Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
  • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
• • 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
  • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6905—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
  • A61K47/6911—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • 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

Definitions

• Transfection agents such as lipid aggregates comprising cationic lipid components have been used to deliver large anionic molecules, such as nucleic acids, into certain types of cells. See Feigner et al., Nature 337:387-388 (1989); Proc. Natl. Acad. Sci. USA 84:7413 (1987). These agents are not, however, universally effective in all cell types. In many cases, cationic lipids alone are not effective or are only partially effective for transfection.
• New compounds, compositions and methods are provided that improve the efficiency of introducing macromolecules, such as nucleic acids, into cells.
• New cationic lipid molecules are provided, together with compositions containing those lipids and methods for using the new lipid molecules and compositions for transfection.
• the cationic lipids may be used alone for transfection, or they may be used in combination with additional reagents in transfection compositions.
• the cationic lipids may be combined with one or more neutral lipids, additional cationic lipids, one or more cell surface ligands, one or more fusion enhancing agents, and/or one or more nuclear localization agents.
• the resulting compositions may be complexed with one or macromolecules, such as DNA or RNA and used to deliver the macromolecule into eukaryotic cells..
• each R independently is C1-C2 3 alkyl, C1-C2 3 alkenyl, -(CO)Ci-C23 alkyl, or (CO)Ci-C 23 alkenyl; and each R 2 independently is -CH 2 -(CHR 3 )!_6-CH 2 -NHR 4 or -CH 2 -
• R 1 may be Ci 4 -C 2 o alkyl or monounsaturated Ci 4 -C 2 o-alkenyl.
• R 2 may be-CH 2 -(CHR 3 )!_6-CH 2 -NHR 4 and R 3 is H or OH.
• no more than 3 R 3 groups are OH in each R 3 moiety.
• each R 1 may be the same or different, and each R 2 may be the same or different.
• one or both R may be H, or one or both R 10 may be C1-C3 alkyl, for example, one or both R 10 may be methyl .
• R 1 may be C] 2 -C 2 o alkenyl, and the alkenyl moieties may be cis alkenyl.
• one or both R 2 may be-CH 2 -CHOH-(CHR 3 ) 0 - 5 -CH 2 -NHR 4 ; for example one or both R 2 may be-CH 2 -CHOH-CH 2 -NH 2 .
• compositions containing a compound as described above and at least one cationic lipid, and/or at least one neutral lipid are also provided.
• the cationic lipid may be
• TMTOS TMTLS, TMTMS, TMDOS
• N-l-dimethyl-N-l-(2,3-diaoleoyloxypropyl)-2- hydroxypropane-l,3-diamine N-l-dimethyl-N-l-(2,3-diamyristyloxypropyl)-2- hydroxypropane-l,3-diamine
• N-l-dimethyl-N-l-(2,3-diaoleoyloxypropyl)-2-(3-amino-2- hydroxypropyloxy)propane-l,3-diamine N-l-dimethyl-N-l-(2,3-diamyristyloxypropyl)-2-(3- amino-2-hydroxypropyloxy)propane
• composition contains a neutral lipid
• that lipid may be, for example, DOPE, DPhPE, cholesterol, DOPC, Lyso-PE ( l-acyl-2-hydroxy-sn-glycero-3- phosphoethanolamine), Lyso-PC ( l-acyl-3-hydroxy-sn-glycero-3-phosphocholine), or 3- alkyloxy-2-hydroxy-l-acetamidopropane.
• the composition may contain more than one of these neutral lipids.
• compositions containing a compound of structure (I) as described above and a polyamine transfection agent may be, for example, selected from the group consisting of dense star dendrimers, PAMAM dendrimers, N3 ⁇ 4 core dendrimers, ethylenediamine core dendrimers, dendrimers of generation 5 or higher, dendrimers with substituted groups, dendrimers comprising one or more amino acids, grafted dendrimers, activated dendrimers, polyethylenimine, and polyethylenimine conjugates.
• compositions containing a compound of formula (I) as described above and a fusion agent are also provided.
• the fusion agent optionally may comprise a polycationic nucleic acid binding moiety.
• One or more cationic and/or neutral lipids may be present in these compositions.
• compositions containing a compound of formula (I) as described above and a cell surface ligand containing a compound of formula (I) as described above and a cell surface ligand .
• the cell surface ligand optionally may comprise a polycationic nucleic acid binding moiety.
• These compositions may also contain one or more cationic and/or neutral lipids and/or a fusion agent.
• compositions are provided containing a compound of formula (I) as described above and a nuclear localization peptide or protein cell surface ligand .
• These compositions may also contain one or more cationic and/or neutral lipids and/or a fusion agent and/or a cell surface ligand.
• the cell surface ligand and/or the nuclear localization peptide optionally may comprise a polycationic nucleic acid binding moiety.
• compositions may also contain an amphipathic peptide, which in some embodiments may also function as a fusion agent.
• Suitable amphipathic peptides include, but are not limited to, peptides comprising a sequence selected from the group consisting of
• amphipathic peptide may optionally be linked to a polycationic nucleic acid binding moiety, for example via a covalent linkage.
• compositions as described above further comprising a nucleic acid.
• the nucleic acid may be, for example, an RNA molecule, such as an RNAi molecule.
• Methods are provided of introducing a nucleic acid into a eukaryotic cell, comprising contacting the cell with a composition as described above, thereby introducing the nucleic acid.
• the cell may be an animal cell, for example a human cell.
• kits containing a compound of formula (I) and: a neutral lipid, a cationic lipid, a cell surface ligand, a fusion agent and/or a nuclear localization peptide or protein.
• expressing a protein in a cell comprising contacting the cell with an expression vector encoding the protein and a compound of formula (I) as described above or a composition as described above.
• Methods are provided for inhibiting expression of a protein in a cell, in which the cell is contacted with an RNAi molecule and a compound according to claim 1.
• R 2 may be-CH 2 -CHOH- (CHR 3 )!_6-NHR 4 , and advantageously may be -CH 2 -CHOH-CH 2 -NH 2 .
• R advantageously is Ci 2 _ 2 o alkyl or monounsaturated alkenyl.
• At least two neutral lipids are present, which may, for example, be selected from the group consisting of DOPE, DPhPE, cholesterol, DOPC, Lyso-PE ( 1- acyl-2-hydroxy-sn-glycero-3-phosphoethanolamine), Lyso-PC ( l-acyl-3-hydroxy-sn- glycero-3-phosphocholine), and 3-alkyloxy-2-hydroxy-l-acetamidopropane.
• compositions also may contain one or more polyamine transfection agent, such as dense star dendrimers, PAMAM dendrimers, N3 ⁇ 4 core dendrimers, ethylenediamine core dendrimers, dendrimers of generation 5 or higher, dendrimers with substituted groups, Attorney docket no. 01338.8007.WO01 dendrimers comprising one or more amino acids, grafted dendrimers, activated dendrimers, polyethylenimine, and/or polyethylenimine conjugates.
• polyamine transfection agent such as dense star dendrimers, PAMAM dendrimers, N3 ⁇ 4 core dendrimers, ethylenediamine core dendrimers, dendrimers of generation 5 or higher, dendrimers with substituted groups, Attorney docket no. 01338.8007.WO01 dendrimers comprising one or more amino acids, grafted dendrimers, activated dendrimers, polyethylenimine, and/or polyethylenimine conjugates
• compositions may contain a fusion agent, a cell surface ligand. a nuclear localization peptide or protein, amphipathic peptide and/or a nuclear localization agent.
• a nucleic acid also may be present.
• the fusion agent, cell surface ligand, nuclear localization peptide and/or amphipathic peptide optionally may comprise a polycationic nucleic acid binding moiety.
• nucleic acid into a eukaryotic cell, comprising contacting the cell with a composition as described above, thereby introducing the nucleic acid, into the cell, for example a human cell or an animal cell.
• the nucleic acid may, for example, be an expression vector.
• Also provided are methods of inhibiting expression of a protein in a cell comprising contacting the cell with a double stranded RNAi molecule and a compound according to formula (I), or a composition containing that compound.
• Also provided are methods of expressing a protein in a cell comprising contacting the cell with an expression vector encoding the protein and a compound of formula (I), or a composition containing that compound.
• compositions comprising an amphipathic peptide optionally comprising a polycationic nucleic acid binding moiety and a compound having the structure
• each R 1 independently is C1-C2 3 alkyl, C1-C2 3 alkenyl, -(CO)Ci-C2 3 alkyl, or - (CO)Ci-C2 3 alkenyl; each R 2 independently is Ci-C 6 alkyl, or Ci-C 6 alkenyl, optionally interrupted by O; each R 3 independently is H, Ci-C 6 alkyl, Ci-C 6 alkenyl, C2-C6 alkynyl, C3- C7 cycloalkyl, C3-C7 cycloalkyl-Ci-C6 alkyl, Cs-C7-cycloalkenyl, Cs-C7-cycloalkenyl-Ci-C6 alkyl, -(CH 2 ) m NR 6 (CH 2 ) n NHR 7 , -(CH 2 ) 2 - 6 NHR 7 , -(CH 2 )3- 6
• each R 4 independently is H, C C 6 alkyl, or C C 6 alkenyl
• each m independently is 2-5, each n independently is 2-5, each x independently is 0 or 1, y is 0-2, z is 1-6, R 6 and R 7
• R 8 independently are H, an amine protecting group, -(CO)Ci-C2 3 alkyl, or -(CO)Ci-C2 3 alkenyl;
• R 8 is H, Ci-C 6 alkyl, or Ci-C 6 alkenyl, and Het is a 5-7 membered monocyclic basic heterocycle, or an 8-11 membered bicyclic basic heterocycle.
• compositions also may include one or more reagents such as a cationic lipid, a cell surface ligand, a cell surface ligand comprising a polycationic nucleic acid binding moiety, a fusion agent, a fusion agent comprising a polycationic nucleic acid binding moiety, a nuclear localization peptide or protein, and a nuclear localization peptide or protein comprising a polycationic nucleic acid binding moiety.
• these compositions contain a compound of structure (II), an amphipathic peptide optionally comprising a polycationic nucleic acid binding moiety, a cationic lipid and a cell surface ligand comprising a polycationic nucleic acid binding moiety.
• Figures 1(A) and 1(B) show the results of transfection of a ⁇ -galactosidase gene into 293 GT and CHO Kl cells respectively, compared to the results obtained using commercial reagents.
• Figures 2(A) and 2(B) show the results of transfection of a ⁇ -galactosidase gene into HeLa and NIH 3T3 cells respectively, compared to the results obtained using commercial reagents.
• Figures 3(A) and 3(B) show the results of transfection of a ⁇ -galactosidase gene into A549 and HMSC-Ad cells respectively, compared to the results obtained using commercial reagents.
• Figures 4(A) and 4(B) show the results of transfection of a ⁇ -galactosidase gene into Nuff and UB Stem cells respectively, compared to the results obtained using commercial reagents.
• Positively charged (cationic) molecules are provided that are useful for improved methods of delivering macromolecules into eukaryotic cells.
• the compositions and methods are effective in a wide variety of cells, and provide a high efficiency of transfection.
• molecules based on a core of N,N'-disubstituted 2,3,- dihydroxy-l,4-butanediamine are useful for efficient delivery of macromolecules into cells.
• These molecules advantageously can be used with one or more neutral lipids and additional components such as fusogenic or fusion-enhancing molecules, additional cationic lipids, cell surface ligands, cell adhesion molecules, and nuclear localization agents, in a complex with the macromolecule.
• the complex is easily prepared by straightforward methods and can be used on a wide variety of cells.
• nucleic acid transfection efficiency of cationic lipids in general, and the new cationic lipids described herein in particular can be dramatically enhanced in many cases by reducing the net positive charge on the lipid by partial acylation of free primary and secondary amine functions on the lipid. Unexpectedly, this reduction in charge has been shown to greatly increase the ability of transfection complexes containing the modified lipid to efficiently transfect cells. Thus, for a lipid with N primary or secondary amines, it is possible to acylate up to N-1 of the amine groups.
• acyl groups in a lipid preparation with distinct amino groups will be statistical, because regiospecific acylation likely will not be possible unless the acylation is carried out as part of a more elaborate synthetic scheme.
• the distribution of acyl groups will be affected not only by the stoichiometry of the acylation reagent with respect to the lipid, but will also be affected by Attorney docket no. 01338.8007.WO01 the reactivity of the amine groups, both initially (in the non-acylated amine) but also during the reaction, as acylation activity at a free amine is potentially affected by acylation at another amine elsewhere in the molecule.
• the enhancement of transfection is particularly marked for lipids containing 4 or more reactive amines, in addition to the possible presence of tertiary or quaternary amines, but is not necessarily limited to these lipids. This observed result is surprising in light of the prejudice in the art that a relatively high charge on a cationic lipid is desirable to enhance binding of negatively charged nucleic acids.
• New lipid molecules having the structure (I):
• each R 1 independently is C 1 -C23 alkyl, C 1 -C23 alkenyl, -(CO)Ci-C 2 3 alkyl, or (CO)CrC 23 alkenyl; and each R 2 independently is -CH ⁇ CHR ⁇ Le-CHz-NHR 4 or -CH 2 - (CHR J )o_6-CH 2 -OH, where each R 3 independently is H, OH, or NH2, R is H or CH 3 ; and R 10 is H or Ci-Cs alkyl.
• R 1 may be Ci 4 -C 2 o alkyl or monounsaturated Ci 4 -C 2 o-alkenyl.
• R 2 may be-CH 2 -(CHR 3 )!_6-CH 2 -NHR 4 and R 3 is H or OH.
• no more than 3 R 3 groups are OH in each R 3 moiety.
• each R 1 may be the same or different, and each R 2 may be the same or different.
• one or both R 10 may be H, or one or both R 10 may be C 1 -C3 alkyl, for example, one or both R 10 may be methyl .
• R 1 may be Ci 2 -C 2 o alkenyl, and the alkenyl moieties may be cis alkenyl.
• one or both R 2 may be-CH 2 -CHOH-(CHR 3 ) 0 - 5 -CH 2 -NHR 4 ; for example one or both R 2 may be-CH 2 -CHOH-CH 2 -NH 2 .
• R advantageously is C 14-18 alkyl or C 14-18 monounsaturated alkenyl.
• Each R 1 , R 2 and/or each R 10 may be the same or different and thus the molecule may be symmetrical or non-symmetrical.
• R 2 may be C1-C3 alkyl and/or R 1 is monounsaturated C12-C2 0 alkenyl.
• One or both C12-C2 0 alkenyl moieties in R 1 when present, may be cis alkenyl.
• each R 1 independently may be C1-C23 alkyl, C1-C23 alkenyl, - (CO)Ci-C23 alkyl, or -(CO)CrC 2 3 alkenyl.
• Each R 2 independently may be Ci-C 6 alkyl, or Ci-C 6 alkenyl, optionally interrupted by up to 2 O atoms.
• the peptide advantageously contains multiple positively charged amino acid side chains.
• the peptide may contain one or more lysine, arginine, and/or histidine residues.
• Other positively charged amino acids also may be used, whether or not naturally occurring.
• ornithine, homo-arginine and other amino acids containing amine, guanidine; imidazole and other basic heterocycles and the like can be used.
• Each m independently may be 2-5, and each n independently may be 2-5, while each x independently may be 0 or 1, each y may be 0-2, and each z may be 1-6.
• R 6 and R 7 independently may be H, an amine protecting group, -(CO)Ci-C23 alkyl, or -(CO)Ci-C23 alkenyl
• R 8 may be H, Ci-C 6 alkyl, or Ci-C 6 alkenyl
• Het may be a 5-7 membered monocyclic basic heterocycle, or an 8- 11 membered bicyclic basic heterocycle.
• the molecule of structure (II) may be symmetrical or non-symmetrical with regard to each or all of the substituents R ] -R 5 independently; that is each R 1 may be the same or different, each R 2 may be the same or different, each R 3 may be the same or different, each R 4 may be the same or different, and/or each R 5 may be the same or different.
• R 2 may be C1-C3 alkyl and/or R 1 may be monounsaturated C12-C2 0 alkenyl; for example one or both C12-C2 0 alkenyl moieties in R 1 may be cis alkenyl.
• R 3 may be -(CH 2 ) m NR 6 (CH 2 ) n NHR 7 and/or R 5 may be -(CH 2 ) 2- 6NHR 7 .
• m may be 3
• n may be 3
• R 5 may be -(CH2) 3 NHR 7
• each R 6 and each R 7 may be H.
• At least one R 6 or R 7 may be -(CO)C C 2 3 alkyl, or -(CO)CrC 2 3 alkenyl and the remainder are H.
• each R 1 may be C12-C2 0 alkyl or C12-C2 0 monounsaturated alkenyl
• each R 2 may be C1-C3 alkyl
• each R 3 may be -(CH 2 ) 3 NH(CH 2 ) 3 NH 2
• each R 4 may be H
• each R 5 may be -(CH 2 ) 3 NH 2 .
• each R 1 may be C12-C2 0 alkyl or C12-C2 0 monounsaturated alkenyl
• each R 2 may be C C 3 alkyl
• each R 3 may be -(CH 2 ) 3 NR 6 (CH 2 ) 3 NHR 7
• each R 4 may be H
• each R 5 may be -(CH 2 ) 3 NHR7
• at least one R 6 or R 7 may be -(CO)Ci-C 23 alkyl, or -(CO)Ci-C23 alkenyl and the remainder are H.
• each R for example may be, but is not limited to, C12-2 0 alkyl or C12-2 0 alkenyl; and each R' and each R" independently may be, but is not limited to, H, an amine protecting group, -(CO)Ci-C 2 3 alkyl, or -(CO)C C 2 3 alkenyl.
• R may be C 14 -C 18 alkyl or C 14 -C 18 alkenyl, and/or each R' and each R" independently may be H, C 14 -C 18 alkyl or C 14 -C 18 alkenyl.
• R may be, but is not limited to, C12-2 0 alkenyl; and/or each R' and each R" independently may be, but is not limited to, H, an amine protecting group, -(CO)Cr C 23 alkyl, or -(CO)Ci-C 23 alkenyl.
• R may be oleyl and each R' and each R"
• R' or R" independently may be H, or oleoyl. In still other embodiments at least one R' or R" may be oleoyl and the remainder are H.
• each R 1 may be Ci 2 -C 2 o alkyl or Ci 2 -C 2 o alkenyl
• each R 2 may be C1-C3 alkyl
• each R 5 may be H.
• Het may be
• alkyl refers to a straight- chain or branch-chain saturated aliphatic hydrocarbon radical containing the specified number of carbon atoms, or where no number is specified, in one embodiment from 1 to about 15 (i.e. (Ci_i5)alkyl), in another embodiment from 1 to about 10 carbon atoms (i.e. (Ci-io)alkyl), and in another embodiment from 1 to about 6 carbon atoms (i.e. (Ci_6)alkyl).
• alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, n-hexyl and the like.
• alkenyl refers to a straight- chain or branched-chain mono- or poly-unsaturated aliphatic hydrocarbon radical containing the specified number of carbon atoms, or where no number is specified, in one embodiment from 2-10 carbon atoms (i.e. (C2-io)alkenyl) and in another embodiment, from 2-6 carbon atoms (i.e. (C2- 6 )alkenyl).
• alkenyl radicals include, but are not limited to, ethenyl, E- and Z-propenyl, isopropenyl, E- and Z-butenyl, E- and Z-isobutenyl, E- and Z- pentenyl, E- and Z-hexenyl, ⁇ , ⁇ -, ⁇ , ⁇ -, ⁇ , ⁇ - and ⁇ , ⁇ -hexadienyl and the like.
• alkynyl refers to a straight- chain or branched-chain hydrocarbon radical having one or more triple bonds containing the specified number of carbon atoms, or where no number is specified, in one embodiment from 2 to about 10 carbon atoms.
• alkynyl radicals include, but are not limited to, ethynyl, propynyl, propargyl, butynyl, pentynyl and the like.
• alkoxy refers to an alkyl ether radical, wherein the term “alkyl” is defined above.
• suitable alkyl ether radicals include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy and the like.
• aryl refers to a carbocyclic aromatic radical (such as phenyl or naphthyl) containing the specified number of carbon atoms, in one embodiment from 6- 15 carbon atoms (i.e. (C6-is)aryl), and in another embodiment from 6- 10 carbon atoms (i.e. (C6-io)aryl), optionally substituted with one or more substituents selected from alkyl, alkoxy, (for example methoxy), nitro, halogen, (for example chloro), amino, carboxylate and hydroxy.
• aryl radicals include, but are Attorney docket no.
• 01338.8007.WO01 not limited to phenyl, p-tolyl, 4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, indenyl, indanyl, azulenyl, fluorenyl, anthracenyl and the like.
• aralkyl alone or in combination, means an alkyl radical as defined above in which one hydrogen atom is phenyl, benzyl, 2-phenylethyl and the like.
• aralkoxycarbonyl alone or in combination, means a radical of the formula
• aralkyl in which the term "aralkyl" has the significance given above.
• An example of an aralkoxycarbonyl radical is benzyloxycarbonyl.
• aryloxy alone or in combination, means a radical of the formula aryl-O- in which the term “aryl” has the significance given above.
• alkanoyl alone or in combination, means an acyl radical derived from an alkanecarboxylic acid, examples of which include acetyl, propionyl, butyryl, valeryl, 4- methylvaleryl, and the like.
• aryloxy alkanoyl means an acyl radical of the formula aryl-O-alkanoyl wherein aryl and alkanoyl have the significance given above.
• aralkanoyl means an acyl radical derived from an aryl-substituted alkanecarboxylic acid such as phenylacetyl, 3-phenylpropionyl (hydrocinnamoyl), 4- phenylbutyryl, (2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4-aminohydrocinnamoyl, 4- phenylbutyryl, (l-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4-aminohydrocinnamoyl, 4- methoxyhydrocinnamoyl, and the like.
• aroyl means an acyl radical derived from an aromatic carboxylic acid.
• radicals include aromatic carboxylic acids, an optionally substituted benzoic or naphthoic acid such as benzoyl, 4-chlorobenzoyl, 4-carboxybenzoyl, 4- benzyloxycarbonyl)benzoyl, 1-naphthoyl, 2-naphthoyl, 6-carboxy-2-naphthoyl, 6- (benzyloxycarbonyl)-2-naphthoyl, 3-benzyloxy-2-naphthoyl, 3-hydroxy-2-naphthoyl, 3- (benzyloxyformamido)-2-naphthoyl, and the like.
• aminocarbonyl alone or in combination, means an amino-substituted carbonyl (carbamoyl) group derived from an amino-substituted carboxylic acid wherein the amino group can be a primary, secondary or tertiary amino group continuing substituents selected from hydrogen, alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl radicals and the like.
• aminoalkanoyl means an acyl radical derived from an amino substituted alkanecarboxylic acid wherein the amino group can be a primary, secondary or tertiary amino group containing substituents selected from the group consisting of hydrogen, cycloalkyl, cycloalkylalkyl radicals and the like, examples of which include N,N-dimethylaminoacetyl and N-benzylaminoacetyl.
• aminoalkanoyl means an acyl radical derived from an amino substituted alkanecarboxylic acid wherein the amino group can be a primary, secondary or tertiary amino group containing substituents selected from the group consisting of hydrogen, cycloalkyl, cycloalkylalkyl radicals and the like, examples of which include N,N-dimethylaminoacetyl and N-benzylaminoacetyl.
• Carbocycle refers to a non-aromatic stable 3- to 8-membered carbon ring which may be saturated, mono-unsaturated or poly-unsaturated.
• the carbocycle may be attached at any endocyclic carbon atom which results in a stable structure.
• Carbocycles in one embodiment have 5-7 carbons.
• cycloalkyl alone or in combination, means an alkyl radical which contains from about 3 to about 8 carbon atoms and is cyclic. Examples of such cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
• cycloalkylalkyl means an alkyl radical as defined above which is substituted by a cycloalkyl radical containing from about 3 to about 8, in one embodiment from about 3 to about 6, carbon atoms.
• cycloalkylcarbonyl means an acyl group derived from a monocyclic or bridged cycloalkanecarboxylic acid such as cyclopropanecarbonyl, cyclohexanecarbonyl, adamantanecarbonyl, and the like, or from a benz- fused monocyclic cycloalkanecarboxylic acid which is optionally substituted by, for example, alkanoylamino, such as 1,2,3,4- tetrahydro-2-naphthoyl, 2-acetamido-l,2,3,4-tetrahydro-2-naphthoyl.
• cycloalkylalkoxycarbonyl means an acyl group derived from a cycloalkylalkoxycarboxylic acid of the formula cycloalkylalkyl-O-COOH wherein cycloalkylalkyl has the significance given above.
• basic heterocycle refers to a stable 5-7 membered monocyclic heterocyclic ring or 8-11 membered bicyclic heterocyclic ring which is either saturated or partially unsaturated, and which may be optionally benzofused if monocyclic and which is optionally substituted on one or more carbon atoms by halogen, alkyl, alkoxy, oxo, and the like, and/or on a secondary nitrogen atom (i.e., -NH-) by alkyl, aralkoxycarbonyl, alkanoyl, phenyl or phenylalkyl or on a tertiary nitrogen atom (i.e., +N-) by oxido and which is attached via a carbon atom.
• a secondary nitrogen atom i.e., -NH-
• Each heterocycle consists of one or more carbon atoms and from one to four heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, provided that at least one heteroatom is a basic nitrogen atom.
• a heterocyclyl radical may be attached at any endocyclic carbon or heteroatom which results in the creation of a stable structure.
• Heterocycles include 5-7 membered monocyclic heterocycles and 8-10 membered bicyclic heterocycles.
• Examples of basic heterocycles include imidazolinoyl, imidazolidinyl, indazolinolyl, perhydropyridazyl, pyrrolinyl, pyrrolidinyl, piperidinyl, pyrazolinyl, piperazinyl, morpholinyl, thiamorpholinyl, thiazolidinyl, thiamorpholinyl sulfone, oxopiperidinyl, oxopyrrolidinyl, and oxoazepinyl.
• halogen means fluorine, chlorine, bromine or iodine.
• surface ligand or "cell surface ligand” refers to a chemical compound or structure which will bind to a surface receptor of a cell.
• cell surface receptor refers to a specific chemical grouping on the surface of a cell to which the ligand can attach. Cell surface receptors can be specific for a particular cell, i.e., found
• asialoglycoprotein receptors are specific for hepatocytes).
• the receptor facilitates the internalization of the ligand and attached molecules.
• a cell surface receptor includes but is not limited to a folate receptor, biotin receptor, lipoic acid receptor, low-density lipoprotein receptor, asialoglycoprotein receptor, insulin- like growth factor type II/cation-independent mannose-6-phosphate receptor, calcitonin gene-related peptide receptor, insulin-like growth factor I receptor, nicotinic acetylcholine receptor, hepatocyte growth factor receptor, endothelin receptor, bile acid receptor, bone morphogenetic protein receptor, cartilage induction factor receptor or glycosylphosphatidylinositol (GPI)- anchored proteins (e.g., .beta, adrenergic receptor, T-cell activating protein, Thy-1 protein, GPI-anchored 5' nucleotidase).
• GPI glycosylphosphatid
• a receptor is a molecule to which a ligand binds specifically and with relatively high affinity. It is usually a protein or a glycoprotein, but may also be a glycolipid, a
• lipidpolysaccharide a glycosaminoglycan or a glycocalyx.
• epitopes to which an antibody or its fragments binds is construed as a receptor since the antigen: antibody complex undergoes endocytosis.
• surface ligand includes anything which is capable of entering the cell through cytosis (e.g. endocytosis, potocytosis, pinocytosis).
• ligand refers to a chemical compound or structure which will bind to a receptor. This includes but is not limited to ligands such as asialoorosomucoid, asialoglycoprotein, lipoic acid, biotin, apolipoprotein E sequence, insulin-like growth factor II, calcitonin gene -related peptide, thymopoietin, hepatocyte growth factor, endothelin- 1, atrial natriuretic factor, RGD-containing cell adhesion peptides and the like.
• ligands such as asialoorosomucoid, asialoglycoprotein, lipoic acid, biotin, apolipoprotein E sequence, insulin-like growth factor II, calcitonin gene -related peptide, thymopoietin, hepatocyte growth factor, endothelin- 1, atrial natriuretic factor, RGD-containing cell adhesion peptides
• the ligand chosen will depend on which receptor is being bound. Since different types of cells have different receptors, this provides a method of targeting nucleic acid to specific cell types, depending on which cell surface ligand is used. Thus, the preferred cell surface ligand may depend on the targeted cell type.
• nuclear localization agent refers to a ligand, such as a peptide, which will cause an agent Attorney docket no. 01338.8007.WO01 covalently or non-covalently linked to it to localize at the cell nucleus, typically by binding a nuclear receptor.
• nuclear receptor refers to a chemical grouping on the nuclear membrane which will bind a specific ligand and help transport the ligand, and accompanying linked moieties, through the nuclear membrane.
• Nuclear receptors can be but are not limited to those receptors which bind nuclear localization sequences.
• Nonlimiting examples of nuclear ligands include GYSTPPKKKRKVEDP, GYSTPPKTRRRP,
• GYSTPGRKKR GYSTPRRNRRRRW
• PDEVKRKKKPPTSYG PRRRTKPPTSYG
• RKKRGPTSYG WRRRRNRRPTSYG
• GYGPPKKKRKVEAPYKA(K) 2CMO K may be used to transport nucleic acid to the nucleus.
• polycationic nucleic acid binding moiety refers to a moiety containing multiple positive charges at physiological pH that allow the moiety to bind a negatively charged nucleic acid.
• a polycationic nucleic acid binding moiety may be linked to, for example, a cell surface ligand, a fusion agent, and/or a muclear localization peptide. The linkage may be covalent.
• Suitable polycationic nucleic acid binding moieties include polyamines and polybasic peptides containing, for example, multiple lysine, ornithine, or histidine residues.
• lysis agent refers to a molecule, compound, protein or peptide which is capable of breaking down an endosomal membrane and freeing the DNA transporter into the cytoplasm of the cell. This term includes but is not limited to viruses, synthetic compounds, lytic peptides, or derivatives thereof.
• lytic peptide refers to a chemical grouping which penetrates a membrane such that the structural organization and integrity of the membrane is lost. As a result of the presence of the lysis agent, the membrane undergoes lysis, fusion or both.
• nucleic acid when not applied to a specific type of molecule such as unmodified DNA or RNA, refers to any type of nucleic acid that presently is known or that may be prepared or identified in the future, provided that the nucleic acid is sufficiently negatively charged to form a lipid aggregate, liposome, or liposome-like complex when admixed with any lipid of Formula (I) or (II).
• Nucleic acid refers to deoxyribonucleotides or ribonucleotides and mixtures and polymers thereof in single- or double- stranded form.
• nucleic acids containing known nucleotide analogs or modified backbone residues or linkages which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as a reference nucleic acid, and which are metabolized in a manner similar to reference nucleotides.
• analogs include, without limitation, phosphorothioates, phosphoramidates, methyl Attorney docket no. 01338.8007.WO01 phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
• the nucleic acid may be in the form of an antisense molecule, for example a "gap- mer" containing an RNA-DNA-RNA structure that activates RNAseH.
• the nucleic acid can be, for example, DNA or RNA, or RNA-DNA hybrid, and can be an oligonucleotide, plasmid, parts of a plasmid DNA, pre-condensed DNA, product of a polymerase chain reaction (PCR), vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups or other form of nucleic acid molecule.
• the nucleic acid may be a double- stranded RNA molecule of the type used for inhibiting gene expression by RNA interference.
• the nucleic acid may be a short interfering double stranded RNA molecule (siRNA).
• siRNA short interfering double stranded RNA molecule
• the nucleic acid molecule can also be a Steal thTMRNAi molecule (Invitrogen Corporation/Life Technologies Corporation, Carlsbad, CA).
• amphipathic peptide refers to a peptide whose secondary structure places hydrophobic and hydrophilic amino acid residues on different faces of the peptide.
• the peptides often adopt a helical secondary structure. In some circumstances an amphipathic peptide may also function as a fusion agent.
• amphipathic peptides suitable for use in the compositions described herein include, but are not limited to, peptides comprising a sequence selected from the group consisting of FEAALAEALAEALA, Ac- LARLLPRLLARL-NHCH 3 , GLLEELLELLEELWEELLEG, GWEGLIEGIEGGWEGLIEG, GLFEALAEFIEGGWEGLIEG, GLFEALLELLESLWELLLEA,
• amphipathic peptide may optionally be linked to a polycationic nucleic acid binding moiety, for example via a covalent linkage.
• Symmetric and asymmetric cationic lipids of general structure (I) and (II) may be synthesized using methods that are well known in the art, as shown, for example in Scheme 1.
• Dimethyl tartrate (1) can be treated with an alkylamine at elevated temperature (e.g. 70°C) in a sealed pressure reactor to obtain compound 2.
• This compound may be alkylated with an alkyl mesylate to obtain compound 3, which is then reduced using lithium aluminum hydride to produce the bis-amine 4.
• Compound 4 may be acylated with a suitably protected amino acid 5 using, for example, a carbodiimide as a coupling agent to obtain the protected precursor of compound 6.
• This precursor is then deprotected to produce the desired symmetric compound 6.
• suitable amino acids for the acylation include Attorney docket no. 01338.8007.WO01
• Boc-protected carboxyspermine, histidine or lysine which generate compounds 7, 8, and 9 respectively, after deprotection.
• asymmetric cationic lipids may be prepared using a tartaric acid monoester, readily prepared from diacetyl tartaric anhydride (see Organic Syntheses, Coll. Vol. 4, p.242 (1963); Vol. 35, p.49 (1955)) or from an erythronolactone such as 10. See Scheme 2. Protection of the diol, followed by DIBAL reduction, reductive amination and amine protection produce compound 11. Mild oxidation, reductive amination and protection produce compound 12. Diol deprotection and alkylation produce compound 13. Stepwise selective amine deprotection and coupling reactions then produces the asymmetric compound 14.. .
• a bis-epoxide is reacted with an alkylamine to produce amino alcohol 15. This can be acylated to produce 16 followed by alkylation and
• Compounds of formula (I) may be prepared from intermediates of structure 4 above by methods that are well known in the art.
• compound 4 where R 2 is alkyl may be reacted with a suitable protected epoxyamine, for example, an epoxyphthalimide, followed by deprotection to provide ⁇ -hydroxyamines such as
• the lipids described above may be formulated by various methods to be used in transfection.
• One of the simplest methods for formulation is reverse evaporation. In this procedure the required amount of the cationic lipid and a co-lipid (if used, e.g. a neutral lipid) are transferred into a round bottom flask. An amount of chloroform that is enough to dissolve the lipids is added, followed with enough molecular biology grade water to make the desired concentration of total lipids/volume ( e.g. 2 mg/ml ). The solution is evaporated in vacuo (e.g. on a rotary evaporator) and the chloroform removed under vacuum. As the chloroform is removed liposomes are formed in the aqueous medium.
• vacuo e.g. on a rotary evaporator
• sonication and microfluidization Other methods for formulation that can be used are sonication and microfluidization. In both cases the required amount of the cationic lipid and the co-lipid are transferred into a flask and an amount of chloroform that will dissolve the lipids to homogenous solution is added. The chloroform is evaporated to leave a thin film of lipid mixture in the flask. The lipid film is then hydrated with molecular biology grade water to make the desired concentration and sonicated or microfluidized.
• the new lipids are formulated with one or more colipids, most advantageously neutral colipids, although the skilled artisan will recognize that other lipids, including cationic lipids, may be used.
• formulations where the molar ratio of cationic lipid : DOPE was varied from 2: 1 to 1: 16 were prepared using the above methods.
• Compounds having the structure 7 above were prepared where the hydrocarbon chain was varied from C1 0 -C2 0 and the histidine and lysine analogs 8 and 9 where the hydrocarbon chain was varied from C 10 - C2 0 were formulated in the same manner.
• the new lipids may be formulated with one ore more cationic lipids and/or one or more neutral lipids.
• the neutral lipid(s) may be, for example, DOPE, DPhPE, cholesterol, DOPC, Lyso-PE ( l-acyl-2-hydroxy-sn-glycero-3-phosphoethanolamine), Lyso-PC ( 1-acyl- 3-hydroxy-sn-glycero-3-phosphocholine), and/or 3-alkyloxy-2-hydroxy-l-acetamidopropane.
• the cationic lipid may be selected from the group consisting of DOTMA, DOTAP, DMRIE, DC-Choi, DDAB, DOSPA, DOSPER, DOGS, TMTPS, TMTOS, TMTLS,
• TMTMS Trimethyl-N-l-(2,3-diaoleoyloxypropyl)-2-hydroxypropane-l,3- diamine
• N- 1-dimethyl-N- 1 -(2,3-diamyristyloxypropyl)-2-hydroxypropane- 1 ,3-diamine N- 1 - dimethyl-N- 1 -(2,3-diapalmityloxypropyl)-2-hydroxypropane- 1 ,3-diamine
• formulations may also include one or more polyamine transfection agents, such as dense star dendrimers, PAMAM dendrimers, N3 ⁇ 4 core dendrimers, ethylenediamine core Attorney docket no. 01338.8007.WO01 dendrimers, dendrimers of generation 5 or higher, dendrimers with substituted groups, dendrimers comprising one or more amino acids, grafted dendrimers, activated dendrimers, polyethylenimine, and/or polyethylenimine conjugates.
• polyamine transfection agents such as dense star dendrimers, PAMAM dendrimers, N3 ⁇ 4 core dendrimers, ethylenediamine core Attorney docket no. 01338.8007.WO01 dendrimers, dendrimers of generation 5 or higher, dendrimers with substituted groups, dendrimers comprising one or more amino acids, grafted dendrimers, activated dendrimers, polyethylenimine, and/or polyethylenimine conjugates
• transfection enhancing agents such as a fusion agent, a cell surface ligand and/or a nuclear localization agent such as a nuclear receptor ligand peptide
• transfection enhancing agents include, but are not limited to, reo virus -related fusogenic peptides (see WO07/130073, which is hereby incorporated by reference in its entirety), insulin, a transferrin, epidermal growth factor, fibroblast growth factor, a cell targeting antibody, a lactoferrin, a fibronectin, an adenovirus penton base, Knob, a hexon protein, a vesicular stomatitis virus glycoprotein, a Semliki Forest Virus core protein, a influenza hemagglutinin, a hepatitis B core protein, an HIV Tat protein, a herpes simplex virus VP22 protein, a histone protein, a arginine rich cell permeability protein, a high mobility group protein,
• compositions in transfection can be carried out by methods that are known in the art. See for example, WO07/130073, at pages 54-60 which describes "before” and “after” protocols for transfection where the components of a transfection complex are mixed in differing orders prior to addition to a cell culture.
• a liposomal preparation of the lipid, with or without colipid is prepared, and is then mixed with a macromolecule, such as a DNA molecule or RNAi molecule to form a transfection complex.
• the complex is then added to a cell culture and transfection is monitored using well known methods. Additional components such as cell surface ligands, fusion agents, nuclear localization agents and the like may be added to the nucleic acid prior to admixture with the liposome, or may be added to the liposome prior to addition of nucleic acid.
• Cells which can be transfected according to these methods include, but are not limited to, virtually any eukaryotic cell including primary cells, cells in culture, a passaged cell culture or a cell line, and cells in cultured tissue. Suitable cells include human cell lines and animal cell lines.
• the cell may be a fibroblast.
• the cells can be attached cells or cells in suspension (suspension cells).
• the cells are suspension CHO-S cells and suspension 293-F cells.
• Other cells that may be used include, without limitation, 293, 293-S, CHO, Cos, 3T3, Hela, primary fibroblasts, A549, Be2C, SW480, CHOK1, Attorney docket no. 01338.8007.WO01
• compositions which includes a lipid of Formula (I) or (II) can be used for transfection of cells.
• compositions are further discussed herein, and include, but are not limited to compositions comprising lipids of Formula (I) or (II), a co-lipid and an optional transfection enhancing agent such as a fusogenic peptide or protein.
• RNAi double stranded RNA molecule
• siRNA siRNA molecule designed to inhibit expression of the protein.
• Methods of designing such RNA molecules are well known in the art.
• Lipids of Formula (I) are particularly suitable for deliver of RNAi molecules in this fashion. The cells are incubated and the phenotypic consequence of inhibiting production of the selected protein is observed. The lipids formulated in this manner were used in transfection. The transfection of 293, CHO-K1, NIH3T3, Hela,, A549, Umbilical Cord Mesenchymal Stem Cells (UB Stem), Adipose Mesenchymal Stem
• kits contain the lipid of formula (I) or (II) above, together with additional components, such as a neutral lipid, a cationic lipid, cell surface ligands, fusion agents, and/or nuclear localization agents and the like.
• additional components such as a neutral lipid, a cationic lipid, cell surface ligands, fusion agents, and/or nuclear localization agents and the like.
• the kit components may be separate or may be premixed in any manner.
• the lipid of formula (I) or (II) may be admixed with one or more neutral lipid.
• Additional components may also be present in the same container or may be present in one or more separate containers.
• the kits typically include vessels, such as vials and/or tubes, which are packaged together, for example in a cardboard box.
• kits 01338.8007.WO01 can be shipped from a supplier to a customer.
• a kit that includes a vial that includes a liposomal formulation as described above and, optionally, a transfection agent and a transfection enhancing peptide.
• the kit can also include, for example, a separate vessel that includes a transfection enhancing agent, such as a transfection enhancing peptide, for example Plus ReagentTM (Invitrogen Corp., Carlsbad, CA).
• the kit can also include in separate containers, cells, cell culture medium, and a reporter nucleic acid sequence, such as a plasmid that expresses a reporter gene.
• the culture medium can be reduced-serum medium and/or protein expression medium.
• kits containing a compound of Formula (I) or(II) and additional reagents such as a cationic lipid, a neutral lipid, an amphipathic peptide, an amphipathic peptide comprising a polycationic nucleic acid binding moiety, a cell surface ligand, a cell surface ligand comprising a polycationic nucleic acid binding moiety, a fusion agent, a fusion agent comprising a polycationic nucleic acid binding moiety, a nuclear localization peptide or protein, and a nuclear localization peptide or protein comprising a polycationic nucleic acid binding moiety.
• the kits may contain one, some, or all of these additional reagents, in any possible combination.
• the additional reagents include a cationic lipid, an amphipathic peptide and a cell surface ligand that contains a polycationic nucleic acid binding moiety.
• the cell surface ligand is a peptide or protein
• the polycationic nucleic acid binding moiety is a polybasic amino acid sequence.
• a kit comprises individual portions of, or a mixture of, cationic lipid, such as a lipid of Formula (I), and peptide, protein or fragment thereof or modified peptide, protein or fragment thereof.
• a kit comprises individual portions of, or a mixture of, polycationic polymers and peptide, protein or fragments thereof or modified peptide, protein or fragments thereof.
• Cationic lipid transfection kits can optionally include neutral lipid as well as other transfection-enhancing agents or other additives, and the relative amounts of components in the kit may be adjusted to facilitate preparation of transfection compositions.
• Kit components can include appropriate medium or solvents for other kit components.
• Nucleic acids that can be transfected by the methods of this invention include DNA and RNA (including RNAi/siRNA) of any size from any source comprising natural bases or non-natural bases, and include those encoding and capable of expressing therapeutic or otherwise useful proteins in cells, those which inhibit undesired expression of nucleic acids in cells, those which inhibit undesired enzymatic activity or activate desired enzymes, those Attorney docket no. 01338.8007.WO01 which catalyze reactions (ribozymes), and those which function in diagnostic assays (e.g., diagnostic nucleic acids).
• Therapeutic nucleic acids include those nucleic acids that encode or can express therapeutically useful proteins, peptides or polypeptides in cells, those which inhibit undesired expression of nucleic acids in cells, and those which inhibit undesired enzymatic activity or activate desired enzymes in cells.
• compositions and methods provided herein can also be readily adapted in view of the disclosure herein to introduce biologic ally- active macromolecules other than nucleic acids including, among others, polyamines, polyamine acids, polypeptides and proteins into eukaryotic cells.
• biologic ally- active macromolecules other than nucleic acids including, among others, polyamines, polyamine acids, polypeptides and proteins into eukaryotic cells.
• the reaction mixture was stirred magnetically and the THF was decanted while the mixture was still hot.
• the THF was removed in vacuo on a rotary evaporator and the residue was dissolved in 400 ml chloroform and extracted with water (2 x 300 ml).
• the solution was concentrated and the resulting gum was dissolved in 5 ml chloroform and applied to a flash silica column (lOOg) that was equilibrated with 1% methanol in chloroform.
• the column was eluted with 1L each of 1%, 5%, 10%, 20% and 30% methanol in
• the liposome was formulated using reverse evaporation.
• the lipid mixture was dissolved in 2 ml of chloroform. 10 ml of water was added to the chloroform solution.
• the chloroform was removed under vacuum on a rotary evaporator to obtain a liposome solution.
• the solution was adjusted to 10 ml to obtain a 2 mg/ml liposome solution. It was designated as VII-97-G.
• Figures 1-4 show the results obtained, and compare those results to two widely used commercial reagents. The results show that the lipids described above provided significantly enhanced transfection efficiency, as measure by ⁇ -gal expression, across all cell types.
• Transfection complexes were formed by combining the appropriate transfection reagent containing a lipid of formula (I) (1, 2, 3, 4, 5, and 6 ⁇ ) and either GAPDH-silencing or negative control RNA (25 nM final) in 200 ⁇ of Opti-MemTM. Complexes were allowed to form for 20 minutes at room temperature.
• each reagent/RNA mixture was transferred to the appropriate well containing the adherent cells and 100 ⁇ of complete culture medium (including serum). Incubation was carried out for -72 hours at 37°C and 5% C0 2 . After this 72 hour incubation period, plates were harvested by aspirating the medium from the wells, followed by 2 washes of 100 ⁇ per well with PBS (with calcium and magnesium), aspirating off the PBS each time. Plates were then frozen at -80° for 30 to 60 minutes.

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