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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
  • C07C271/06—Esters of carbamic acids
  • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
  • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
  • C07C271/20—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
  • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
  • C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
  • C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J51/00—Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof

Definitions

• the present invention relates to the field of cationic lipid compounds and their uses including the delivery of macromolecules into cells.
• Lipid aggregates such as liposomes
• macromolecules such as DNA, RNA
• lipid aggregates which include charged as well as uncharged lipids, have been described as being especially effective for
• cationic lipids may result from charge inmteractions with cells which are said to bear a net negative charge. It has also been postulated that the net positive charge on the cationic lipid aggregates may enable them to bind polyanions, such as nucleic acids. For examples, lipid aggregates containing DNA have been reported to be effective agents for efficient
• lipid aggregates include, for example, liposomes, unilamellar vesicles, multilamellar vesicles, micelles and the like, and may have particle sizes in the nanometer to micrometer range.
• lipid aggregates include phospholipid containing liposomes.
• An important drawback to the use of this type of aggregate as a cell delivery vehicle is that the liposome has a negative charge that reduces the efficiency of binding to a negatively charged cell surface.
• DOTMA N[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium chloride
• DOPE dioleoylphosphatidylethanolamine
• DOTAP 1,2- bis (oleoyloxy)-3,3-(trimethylammonia) propane
• DOTAP is reported to be more readily degraded by target cells.
• Other cationic lipids which represent structural modifications of DOTMA and DOTAP have also been
• cationic lipid compounds include those in which carboxyspermine has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) and dipalmitoyl-phosphatidylethanolamine 5- carboxyspermyl-amide (“DPPES”) (See, e . g. Behr et al . , U.S. Patent No. 5,171,678).
• DOGS 5-carboxyspermylglycine dioctaoleoylamide
• DPES dipalmitoyl-phosphatidylethanolamine 5- carboxyspermyl-amide
• DC-Choi cationic cholesterol derivative
• these liposomes were said to exhibit lower toxicity and provide more efficient transfection than the DOTMA- containing compositions.
• Lipopolylysine made by conjugating polylysine to DOPE has been reported to be effective for transfection in the presence of serum. (Zhou, X. et al . , Biochim. Biophys . Acta 1065 : 8 , 1991).
• novel carbamate-based cationic lipids having the structure:
• R 1 is a lipophilic moiety
• R 2 is a positively charged moiety
• n is an integer from 1 to 8
• X- is an anion or polyanion
• m is an integer from 0 to a number equivalent to the positive charge (s) present on the lipid.
• R 1 may be selected from a variety of lipophilic moieties including a straight chain alkyl of 1 to about 24 carbon atoms, a straight chain alkenyl of 2 to about 24 carbon atoms, a symmetrical branched alkyl or alkenyl of about 10 to about 50 carbon atoms (preferably 25-40), an unsymmetrical branched alkyl or alkenyl of about 10 to about 50 carbon atoms, a straight chain alkyl of 1 to about 24 carbon atoms, a straight chain alkenyl of 2 to about 24 carbon atoms, a symmetrical branched alkyl or alkenyl of about 10 to about 50 carbon atoms (preferably 25-40), an unsymmetrical branched alkyl or alkenyl of about 10 to about 50 carbon atoms, a
• steroidyl moiety a glyceryl derivative or CH(R 3 R 4 ), wherein R 3 and R 4 are independently a straight chain alkyl moiety of about 10 to about 30 carbon atoms, or a branched alkyl moiety of about 10 to about 30 carbon atoms.
• R 1 is a steroidyl moiety it is a cholesteryl moiety or a non-cholesteryl moiety.
• R 1 is 3-(1,2-diacyl) propane 1,2-diol moiety or a 3-(1,2-dialkyl) propane 1,2-diol moiety.
• the diacyl group be alkanoic acid of about 10 to about 30 carbon atoms or an alkenoic acid of about 10 to about 30 carbon atoms.
• the alkyl moieties be an alkyl group of about 10 to about 30 carbon atoms or an alkenyl group of about 10 to about 30 carbon atoms.
• R 1 is a glyceryl derivative it is prefered that it be a 3-0-1,2-diacylglyceryl moiety or a 3-O-1,2- dialkylglyceryl moiety.
• R 1 is a 3-O- 1,2-diacylglyceryl moiety it is preferable that the diacyl group be an alkanoic acid of about 10 to about 30 carbon atoms or an alkenoic acid of about 10 to about 30 carbon atoms.
• R 1 is 18-pentatriacontane, 3- (3 ⁇ )-cholest-5-ene, or 3-(1,2 distearyl) propane 1,2- diol.
• R 2 may be selected from a variety of positively charged moieties including an amino acid residue having a positively charged group on the side chain, an alkylamine moiety of about 3 to about 10 carbon atoms, a fluoroalkylamine moiety, or a
• R 6 and R 7 are independently selected from an alkyl moiety of 1 to about 24 carbon atoms, an alkenyl moiety of 2 to about
• R 2 is an amino acid residue it is a lysine, arginine, histidine, ornithine, or an amino acid analog.
• the amino acid residue be 3-carboxyspermidine, 5- carboxyspermidine, 6-carboxyspermine or monoalkyl, dialkyl or peralkyl substituted derivatives which are substituted on one or more amine nitrogens with an alkyl group of 1 to about 6 carbon atoms.
• n be an integer from 2 to 6, or more preferably from 2 to 4 and that X- be a pharmaceutically acceptable anion or polyanion.
• carbamate-based cationic lipid has the structure
• compositions comprising a polyanionic macromolecule and any of the lipids described above are provided.
• the macromolecule may be a variety of polyanionic macromolecules including an expression vector capable of expressing a polypeptide in a cell.
• the polyanionic macromolecule is an
• oligomucleotide or an oligomer and most preferably DNA are oligomucleotide or an oligomer and most preferably DNA.
• a method for delivering a polyanionic macromolecule into a cell by contacting any of the compositions above with the cellare provided.
• a method is provided to interfere with the expression of a protein in a cell by contacting any of the the compositions described above with a cell wherein the composition comprises an oligomer having a base sequence that is substantially complimentary to an RNA sequence in the cell that encodes the protein.
• the present invention further provides a kit for delivering a polyanionic macromolecule into a cell comprising any of the compositions described above.
• Lipophilic Moiety refers to a moiety which demonstrates one or more of the following
• A. tend to be water insoluable
• lipophilic moieties having an octanol/water partition coefficinet of 0.5 or lower are preferable, where octanol/water partition coefficient is measured by the concentration in water divided by concentration in octanol.
• "Positively Charged Moiety” refers to "positively charged moiety and negatively charged moiety” means a moiety, independent of the cationic lipid for which it is a substituent, having a net positive or negative charge within the pH range of 2 to 12. The net charge of the cationic lipid is the summation of all charged moieties occuring on the lipid, such that the net charge may be positive, neutral or negative.
• alkyl refers to saturated aliphatic groups including straight-chain, branched-chain and cyclic groups. Suitable alkyl groups include, but are not limited to, cycloalkyl groups such as cyclohexyl and cyclohexylmethyl. "Lower alkyl” refers to alkyl groups of 1 to 6 carbon atoms. Fluoroalkyl or perfluoroalkyl refers to singly, partially, or fully fluorinated alkyl groups.
• alkenyl refers to an unsaturated
• arylamine refers to aromatic groups that have at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which are substituted with an amine .
• aralkylamine refers to an alkylamine group substituted with an aryl group. Suitable aralkyl groups include benzyl, picolyl, and the like, all of which may be optionally substituted.
• heterocyclic amine refers to groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and suitable heterocyclic aryls include furanyl, thienyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, and the like.
• oligonucleoside or “oligomer” refers to a chain of nucleosides that are linked by
• internucleoside linkages that is generally from about 4 to about 100 nucleosides in length, but which may be greater than about 100 nucleosides in length. They are usually synthesized from nucleoside monomers, but may also be obtained by enzymatic means.
• oligomer refers to a chain of oligonucleosides that have internucleosidyl linkages linking the nucleoside monomers and, thus, includes oligonucleotides, nonionic oligonucleoside alkyl- and aryl-phosphonate analogs, alkyl- and aryl-phosphonothioates, phosphorothioate or phosphorodithioate analogs of oligonucleotides,
• phosphoramidate analogs of oligonucleotides neutral phosphate ester oligonucleoside analogs, such as phosphotriesters and other oligonucleoside analogs and modified oligonucleosides, and also includes
• nucleoside/non-nucleoside polymers are nucleoside/non-nucleoside polymers.
• the term also includes nucleoside/non-nucleoside polymers wherein one or more of the phosphorus group linkages between monomeric units has been replaced by a non-phosphorous linkage such as a formacetal linkage, a thioformacetal linkage, a morpholino linkage, a sulfamate linkage, a silyl linkage, a carbamate linkage, an amide linkage, a guanidine linkage, a nitroxide linkage or a substituted hydrazine linkage.
• a non-phosphorous linkage such as a formacetal linkage, a thioformacetal linkage, a morpholino linkage, a sulfamate linkage, a silyl linkage, a carbamate linkage, an amide linkage, a guanidine
• nucleoside/non- nucleoside polymers wherein both the sugar and the phosphorous moiety have been replaced or modified such as morpholino base analogs, or polyamide base analogs. It also includes nucleoside/non-nucleoside polymers wherein the base, the sugar, and the phosphate backbone of the non-nucleoside are either replaced by a nonnucleoside moiety or wherein a non-nucleoside moiety is inserted into the nucleoside/non-nucleoside polymer.
• said non-nucleoside moiety may serve to link other small molecules which may interact with target sequences or alter uptake into target cells.
• Lipid Aggregate is a term that includes liposomes of all types both unilamellar and multilamellar as well as micelles and more amorphous aggregates of cationic lipid or lipid mixed with amphipathic lipids such as phospholipids.
• Target Cell refers to any cell to which a desired compound is delivered, using a lipid aggregate as carrier for the desired compound.
• Transfection is used herein to mean the delivery of expressible nucleic acid to a target cell, such that the target cell is rendered capable of expressing said nucleic acid. It will be understood that the term
• 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.
• 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 is a means for getting the desired compound into the cell.
• delivery to a specific target cell type is preferable and can be facilitated by compounds of the invention.
• the generic structure of functionally active cationic lipids requires three contiguous moities, e.g. cationic-head-group/ linker/lipid-tail group. While a wide range of structures can be envisioned for each of the three moieties, it has been demonstrated that there is no a priori means to predict which cationic lipid will successfully transfect anionic macromolecules into a particular cell line. The property of a cationic lipid to be formulated with an anionic macromolecule which will then successfully transfect a cell line is empirical. We demonstrate the abilities of novel cationic lipids which are chemically linked into
• novel carbamate-based cationic lipids of the present invention have the general structure: comprising any salt, solvate, or enantiomers thereof.
• the symbols R 1 , R 2 , X, n, and m are described as follows.
• R 1 represents the lipid-tail group of the carbamate- based cationic lipid and may be a variety of lipophilic moieties, in particular, these include for example, a straight chain alkyl of 1 to about 24 carbon atoms, a straight chain alkenyl of 2 to about 24 carbon atoms, a symmetrical branched alkyl or alkenyl of about 10 to about 50 carbon atoms, a unsymmetrical branched alkyl or alkenyl of about 10 to about 50 carbon atoms, a
• steroidyl moiety a glyceryl derivative, a amine derivative, or OCH(R 4 R 5 ) wherein R 4 and R 5 are straight chain or branched alkyl moieties of about 10 to about 30 carbon atoms.
• R 2 is a steriodal moiety
• moieties including for example pregnenolone, progesterone, cortisol, corticosterone, aldosterone, androstenedione, testosterone, or
• a cholesteryl moiety is particularly prefered.
• R 1 may be a 3-(1,2-diacyl) propane 1,2-diol moiety, a 3-(1,2-dialkyl) propane 1,2-diol moiety, a 3-O-1,2-diacylglyceryl moiety, or 3-O-1,2- dialkylglyceryl moiety.
• the diacyl group may be an
• alkanoic acid of about 10 to about 30 carbon atoms or an alkenoic acid of about 10 to about 30 carbon atoms.
• R 1 is a 3-(1,2-dialkyl) propane 1,2-diol moiety or a 3-O-1,2-diacylglyceryl moiety
• the alkyl moieties may be an alkyl group of about 10 to about 30 carbon atoms or an alkenyl group of about 10 to about 30 carbon atoms. These groups may be straight chain, symmetrically, or unsymmetrically branched alkyl and alkenyl groups.
• R 1 is a propanediol moiety or a glyceryl derivative comprising an alkanoic acid
• the acid is preferebly a stearic acid.
• these derivative comprise an alkenoic acid the acid is
• a palmitoic acid or an oleic acid preferably a palmitoic acid or an oleic acid.
• R 2 represents the cationic head group of the carbamate-based cationic lipid and may be a variety of positively charged moieties including, for example, an amino acid residue having a positively charged group on the side chain, an alkylamine moiety of about 3 to about 10 carbon atoms, a fluoroalkylamine moiety or a
• R 2 when R 2 is an amino acid residue it may be, for example, lysine, arginine, histidine, ornithine, or an amino acid analog.
• R 2 may be a variety of positively charged amino acid analogs, specific examples include 3-carboxyspermidine, 5- carboxyspermidine, 6-carboxyspermine and a monoalkyl, dialkyl, or peralkyl substituted derivative which is substituted on one or more amine nitrogens with an alkyl group of 1 to about 6 carbon atoms.
• the linker comprises the structure joining the head group, R 1 to the lipid-tail group, R 2 .
• This structure includes Y which may be an oxygen or a nitrogen and a series of -CH 2 - groups, the number of which, is indicated by the letter n.
• n is an integer ranging from 1 to 8, in particular cases it ranges from 2 to 6 and in specific instances the integer is 2 to 4.
• the counterion represented by X- is an anion or a polyanion that binds to the positively charged groups present on the phosphonic acid-based cationic lipid via charge-charge interactions.
• the anion or polyanion should be pharmaceutically acceptable.
• m is an integer indicating the number of anions or polyanions associated with the cationic lipid. In particular this integer ranges in magnitude from 0 to a number equivalent to the positive charge (s) present on the lipid.
• the cationic lipids of the present invention include salts, solvates, or enatiomeric isomers
• racemic mixtures may be separated into their individual, substantially optically pure isomers by techniques known in the art, such as, for example, the separation of diastereomeric salts formed with optically active acid or base adjuncts followed by conversion back to the optically active substances.
• desired optical isomer is synthesized by means of stereospecific reactions, beginning with the appropriate stereoisomer of the desired starting material. Methods and theories used to obtain enriched and resolved isomers have been described (Jacques et al., "Enantiomers, Racemates and Resolutions.” Kreiger, Malabar, FL, 1991).
• the salts include pharmaceutically or
• salts may include those derived by combination of appropriate cations such as alkali and alkaline earth metal ions or ammonium and quaternary amino ions with the acid anion moiety of the phosphate or phosphorothioate acid group present in
• Suitable salts include for example, acid addition salts such as HCl, HBr, HF, HI, H 2 SO 4 , and trifluoroacetate.
• the salts may be formed from acid addition of certain organic and inorganic acids, e.g., HCl, Hbr, H 2 SO 4, amino acids or organic sulfonic acids, with basic centers, (e.g. amines), or with acidic groups.
• the composition herein also comprise compounds of the invention in their un-ionized, as well as
• Exemplary invention cationic lipids have the structures shown in the Summary of the Invention above.
• the cationic lipids form aggregates with
• polyanionic macromolecules such as oligonucleotides, oligomers, peptides, or polypeptides through attraction between the positively charged lipid and the negatively charged polyanionic macromolecule.
• the aggregates may comprise multiamellar or unilamellar liposomes or other particles. Hydrophobic interactions between the
• cationic lipids and the hydrophobic substituents in the polyanionic macromolecule such as aromatic and alkyl moieties may also facilitate aggregate formation.
• Cationic lipids have been shown to efficiently deliver nucleic acids and peptides into cells in the presence of serum and thus are suitable for use in vivo or ex vivo .
• Cationic lipid-polyanionic macromolecule aggregates may be formed by a variety of method known in the art. Representative methods are disclosed by Feigner et al . , supra; Eppstein et al . supra; Behr et al . supra; Bangham, A. et al . M. Mol . Biol. 23 : 238 , 1965; Olson, F. et al . Biochim. Biophys . Acta 557:9, 1979; Szoka, F. et al . Proc. Natl . Acad. Sci . 75 : 4194, 1978; Mayhew, E. et al . Biochim. Biophys . Acta 775:169, 1984; Kim, S. et al. Biochim . Biophys . Acta 728 : 339 , 1983; and Fukunaga, M. et al . Endocrinol . 115:757, 1984. Commonly used
• lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion. See, e . g. , Mayer, L. et al . Biochim. Biophys . Acta 858 : 161, 1986. Microfluidization is used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, E., supra) . In general aggregates may be formed by
• lipid particles consisting of either (1) a cationic lipid of the invention or (2) a cationic lipid mixed with a colipid, followed by adding a polyanionic macromolecule to the lipid particles at about room temperature (about 18 to 26° C).
• room temperature about 18 to 26° C.
• conditions are chosen that are not conducive to deprotection of protected groups.
• the mixture is then allowed to form an aggregate over a period of about 10 minutes to about 20 hours, with about 15 to 60 minutes most conveniently used.
• the complexes may be formed over a longer period, but additional enhancement of transfection efficiency will not usually be gained by a longer period of
• Colipids that are suitable for preparing lipid aggregates with the cationic lipids of the present invention are dimyristoylphosphatidylethanolamine, dipalmitoyl-phosphatidylethanolamine,
• palmitoyloleolphosphatidyl-ethanolamine palmitoyloleolphosphatidyl-ethanolamine, cholesterrl, distearoyalphosphatidyl-ethanolamine,
• polyethylene glycol and mixtures of these colipids.
• the optimal cationic lipid: colipid ratios for a given cationic lipid is determined by mixing
• lipid colipid ratios between about 1:0 and 1:10.
• Suitable molar ratios of cationic lipid: colipid are about 0.1:1 t ⁇ l:0.1, 0.2:1 to 1:0.2, 0.4:1 to 1:0.4, or 0.6:1 to 1:0.6.
• Lipid particle preparation containing increasing molar proportions of colipid have been found to enhance oligonucleotide transfection into cells with increasing colipid concentrations.
• the cationic lipids can be used together in admixture, or different concentrations of two or more cationic lipids in admixture, with or without colipid.
• Liposomes or aggregates are conveniently prepared by first drying the lipids in solvent (such as
• the lipids are then hydrated and converted to liposomes or aggregates by adding water or low ionic strength buffer (usually less than about 200 mM total ion concentration) followed by agitating (such as vortexing and/or sonication) and/or freeze/thaw treatments.
• water or low ionic strength buffer usually less than about 200 mM total ion concentration
• agitating such as vortexing and/or sonication
• freeze/thaw treatments such as freeze/thaw treatments.
• the size of the aggregates or liposomes formed range from about 40 nm to 600 nm in diameter.
• the amount of an oligonucleotide delivered to a representative cell by at least some of the cationic lipids was found to be significantly greater than the amount delivered by commercially available transfection lipids.
• the amount of oligonucleotide delivered into cells was estimated to be about 2 - to 100-fold greater for these cationic lipids of the invention based on the observed fluorescence intensity of transfected cells after transfection using a fluorescently labeled oligonucleotide.
• the cationic lipids described herein also transfect some cell types that are not detectably transfected by commercial lipids. Functionality of cationic lipid-DNA aggregates was demonstrated by assaying for the gene product of the exogenous DNA.
• the cationic lipids described herein also differed from commercially available lipids by efficiently delivering an oligonucleotide into cells in tissue culture over a range of cell confluency from about 50 to 100%. Most commercially available lipids require cells that are at a relatively narrow confluency range for optimal transfection efficiency. For example,
• LipofectinTM requires cells that are 70-80% confluent for transfecting the highest proportion of cells in a population.
• the cationic lipids described herein may be used to transfect cells that are about 10-50% confluent, but toxicity of the lipids was more pronounced, relative to that seen using cells that are about 50-100%
• the cationic lipids transfected cells that were about 60-100% confluent with minimal toxicity and optimal efficiency. Confluency ranges of 60-95% or 60-90% are thus convenient for transfection protocols with most cell lines in tissue culture.
• the cationic lipid aggregates may be formed with a variety of macromolecules such as oligonucleotides and oligomers. Oligonucleotides used in aggregate formation may be single stranded or double stranded DNA or RNA, oligonucleotide analogs, and plasmids.
• oligonucleotides such as plasmids or mRNAs will carry one or more genes that are to be expressed in a transfected cell, while
• oligonucleotides will comprise (1) a base sequence that is complementary (via Watson Crick or Hoogsteen binding) to a DNA or RNA sequence present in the cell or (2) a base sequence that permits
• RNAs include ribozymes and antisense RNA sequences that are complementary to a target RNA sequence in a cell.
• An Oligonucleotide may be a single stranded unmodified DNA or RNA comprising (a) the purine or pyrimidine bases guanine, adenine, cytosine, thymine and/or uracil: (b) ribose or deoxyribose; and (c) a phosphodiester group that linkage adjacent nucleoside moieties.
• Oligonucleotides typically comprise 2 to about 100 linked nucleosides. Typical oligonucleotides range in size from 2-10, 2-15, 2-20, 2-25, 2-30, 2-50, 8-20, 8-30 or 2-100 linked nucleotides. Oligonucleotides are usually linear with uniform polarity and, when regions of inverted polarity are present, such regions comprise no more than one polarity inversion per 10 nucleotides. One inversion per 20 nucleotides is typical.
• Oligonucleotides can also be circular, branched or double-stranded.
• Antisense oligonucleotides generally will comprise a sequence of about from 8-30 bases or about 8-50 bases that is substantially complementary to a DNA or RNA base sequence present in the cell.
• the size of oligonucleotide that is delivered into a cell is limited only by the size of polyanionic macromolecules that can reasonably be prepared and thus DNA or RNA that is 0.1 to 1 Kilobase (Kb), 1 to 20 Kb, 20 Kb to 40 Kb or
• 40 Kb to 1,000 Kb in length may be delivered into cells.
• Oligonucleotides also include DNA or RNA comprising one or more covalent modifications.
• modifications include (a) substitution of an oxygen atom in the phosphodiester linkage of an polynucleotide with a sulfur atom, a methyl group or the like, (b)
• nonphosphorus moiety such as -O-CH 2 O-, -S-CH 2 O- or -O- CH 2 O-S, and (c) replacement of the phosphodiester grrup with a phosphate analog such as -O-P(S) (O) -O, -O- P(S) (S)-O-, -O-P(CH 3 ) (O)-O or -O-P (NHR 10 ) (O) -O- where R 10 is alkyl (C 1-6 ), or an alkyl ether (C 1-6 ) .
• a phosphate analog such as -O-P(S) (O) -O, -O- P(S) (S)-O-, -O-P(CH 3 ) (O)-O or -O-P (NHR 10 ) (O) -O- where R 10 is alkyl (C 1-6 ), or an alkyl ether (C 1-6 ) .
• substitutions may constitute from about 10% to 100% or about 20 to about 80% of the phosphodiester groups in unmodified DNA or RNA.
• Other modifications include substitutions of or on sugar moiety such as morpholino, arabinose 2'-fluororibose, 2'-fluoroarabinose, 2'-O- methylribose or 2'-O-allylribose. Oligonucleotides and methods to synthesize them have been described (for example see: U.S. Patent Application Serial Nos.
• PCT/US90/0612 PCT/US90/06090, PCT/US90/06110,
• PCT/US91/0685 PCT/US91/01141, PCT/US92/10115,
• Oligonucleotides are usually linear with uniform polarity and, when regions of inverted polarity are present, such regions comprise no more than one polarity inversion per 10 nucleotides. One inversion per 20 nucleotides is typical.
• Oligonucleotides may also be circular, branched or double-stranded.
• oligonucleotide may be a variety of moieties including both phosphorus-containing moieties and non phosphorus- containing moieties such as formacetal, thioformacetal, riboacetal and the like.
• a linkage usually comprises 2 or 3 atoms between the 5' position of a nucleotide and the 2' or 3' position of an adjacent nucleotide.
• the bases contained in the oligonucleotide may be unmodified or modified or natural or unatural purine or pyrimidine bases and may be in the ⁇ or ⁇ anomer. Such bases may be selected to enhance the affinity of
• modified bases are not
• oligonucleotide incorporated into an oligonucleotide to an extent that it is unable to bind to complementary sequences to produce a detectably stable duplex or triplex.
• Exemplary bases include adenine, cytosine, guanine, hypoxanthine, inosine, thymine, uracil, xanthine, 2- aminopurine, 2,6-diaminopurine, 5-(4-methylthiazol-2- yl) uracil, 5-(5-methylthiazol-2-yl) uracil, 5-(4- methylthiazol-2-yl) cytosine, 5-(5-methylthiazol-2- yl) cytosine and the like.
• exemplary bases include alkylated or alkynylated bases having substitutions at, for example, the 5 position of pyrimidines that results in a pyrimidine base other than uracil, thymine or cytosine, ( i . e . , 5-methylcytosine, 5-(1- propynyl) cytosine, 5-(1-butynyl) cytosine, 5-(1- butynyl) uracil, 5-(1-propynyl) uracil and the like).
• modified bases or base analogs in oligonucleotides have been previously described (see PCT/US92/10115; PCT/US91/08811; PCT/US92/09195; WO
• Aggregates may comprise oligonucleotides or
• polypeptide examples include histocompatibility antigens, cell adhesion molecules, cytokines, antibodies, antibody fragments, cell receptor subunits, cell receptors, intracellular enzymes and extracellular enzymes or a fragment of any of these.
• the oligonucleotides also may optionally comprise expression control sequences and generally will comprise a transcriptional unit comprising a transcriptional promoter, an enhancer, a transcriptional terminator, an operator or other expression control sequences.
• Oligonucleotides used to form aggregates for transfecting a cell may be present as more than one expression vector.
• 1, 2, or 3 or more different expression vectors may be delivered into a cell as desired.
• Expression vectors will typically express 1, 2, or 3 genes when transfected into a cell, although many genes may be present such as when a herpes virus vector or a yeast artificial chromosome is delivered into a cell.
• Expression vectors that are introduced into a cell can encode selectable markers (e.g. neomycin phosphotransferase, thymidine kinase, xanthine-guanine phosphoribosyl-transferase, and the like) or
• biologically active proteins such as metabolic enzymes or functional proteins (e.g. immunoglobulin genes, cell receptor genes, cytokines (e.g. IL-2, IL-4, GM-CSF, ⁇ - INF and the like), or genes that encode enzymes that mediate purine or pyrimidine metabolism and the like).
• metabolic enzymes or functional proteins e.g. immunoglobulin genes, cell receptor genes, cytokines (e.g. IL-2, IL-4, GM-CSF, ⁇ - INF and the like), or genes that encode enzymes that mediate purine or pyrimidine metabolism and the like).
• nucleic acid sequence of the oligonulcleotide coding for specific genes of interest may be retrieved, without undue experimentation, from the GenBank of EMBL DNA libraries.
• sequences may include coding sequences, for example, the coding sequences for
• transcriptional and translational regulatory elements promoters, enhancers, terminators, signal sequences and the like
• vectors integrating or
• Non-limiting examples of DNA sequences which may be introduced into cells with the reagent of the invention include those sequences coding for fibroblast growth factor ( see WO 87/01728); ciliary neurotrophic factor (Lin et al . , Science, 246 : 1023 , 1989; human interferon- ⁇ receptor (Uze, et al . , Cell , 60 : 225 , 1990; the interleukins and their receptors
• Aggregates can be used in antisense inhibition of gene expression in a cell by delivering an antisense oligonucleotide into the cell (see Wagner, Science
• Such oligonucleotides will generally comprise a base sequence that is
• the oligonucleotide may regulate intracellular gene expression by binding to an
• nucleic acid binding protein see Clusel, Nucl . Acids Res . 21:3405, 1993
• intracellular protein or organelle that is not known to bind to nucleic acids
• a cell that is blocked for expression of a specific gene(s) is useful for manufacturing and therapeutic applications.
• Exemplary manufacturing uses include inhibiting protease synthesis in a cell to increase production of a protein for a therapeutic or diagnostic application (e . g. , reduce target protein degradation caused by the
• Exemplary therapeutic applications include inhibiting synthesis of cell surface antigens to reduce rejection and/or to induce immunologic tolerance of the cell either after it is implanted into a subject or when the cell is trnsfected in vivo (e . g. histocompatibility antigens, such as MHC class II genes, and the like).
• Entry of liposomes or aggregates into cells may be by endocytosis or by fusion of the liposome or aggregate with the cell membane.
• the liposomal membrane is integrated into the cell membrane and the aqueous contents of the liposome merge with the fluid in the cell.
• Endocytosis of liposomes occurs in a limited class of cells; those that are phagocytic, or able to ingest foreign particles.
• phagocytic cells take up liposomes or aggregates, the cells move the spheres into subcellular organelles known as lysosomes, where the liposomal membranbes are thought to be degraded.
• the liposomal lipid components probably migrate outward to become part of cell's membranes and other liposomal components that resist lysosomal degradation (such as modified oligonucleotides or oligomers) may enter the cytoplasm.
• Lipid fusion involves the transfer of individual lipid molecules from the liposome or aggregate into the plasma membrane (and vice versa); the aqueous contents of the liposome may then enter the cell.
• the liposomal lipid must have a particular chemistry in relation to the target cell. Once a liposomal lipid joins the cell membrane it can either remain in the membrane for a period of time or be redistributed to a variety of intracellular membranes.
• the invention lipids can be used to deliver an
• the expression vectors can be used in gene therapy protocols to deliver a therapeutically useful protein to a cell or for delivering nucleic acids encoding molecules that encode therapeutically useful proteins or proteins that can generate an immune
• the vector-transformed cell can be used to produce commercially useful cell lines, such as a cell line for producing therapeutic proteins or enzymes (e.g. erythropoietin, and the like), growth factors (e.g.
• the aggregates may be utilized to develop cell lines for gene therapy applications in humans or other species including murine, feline, bovine, equine, ovine or non- human primate species.
• the aggregates may be used in the presence of serum and will thus deliver polyanionic macromolecules into cells in tissue culture medium containing serum in vitro or in an animal in vivo.
• Figure 1 depicts synthetic route for the synthesis of 1-2.
• Figure 2 depicts synthetic route for the synthesis of 2-4.
• Figure 3 depicts synthetic route for the syntheses of 3-3 and 3-4.
• Figure 4 depicts structure of 4-1 ( (3 ⁇ - cholest-5- en-3-ol, 2-(dimethylamino) ethyl methylphosphonate), 4-2.
• the syntheses of 4-1 and 4-2 are described in the commonly assigned and concurrently filed US Patent
• Figure 5 depicts the combination of cationic lipids 1-2/4-1 delivers plasmids and oligodeoxynucleotides into cells as demonstrated by the dose dependent protein translation arrest.
• N-Cholesteryloxycarbonylethylenediamine (1-1) was prepared as follows and used without further
• succinimidyl ester (Boc-orn(Boc) -OSu) (1-3) was prepared as follows and used without further
• 18-Hydroxypentatriacontane (2-1) was prepared as follows and used without further purification.
• a solution of lithium aluminum hydride (0.75 g, (19.8 mmol)) in freshly distilled tetrahydrofuran (400 mL).
• stearone 5.0 g, (9.9 mmol)
• the reaction mixture was slowly brought to reflux (and allowed to stir at reflux overnight).
• the remaining lithium aluminum hydride was quenched by the dropwise addition of water (0.76 mL), followed by 15 % NaOH (0.76 mL), and water again (2.2 mL).
• the reaction mixture was allowed to stir for 1 hour.
• N-t-Butyloxycarbonylethylenediamine (2-3) was prepared as follows and used without further
• the purified product (0.2g, (0.3 mmol) was then deprotected with trifiuoroacetic acid/dichloromethane 1:1 (2 mL) for 20 minutes. After coevaporation with 1,2-dichloroethane the desired product 2-4 was obtained (175 mg, 87% yield) .
• N, N 2 , N 3 , N 4 -Tetrakis- ( tert-butoxycarbonyl) spermine carboxylic acid N-hydroxysuccinimydyl ester (3-2) was prepared as follows and used without further
• COS 7 cells (ATCC # CRL 1651) were plated at 1.5 x 10 5 cells/well in a 12 well plate format on the day before transfections began. All cultures were
• the transfection mixes were prepared as follows: 4 ⁇ g of the target CAT plasmid (see Example 7), was combined with 10, 50, 100, or 400 nanomolar final
• phosphorothioate oligomer (synthesized by JBL Scientific, San Luis Obispo, CA) in 2 mL Opti-MEM ® (Gibco/BRL, Gaithersburg, MD) The oligomer/plasmid mixes were combined with 24 micrograms of lipid and gently
• the final plasmid concentration was 2 ⁇ g/mL and the final lipid concentration was 12 mg/mL.
• the final result was four sets of transfection mixes tested in duplicate: pG1040 plasmid + 2519-1 oligomer + Lipofectin pG1040 plasmid + 2520-1 oligomer + Lipofectin pG1040 plasmid + 2519-1 oligomer + GC-001/GC-003 lipid mix
• Phosphorothioate oligomer 2519-1 (5 ' -tag-ctt-cct- tag-ctc-ctg-cat) is a perfect antisense match to pG1040 from +1 to +21 on the CAT mRNA.
• Phosphorthioate oligomer 2520-1 (5' tag-ctt-ccg- caa-ctc-ttg-cat) is a 4 mismatch control
• phosphorothioate also from +1 to +21 on the CAT mRNA.
• the culture medium was aspirated off and the cells were rinsed twice in one mL Opti-MEM ® per well, and then 1 mL of transfection mix was added to each well. The cells were cultured in the transfection mix for 16 hours. The mix was removed and replaced with 1 mL of complete culture medium (DMEM plus 10% fetal bovine serum and 1/100 dilution of penicillin/streptomycin stock, all from Gibco/BRL, Gaithersburg, MD) and the cells were incubated another 5 hours.
• complete culture medium DMEM plus 10% fetal bovine serum and 1/100 dilution of penicillin/streptomycin stock, all from Gibco/BRL, Gaithersburg, MD
• Cell lysates were prepared by rinsing twice in PBS and then treated with 0.5 mL of IX Reporter Lysis Buffer (Promega, Madison, WI). The lysed cells were piptetted into 1.5 mL tubes and frozen in CO 2 /EtOH once and thawed. The crude lysate was then clarified by
• the cell lysates were then assayed for CAT activity and the total protein concentration was determined as described below.
• the CAT activity was normalized to total protein and plotted as shown.
• a CAT activity standard curve was prepared by serially diluting CAT stock (Promega, Madison, WI) 1:1000, 1:10,000 and 1:90,000 in 0.25 M Tris, pH 8/0.5% BSA. The original stock CAT was at 7000 Units/mL. CAT lysate was then added in a labeled tube with Tris/BSA buffer for final volume of 50 ⁇ L.
• reaction mixture Approximately 74 ⁇ L of reaction mixture was added to each tube, which was then incubated for approximately 1 hour in a 37°C oven. The reaction was terminated by adding 500 ⁇ L pristane/mixed xylenes (2:1) (Sigma, St. Louis, MO) to each tube. The tubes were then vortexed for 2 minutes and spun for 5 minutes. Approximately, 400 ⁇ L of the upper phase was transferred to a
• Coomassie Protein Assay The total protein content of the clarified cell lysates was determined by mixing 6 ⁇ L of each cell lysate to 300 ⁇ L of Coomassie protein assay reagent (Pierce, Rorkford, MD) in the wells of an untreated microtiter assay plate. Concentration curve standards were prepared using 6 ⁇ L of 0, 75, 100, 200, 250, 400, 500, 1000, and 1500 mg/mL BSA stock solutions and 300 ⁇ L of the Coomassie reagent. The assay samples were allowed to sit for approximately 30 minutes before reading the optical absorbance at 570 nm in a microplate reader (Molecular Probes).
• Results indicate that the combination of 1 and 4 was able to deliver oligo/plasmid better than
• oligonucleotides used for the determination of cationic lipid mediated oligonucleotide uptake in all cell lines tested were:
• #3498-PS 5' FITC-ggt-ata-tcc-agt-gat-ctt-ctt-ctc, all-phosphorothioate backbone. This oligonucleotide has 23 negative charges on the backbone, and is considered 100% negatively charged.
• #3498 5' FITC-ggt-ata-tcc-agt-gat-ctt-ctt-ctc, chimeric oligonucleotide.
• the underlined bases were linked by a phosphorothioate backbone, while the other linkages in the oligomer consisted of alternating methylphosphonates and phosphodiesters.
• the oligomer had 11 methylphonate, 7 diester, and 5 phosphorothioates linkages. The total charge density was 57% of 3498-PS.
• the total charge density was 50% of 3498-PS.
• Oligonucleotides 3498-PS and 3498 stocks were at 300 ⁇ m, while the 3793-2 stock was at 440 ⁇ m.
• glioblastoma C8161 (human tumor of unknown tissue origin), RD (human rhabdomyosarcoma, ATCC # CCL-136) and COS-7 (African green monkey kidney cells, ATCC # 1651) were maintained in standard cell culture media.
• DMEM /F12 (1:1) mix from Mediatech, Lot#150901126, 10% fetal bovine serum from Gemini Bioproducts, Lot#A1089K, 100 units/mL penicillin and 100 micrograms/mL streptomycin, from Mediatech, Lot#30001044 and 365 micrograms/mL L- glutamine.
• the cells were maintained under standard conditions at all times, 37 degrees C, 5% CO 2 atmosphere prior to fixation and microscopic examination.
• Oligonucleotide transfection mixes were made up in Opti-MEM ® without antibiotics as follows: 500 ⁇ L aliquotes of Opti-MEM ® containing 0.25 ⁇ m of 3498-PS, 3498, or 3793-2 (2 ⁇ g of oligonucleotide) were pipetted into 1.5 mL Eppendorf tubes. Each cationic lipid or lipid mixture was then added to the oligonucleotide solution to give a final 9:1 or 6:1 ratio (18 or 12 ⁇ g of lipid total) of cationic lipid to oligonucleotide by weight, as listed in the data tables. The tubes were mixed by vortexing immediately after the addition of lipids.
• Intracellular fluorescence was evaluated under 200X magnification with a Nikon Labophot-2 microscope with an episcopic-fluorescence attachment. Using this
• the cells were scored for uptake as follows: No nuclear fluorescence, 0; up to 20% fluorescent nuclei, 1; up to 40%
• fluorescent nuclei 2; up to 60% fluorescent nuclei, 3; up to 80% fluorescent nuclei, 4; and up to 100%
• pG1035 Splicer CAT inserted into a pRc/CMV vector pG1036 Wild-type CAT, inserted into a pRc/CMV vector pG1040 UCAT, inserted into a pRc/CMV vector
• pGL2 Luciferase expressing plasmid (Promega)
• pSVb b-galactosidase expressing plasmid (Clonetech)
• the region of the CAT gene into which the intron was inserted is shown in sequence A above. Wild type CAT DNA (Pharmacia) was inserted into pRc/CMV
• plasmid pG1036 The sequence is shown as the mRNA. Bases 409 and 410 are labeled for comparison to pG1035.
• the canonical guanosine of the splice donor is labeled +409, which corresponds to base 409 of the CAT open reading frame.
• the first base of the intron is labeled 1.
• the canonical branchpoint adenosine is base 39 and the canonical intronic splice acceptor guanosine is base 87 of the intron.
• Base 410 marks the resumption of the CAT open reading frame. The sequences against which the oligomers are targeted are underlined. The consensus splice site bases are given in bold face italics (Smi th et al . , Ann . Rev. Genet . 23 : 527, 1989; Green, Ann . Rev. Genet . 20 : 671, 1986).
• the clone pG1035 was created using synthetic DNA PCR primers to create a Hind III-Spe I 5 ' fragment containing the first 2/3 of the open reading frame and half of the synthetic intron and an Spe I-Not I fragment containing the second half of the intron and the last 1/3 of the open reading frame. These were combined with Hind III-Not I cut pRc/CMV in a 3 -way ligation to yield the final plasmid.
• the artificial CAT gene containing the intron is named SplicerCAT. References applicable to the foregoing include Smith et. al . supra and Green supra .
• the sequences of wild type and pG1040 UCAT around the AUG start codon are shown.
• the target sites for the oligomers are named and underlined, and the numbers of the chimeric oligomers against each target site are shown beneath.
• UCAT was made from wild-type CAT DNA (Pharmacia, piscataway, NJ) using synthetic DNA PCR primers. The resulting fragment was cloned as a Hind III (5' end), Not I (3' end) fragment into the vector pRc/CMV
• the first adenosine of the open reading frame is designated +1.
• the amino acid changes between wild-type and pG1040 are conservative.

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