucleic acid aptamer, an organic molecule, a polysaccharide, or any other molecule that is capable of targeting a transfection complex to specific tissue or cell type for targeted delivery of a biologically agent thereto, such as will be readily apparent to have having ordinary skill level in the art.
  • modification of a peptide, an antibody, a nucleic acid, an aptamer, and the like may include conjugating the peptide, antibody, nucleic acid, aptamer, and the like to a PEG moiety.
  • modification of a peptide, an antibody, a nucleic acid, an aptamer, and the like may include conjugating the peptide, antibody, nucleic acid, aptamer, and the like to a PEG-lipid moiety
  • targeting moieties are widely known to those skilled in the art, and all are contemplated for use with the presently described embodiments, without limitation.
  • the transfection complexes provided for herein may be stable for up to 1 year and may either be contacted with the cells or tissues to be transfected, or be administered to an animal or to a human immediately or shortly after being formed, or optionally may stored for a period of time prior to being contacted with the cells or tissues, or being administered to an animal or a human.
  • the transfection complexes are stable and may be stored for a time period of at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 5 days, at least 7 days, at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months or at least 1 year at room temperature, or at a temperature greater than freezing, up to about room temperature.
  • the formulation described herein may include one or more stabilizing agents, preservatives, buffers, etc, that aid in the long-term stabilization and storage of bioactive formulation, such as will be readily understood by the skilled practitioner of the biological and pharmaceutical arts, and without requiring undue experimentation to achieve. It is also understood, that the storage period can be between any of these time periods, for example between 31 minutes and 1 hour or between 1 hour and 24 hours.
  • the bioactive agent-lipid aggregate complex is stored for a period prior to being contacted with the cell or cells.
  • the polyanion-lipid aggregate complex is stable and can be stored for a time period of at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 5 days, at least 7 days, at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months or at least 1 year, or for a time period between any of these time periods.
  • RNAi components including siRNA, short hairpin RNA (shRNA), microRNAs (miRNA) and small temporally regulated RNA (stRNA), which optionally are chemically modified, to cells or to tissues in vitro or in vivo.
  • shRNA short hairpin RNA
  • miRNA microRNAs
  • stRNA small temporally regulated RNA
  • the delivery methods employing the transfection complexes of the present invention or mixtures thereof can be applied to cells in vitro, ex vivo, and in vivo, particularly for transfection of eukaryotic cells or tissues including animal cells, human cells, non-human animal cells, insect cells, plant cells (including algae), avian cells, fish cells, mammalian cells and the like.
  • the bioactive agent that is to be delivered into the cell is contacted with lipid aggregates of this invention to form a transfection complex comprising a bioactive agent-lipid aggregate complex.
  • the target cell or cells or the target tissues are then incubated with the complex, or, for in vivo applications, the complex is administered to the organism by an appropriate route (e.g., intravenous, intramuscular, subcutaneous, transdermal, transmucosal, etc) so that the complex contacts the target cells or tissue.
  • the compounds of may also be conjugated to or mixed with or used in conjunction with a variety of useful molecules and substances, also referred to as transfection helpers or targeting moieties, such as proteins, peptides, growth factors, antibodies, nucleic acids, aptamers, or modified versions thereof (such as, e.g., conjugating said transfection helpers or targeting moieties to PEG or PEG-lipids) and the like to enhance cell-targeting, uptake, internalization, nuclear targeting and expression, all of which are likewise within the skill level of the skilled practitioner.
  • transfection helpers or targeting moieties such as proteins, peptides, growth factors, antibodies, nucleic acids, aptamers, or modified versions thereof (such as, e.g., conjugating said transfection helpers or targeting moieties to PEG or PEG-lipids) and the like to enhance cell-targeting, uptake, internalization, nuclear targeting and expression, all of which are likewise within the skill level of the skilled practitioner.
  • a further embodiment provides a method of transfecting a cell or tissue with a nucleic acid in vivo wherein the method comprises forming a lipid aggregate, such as a liposome, comprising one or more amine-containing transfection compounds, one or more pegylated lipids and optionally one or more helper lipids, contacting the lipid aggregate with the nucleic acid to form a neutral or positively charged lipid aggregate-nucleic acid complex, and administering the lipid aggregate-nucleic acid complex to the cells or tissues in vitro or to an organism so that the complex contacts the target cells or tissue.
  • Administration of the lipid aggregate-nucleic acid complex can be achieved orally, intravenously, or by subcutaneous or intramuscular injection or applied topically to the tissue as further described below.
  • the bioactive agent-lipid aggregate complex is stored for a period prior to being contacted with the cell or cells for transfection.
  • the polyanion-lipid aggregate complex is stable and can be stored for a time period of at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 5 days, at least 7 days, at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months or at least 1 year, or for a time period between any of these time periods.
  • transfection complexes of the present invention are provided in the wells of a multiwell plate.
  • Bioactive molecules to be delivered into target cells are selected and added to the wells to form polyanion-lipid aggregate complexes, which are subsequently contacted with the target cells in vitro or in vivo.
  • the lipid aggregates can have the same composition and concentration in each well, or the lipid aggregate composition and/or concentration can vary from well to well (for example, the amount of pegylation in the lipid aggregate can vary across the wells to determine the range for delivery and transfection).
  • the bioactive agents are nucleic acids such as RNAi
  • the nucleic acids can be added to the wells and optionally stored before contacting with the target cells.
  • the methods of this invention optionally comprise the step of contacting the one or more amine-containing transfection compounds with one or more helper lipids and one or more pegylated lipids before or at the same time as contacting the bioactive agent with the one or more amine-containing transfection compounds to form lipid aggregates encapsulating the bioactive agent.
  • the methods also optionally comprise forming the lipid aggregates into liposomes prior to contact with the bioactive agent.
  • the liposomes are formed by microfluidization, extrusion or other means known in the art.
  • the bioactive agents may include nucleic acid molecules such as DNA or RNA that inhibit or promote the expression of a target gene.
  • the bioactive agent associates with a transcript of the gene to effect inhibition thereof.
  • the bioactive agent is a nucleic acid, such as a DNA an mRNA, RNAi, siRNA, shRNA, miRNA or stRNA, and is optionally chemically modified.
  • the invention also provides medicaments prepared by combining liposome comprising one or more amine-containing transfection compounds optionally with one or more helper lipids, optionally with one or more pegylated lipids and optionally with one or more targeting moieties or transfection helpers (as described above) or a salt thereof, with a bioactive agent such as, e.g., a nucleic acid, wherein introduction of the bioactive agent into a cell or tissue modulates expression of one or more target genes therein thereby effecting at least one biological response and/or at least one therapeutic benefit.
  • the medicament further comprises an additional excipient or pharmaceutical carrier, such as will be readily apparent to the practitioner having ordinary skill level in the pharmaceutical and/or the medical arts.
  • the bioactive agent may be a nucleic acid, such as, e.g., a DNA or RNA.
  • the nucleic acid is mRNA, RNAi, siRNA, shRNA, miRNA or stRNA, and is optionally chemically modified.
  • medicaments or pharmaceutical preparations are provided for treatment of a disease, condition, or disorder that relates to the expression of one or more genes in a cell or a tissue.
  • Formulation of pharmacologically acceptable medicaments is known in the art.
  • Administration of the medicament delivers an effective amount of the polyanion, for example RNA or a DNA, to the cells or tissue associated with the disease, condition, or disorder to provide amelioration of the disease, condition or disorder.
  • such medicaments can be administered orally, intravenously, or by subcutaneous or intramuscular injection or applied topically to the tissue as further described below.
  • kits for the preparation of one or more transfection complexes of the presently described invention invention may, for example, comprise one or more liposomal compositions of this invention.
  • kits typically comprise a carrier, such as a box, carton, tube or the like, having in close confinement therein one or more containers, such as vials, tubes, ampules, bottles, and the like, wherein containers contain one or more amine-containing transfection compounds, optionally with one or more helper lipids, optionally with one or more pegylated or cationic lipids (or acceptable salts thereof) in accordance with the embodiments described above, or a liposomal compositions of the present invention.
  • kits encompassed by this aspect of the present invention may further comprise one or more additional components (e.g., reagents and compounds) necessary or beneficial for carrying out one or more particular applications of the compositions of the present invention.
  • the kit may optionally contain one or more multiwell plates suitable for holding the lipid aggregates or transfection complexes of the present invention.
  • the kits may also contain one or more components useful in carrying out a desired transfection of cells.
  • the kit may also contain one or more components useful in carrying out diagnosis, treatment or prevention of a particular disease or physical disorder (e.g., one or more additional therapeutic compounds or compositions, one or more diagnostic reagents).
  • kits may also contain one or more buffers, positive or negative control samples, carriers or excipients, and the like, one or more additional compositions of the invention, one or more sets of instructions, and the like.
  • lipid aggregate-bioactive agent complex i.e., a lipid aggregate-bioactive agent complex
  • methods may include forming a lipid aggregate comprising one or more amine-containing transfection compounds, such as those represented in structures I and II and in formulae 1-87, one or more helper lipids, one or more pegylated lipids, and optionally one or more transfection helpers or targeting moieties under any one or more of the following conditions:
  • Transfection complexes of embodiments described herein may be administered via the following routes for in vivo administration, for example, intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraventricular, intraprostatica, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrauterine, intrarectal, topical, intratumoral, intrathecal, intramuscular, subcutaneous, subconjunctival, intravesicular, mucosal, intrapericardial, intraumbilical, intraocular, oral, topical, local, via inhalation (e.g.
  • a dosage for ex vivo or in vivo use is from 0.01 ⁇ g to 1 g/kg of body weight, 0.1 ⁇ g to 0.1 g/kg of body weight, 1 ⁇ g to 0.01 g/kg of body weight, 10 ⁇ g to 0.01 g/kg of body weight, 0.1 mg to 10 mg/kg of body weight, or ranges between and including any of 0.1 mg, 0.25 mg, 0.5 mg, 1.0 mg, 1.5 mg, 2 mg, and 10 mg/kg of body weight.
  • Administration may be once or more per day, week, month or year. Administration may be in bolus form.
  • an effective amount of the lipoplexes of embodiments herein is an amount sufficient to reduce expression of the targeted gene and results in an extracellular concentration at the surface of the target cell of from 100 pM to 1 ⁇ M, or from 1 nM to 100 nM, or from 2 nM to about 25 nM, or to about 10 nM.
  • the amount required to achieve this local concentration will vary depending on a number of factors including the delivery method, the site of delivery, the number of cell layers between the delivery site and the target cell or tissue, whether delivery is local or systemic, etc.
  • the concentration at the delivery site may be considerably higher than it is at the surface of the target cell or tissue.
  • transfection complexes are prepared at a final concentration of 0.5-1 mg/ml.
  • the ratio of lipid aggregate to nucleic acid is between about 0.7:1 and 1.3:1 (v:w) depending upon the targeted organ.
  • v:w 1.3:1
  • administration may be intravenous thereby targeting the lung, kidney, liver, tumor, or spleen (using 50-200 ⁇ l), intraperitoneal thereby targeting a tumor or inflammation (using 100 l), intranasal thereby targeting the lung (using 50 ⁇ l), intratumoral or retro-orbital (local) thereby targeting the eye, a tumor, knee-joint, or the ear (using 10-100 ⁇ l), intracerebral (local) thereby targeting the brain (using 0.5-5 ⁇ l), intrathecal thereby targeting the spinal cord (using 10 ⁇ l), or hydrodynamic thereby targeting the liver, kidney, or virus (using 0.8-2.5 ml), for example.
  • Such methods may include preparing a plurality of transfection complexes containing a compound that readily facilitates the detection of a marker in combination with a test transfection compound, delivering each of the plurality of transfection complexes to a test animal, and detecting the marker.
  • a method for screening tissue-biased delivery of a transfection complex may include preparing a plurality of transfection complexes, each transfection complex having at least one test transfection compound in combination with at least one nucleic acid that facilitates detection of delivery to a tissue.
  • the nucleic acid may be an RNA molecule or a DNA molecule that encodes a protein that can be directly detected (such as, e.g., Green Fluorescent Protein (GFP), red Fluorescent Protein, luciferase, or the like), or encode a protein that effects expression of a protein that can be directly detected.
  • GFP Green Fluorescent Protein
  • red Fluorescent Protein red Fluorescent Protein
  • luciferase or the like
  • a method for screening tissue-biased delivery of a transfection complex may include preparing a plurality of unique transfection complexes, each transfection complex having at least one test transfection compound in combination with an mRNA or a cDNA that encodes Green Fluorescent Protein.
  • Each unique transfection complex may be delivered either intravenously, subcutaneously, or to a tissue to a test animal, such as a mouse. After a predetermined amount of time, tissues from the mouse may be harvested and the expression of GFP in various tissues may be detected by gross examination, histological examination or by molecular detection (PCR, Western blotting, or the like) to determine which to tissue or tissues transfection complexes containing specific transfection compounds are delivered to.
  • a method for screening tissue-biased delivery of a transfection complex may include preparing a plurality of unique transfection complexes, each transfection complex having at least one test transfection compound in combination with an mRNA or a cDNA that encodes Luciferase.
  • Each unique transfection complex may be delivered either intravenously, subcutaneously, or to a tissue to a test animal, such as a mouse. After a predetermined amount of time, tissues from the mouse may be harvested and the expression of Luciferase in various tissues may be detected by gross examination, histological examination or by molecular detection (PCR, Western blotting, or the like), or imaged in-vivo using the IVIS® Imaging System (Caliper).
  • a method for screening tissue-biased delivery of a transfection complex may include preparing a plurality of unique transfection complexes, each transfection complex having at least one test transfection compound in combination with an mRNA or a cDNA that encodes a specific transcription factor.
  • Each unique transfection complex may be delivered either intravenously, subcutaneously, or to a tissue to a transgenic animal that expresses a reporter gene (such as, e.g., luciferase) under the control of the specific transcription factor.
  • a reporter gene such as, e.g., luciferase
  • tissues from the transgenic animal may be harvested and the expression of reporter gene in various tissues may be detected by gross examination, histological examination or by molecular detection (PCR, Western blotting, or the like). If the reporter gene is luciferase, detection may be accomplished in-vivo using the IVIS® Imaging System (Caliper).
  • a method for screening tissue-biased delivery of a transfection complex may include preparing a plurality of unique transfection complexes, each unique transfection complex containing at least one unique test transfection compound in combination with an mRNA or DNA molecule that encodes Cre recombinase.
  • the plurality of unique transfection complexes may also optionally include one or more transfection enhancers, one or more helper lipids, one or more pegylated lipids or one or more targeting moieties as described above and incorporated herein.
  • Each of the unique transfection complexes may be delivered either intravenously (for example by tail vein injection), subcutaneously or via intra-tissue injection to a transgenic mouse that expresses a reporter gene in the presence of Cre recombinase.
  • each of the unique transfection complexes may be delivered to a transgenic mouse bearing a loxP-STOP-loxP-Luciferase gene, such as, e.g. any of the 129S6(B6)-Gt(ROSA)26 transgenic mouse strains, in which the firefly luciferase gene is inserted into the Gt(ROSA)26Sor locus.
  • the luciferase gene is blocked by a loxP-flanked STOP sequence located in between the luciferase transgene and its promoter.
  • Cre recombinase i.e., in tissues to which the Cre-mRNA or DNA containing transfection complexes are delivered
  • the STOP sequence is excised and luciferase is expressed.
  • expression of the luciferase transgene may be accomplished according to any of the widely-used techniques for assessing gene expression known to the skilled artisan (such as PCR, Northern blotting, Western blotting, or the like or directly measuring luciferase activity).
  • the transgewhole mount excised tissues or histologic sections may be examined, for example, using the IVIS® In Vivo
  • reaction mixture was transferred to a separatory funnel and the organic layer was washed with saturated sodium bicarbonate (x3), water (x2), brine (x1). The organic layer was dried over sodium sulfate, filtered and concentrated to give the desired product, pentadecyl-4-bromobutanoate as colorless oil (88% yield).
  • reaction mixture was transferred to a separatory funnel and the organic layer was washed with saturated sodium bicarbonate (x3), water (x2), brine (x1). The organic layer was dried over sodium sulfate, filtered and concentrated to give the desired product, pentadecyl-4-bromobutanoate as colorless oil (88% yield).
  • a dry microwave reaction synthesis vessel containing a small magnetic stir bar was charged with N1-methylpropane-1,3-diamine (42 mg, 0.48 mmole), pentadecyl-4-bromobutanoate (2.1 eq), N,N-diisopropylethyl amine (2.1 eq.) and dimethylformamide (1.0 mL).
  • the vessel was sealed and the reaction mixture was heated up to 90° C. in a CEM microwave synthesizer. After about 20 minutes, the reaction was stopped and the vessel was allowed to cool down to about 40° C.
  • the analytical LCMS traces of the reaction crude showed the formation of the desired products.
  • the crude material was purified by preparative LCMS.
  • lipids of the present invention were formulated into lipid compositions, which also comprise cholesterol, poly(ethylene glycol) (PEG) lipids, buffers and ethanol.
  • Animal origin-free cholesterol was purchased from Sigma-Aldrich (St Louis, Mo.), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (C16 PEG2000 PE) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (C14 PEG2000 PE) were purchased from Avanti Lipids (Alabaster, Ala.).
  • RNAi was STEALTHTM RNAi having 25-base-pair double-stranded RNA oligonucleotides with stabilizing chemical modifications.
  • STEALTHTM RNAi is commercially available from Invitrogen (Carlsbad, Calif.).
  • Dry powder lipids were re-suspended in ethanol and the cationic lipid: cholesterol and a PEG lipid were mixed at a weight ratio of 52:20:28 respectively.
  • the lipid ethanolic mixture was mixed very rapidly in 200 mM Sodium Acetate pH 5.2 solution at a 1:4 ratio.
  • the final formulation i.e., the total the lipid of the invention plus PEG lipid plus cholesterol
  • the formulations that were made are described in Table 1.
  • the lipoplex (siRNA preliposome complex) was prepared by mixing equal volumes of preliposome and STEALTHTM RNAi solution diluted to 1.5 mg/ml in water and 25% ethanol. After mixing, the complex was incubated at about 50° C. for about 30 minutes. After incubation, the lipoplex was Dialysed for about 2 hours in 1 liter PBS 1 ⁇ pH7.4 using SPECTRA/POR® FLOAT-A-LYZER® G2 8 KDa. After dialysis the volume was measured and adjusted with PBS to the desired siRNA concentration. The lipoplex was incubated at about 4° C. until in vivo tail vein injection.
  • RNAi against Factor VII (FVII) mouse gene having the Antisense sequence 5′-AUUUGCACAGAUCAGCUGCUCAUUC-3′ and the negative control Medium GC content-RNAi were complexed with pre-liposomes as previously described and tested invivo. 200 ⁇ l of lipoplexes containing FVII or CTRL RNAi in PBS were injected per 20 g mouse, by low pressure tail vein injection at a dose of 10 mg/kg and 2 mg/kg (siRNA dose). Thirty six hours after tail vein injection liver tissue and serum was collected for mRNA and protein knock down analysis respectively.
  • FVII Factor VII
  • RNA Frozen liver tissue was ground into powder and RNA was extracted using TRIZOL® PLUS RNA Purification System (Invitrogen). Total RNA (about 750 ng) for the first strand synthesis was determined using SUPERSCRIPT® III RT kit (Invitrogen) and QPCR analysis was performed using taqman assay using EXPRESS qPCR Supermix Universal (cat# 11785-01K).
  • Factor VII serum protein level was determined as follows. Animals were anesthetized by intramuscular injection of Ketamine/xylazine/Acepromazine (75/5/1 mg/kg respectively), blood was collected by retroorbital bleed, and serum was processed to measure Factor VII protein level using a chromogenic assay (Biophen FVII, Aniara Corporation) according to manufacturers' protocols.
  • FIG. 1 provides a graph summarizing the relative % of Factor VII protein remaing activity, as measured by chromogenic assay, of the different lipoplex formulations in liver. Referring to the x axis, the numbers refer to the cationic lipid compounds tested, as shown in Table 1. The data demonstrate that such formulations do possess activity in vivo. No knockdown was observed when the CTRL negative control was injected in the liver.
  • FIG. 2 provides a graph summarizing the relative % of mRNA Factor VII as measured by qRT-PCR using 2 Taqman assays.
  • the left-hand bar refers to Taqman 29 assay and the right hand bar refers to Taqman 33 assys.
  • the numbers along the x axis refer to the cationic lipid compounds tested, as shown in Table 1. The data demonstrate that such formulations do possess activity in vivo. No knockdown was observed when the CTRL negative control was injected in the liver.
  • FIG. 3 provides a graph summarizing the relative % of Factor VII mRNA as measured by qRT-PCR as described above. The results are normalized and are expressed as a % remaining FVII expression (y axis) as a function of administered dosage (mg/kg body weight).
  • lipids of the present invention were formulated into lipid compositions, which also comprise cholesterol or DOPE, buffers and ethanol.
  • Animal origin-free cholesterol was purchased from Sigma-Aldrich (St Louis, Mo.) and DOPE was purchased from Avanti Lipids (Alabaster, Ala.).
  • Silencer® Select CSNK2A1 siRNA and Silencer® Select negative control siRNA (cat #4390824 siRNA id# s3637 and cat #4390843) having the antisense sequence AAACUAUAAUCGUACAUCUGA and UUACGUCGUCGCGUCGUUATT, respectively, were resuspended with nuclease-free water to a stock concentration of 50 ⁇ M, which were further diluted to meet downstream experiments and is commercially available from Life Technologies (Carlsbad, Calif.).
  • Primary rat cortex neuronal cells were purchased from GIBCO (Cat. No. A10840) and maintained in NeurobasalTM medium (cat #21103-049) supplemented with 2% B-27® Serum-free (50 ⁇ cat #17504-044) and 0.5 mM GlutaMAXTM (cat #35050-061).
  • Primary rat hippocampal neuronal cells were purchased from GIBCO (Cat. No. A10841) and maintained in NeurobasalTM medium (cat #21103-049) supplemented with 2% B-27® Serum-free (50 ⁇ cat #17504-044), 0.5 mM GlutaMAXTM (cat #35050-061) and 25 nM L-Glutamate (Sigma cat #G-2834).
  • Hela cells were purchased from ATCC (ATCC # CCL-2) and maintained DMEM, high glucose, GlutaMAXTM, pyruvate (cat #10569-010) supplemented with 10% fetal bovine serum, US certified, heat inactivated (cat #10082-139) without antibiotics
  • Neuronal cells were plated in 96-well plates coated poly-d-lysine two days before transfection at 10K cells/well. Hela cells were plated in 96-well plates one day prior to transfection at 25K/well.
  • siRNAs (3 pmol) were diluted 20 ul nuclease-free water at a final concentration of 30 nM in 1.5 mL tubes. Master mixes were made to cover all wells to be transfected for each siRNA. Lipid dilutions were prepared by pipetting 0.15, 0.3 and/or 0.6 ⁇ l/well or 0.3 ul LIPOFECTAMINETM RNAiMAX as a control in Opti-MEM® I in total final volume of 10 ⁇ l/well.
  • the lipid and siRNA complexes were prepared by adding 10 ul of the lipid mixture to 20 ⁇ l of diluted siRNA. The complexes were mixed by pipetting up and down and incubated at room temperature for 10 minutes. After the 10 minute incubation, 30 ⁇ l of the complex was then added to the pre-plated cells containing 120 ul of growth medium cultured in 96-well plates containing 150 ul. After 24 hours, cells were harvested with the TaqMan° Gene Expression Cells-to-CTTM kit (cat #AM1728, Life Technologies) according to the manufacturer's protocol.
  • RT reverse transcription
  • FIG. 4 depicts a graph summarizing the relative percent of CSNK2A1 remaining activity normalized to negative control as measured by qPCR assay of the Compound 83 lipoplex formulations in HeLa cells, rat primary cortical neurons and rat primary hippocampal neurons.
  • the numbers refer to the final volume of cationic lipid formulation summarized in Table 3 for each well.
  • FIG. 5 provides a graph summarizing the relative percent of CSNK2A1 remaining activity normalized to negative control, as measured by qPCR assay of Compound 67 lipoplex formulations in Hela, primary cortical neurons and primary hippocampal neurons. Referring to the x axis, the numbers refer to the final volume of cationic lipid per well tested, as shown in Table 3. The data demonstrate that such formulations do possess activity in vitro.
  • Cationic lipid 87 (shown above) was synthesized at Life Technologies, Carlsbad, Calif. according to methods described herein; Animal origin-free Cholesterol was purchased from Sigma-Aldrich (StLouis), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (C16PEG)) was purchased from Avanti Lipids Alabaster. CB547, Cholesterol and C16PEG were diluted in 100% ethanol at 40° C. The lipid mixture was then mixed in 200 mM Sodium Acetate, pH 5.2 using a syringe equipped with a 27G needle at a flow rate of 20 ml/minute. The formulation was then stored at 4° C.
  • a DNA plasmid containing the cDNA sequence was synthesis (GENEART® Gene Synthesis) and cloned into a plasmid DNA vector.
  • Purified plasmid DNA is digested with Ase I restriction enzyme (New England Biosciences, Cat. No. R0526) according to manufacturer's protocol.
  • the linearized DNA was purified using PureLinkTM PCR purification kit (Life Technologies, Cat. No. K310001) according to manufacturer's protocol, and eluted with purified water. DNA concentration was determined by UV absorbance.
  • the Promega RiboMAXTTM Large Scale RNA production System-T7 (Cat. No. P1300) was utilized to synthesize mRNA according to manufacture's protocol. In each reaction, 5-10 ⁇ g of linearized DNA yielded 200-250 ⁇ g of mRNA.
  • mRNA was purified using phenol:chloroform extraction followed by ethanolic precipitation.
  • the mRNA product was resuspended in purified water and the concentration was determined
  • the mRNA was capped utilizing ScriptCapTM m 7 G Capping System (Cat. No. C-SCCE0625) and ScriptCapTM 2′-O-Methyltransferase Kit (Cat. No. C-SCMT0625), both from CellScriptTM.
  • the capped mRNA was polyadenylated using Epicentre® Poly(A)Polymerase Tailing Kit (Cat. No. PAP5104H).
  • the final product is purified again via phenol:chloroform extraction followed by nucleic acid precipitation.
  • the purified mRNA is resuspended in purified water and concentration determined Concentration was adjusted to 3 mg/ml in water and stored at ⁇ 80° C.
  • the lipoplex (mRNA preliposome complex) was prepared by mixing equal volumes of preliposome and mRNA solution. After mixing, the complex was incubated at 50° C. for 30 minutes, then dialysed for 2 hours in 1 liter phosphate buffered saline (PBS), pH 7.4 using Spectra/Por® Float-A-Lyzer® G2 8 kDa. After dialysis, the volume was measured and adjusted with PBS to the desired mRNA concentration. The lipoplex was stored at 4° C. until in vivo injection.
  • PBS phosphate buffered saline
  • mice were purchased from Jackson laboratories (B6.129S4-Gt(ROSA)26Sor tm3(CAG-luc)Tyj /J). This mouse bears a CMV driven luciferase reporter gene with a loxP-flanked STOP codon under the control of a CMV promoter. In the presence of Cre recombinase, the loxP sites recombine to excise the STOP codon, thereby allowing translation of the luciferase reporter protein.
  • IACUC Institutional Animal Care and Use Committee
  • mice aged from 4 to 6 weeks carrying lacZ gene (B6.129S4-Gt(ROSA)26Sor tm1Sor /J) were purchased. Expression of lacZ was determined by qRT-PCR using standard methods. Two 200 ⁇ l of lipoplex containing mRNA CRE in PBS prepared as described above were injected per mouse by low pressure tail vein injection at a dose of 1.5 mg/kg and 0.5 mg/kg (mRNA dose). For luminescence imaging, mice received 150 mg of firefly luciferase (Biosynth AG, Staad, Switzerland) per kg body weight given i.p.
  • mice After anesthesia with isoflurane gas (Abbott Laboratories, North Chicago, Ill.), the mice were placed into a Xenogen IVIS imaging station (Xenogen Corp., Alameda, Calif.) and imaged using Living Image Software (Xenogen Corporation).
  • Xenogen IVIS imaging station Xenogen Corp., Alameda, Calif.
  • Living Image Software Xenogen Corporation
  • FIG. 6A shows whole body images of mice treated with a transfection complex containing Cre mRNA and lipid 87 at 1.5 mg/kg (left), 0.5 mg/kg (middle) or PBS (right). Luciferase expression was measured using Xenogen IVIS and signal was quantified.
  • FIG. 6B shows whole mount lung, heart, spleen and liver (as indicated) organs dissected from each of the two treated mice shown in FIG. 6A .
  • FIG. 7 A-F is a graphical representation showing analysis of LacZ gene expression as measured by qRT-PCR.
  • Mice treated with a transfection complex containing Cre mRNA and lipid 87 displayed a much stronger expression of the lacZ gene in Spleen (compare spleen control HMBS expression FIG. 7A with spleen LacZ expression FIG. 7B ), Liver (compare liver control HMBS expression FIG. 7C with liver LacZ expression FIG. 7D ) compared to control as determined by Cts.
  • cDNA input was normalized using HMBS. No increase expression of lacZ was observed in Kidney ( FIG. 7E and FIG. 7F ).

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Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090291131A1 (en) * 2007-12-27 2009-11-26 Protiva Biotherapeutics, Inc. Silencing of polo-like kinase expression using interfering rna
US20110060032A1 (en) * 2004-06-07 2011-03-10 Protiva Biotherapeutics, Inc. Lipid encapsulating interfering rna
US20110091525A1 (en) * 2003-09-15 2011-04-21 Protiva Biotherapeutics, Inc. Polyethyleneglycol-modified lipid compounds and uses thereof
US20110117125A1 (en) * 2008-01-02 2011-05-19 Tekmira Pharmaceuticals Corporation Compositions and methods for the delivery of nucleic acids
US20110189300A1 (en) * 2004-11-17 2011-08-04 Protiva Biotherapeutics, Inc. siRNA SILENCING OF APOLIPOPROTEIN B
US20110201667A1 (en) * 2009-07-20 2011-08-18 Protiva Biotherapeutics, Inc. Compositions and methods for silencing ebola virus gene expression
US8455455B1 (en) 2010-03-31 2013-06-04 Protiva Biotherapeutics, Inc. Compositions and methods for silencing genes involved in hemorrhagic fever
US8466122B2 (en) 2010-09-17 2013-06-18 Protiva Biotherapeutics, Inc. Trialkyl cationic lipids and methods of use thereof
US8492359B2 (en) 2008-04-15 2013-07-23 Protiva Biotherapeutics, Inc. Lipid formulations for nucleic acid delivery
US8513403B2 (en) 2005-11-02 2013-08-20 Protiva Biotherapeutics, Inc. Modified siRNA molecules and uses thereof
US8569256B2 (en) 2009-07-01 2013-10-29 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
WO2013166339A1 (en) 2012-05-02 2013-11-07 Life Technologies Corporation High yield transient expression in mammalian cells using unique pairing of high density growth and transfection medium and expression enhancers
US9006417B2 (en) 2010-06-30 2015-04-14 Protiva Biotherapeutics, Inc. Non-liposomal systems for nucleic acid delivery
US9018187B2 (en) 2009-07-01 2015-04-28 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
US9023820B2 (en) 2009-01-26 2015-05-05 Protiva Biotherapeutics, Inc. Compositions and methods for silencing apolipoprotein C-III expression
US9035039B2 (en) 2011-12-22 2015-05-19 Protiva Biotherapeutics, Inc. Compositions and methods for silencing SMAD4
US9139554B2 (en) 2008-10-09 2015-09-22 Tekmira Pharmaceuticals Corporation Amino lipids and methods for the delivery of nucleic acids
US9492386B2 (en) 2002-06-28 2016-11-15 Protiva Biotherapeutics, Inc. Liposomal apparatus and manufacturing methods
WO2017011598A1 (en) 2015-07-13 2017-01-19 Life Technologies Corporation System and method for improved transient protein expression in cho cells
WO2017176974A1 (en) * 2016-04-06 2017-10-12 Ohio State Innovation Foundation Biodegradable amino-ester nanomaterials for nucleic acid delivery
US9868692B2 (en) 2015-09-17 2018-01-16 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US9878042B2 (en) 2009-07-01 2018-01-30 Protiva Biotherapeutics, Inc. Lipid formulations for delivery of therapeutic agents to solid tumors
US10195156B2 (en) 2015-12-22 2019-02-05 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
US20190062749A1 (en) * 2015-06-26 2019-02-28 Suzhou Ribo Life Science Co., Ltd. Sirna, pharmaceutical composition and conjugate which contain sirna, and uses thereof
US10323076B2 (en) 2013-10-03 2019-06-18 Modernatx, Inc. Polynucleotides encoding low density lipoprotein receptor
US10857105B2 (en) 2017-03-15 2020-12-08 MordernaTX, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
EP3756690A2 (de) 2013-12-12 2020-12-30 Life Technologies Corporation Membranpenetrierende peptide zur erhöhung der transfektion und zusammensetzungen und verfahren zur verwendung davon
US11066355B2 (en) 2019-09-19 2021-07-20 Modernatx, Inc. Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents
US11203569B2 (en) 2017-03-15 2021-12-21 Modernatx, Inc. Crystal forms of amino lipids
US11246933B1 (en) 2011-12-07 2022-02-15 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
US11583504B2 (en) 2016-11-08 2023-02-21 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
US11591544B2 (en) 2020-11-25 2023-02-28 Akagera Medicines, Inc. Ionizable cationic lipids
WO2023045036A1 (zh) * 2021-09-23 2023-03-30 深圳市人民医院 一种mRNA递送系统及其制备方法和应用
US11969506B2 (en) 2017-03-15 2024-04-30 Modernatx, Inc. Lipid nanoparticle formulation
US12064479B2 (en) 2022-05-25 2024-08-20 Akagera Medicines, Inc. Lipid nanoparticles for delivery of nucleic acids and methods of use thereof
US12077501B2 (en) 2017-06-14 2024-09-03 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
US12129467B2 (en) 2024-01-11 2024-10-29 Arbutus Biopharma Corporation Non-liposomal systems for nucleic acid delivery

Families Citing this family (59)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103380113B (zh) * 2010-11-15 2018-03-30 生命科技公司 含胺的转染试剂及其制备和使用方法
US20140308304A1 (en) * 2011-12-07 2014-10-16 Alnylam Pharmaceuticals, Inc. Lipids for the delivery of active agents
JP6817194B2 (ja) * 2014-05-12 2021-01-20 ルースタービオ インコーポレイテッド 即時プリント可能な細胞及び一体化されたデバイス
WO2015178343A1 (ja) * 2014-05-20 2015-11-26 国立大学法人北海道大学 ｓｉＲＮＡ細胞内送達のための脂質膜構造体
HRP20221536T1 (hr) 2014-06-25 2023-02-17 Acuitas Therapeutics Inc. Novi lipidi i formulacije lipidnih nanočestica za isporuku nukleinskih kiselina
SI3313829T1 (sl) 2015-06-29 2024-09-30 Acuitas Therapeutics Inc. Lipidi in formulacije lipidnih nanodelcev za dostavo nukleinskih kislin
CN108431224B (zh) * 2015-09-01 2020-01-17 苏州瑞博生物技术有限公司 一种小干扰核酸和药物组合物及其用途
CN113636947A (zh) 2015-10-28 2021-11-12 爱康泰生治疗公司 用于递送核酸的新型脂质和脂质纳米颗粒制剂
CN108779074A (zh) * 2016-03-01 2018-11-09 分子传递有限公司 植物病毒移动蛋白和其使用方法
AU2017286606A1 (en) 2016-06-14 2018-12-13 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
CN113652431A (zh) * 2016-12-21 2021-11-16 苏州瑞博生物技术股份有限公司 一种小干扰核酸和药物组合物及其用途
CN108210510B (zh) * 2016-12-21 2021-04-27 苏州瑞博生物技术股份有限公司 一种小干扰核酸药物组合物及其用途
CN108239644B (zh) * 2016-12-23 2021-05-28 苏州瑞博生物技术股份有限公司 一种小干扰核酸和药物组合物及其用途
CN108239643A (zh) * 2016-12-23 2018-07-03 苏州瑞博生物技术有限公司 抑制人和动物中TIMP-1基因表达的siRNA、包含其的组合物及其应用
CN108251421B (zh) * 2016-12-28 2022-01-04 苏州瑞博生物技术股份有限公司 抑制人和动物中COL1A1基因表达的siRNA、包含其的组合物及其应用
CN108251420B (zh) * 2016-12-28 2024-08-02 苏州瑞博生物技术股份有限公司 抑制人和动物中CTGF基因表达的siRNA、包含其的组合物及其应用
CN108265052B (zh) * 2016-12-30 2021-05-28 苏州瑞博生物技术股份有限公司 一种小干扰核酸和药物组合物及其用途
BR112019014314A2 (pt) 2017-01-11 2020-02-18 The Trustees Of The University Of Pennsylvania Composição, e, método de indução de uma resposta imune adaptativa contra vírus zika.
WO2018191657A1 (en) 2017-04-13 2018-10-18 Acuitas Therapeutics, Inc. Lipids for delivery of active agents
AU2018256877B2 (en) 2017-04-28 2022-06-02 Acuitas Therapeutics, Inc. Novel carbonyl lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2019036008A1 (en) * 2017-08-16 2019-02-21 Acuitas Therapeutics, Inc. LIPIDS FOR USE IN LIPID NANOPARTICULAR FORMULATIONS
WO2019036028A1 (en) * 2017-08-17 2019-02-21 Acuitas Therapeutics, Inc. LIPIDS FOR USE IN LIPID NANOPARTICULAR FORMULATIONS
US11524932B2 (en) 2017-08-17 2022-12-13 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
WO2019036000A1 (en) 2017-08-17 2019-02-21 Acuitas Therapeutics, Inc. LIPIDS FOR USE IN LIPID NANOPARTICLE FORMULATIONS
US20190060482A1 (en) * 2017-08-31 2019-02-28 Life Technologies Corporation Cationic lipid compositions for tissue-specific delivery
JP7365052B2 (ja) 2017-12-01 2023-10-19 スーチョウ リボ ライフ サイエンス カンパニー、リミテッド 核酸、当該核酸を含む組成物及び複合体ならびに調製方法と使用
WO2019105414A1 (zh) 2017-12-01 2019-06-06 苏州瑞博生物技术有限公司 一种核酸、含有该核酸的组合物与缀合物及制备方法和用途
WO2019105435A1 (zh) 2017-12-01 2019-06-06 苏州瑞博生物技术有限公司 一种核酸、含有该核酸的组合物与缀合物及制备方法和用途
CN118236391A (zh) 2017-12-01 2024-06-25 苏州瑞博生物技术股份有限公司 一种核酸、含有该核酸的组合物与缀合物及制备方法和用途
US11492620B2 (en) 2017-12-01 2022-11-08 Suzhou Ribo Life Science Co., Ltd. Double-stranded oligonucleotide, composition and conjugate comprising double-stranded oligonucleotide, preparation method thereof and use thereof
US11633482B2 (en) 2017-12-29 2023-04-25 Suzhou Ribo Life Science Co., Ltd. Conjugates and preparation and use thereof
CN110283223B (zh) * 2018-03-19 2022-01-04 广州市锐博生物科技有限公司 一种阳离子脂质分子及其在核酸递送中的应用
CN111655849B (zh) 2018-08-21 2024-05-10 苏州瑞博生物技术股份有限公司 一种核酸、含有该核酸的药物组合物和缀合物及其用途
CN111655297A (zh) 2018-09-30 2020-09-11 苏州瑞博生物技术有限公司 一种siRNA缀合物及其制备方法和用途
AU2019383413A1 (en) * 2018-11-21 2021-05-27 Translate Bio, Inc. Cationic lipid compounds and compositions thereof for use in the delivery of messenger RNA
WO2020135673A1 (zh) 2018-12-28 2020-07-02 苏州瑞博生物技术有限公司 一种核酸、含有该核酸的组合物与缀合物及制备方法和用途
MX2021008358A (es) 2019-01-11 2021-09-30 Acuitas Therapeutics Inc Lipidos para la administracion de agentes activos en nanoparticulas lipidicas.
BR112021020664A2 (pt) 2019-04-16 2021-12-07 Genfit Composições e métodos para a estabilização de micro-rna
EP3974532A4 (de) 2019-05-22 2024-01-24 Suzhou Ribo Life Science Co., Ltd. Nukleinsäure, pharmazeutische zusammensetzung, konjugat, herstellungsverfahren und verwendung
CN113286888B (zh) 2019-05-22 2023-12-22 苏州瑞博生物技术股份有限公司 核酸、药物组合物与缀合物及制备方法和用途
US20240200060A1 (en) * 2019-05-22 2024-06-20 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, pharmaceutical composition, conjugate, preparation method, and use
US20240200076A1 (en) 2019-05-22 2024-06-20 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, pharmaceutical composition, conjugate, preparation method, and use
CN111973618B (zh) * 2019-05-23 2024-02-02 苏州瑞博生物技术股份有限公司 核酸、药物组合物与siRNA缀合物及制备方法和用途
AU2020282453A1 (en) 2019-05-24 2021-12-16 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, pharmaceutical composition and conjugate, preparation method and use
CN113795582B (zh) 2019-05-24 2024-05-24 苏州瑞博生物技术股份有限公司 核酸、药物组合物与缀合物及制备方法和用途
JP2022534069A (ja) 2019-05-24 2022-07-27 スーチョウ リボ ライフ サイエンス カンパニー、リミテッド 核酸、薬物組成物及び複合体並びに調製方法と使用
WO2021114227A1 (zh) * 2019-12-13 2021-06-17 苏州瑞博生物技术股份有限公司 一种含胺转染试剂和制备方法及转染复合物
WO2021255262A1 (en) 2020-06-19 2021-12-23 Sylentis Sau siRNA AND COMPOSITIONS FOR PROPHYLACTIC AND THERAPEUTIC TREATMENT OF VIRUS DISEASES
US11976019B2 (en) 2020-07-16 2024-05-07 Acuitas Therapeutics, Inc. Cationic lipids for use in lipid nanoparticles
EP4015634A1 (de) 2020-12-15 2022-06-22 Sylentis, S.A.U. Sirna und zusammensetzungen zur prophylaktischen und therapeutischen behandlung von viruskrankheiten
WO2022143531A1 (zh) 2020-12-29 2022-07-07 苏州瑞博生物技术股份有限公司 一种核酸、含有该核酸的药物组合物与siRNA缀合物及制备方法和用途
EP4342881A1 (de) * 2021-05-20 2024-03-27 Beijing Institute Of Technology Verbindung, liposom und verwendungen davon
WO2023284763A1 (zh) 2021-07-16 2023-01-19 苏州瑞博生物技术股份有限公司 双链寡核苷酸、含双链寡核苷酸的组合物与缀合物及制备方法和用途
KR20240034787A (ko) 2021-07-16 2024-03-14 쑤저우 리보 라이프 사이언스 컴퍼니, 리미티드 핵산, 이를 함유하는 조성물 및 접합체, 제조 방법 및 용도
CN114262275B (zh) * 2021-12-15 2023-09-29 华中师范大学 一种高效低毒性dna及rna脂质递送载体
KR20240117104A (ko) 2021-12-21 2024-07-31 쑤저우 리보 라이프 사이언스 컴퍼니, 리미티드 핵산, 핵산을 포함하는 조성물 및 접합체, 이들의 제조 방법 및 용도
CN114306641A (zh) * 2022-02-17 2022-04-12 大连民族大学 一种胆固醇氨基衍生物-蔗糖酯型阳离子脂质体/基因复合物及其制备方法
WO2024145435A2 (en) 2022-12-29 2024-07-04 Life Technologies Corporation Lipid compositions and methods for delivery to immune cells
CN115745820B (zh) * 2023-01-05 2023-04-28 北京悦康科创医药科技股份有限公司 长效低毒的新型阳离子脂质化合物及其组合物

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130022665A1 (en) * 2011-06-08 2013-01-24 Yoshiro Niitsu Cationic lipids for therapeutic agent delivery formulations

Family Cites Families (50)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA780214A (en) 1968-03-12 Wean Industries Belt wrapper
US2580922A (en) 1942-09-19 1952-01-01 Nat Aluminate Corp Prevention of foaming in steam generation
US2844629A (en) * 1956-04-25 1958-07-22 American Home Prod Fatty acid amides and derivatives thereof
DE1129950B (de) * 1960-06-11 1962-05-24 Knoll Ag Verfahren zur Herstellung von antimikrobiell wirksamen Derivaten des Glycinamids
DE1165038B (de) * 1962-03-03 1964-03-12 Knoll Ag Verfahren zur Herstellung von antimikrobiell wirksamen Derivaten des Glycinamids
DE1162380B (de) * 1962-03-03 1964-02-06 Knoll Ag Verfahren zur Herstellung von antimikrobiell wirksamen Derivaten des Glycinamids
CH575909A5 (de) 1972-08-21 1976-05-31 Ciba Geigy Ag
US5334761A (en) 1992-08-28 1994-08-02 Life Technologies, Inc. Cationic lipids
US5316948A (en) 1992-09-04 1994-05-31 Life Technologies, Inc. N4 -methyl-2'-deoxycytidine 5'-triphosphate and its use in polymerase-catalyzed nucleic acid syntheses
WO1995002698A1 (en) 1993-07-12 1995-01-26 Life Technologies, Inc. Composition and methods for transfecting eukaryotic cells
US5674908A (en) 1993-12-20 1997-10-07 Life Technologies, Inc. Highly packed polycationic ammonium, sulfonium and phosphonium lipids
US6075012A (en) 1994-02-11 2000-06-13 Life Technologies, Inc. Reagents for intracellular delivery of macromolecules
US5627159A (en) 1994-10-27 1997-05-06 Life Technologies, Inc. Enhancement of lipid cationic transfections in the presence of serum
US6071890A (en) 1994-12-09 2000-06-06 Genzyme Corporation Organ-specific targeting of cationic amphiphile/DNA complexes for gene therapy
WO1996040961A1 (en) 1995-06-07 1996-12-19 Life Technologies, Inc. Peptide-enhanced cationic lipid transfections
US6051429A (en) 1995-06-07 2000-04-18 Life Technologies, Inc. Peptide-enhanced cationic lipid transfections
JPH11510489A (ja) 1995-07-21 1999-09-14 ジンタ・インコーポレイテッド 新規アミド基本カチオン性脂質
FR2741066B1 (fr) 1995-11-14 1997-12-12 Rhone Poulenc Rorer Sa Nouveaux agents de transfection et leurs applications pharmaceutiques
DE19621871A1 (de) 1996-05-31 1997-12-04 Huels Chemische Werke Ag Ester- und amidgruppenhaltige Polyamine sowie ein Verfahren zu ihrer Herstellung und deren Verwendung
CA2217550A1 (en) 1996-10-22 1998-04-22 F. Hoffmann-La Roche Ag Cationic lipids for gene therapy
AU739998B2 (en) * 1997-06-12 2001-10-25 Transgene S.A. Novel lipid complexes for transferring at least one therapeutically active substance, in particular a polynucleotide, into a target cell, and use in gene therapy
US6369268B1 (en) 1997-11-06 2002-04-09 Ciba Specialty Chemicals Corporation Diester amine adducts
NZ512244A (en) 1998-11-12 2003-12-19 Invitrogen Corp Polycationic transfection reagents for introducing anions into a cell
US6238008B1 (en) 1999-09-08 2001-05-29 Paul D. Forsythe Wheel and tension spoke system
GB0031068D0 (en) 2000-12-19 2001-01-31 Smithkline Beecham Plc Novel compounds
US20060009409A1 (en) 2002-02-01 2006-01-12 Woolf Tod M Double-stranded oligonucleotides
WO2003064625A2 (en) 2002-02-01 2003-08-07 Sequitur, Inc. Oligonucleotide compositions with enhanced efficiency
CN1308292C (zh) * 2002-09-19 2007-04-04 西巴特殊化学品控股有限公司 作为抗蚀剂的琥珀酸半酰胺
EP1587908A4 (de) 2003-01-09 2008-02-20 Invitrogen Corp Zelluläre zuführung und aktivierung von polypeptid-nukleinsäurekomplexen
WO2004105697A2 (en) 2003-05-22 2004-12-09 Molecular Transfer, Inc. Novel lipids for transfection of nucleic acids
GB0502482D0 (en) 2005-02-07 2005-03-16 Glaxo Group Ltd Novel compounds
US7915230B2 (en) 2005-05-17 2011-03-29 Molecular Transfer, Inc. Reagents for transfection of eukaryotic cells
US9006487B2 (en) 2005-06-15 2015-04-14 Massachusetts Institute Of Technology Amine-containing lipids and uses thereof
US7199267B1 (en) * 2005-10-21 2007-04-03 Mediquest Therapeutics, Inc. Recognition of oligiosaccaride molecular targets by polycationic small molecule inhibitors and treatment of immunological disorders and infectious diseases
EP2004141A2 (de) 2006-03-17 2008-12-24 Novosom AG Effizientes verfahren zum beladen von amphoteren liposomen mit nukleinsäure-wirkstoffen
EP2604255B1 (de) 2006-05-05 2017-10-25 Molecular Transfer, Inc. Neuartige Reagenzien zur Transfektion von eukaryotischen Zellen
KR101129509B1 (ko) * 2006-10-03 2012-04-13 알닐람 파마슈티칼스 인코포레이티드 지질 함유 조성물
EP2548962B1 (de) 2007-09-19 2016-01-13 Applied Biosystems, LLC Sirna-sequenz-unabhängige Modifikationsformate zur Verringerung von das Ziel verfehlenden phänotypischen Effekten bei RNAI und stabilisierte Formen davon
NZ584048A (en) * 2007-10-02 2012-08-31 Marina Biotech Inc Lipopeptides for delivery of nucleic acids
WO2009086558A1 (en) 2008-01-02 2009-07-09 Tekmira Pharmaceuticals Corporation Improved compositions and methods for the delivery of nucleic acids
EP2296711A2 (de) * 2008-05-29 2011-03-23 MDRNA, Inc. Mehrarmige amine und ihre verwendungen
SI2344639T1 (sl) 2008-10-20 2015-09-30 Alnylam Pharmaceuticals, Inc. Sestavki in postopki inhibicije izražanja transtiretina
US20120009222A1 (en) * 2008-10-27 2012-01-12 Massachusetts Institute Of Technology Modulation of the immune response
CA2742954C (en) 2008-11-07 2018-07-10 Massachusetts Institute Of Technology Aminoalcohol lipidoids and uses thereof
SG10201901089TA (en) * 2008-11-10 2019-03-28 Arbutus Biopharma Corp Novel lipids and compositions for the delivery of therapeutics
AU2010226434A1 (en) 2009-03-20 2011-10-13 Egen, Inc. Polyamine derivatives
US8273869B2 (en) 2009-06-15 2012-09-25 Alnylam Pharmaceuticals, Inc. Lipid formulated dsRNA targeting the PCSK9 gene
US8569256B2 (en) * 2009-07-01 2013-10-29 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
US8466122B2 (en) * 2010-09-17 2013-06-18 Protiva Biotherapeutics, Inc. Trialkyl cationic lipids and methods of use thereof
CN103380113B (zh) * 2010-11-15 2018-03-30 生命科技公司 含胺的转染试剂及其制备和使用方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130022665A1 (en) * 2011-06-08 2013-01-24 Yoshiro Niitsu Cationic lipids for therapeutic agent delivery formulations

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
Guillot-Nieckowski et al."Dendritic vectors...." N. J. Chem. v. 31, p. 1111-1127 (2007) *
Hann et al. "On double bond........" J.C.S. Perkin I. p.307-314 (1982) *
Improper Markush , Fed. Reg. vo. 76(27) 7162-7175, training slides p.1, 64-47 (2001) *
Patani et al. "Bioisosterism....." Chem Rev. 96, p.3147-3176 (1996) *
Perrine "Symmetrical....." CA48:64107 (1954) *
Rubini et al. "Synthesis of isosteric......" Tetrhedron 42(21) 6039-45 (1986) *

Cited By (78)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11298320B2 (en) 2002-06-28 2022-04-12 Arbutus Biopharma Corporation Liposomal apparatus and manufacturing methods
US11318098B2 (en) 2002-06-28 2022-05-03 Arbutus Biopharma Corporation Liposomal apparatus and manufacturing methods
US9504651B2 (en) 2002-06-28 2016-11-29 Protiva Biotherapeutics, Inc. Lipid compositions for nucleic acid delivery
US9492386B2 (en) 2002-06-28 2016-11-15 Protiva Biotherapeutics, Inc. Liposomal apparatus and manufacturing methods
US8936942B2 (en) 2003-09-15 2015-01-20 Protiva Biotherapeutics, Inc. Polyethyleneglycol-modified lipid compounds and uses thereof
US20110091525A1 (en) * 2003-09-15 2011-04-21 Protiva Biotherapeutics, Inc. Polyethyleneglycol-modified lipid compounds and uses thereof
US20110060032A1 (en) * 2004-06-07 2011-03-10 Protiva Biotherapeutics, Inc. Lipid encapsulating interfering rna
US9926560B2 (en) 2004-06-07 2018-03-27 Protiva Biotherapeutics, Inc. Lipid encapsulating interfering RNA
US9181545B2 (en) 2004-06-07 2015-11-10 Protiva Biotherapeutics, Inc. Lipid encapsulating interfering RNA
US20110189300A1 (en) * 2004-11-17 2011-08-04 Protiva Biotherapeutics, Inc. siRNA SILENCING OF APOLIPOPROTEIN B
US8513403B2 (en) 2005-11-02 2013-08-20 Protiva Biotherapeutics, Inc. Modified siRNA molecules and uses thereof
US9074208B2 (en) 2005-11-02 2015-07-07 Protiva Biotherapeutics, Inc. Modified siRNA molecules and uses thereof
US20090291131A1 (en) * 2007-12-27 2009-11-26 Protiva Biotherapeutics, Inc. Silencing of polo-like kinase expression using interfering rna
US9006191B2 (en) 2007-12-27 2015-04-14 Protiva Biotherapeutics, Inc. Silencing of polo-like kinase expression using interfering RNA
US20110117125A1 (en) * 2008-01-02 2011-05-19 Tekmira Pharmaceuticals Corporation Compositions and methods for the delivery of nucleic acids
US9364435B2 (en) 2008-04-15 2016-06-14 Protiva Biotherapeutics, Inc. Lipid formulations for nucleic acid delivery
US8822668B2 (en) 2008-04-15 2014-09-02 Protiva Biotherapeutics, Inc. Lipid formulations for nucleic acid delivery
US8492359B2 (en) 2008-04-15 2013-07-23 Protiva Biotherapeutics, Inc. Lipid formulations for nucleic acid delivery
US11141378B2 (en) 2008-04-15 2021-10-12 Arbutus Biopharma Corporation Lipid formulations for nucleic acid delivery
US9139554B2 (en) 2008-10-09 2015-09-22 Tekmira Pharmaceuticals Corporation Amino lipids and methods for the delivery of nucleic acids
US10653780B2 (en) 2008-10-09 2020-05-19 The University Of British Columbia Amino lipids and methods for the delivery of nucleic acids
US9428751B2 (en) 2009-01-26 2016-08-30 Protiva Biotherapeutics, Inc. Compositions and methods for silencing apolipoprotein C-III expression
US9023820B2 (en) 2009-01-26 2015-05-05 Protiva Biotherapeutics, Inc. Compositions and methods for silencing apolipoprotein C-III expression
US11786598B2 (en) 2009-07-01 2023-10-17 Arbutus Biopharma Corporation Lipid formulations for delivery of therapeutic agents
US12016929B2 (en) 2009-07-01 2024-06-25 Arbutus Biopharma Corporation Lipid formulations for delivery of therapeutic agents
US11446383B2 (en) 2009-07-01 2022-09-20 Arbutus Biopharma Corporation Lipid formulations for delivery of therapeutic agents
US9018187B2 (en) 2009-07-01 2015-04-28 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
US9878042B2 (en) 2009-07-01 2018-01-30 Protiva Biotherapeutics, Inc. Lipid formulations for delivery of therapeutic agents to solid tumors
US8569256B2 (en) 2009-07-01 2013-10-29 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
US9187748B2 (en) 2009-07-20 2015-11-17 Protiva Biotherapeutics, Inc. Compositions and methods for silencing ebola virus gene expression
US20110201667A1 (en) * 2009-07-20 2011-08-18 Protiva Biotherapeutics, Inc. Compositions and methods for silencing ebola virus gene expression
US8716464B2 (en) 2009-07-20 2014-05-06 Thomas W. Geisbert Compositions and methods for silencing Ebola virus gene expression
US8455455B1 (en) 2010-03-31 2013-06-04 Protiva Biotherapeutics, Inc. Compositions and methods for silencing genes involved in hemorrhagic fever
US11718852B2 (en) 2010-06-30 2023-08-08 Arbutus Biopharma Corporation Non-liposomal systems for nucleic acid delivery
US9518272B2 (en) 2010-06-30 2016-12-13 Protiva Biotherapeutics, Inc. Non-liposomal systems for nucleic acid delivery
US9404127B2 (en) 2010-06-30 2016-08-02 Protiva Biotherapeutics, Inc. Non-liposomal systems for nucleic acid delivery
US9006417B2 (en) 2010-06-30 2015-04-14 Protiva Biotherapeutics, Inc. Non-liposomal systems for nucleic acid delivery
US8466122B2 (en) 2010-09-17 2013-06-18 Protiva Biotherapeutics, Inc. Trialkyl cationic lipids and methods of use thereof
US11382979B2 (en) 2011-12-07 2022-07-12 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
US11633480B2 (en) 2011-12-07 2023-04-25 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
US11400158B2 (en) 2011-12-07 2022-08-02 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
US11246933B1 (en) 2011-12-07 2022-02-15 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
US11679158B2 (en) 2011-12-07 2023-06-20 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
US9035039B2 (en) 2011-12-22 2015-05-19 Protiva Biotherapeutics, Inc. Compositions and methods for silencing SMAD4
EP3404095A1 (de) 2012-05-02 2018-11-21 Life Technologies Corporation Transiente expression mit hoher ausbeute in säugetierzellen mit einmaliger paarung von hochdichtem wachstum sowie einem transfektionsmedium und expressionsverstärkern
EP4148122A1 (de) 2012-05-02 2023-03-15 Life Technologies Corporation Transiente expression mit hoher ausbeute in säugetierzellen mit einmaliger paarung von hochdichtem wachstum sowie einem transfektionsmedium und expressionsverstärkern
WO2013166339A1 (en) 2012-05-02 2013-11-07 Life Technologies Corporation High yield transient expression in mammalian cells using unique pairing of high density growth and transfection medium and expression enhancers
US10323076B2 (en) 2013-10-03 2019-06-18 Modernatx, Inc. Polynucleotides encoding low density lipoprotein receptor
EP3756690A2 (de) 2013-12-12 2020-12-30 Life Technologies Corporation Membranpenetrierende peptide zur erhöhung der transfektion und zusammensetzungen und verfahren zur verwendung davon
US20190062749A1 (en) * 2015-06-26 2019-02-28 Suzhou Ribo Life Science Co., Ltd. Sirna, pharmaceutical composition and conjugate which contain sirna, and uses thereof
WO2017011598A1 (en) 2015-07-13 2017-01-19 Life Technologies Corporation System and method for improved transient protein expression in cho cells
US11220476B2 (en) 2015-09-17 2022-01-11 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US9868693B2 (en) 2015-09-17 2018-01-16 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US9867888B2 (en) 2015-09-17 2018-01-16 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US9868692B2 (en) 2015-09-17 2018-01-16 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US9868691B2 (en) 2015-09-17 2018-01-16 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US10442756B2 (en) 2015-09-17 2019-10-15 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US10392341B2 (en) 2015-09-17 2019-08-27 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US10266485B2 (en) 2015-09-17 2019-04-23 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US10195156B2 (en) 2015-12-22 2019-02-05 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
US10799463B2 (en) 2015-12-22 2020-10-13 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
WO2017176974A1 (en) * 2016-04-06 2017-10-12 Ohio State Innovation Foundation Biodegradable amino-ester nanomaterials for nucleic acid delivery
US10457631B2 (en) 2016-04-06 2019-10-29 Ohio State Innovation Foundation Biodegradable amino-ester nanomaterials for nucleic acid delivery
US11583504B2 (en) 2016-11-08 2023-02-21 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
US10857105B2 (en) 2017-03-15 2020-12-08 MordernaTX, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US11203569B2 (en) 2017-03-15 2021-12-21 Modernatx, Inc. Crystal forms of amino lipids
US11969506B2 (en) 2017-03-15 2024-04-30 Modernatx, Inc. Lipid nanoparticle formulation
US12077501B2 (en) 2017-06-14 2024-09-03 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
US11597698B2 (en) 2019-09-19 2023-03-07 Modernatx, Inc. Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents
US11066355B2 (en) 2019-09-19 2021-07-20 Modernatx, Inc. Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents
US11591544B2 (en) 2020-11-25 2023-02-28 Akagera Medicines, Inc. Ionizable cationic lipids
US12077725B2 (en) 2020-11-25 2024-09-03 Akagera Medicines, Inc. Ionizable cationic lipids
WO2023045036A1 (zh) * 2021-09-23 2023-03-30 深圳市人民医院 一种mRNA递送系统及其制备方法和应用
US12064479B2 (en) 2022-05-25 2024-08-20 Akagera Medicines, Inc. Lipid nanoparticles for delivery of nucleic acids and methods of use thereof
US12129467B2 (en) 2024-01-11 2024-10-29 Arbutus Biopharma Corporation Non-liposomal systems for nucleic acid delivery

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