WO1999015206A1 - Procedes de preparation de complexes de transfection lipidiques/polynucleotidiques - Google Patents

Procedes de preparation de complexes de transfection lipidiques/polynucleotidiques Download PDF

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Publication number
WO1999015206A1
WO1999015206A1 PCT/US1998/019936 US9819936W WO9915206A1 WO 1999015206 A1 WO1999015206 A1 WO 1999015206A1 US 9819936 W US9819936 W US 9819936W WO 9915206 A1 WO9915206 A1 WO 9915206A1
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polynucleotide
cationic lipid
complexes
lipid
dna
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PCT/US1998/019936
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English (en)
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Todd Wedeking
Ralph W. Niven
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Megabios Corporation
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Priority to AU94043/98A priority Critical patent/AU9404398A/en
Publication of WO1999015206A1 publication Critical patent/WO1999015206A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A61K47/544—Phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids

Definitions

  • This invention relates to preparation of polynucleotide transfection complexes and their use in delivering polynucleotides to cellshelper lipids, used in conjunction with cationic lipids, for the preparation of liposomes and other lipid-containing carriers of nucleic acids and other substances, for delivery to cells.
  • the invention relates to methods for preparing complexes of polynucleotides and cationic lipids suitable for transfecting eukaryotic cells in vivo and in vitro.
  • Ex vivo genetic modification involves the removal of specific cells from an animal, including humans, introduction of the exogenous genetic material, and then re-introduction of the genetically modified cells into the animal.
  • in vivo genetic modification involves the introduction of genetic material directly to the animal, including humans, using an appropriate delivery vehicle, where it is taken up directly by the target cells.
  • the various methods used to introduce nucleic acids into cells have as a goal the efficient uptake and expression of foreign genes.
  • the delivery of exogenous nucleic acids in humans and/or various commercially important animals will ultimately permit the prevention, amelioration and cure of many important diseases and the development of animals with commercially important characteristics.
  • the exogenous genetic material either DNA or RNA, may provide a functional gene which, when expressed, produces a protein lacking in the cell or produced in insufficient amounts, or may provide an antisense DNA or RNA or ribozyme to interfere with a cellular function in, e.g., a virus-infected cell or a cancer cell, thereby providing an effective therapeutic for a disease state.
  • Cationic lipids have been developed that greatly facilitate nucleic acid delivery to cells, both in vitro and in vivo. See, for example, U.S. Patent No. 5,264,618, which describes techniques for using lipid carriers, including the preparation of liposomes and pharmaceutical compositions and the use of such compositions in clinical situations.
  • a number of cationic lipids have been developed, which are generally amphipathic molecules comprising a positively charged headgroup, varying from single to multiple positive charges, linked to hydrophobic lipid tail groups or steroidal groups.
  • the cationic lipids typically are mixed with a non-cationic lipid, usually a neutral lipid, and allowed to form stable liposomes, which liposomes are then mixed with the nucleic acid to be delivered.
  • the liposomes may be large unilamellar vesicles (LUVs), multilamellar vesicles (MLVs) or small unilamellar vesicles (SUVs).
  • LUVs large unilamellar vesicles
  • MLVs multilamellar vesicles
  • SUVs small unilamellar vesicles
  • the liposomes are mixed with nucleic acid in solution, at concentrations and ratios optimized for the target cells to be transfected, to form cationic lipid-nucleic acid transfection complexes. Alterations in the lipid formulation allow preferential delivery of nucleic acids to particular tissues in vivo. See PCT patent application numbers WO 96/40962 and WO 96/4
  • Nucleic acids are generally large polyanionic molecules which, therefore, bind cationic lipids and other positively-charged carriers through charge interactions. It is believed that the positively charged carriers such as cationic lipids form tight complexes with the nucleic acid, thereby condensing it and protecting it from nuclease degradation. In addition, cationic lipid carriers may act to mediate transfection by improving association with negatively-charged cellular membranes by giving the complexes a positive charge, and/or enhancing transport from the cytoplasm to the nucleus where DNA may be transcribed.
  • transfection efficiency is highly dependent on the characteristics of the cationic lipid/nucleic acid complex.
  • the nature of the complex that yields optimal transfection efficiency depends upon the mode of delivery, e.g. ex vivo or in vivo; for in vivo delivery, the route of administration, e.g., intravenous, intraperitoneal, inhalation, etc.; the target cell type, etc. Depending on the use, therefore, different carriers will be preferred.
  • transfection efficiency will depend on certain physical characteristics of the complexes as well, such as charge and size.
  • the stability of the complexes during storage will be highly dependent on the physical nature of the complexes.
  • the present invention provides these and related advantages as well.
  • Cationic lipid carriers have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al., (1987) Proc. Natl. Acad. Sci. (USA), 84:7413-7416); mRNA ( alone et al, (1989) Proc. Natl. Acad. Sci. (USA) 86:6077-6081); and purified transcription factors (Debs et al., (1990) J. Biol. Chem. 265:10189-10192), in functional form.
  • Literature describing the use of lipids as carriers for DNA include the following: Zhu et al., (1993) Science, 261:209-211; Vigneron et al., (1996) Proc.
  • a method of preparing cationic lipid nucleic acid transfection complexes by first forming lipid micelles in the presence of detergent is described in WO 96/37194.
  • the effect of surfactants on DNA/lipid complexes and transfection activity are described in Liu et al., (1996) Pharm. Res. 13(11):1642-1646.
  • the effect of liposome preparation and complex size on cationic lipid-mediated gene delivery is described in
  • the invention provides a method of preparing a cationic lipid/polynucleotide transfection complex, the method comprising co-solubilizing a cationic lipid carrier and a polynucleotide in an aqueous solution in the presence of a co-solvent, then removing the co-solvent such that the polynucleotide and cationic lipid carrier molecules "nucleate" in solution, forming an aqueous dispersion of cationic lipid/polynucleotide transfection complexes.
  • the polynucleotide, cationic lipid and, optionally, non-cationic lipid are co-solubilized in the ratios desired in the final transfection complex, which ratios will be dependent upon the lipids used, the target cell type and, if administered in vivo, the route of administration.
  • the polynucleotide is typically plasmid DNA, generally including a recombinant expression construct, the DNA encoding a transcription product and operatively linked regulatory elements, whereby the DNA is capable of transcription in the target cells.
  • transcription product is intended to encompass an RNA product resulting from transcription of a nucleic acid sequence, and includes RNA sequences that are not translated into protein (such as antisense RNA or ribozymes) as well as RNAs that are subsequently translated into polypeptides or proteins. Also included is the preparation of complexes including polycationic carriers and oligonucleotides.
  • the cationic lipid is DOTIM
  • the neutral lipid is cholesterol
  • the lipid and DNA are co-solubilized in a solution of about 70% ethanol and 10% ethyl acetate.
  • the co-solvents are removed by dialysis, and the resulting cationic lipid/polynucleotide transfection complexes have an average size of less than about 500 nm.
  • Figure 1 shows the density gradient profiles of DNAxationic lipid complexes. Profiles are measured by flow-cell UV spectrophotometer at 237nm, which is the absorbarice of DOTIM. The contents of the centrifuged samples (approx. 13ml) are pumped through the flow-cell at a rate of 1 ml/min. The ordinate represents the approximate location within the centrifuge tube.
  • FIG. 1 shows the density gradient profiles of complex preparations: a) prepared by the standard mixing method in a 1:5 DNA/cationic lipid ratio ( ⁇ g DNA:nmoles cationic lipid); b) prepared by the solution nucleation method in a 1:1 DNA/cationic lipid ratio; and c) prepared by the standard mixing method in a 1:1 DNA/cationic lipid ratio.
  • polynucleotide/cationic lipid transfection complexes The physical nature of polynucleotide/cationic lipid transfection complexes is highly dependent on the method in which they are prepared. The physical nature of the transfection complexes, in turn, is important for the ability of the complexes to transfect target cells. In addition, the stability of the complexes during storage is highly dependent upon their physical nature. Typically, more homogenous compositions are more stable during storage than heterogeneous compositions. Also, compositions containing a higher proportion of active particles utilize starting materials more efficiently, and, in many cases less material will be necessary to achieve desired transfection rates, thereby decreasing any undesirable toxicities associated with excess material.
  • transfection complexes are prepared by adding one solution to the other, i.e. nucleic acid to pre-formed liposomes or liposomes to nucleic acid, with constant stirring.
  • Transfection complexes prepared by prior art methods of adding one solution to the other results in a heterogeneous mix of complexes because the environment under which the complexes are formed is constantly changing.
  • most prior art methods of complex preparation involve first preparing the lipids in a dispersed liposomal form.
  • most cationic lipid carriers are formulated with a neutral lipid to aid in forming stable liposomal intermediates.
  • the lipids are usually dried as a film in an organic solution such as chloroform, then dispersed in an aqueous solution.
  • the lipids spontaneously form liposomes.
  • the liposomes are formed as a heterogeneous mix unilamellar and multilamellar vesicles, in a range of sizes. They are then usually sonicated or extruded through membranes with specified pore sizes, usually to form small unilamellar vesicles (SUVs).
  • SUVs small unilamellar vesicles
  • the method of the present invention allows the formation of nucleic acid/cationic lipid transfection complexes by a process termed "solution nucleation.”
  • solution nucleation involves the co-solubilization of the desired components in the ratios desired in the final transfection complex.
  • the solution will contain an aqueous component and one or more co-solvents.
  • co-solvent(s) may then be removed, resulting in a substantially aqueous environment. The loss of hydration within the co-solvent(s) may cause the polynucleotides to compact or condense more readily than within a more aqueous environment.
  • Transfection means the delivery of exogenous nucleic acid molecules to a cell, either in vivo or in vitro, whereby the nucleic acid is taken up by the cell and is functional within the cell.
  • a cell that has taken up the exogenous nucleic acid is referred to as a "host cell”, “target cell” or “transfected cell.”
  • a nucleic acid is functional within a host cell when it is capable of functioning as intended.
  • the exogenous nucleic acid will comprise an expression cassette which includes DNA coding for a gene of interest, with appropriate regulatory elements, which will have the intended function if the DNA is transcribed and translated, thereby causing the host cell to produce the peptide or protein encoded therein.
  • DNA may encode a protein lacking in the transfected cell, or produced in insufficient quantity or less active form, or secreted, where it may have an effect on cells other than the transfected cell.
  • exogenous nucleic acid to be delivered include, e.g., antisense oligonucleotides, mRNA ribozymes, or DNA encoding antisense RNA or ribozymes.
  • Nucleic acids of interest also include DNA coding for a cellular factor which, when expressed, activates the expression of an endogenous gene.
  • Transfection efficiency refers to the relative number of cells of the total within a cell population that are transfected and/or to the level of expression obtained in the transfected cells. It will be understood by those of skill in the art that, by use of appropriate regulatory control elements such as promoters, enhancers and the like, the level of gene expression in a host cell can be modulated. The transfection efficiency necessary or desirable for a given purpose will depend on the purpose, for example the disease indication for which treatment is intended, and on the level of gene expression obtained in the transfected cells.
  • Polycation refers to any molecular entity having multiple positive charges. Because of their positive charges, polycations interact electrostatically with negatively charged polynucleotides, usually condensing the polynucleotide molecules.
  • Polycationic carrier refers to a polycation which, when combined with a polynucleotide, forms a complex suitable for transfecting eukaryotic cells. Cationic lipids have been shown to be efficient polycationic carriers for nucleic acid delivery to cells. Typically, cationic lipid carriers are formulated with both cationic and non-cationic lipid (usually neutral lipid) components.
  • lipid carrier or “cationic lipid carrier” refers to a lipid composition of one or more cationic lipids and, optionally, one or more non-cationic lipids for delivering agents to cells.
  • a lipid carrier may be complexed with other polycations or additional transfection facilitating agents.
  • Transfection complex or “polynucleotide transfection complex” refers to a combination of a polycationic carrier and a nucleic acid, in any physical form, for use in transfecting eukaryotic cells.
  • a transfection complex may include additional moieties, e.g., targeting molecules such as receptor ligands or antibody fragments, or other accessory molecules.
  • targeting molecules such as receptor ligands or antibody fragments, or other accessory molecules.
  • nuclear localizing peptides may be included for facilitating transport of the polynucleotide to the cell nucleus. Kalderon et al., (1984) Cell 39:499-509; Chelsky et al., (1989) Mol. Cell Biol. 9:2487-2492; Dingwall & Laskey (1991) Trends Biochem. Sci.
  • Proteins or peptides may be included in the transfection complex to facilitate release of the transfection complex from the endosome after internalization.
  • enzymes involved in transcription and/or translation may be included to facilitate gene expression in the cell cytoplasm without transport to the cell nucleus.
  • the transfection complexes may also be prepared to include a targeting moiety, to specifically deliver complexes to the desired target cell in vivo.
  • a targeting moiety to specifically deliver complexes to the desired target cell in vivo.
  • strategies are known in the art for including receptor ligands for delivery to cells expressing the appropriate receptor, or using antibodies or antibody fragments to target transfection complexes to cells expressing a specific cell surface molecule. See WO 96/37194; Ferkol et al., (1993) J. Clin. Invest. 92:2394-2400.
  • cationic lipid is intended to encompass lipids that are positively charged at physiological pH, and more particularly, constituitively positively charged lipids comprising, for example, a quarternary ammonium salt moiety.
  • Cationic lipids used for gene delivery typically consist of a hydrophilic polar head group and lipophilic aliphatic chains. Alternatively, cholesterol derivatives having a cationic polar head group are used in a similar manner.
  • Cationic lipids of interest include, for example, imidazolinium derivatives (WO 95/14380), guanidine derivatives (WO 95/14381), phosphatidyl choline derivatives (WO 95/35301), and piperazine derivatives (WO 95/14651).
  • Examples of cationic lipids that may be used in the present invention include DOTIM (also called BODAI) (Solodin et al., (1995) Biochem. 34: 13537-13544), DDAB (Rose et al.,
  • MBOP also called MeBOP
  • EDMPC aerosolized delivery to airway epithelial cells
  • DOTIM for aerosolized delivery to airway epithelial cells
  • DOTIM for aerosolized delivery to airway epithelial cells
  • DOTIM for aerosolized delivery to airway epithelial cells
  • DOTAP for intravenous delivery to vascular endothelial cells of various organs, particularly the lung.
  • cationic lipid carriers having more than one cationic lipid species may be used to produce complexes according to the method of the present invention.
  • Non-cationic lipids of use in transfection complexes include, for example, dioleoyl phosphatidylethanolamine (DOPE), Hui et al., (1996) Biophys. J. (71):590-599; cholesterol, Liu et al., (1997) Nat. Biotech. (15):167-173; and dilauroyl phosphatidylethanolamine (DLPE) (co-pending patent application serial no. 08/832,749).
  • DOPE dioleoyl phosphatidylethanolamine
  • Hui et al. Hui et al., (1996) Biophys. J. (71):590-599
  • cholesterol Liu et al., (1997) Nat. Biotech. (15):167-173
  • DLPE dilauroyl phosphatidylethanolamine
  • DLPE dilauroyl phosphatidylethanolamine
  • Additional polycationic carriers include positively charged peptides and proteins, both naturally occurring and synthetic, as well as polyamines, carbohydrates or synthetic polycationic polymers. Examples include polylysine, polyarginine, protamine, polybrene, histone, cationic dendrimer, and synthetic polypeptides based on viral peptides, e.g., having cell binding, endosomal release or nuclear localizing functions, etc.
  • polycationic carriers may include cationic lipid as well as peptide moieties. See, e.g., WO 96/22765.
  • the nucleic acid may be in any physical form, e.g., linear, circular or supercoiled; single-stranded, double-, triple-, or quadruple-stranded; and further including those having naturally occurring nitrogenous bases and phosphodiester linkages as well as non-naturally occurring bases and linkages, e.g. for stabilization purposes.
  • it is in the form of supercoiled plasmid DNA.
  • Plasmid DNA is conveniently used for DNA transfections since there are no size constraints on the DNA sequences that may be included, and it can be produced in large quantity by growing and purifying it from bacterial cells.
  • Polynucleotide transfection complexes are formed by the electrostatic binding between the polynucleotide and the polycationic carrier. In addition to the mixing conditions, the physical structure of such complexes depends on the polycationic carrier and nucleic acid components, the ratios between them, concentrations of each, buffer ionic strength, and the like. Smith et al., (1997) Adv. Drug Deliv. Rev. 26:135-150. Initially, the components of the transfection complex must be co-solubilized in a solution in the ratios desired in the final complex. The solution will contain water and one or more co-solvents. As used herein, a co-solvent is any solvent other than water. Typically, co-solvents will include polar organic solvents. The solution should be prepared in the absence of salts, which would decrease the solubility of the lipid and polynucleotide components and interfere with the complexation process.
  • the co-solvent is usually one that is miscible with water and, when combined with the aqueous component, the polynucleotide and lipids are both soluble.
  • the selection of co-solvent will depend on the lipid(s) used. Solubility of many lipids are known or can be determined from, for example, CRC Handbook of Chemistry and
  • lipid component for example a cationic lipid and a neutral lipid, the least soluble lipid is the preferred starting point for selecting a co-solvent or combination of co-solvents. Once the solubility of the individual components is determined, the choice of co-solvents and relative amounts may be determined empirically.
  • the solvents should be selected to minimize any safety or regulatory issues.
  • Short chain alcohols are useful co-solvents because nucleic acids remain soluble up to high levels.
  • a preferred alcohol is ethanol.
  • Other useful co-solvents include chloroform, methanol, propanol, butanol (e.g.
  • the components are co-solubilized in a solution containing at least about 50% ethanol and less than about 20% ethyl acetate, most preferably, the solution contains about 70% ethanol and about 10% ethyl acetate.
  • the components may be mixed in any order that avoids precipitation, although the mixing process described in the examples that follow is preferred.
  • the solution will be prepared in the absence of detergent. In a detergent solution, the lipid component will tend to form micelles. It is desirable to simplify the process to avoid issues relating to detergent removal.
  • lipid/polynucleotide complexes Upon continued removal of the co-solvent, lipid/polynucleotide complexes will form around such "nucleation" sites, resulting in an aqueous solution comprising substantially homogeneous lipid/polynucleotide transfection complexes.
  • the co-solvent is preferably removed by dialysis. Other means of co-solvent removal include, for example, diafiltration, tangential flow filtration, dilution, heating, freezing, etc.
  • any dialysis membrane may be used that is compatible with the solvent system used.
  • a wide variety of dialysis membranes are available commercially, and solvent compatibility is generally available from the manufacturers' specifications.
  • the dialysis membrane has the smallest molecular weight cutoff available.
  • a regenerated cellulose membrane with a molecular weight cutoff of about 12,000 to 14,000 daltons may be used, such as the Spectra/Por #2 dialysis membrane (Spectrum, Houston, Texas).
  • the dialysis buffer may be any buffer suitable for the subsequent uses of the complexes, and may include any physiologically acceptable buffer or no buffer. If the complexes will not be used immediately, but will be stored before use, the dialysis buffer selected will depend primarily on the lipid components of the complexes and will be of a composition and pH designed to preserve the stability of the complexes.
  • the dialysis buffer is a low ionic strength buffer to minimize interference by any additional ions in the complexation process.
  • a low-ionic strength solution means a solution having a conductivity less than about 35 mS, preferably less than about 10 mS, and most preferably less than about 1 mS.
  • the dialysis solution will contain no salts.
  • a preferred dialysis buffer is 5% dextrose in 5 mM Tris-HCl (pH 8.0). Dialysis should be performed against a large excess of dialysis buffer, e.g., at least about 500-fold, and may be 1000-fold or greater.
  • the complexes may be concentrated after dialysis.
  • the degree of concentration will depend on the desired use of the complexes, for example, any limitations in volume due to the intended route of administration. Methods for concentration include, for example, vacuum dialysis, centrifugation, lyophilization, evaporation, and tangential flow filtration.
  • a number of analytical methods are known for characterizing the complexes prepared according to the method of the invention.
  • Visual inspection may provide initial information as to aggregation of the complexes.
  • Spectrophotometric analysis may be used to measure the optical density, giving information as to the aggregated state of the complexes; surface charge may be determined by measuring zeta potential; agarose gel electrophoresis may be utilized to examine the amounts and physical condition of the polynucleotide molecules in the complexes; particle sizing may be performed using commercially available instruments; HPLC analysis will give additional information as to resulting component ratios and any component degradation; and dextrose or sucrose gradients may be used to analyze the composition and heterogeneity of complexes formed.
  • the preferred size of the resulting complexes will depend on the desire use. For intravenous administration, the size is preferably less than about five microns, more preferably less than about 500 nm. For aerosol administration, the size should be less than about 500 ran, preferably around 100 nm or less.
  • the size of the resulting complexes may be altered by adjusting the pH, the lipid :polynucleo tide concentrations or ratios, the ratios of the lipid components, or by adjusting physical parameters such as temperature, viscosity or agitation as known for other nucleation processes. See, e.g, Mullin, J.W., Crystallization, 3 rd P,H. (Butterworth-Heinemann, Oxford, 1993)
  • polynucleotide transfection complexes may be prepared in a variety of formulations depending of the desired use.
  • Uses contemplated for the complexes of the invention include both in vivo and in vitro transfection procedures corresponding to those presently known that use cationic lipid carriers, including those using commercial cationic lipid preparations, such as Lipofectin(, and various other published techniques using conventional cationic lipid technology and methods. See, generally, Lasic and Templeton (1996) Adv. Drug Deliv. Rev. 20: 221-266 and references cited therein.
  • the ratios of each component in the complexes, final concentrations, buffer solutions, and the like are easily adjusted by adjusting the starting components.
  • the method allows the resulting transfection complexes to the prepared in a highly controlled fashion, efficiently using starting materials and yielding active transfection complexes.
  • Cationic lipid-nucleic acid transfection complexes can be prepared in various formulations depending on the target cells to be transfected. See, e.g., WO 96/40962 and WO 96/40963. While a range of lipid/polynucleotide complex formulations will be effective in cell transfection Since the activity of a given cationic lipid-nucleic acid transfection complex in transfecting cells in vitro does not correlate with in vivo activity, optimum conditions are determined empirically in the desired experimental system. Lipid carrier compositions may be evaluated by their ability to deliver a reporter gene (e.g.
  • CAT which encodes chloramphenicol acetyltransferase, luciferase, or (-galactosidase) in vitro, or in vivo to a given tissue in an animal, such as a mouse.
  • in vitro transfections the various combinations are tested for their ability to transfect target cells using standard molecular biology techniques to determine DNA uptake, RNA and/or protein production.
  • in vitro cell transfection involves mixing nucleic acid and lipid, in cell culture media, and allowing the lipid-nucleic acid transfection complexes to form for about 10 to 15 minutes at room temperature. The transfection complexes are added to the cells and incubated at 37°C for about four hours.
  • the complex-containing media is removed and replaced with fresh media, and the cells incubated for an additional 24 to 48 hours.
  • particular cells can be preferentially transfected by the use of particular cationic lipids for preparation of the lipid carriers, for example, by the use of EDMPC to transfect airway epithelial cells (WO 96/40963) or by altering the cationic lipid-nucleic acid formulation to preferentially transfect the desired cell types (WO 96/40962).
  • EDMPC to transfect airway epithelial cells
  • WO 96/40962 altering the cationic lipid-nucleic acid formulation to preferentially transfect the desired cell types
  • relatively less cationic lipid will be complexed to the nucleic acid resulting in a higher nucleic acid: cationic lipid ratio.
  • nucleic acid in circumstances where a positively charged complex is desired, relatively more cationic lipid will be complexed with the nucleic acid, resulting in a lower nucleic acid: cationic lipid ratio.
  • the lipid mixtures are complexed with DNA in different ratios depending on the target cell type, generally ranging from about 6: 1 to 1 :20 ⁇ g DNA:nmole cationic lipid.
  • DNA ratios are from about 10:1 to about 1:20, preferably about 3:1.
  • DNA ratios range from about 1:3 to about 1:20 ⁇ g DNA: nmole cationic lipid, most preferably, about 1:6 to about 1:15 ⁇ g DNA: nmole cationic lipid. Additional parameters such as nucleic acid concentration, buffer type and concentration, etc., will have an effect on transfection efficiency, and can be optimized by routine experimentation by a person of ordinary skill in the art.
  • Delivery can be by any means known to persons of skill in the art, e.g., intravenous, intraperitoneal, intratracheal, intranasal, intramuscular, intradermal, etc.
  • PCT patent application WO 96/40962 describes the preparation and use of cationic lipid carriers for in vivo DNA delivery.
  • the cationic lipid-nucleic acid transfection complex will withstand both the forces of nebulization and the environment within the lung airways and be capable of transfecting lung cells.
  • Techniques for delivering genes via aerosol administration of cationic lipid-DNA transfection complexes is described in U.S. Patent No. 5,641,662.
  • Plasmid p4119 containing the CAT reporter gene under the control of the HCMV promoter was prepared at a concentration of 8.2 mg/ml in 10 mM Tris, pH 8.0. 100 mg cholesterol (Sigma, St. Louis, MO) and 100 mg DOTIM (Sigma) were each dissolved into 5 ml ethyl acetate solutions, making 20 mg/ml solutions (final concentrations of 28.75 mM DOTIM and 51.7 mM cholesterol).
  • the DNA/lipid solution was prepared in a 4 ml glass vial by mixing 1400 ⁇ l EtOH, 321 ⁇ l water, and 76.2 ⁇ l DNA. Then 165.16 ⁇ l ethyl acetate was added, followed by addition of 12.1 ⁇ l 51.7 mM cholesterol and 21.74 ⁇ l 28.75 mM DOTIM. The final solution contained 70% EtOH and 10% ethyl acetate; 3.125 mM DOTIM and 3.125 mM cholesterol for a 1:1 molar ratio; and DNA in a final concentration of
  • DNA/lipid complexes were also prepared by standard mixing methods as follows. Liposomes were prepared by first dissolving the lipids (DOTIM and cholesterol) in a mixture of chloroform and methanol (1:1 molar ratio) and lipid films were formed with a rotary evaporator. The films were hydrated with 5% dextrose in water (D5W) at room temperature and the resulting liposomes extruded through a series of membranes having pore sizes of 400nm, 200nm, and 50nm.
  • DOTIM and cholesterol lipids
  • D5W dextrose in water
  • DNA-liposome complexes were prepared at a 1:5 DNAxationic lipid ratio (mg DNA:umole cationic lipid) by adding the DNA, in a solution at 0.625 mg/ml concentration in D5W to the solution containing liposomes, in an equal volume, with constant stirring, using a Hamilton Dilutor 540B (Hamilton, Reno, Nevada). DNA-liposome complexes were also prepared at a 1:1 DNAxationic lipid ratio (mg DNA:umole cationic lipid) in a similar manner except that the solution containing liposomes was added to the solution containing the DNA.
  • DNA/lipid complexes were sized using a NiComp 370 particle sizer and found to be 135 nm ( 78 in diameter.
  • HPLC analysis was performed using a 10 ⁇ l sample, analyzed on a Shimadzu LC-IOAD HPLC equipped with an Altima C8 column, 250 cm x 4.6 mm, ID 5 (m, Model #88075. The column was previously equilibrated with a mobile phase consisting of 65% acetonitrile, 25% v/v methanol, 9.9% v/v water, and 0.1%) v/v trifluoroacetic acid. Following injection, mobile phase is run at a rate of 1 ml/min, 37(C for 20 minutes. Elution peaks are detected by UV absorbance at 215 nm.
  • Figure 1 shows the density gradient profile of DNA/lipid complexes prepared according to the methods described above. From top to bottom, Figure 1 shows the density gradient profiles of complex preparations: a) prepared by the standard mixing method in a 1 :5 DNA/cationic lipid ratio ( ⁇ g DNA:nmoles cationic lipid); b) prepared by the solution nucleation method in a 1:1 DNA/cationic lipid ratio; and c) prepared by the standard mixing method in a 1 : 1 DNA/cationic lipid ratio.
  • the density gradient profile shows that the solution nucleation method produces a less heterogeneous mixture of DNA-lipid complexes than that prepared by standard mixing methods.

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Abstract

L'invention concerne des procédés de préparation de complexes de transfection constitués de polynucléotides et de lipides cationiques, convenant à l'administration de polynucléotides aux cellules. On prépare lesdits complexes de transfection à polynucléotides/lipides cationiques, en l'absence de détergent, en utilisant des procédés dans lesquels des polynucléotides et les lipides cationiques sont solubilisés dans une solution aqueuse en présence de co-solvant, et dans lesquels la suppression du co-solvant s'effectue dans des conditions permettant l'assemblage des polynucléotides et des lipides cationiques sous forme de complexes de transfection. Ledit procédé permet de produire une population sensiblement homogène de complexes de transfection et peut être modulé.
PCT/US1998/019936 1997-09-23 1998-09-23 Procedes de preparation de complexes de transfection lipidiques/polynucleotidiques WO1999015206A1 (fr)

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Cited By (6)

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US7320963B2 (en) 1999-09-01 2008-01-22 Genecure Pte Ltd Methods and compositions for delivery of pharmaceutical agents
DE102012219954A1 (de) 2012-10-31 2014-04-30 Henkel Ag & Co. Kgaa Polymere zur allergen-repulsiven Ausrüstung
DE102012219948A1 (de) 2012-10-31 2014-04-30 Henkel Ag & Co. Kgaa Polymere zur allergen-adhäsiven Ausrüstung
WO2017212007A1 (fr) * 2016-06-09 2017-12-14 Curevac Ag Supports cationiques destinés à l'administration d'acides nucléiques
US11478552B2 (en) 2016-06-09 2022-10-25 Curevac Ag Hybrid carriers for nucleic acid cargo
WO2023133922A1 (fr) * 2022-01-14 2023-07-20 苏州尔生生物医药有限公司 Complexe lipidique à l'échelle micrométrique, son procédé de préparation et son utilisation

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7320963B2 (en) 1999-09-01 2008-01-22 Genecure Pte Ltd Methods and compositions for delivery of pharmaceutical agents
US7709457B2 (en) 1999-09-01 2010-05-04 Genecure Pte Ltd. Methods and compositions for delivery of pharmaceutical agents
DE102012219954A1 (de) 2012-10-31 2014-04-30 Henkel Ag & Co. Kgaa Polymere zur allergen-repulsiven Ausrüstung
DE102012219948A1 (de) 2012-10-31 2014-04-30 Henkel Ag & Co. Kgaa Polymere zur allergen-adhäsiven Ausrüstung
EP2727986A2 (fr) 2012-10-31 2014-05-07 Henkel AG & Co. KGaA Polymères pour équipement adhésif pour les allergènes
EP2727985A2 (fr) 2012-10-31 2014-05-07 Henkel AG & Co. KGaA Polymères pour équipement répulsif pour les allergènes
WO2017212007A1 (fr) * 2016-06-09 2017-12-14 Curevac Ag Supports cationiques destinés à l'administration d'acides nucléiques
US11478552B2 (en) 2016-06-09 2022-10-25 Curevac Ag Hybrid carriers for nucleic acid cargo
WO2023133922A1 (fr) * 2022-01-14 2023-07-20 苏州尔生生物医药有限公司 Complexe lipidique à l'échelle micrométrique, son procédé de préparation et son utilisation

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