EP0975332A1 - Methodes ameliorees de transport utilisant des lipides cationiques et des lipides assistants - Google Patents

Methodes ameliorees de transport utilisant des lipides cationiques et des lipides assistants

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Publication number
EP0975332A1
EP0975332A1 EP98915263A EP98915263A EP0975332A1 EP 0975332 A1 EP0975332 A1 EP 0975332A1 EP 98915263 A EP98915263 A EP 98915263A EP 98915263 A EP98915263 A EP 98915263A EP 0975332 A1 EP0975332 A1 EP 0975332A1
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EP
European Patent Office
Prior art keywords
lipid
dlpe
cationic
cationic lipid
transfection complex
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EP98915263A
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German (de)
English (en)
Inventor
Jinkang Wang
Yi-Lin Zhang
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Urigen Pharmaceuticals Inc
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Urigen Pharmaceuticals Inc
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Publication of EP0975332A1 publication Critical patent/EP0975332A1/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
    • A61K47/544—Phospholipids
    • 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 helper 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 the use of certain phosphatidylethanolamines as helper lipids for improving cationic-lipid mediated nucleic acid delivery.
  • lipid-based materials such as liposomes have been used as biological carriers for many pharmaceutical and other biological applications, particularly to introduce drugs, radiotherapeutic agents, enzymes, viruses, transcriptional factors and other cellular vectors into a variety of cultured cell lines and animals.
  • Clinical trials have demonstrated the effectiveness of liposome- mediated drug delivery for targeting liposome-entrapped drugs to specific tissues and specific cell types. 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.
  • cationic lipids have been used to deliver nucleic acids to cells, allowing uptake and expression of foreign genes.
  • cationic lipid- mediated delivery of exogenous nucleic acids in vivo 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 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.
  • Nucleic acids are generally large polyanionic molecules which, therefore, bind cationic lipids through charge interactions. While lipid carriers have been shown to enhance nucleic acid delivery in vitro and in vivo, the mechanism by which they facilitate transfection is not clearly understood. While it was initially believed that lipid carriers mediated transfection by promoting fusion with plasma membranes, allowing delivery of the DNA complex into the cytoplasm, it is now generally accepted that the primary mechanism of cellular uptake is by endocytosis.
  • cationic lipid carriers While the mechanism by which cationic lipid carriers act to mediate transfection is not clearly understood, they are postulated to act in a number of ways with respect to both cellular uptake and intracellular trafficking.
  • Some of the proposed mechanisms by which cationic lipids enhance transfection include: (i) compacting the DNA, protecting it from nuclease degradation and enhancing receptor- mediated uptake, (ii) improving association with negatively-charged cellular membranes by giving the complexes a positive charge, (iii) promoting fusion with endosomal membranes facilitating the release of complexes from endosomal compartments, and (iv) enhancing transport from the cytoplasm to the nucleus where DNA may be transcribed.
  • the role of the cationic lipid carriers is further complicated by the interactions between the lipid-nucleic acid complexes and host factors, e.g., the effects of the lipids on binding of blood proteins, clearance and/or destabilization of the complexes.
  • cationic lipids 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 and mode of delivery allow preferential delivery of nucleic acids to particular tissues in vivo.
  • DOPE dioleoylphosphatidylethanolamine
  • cationic lipid carriers While the use of cationic lipid carriers for transfection is well-known, structure activity relationships are not well understood. It is postulated that different lipid carriers will affect each of the various steps in the transfection process (e.g., condensation, uptake, nuclease protection, endosomal release, nuclear trafficking, and decomplexation) with greater or lesser efficiency, thereby making the overall transfection rate difficult to correlate with lipid structures. Thus, alterations in either the cationic or helper lipid component do not have easily predictable effects on activity. For the most part, therefore, improvements to known cationic lipid-mediated delivery systems are dependent on empirical testing. When intended for in vivo transfection, new lipids and lipid formulations should be screened in vivo to accurately predict optimal lipids and formulations for transfection of target cells.
  • lipid delivery systems e.g., to achieve higher levels of in vivo gene transfection.
  • Improved levels of gene transfection will allow the treatment of disease states for which higher levels of expression are needed for therapeutic effect than achievable with prior art lipid delivery systems.
  • higher transfection levels allow use of smaller amounts of material to achieve comparable expression levels, thereby decreasing potential lipid-associated toxicities and decreasing cost.
  • neutral lipid the toxicity of particular cationic lipids can be decreased.
  • 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 (Malone 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.
  • helper lipids in cationic lipid-mediated gene delivery is described in Feigner et al, (1994) J. Biol. Chem. 269(4): 2550-2561 (describing improved transfection using DOPE); Hui et al., (1996) Biophys. J. 71 : 590-599; and Wheeler et al., (1996) Biochim. Biophys. Acta 1280:1-11.
  • the effect of cholesterol on liposomes in vivo is described in Semple et al., (1996) Biochem. 35(8): 2521-2525.
  • Lipid carrier compositions comprising a cationic lipid and certain neutral phosphatidyl ethanolamines are provided, for delivery polyanionic molecules to cells.
  • the neutral lipid may be l,2-dilauroyl-_? «-glycero-3-phosphoethanolamine (DLPE) or 1 ,2-diphytanoyl-5 «-glycero-3-phosphoethanolamine (DiPPE), as the sole neutral lipid or in combination with other neutral lipids.
  • DLPE l,2-dilauroyl-_? «-glycero-3-phosphoethanolamine
  • DiPPE 1 ,2-diphytanoyl-5 «-glycero-3-phosphoethanolamine
  • liposomes are provided, comprising a cationic lipid and a neutral lipid, where the neutral lipid is DLPE or DiPPE, as the sole neutral lipid or in combination with other neutral lipids.
  • the liposomes are useful as carriers for nucleic acid molecules, particularly plasmid DNA, to cells, whereby the DNA is taken up by the cells in functional form.
  • the plasmid DNA typically comprises 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 direct delivery of RNA molecules, e.g., antisense RNA or ribozymes.
  • the invention also provides methods of in vivo and in vitro transfection of a target cell with a nucleic acid of interest.
  • the methods include delivery of cationic lipid-nucleic acid complexes to cells in vitro, or in vivo by various routes of administration, where the complexes include a cationic lipid and a neutral lipid selected from the group consisting of DLPE and DiPPE.
  • Preferred means of in vivo delivery include intravenous administration, intraperitoneal administration and inhalation of aerosolized complexes.
  • the cationic lipid used in combination with DLPE is DOTIM, MBOP or DOTAP, and the cationic lipid and DLPE are used in molar ratios of about 3:1 to 1:3, most preferably, molar ratios of about 1:1.
  • liposomes comprising DLPE are complexed to DNA in ratios ranging from about 6:1 to 1:20 ⁇ g DNA:nmole cationic lipid, most preferably ratios from 1 :6 to 1 :15 ⁇ g DNA:nmole cationic lipid.
  • Figure 1 shows the chemical structures of nine phosphatidyl ethanolamine derivatives that were tested in combination with cationic lipids for the ability to transfect cells.
  • Figure 2 is a histogram showing the levels of transfection obtained in lung tissue, as measured by CAT expression, resulting from transfection of a CAT reporter plasmid using various lipid formulations as described in Example 1.
  • Figure 3 is a histogram showing the levels of transfection obtained in spleen tissue, as measured by CAT expression, resulting from transfection of a CAT reporter plasmid using various lipid formulations as described in Example 1.
  • Figure 4 is a histogram showing the levels of transfection obtained in liver tissue, as measured by CAT expression, resulting from transfection of a CAT reporter plasmid using various lipid formulations as described in Example 1.
  • Figure 5 is a histogram showing the levels of transfection obtained in heart tissue, as measured by CAT expression, resulting from transfection of a CAT reporter plasmid using various lipid formulations as described in Example 1.
  • Figure 6 is a histogram showing the DNA uptake with lipid/DNA complexes prepared with different neutral lipids, at varying lipid/DNA ratios, in C57, CHO and COS cells.
  • Figure 7 is a histogram showing levels of GFP expression with lipid/DNA complexes prepared with different neutral lipids, at varying lipid/DNA ratios, in C57, CHO and COS cells.
  • Figure 8 is a histogram showing levels of CAT expression with lipid/DNA complexes prepared with different neutral lipids, at varying lipid/DNA ratios, in C57, CHO and COS cells.
  • neutral lipids are useful as helper lipids in conjunction with cationic lipids for nucleic acid delivery, and can have a dramatic effect on gene expression levels.
  • Useful neutral lipids include DLPE (1,2-dilauroyl- sH-glycero-3-phosphoethanolamine) and DiPPE (l,2-diphytanoyl-5 «-glycero-3- phosphoethanolamine). These neutral lipids may be used as the sole neutral lipid, or in combination with one or more additional neutral lipids.
  • the lipid carrier (also called cationic lipid carrier) compositions of the invention are useful in any of the several applications in which cationic lipid carriers find use.
  • Lipid carrier compositions of the invention may be used as carriers for biologically active molecules such as antibiotics or nucleic acids in cell transfection processes.
  • the compositions are particularly useful in the preparation of lipid carriers for nucleic acid delivery, mediating mammalian cell transfection in vitro and in vivo.
  • lipid carrier or "cationic lipid carrier” refers to a lipid composition of one or more cationic lipids and one or more non-cationic lipids for delivering agents to cells.
  • a cationic lipid carrier of the present invention includes as a helper lipid a neutral lipid selected from the group consisting of DLPE and DiPPE.
  • the lipid earner may be in any physical form including, e.g., liposomes, micelles, interleaved bilayers, etc.
  • cationic lipid is intended to encompass lipids that are positively charged at physiological pH, and more particularly, constiuiitively 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.
  • Transfection is intended to mean 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” 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 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 DNA or RNA, mRNA 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.
  • Lipid carriers usually contain a cationic lipid and a neutral lipid; most prior art lipid carriers contain DOPE or cholesterol as the neutral lipid. Most protocols involve forming liposomes made up of a mixture of cationic and neutral lipid. The neutral lipid is often helpful in maintaining a stable lipid bilayer in liposomes, and can significantly affect transfection efficiency.
  • the liposomes may have a single lipid bilayer (unilamellar) or more than one bilayer (multilamellar).
  • liposomes are typically referred to as large unilamellar vesicles (LUVs), multilamellar vesicles (MLVs) or small unilamellar vesicles (SUVs).
  • LUVs large unilamellar vesicles
  • MLVs multilamellar vesicles
  • SUVs small unilamellar vesicles
  • the cationic liposomes are mixed with polyanionic compounds, and complexes form by charge interactions between the cationic lipid components and the negative charges of the polyanionic compounds.
  • Polyanions of particular interest include nucleic acids, e.g., DNA, RNA or combinations of the two.
  • "Cationic lipid- nucleic acid transfection complex” or “transfection complex” refers to a combination of a lipid carrier and a nucleic acid, in any physical form, for use in transfecting cells.
  • a transfection complex may include additional moieties, e.g., targeting molecules such as receptor ligands or antibody fragments, or other accessory molecules such as, for example, transcription factors, polymerases, integrases, nuclear localizing peptides, and the like.
  • 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.
  • the cationic lipid carriers and polynucleotide molecules are mixed, resulting in cationic lipid-polynucleotide transfection complexes, the physical structure of which depends on the lipid and nucleic acid components, the ratios between them, concentrations, mixing conditions and the like.
  • the lipids are mixed with nucleic acids in solution, at concentrations and ratios optimized for the target cells to be transfected.
  • the process of forming cationic lipid-nucleic acid transfection complexes is generally as described in PCT patent application number WO 93/25673. For in vivo administration, care is taken to prevent complex aggregation.
  • DLPE and DiPPE are commercially available, e.g., from Avanti Polar Lipids (Alabaster, Alabama). Alternatively, they may be synthesized by methods known in the art. Several methods are described, e.g., in Eibl, (1980) "Synthesis of Glycerophospholipids," Chemistry and Physics of Lipids, 26:405-429. See also the references cited therein. For instance, DLPE may be synthesized starting from 1,2- dilauroylglycerol. Phosphorylation may be achieved by subsequent reactions with phosphorous oxychloride and t-butyloxycarbonylaminoethanol. The condensation product is then dissolved in formic acid to remove the protecting group.
  • l,2-dilauroyl-_?n-glycerol may be converted to l,2-dilauroyl-5 «-glycero- 3 -phosphoric acid dichloride by phosphorylation with phosphorus oxychloride in the presence of triethylamine, in molar ratios of diacylglycero phosphorus oxychloride:base, 1 :1.5:1.5.
  • the excess phosphorous oxychloride is removed by evaporation and the l,2-dilauroyl-_s- «-glycero-3-phosphoric acid dichloride thus obtained is reacted with ethanolamine in the presence of triethylamine (molar ratios 1 : 1 :2).
  • the shore phosphatidylethanolamines may be used with any of a variety of cationic lipids, in place of or in addition to other known helper lipid components, to form lipid carriers, which are useful as carriers for various biological molecules, such as nucleic acids.
  • the lipids can be used in formulations for the preparation of lipid vesicles or liposomes for use in intracellular delivery systems. See Lasic, D., Liposomes: From Physics to Applications, Elsevier: Amsterdam, 1993.
  • lipids 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 LipofectinTM, 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 lipid carriers of the invention can be used in pharmaceutical formulations to deliver therapeutic agents by various routes of administration, and to various sites in an animal body, to achieve a desired therapeutic effect. As an example, by substituting DLPE for cholesterol as the helper lipid for transfection by intravenous administration, transfection efficiency can be improved by approximately ten-fold.
  • Cationic lipids useful in combination with the neutral lipids of the invention include, for example, imidazolinium derivatives (WO 95/14380), guanidine derivatives (WO 95/14381), phosphatidyl choline derivatives (WO 95/35301), piperazine derivatives (WO 95/14651), and biguanide derivatives (as disclosed in co- owned and co-pending U.S. patent application Serial No. 08/825,854, Attorney Docket No. 97,171).
  • Examples of cationic lipids that may be used in the present invention include DOTIM (also called BODAI) (Solodin et al., (1995) Biochem.
  • EDMPC aerosolized delivery to airway epithelial cells and for intraperitoneal delivery
  • DOTIM DOTAP or MBN222
  • compositions of the present invention will be usable in the manner described for other known neutral lipids, e.g., DOPE or cholesterol, in conjunction with the various cationic lipids, although optimization of operating parameters will improve results, using the specific information provided in this specification along with the knowledge of a person of skill in the art of lipid preparation and use.
  • a reader unfamiliar with this background information is referred to the publications under the heading Relevant Literature above and further to PCT patent application numbers WO 96/40962 and WO 96/40963.
  • These last-cited patent applications describe a number of therapeutic formulations and methods in detail, including examples of the use of specific cationic and neutral lipids that can be followed substantially be substituting, e.g., DLPE or DiPPE for the neutral lipids described.
  • the lipid carriers of the invention will generally be a mixture of cationic lipid and helper lipid in a molar ratio of from about 3:1 to 1:3, preferably about 1 :1.
  • the lipid carriers may include one or more cationic lipid, and may include DLPE or DiPPE alone as the helper lipid, or may include additional non-cationic helper lipids, which may be either anionic or neutral lipids.
  • the lipid carriers will have as the lipid components a single cationic lipid and a single neutral lipid, preferably in approximately equimolar amounts.
  • the lipid mixtures typically are prepared in chloroform, dried, and rehydrated in, e.g., 5% dextrose in water or a physiologic buffer to form liposomes.
  • Low ionic strength solutions are preferred.
  • Liposomes may be LUVs, MLVs, or SUVs.
  • the liposomes formed upon rehydration are predominantly MLVs, and SUVs are formed from them by sonication or by extrusion through membranes with pore sizes ranging from 50 to 600nm to reduce their size.
  • the liposomes are extruded through a series of membranes with decreasing pore sizes, e.g., 400nm, 200nm and 50nm.
  • the resulting liposomes are mixed with a nucleic acid solution with constant agitation to form the cationic lipid-nucleic acid transfection complexes.
  • the preferred size will vary depending on use. For example, smaller transfection complexes are preferred for aerosol administration, thereby reducing shear caused by the aerosohzation process.
  • Preferred transfection complex size for aerosol administration is less than 5000nm, most preferably from 50 to 300nm.
  • Preferred transfection complex size for intravenous administration is from 50 to 5000nm, most preferably from 100 to 400nm.
  • 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.
  • DLPE or DiPPE may be substituted into a formulation in place of a different neutral lipid, and used in the same concentration, DNA-lipid ratio, etc.
  • additional formulations be tested empirically to obtain optimal results. While a range of lipid-nucleic acid complex formulations will be effective in cell transfection, 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.
  • a reporter gene e.g. CAT which encodes chloramphenicol acetyltransferase, luciferase, or ⁇ -galactosidase
  • 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 By 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.
  • net positively charged complexes are generally prepared by adding nucleic acid to the liposomes, and net negatively charged complexes are prepared by adding liposomes to the nucleic acid, in either case with constant agitation.
  • the lipid mixnires 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:nmole cationic lipid For transfection of airway epithelial cells, e.g., via aerosol, intratracheal or intranasal administration, net negatively charged complexes are preferred.
  • preferred DNAxationic lipid ratios are from about 10:1 to about 1:20, preferably about 3:1.
  • preferred DNAxationic lipid ratios range from about 1:3.5 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. Preferred conditions are described in the Examples that follow.
  • a preferred formulation consists of EDMPC and DiPPE in a 1:1 molar ratio, 1:8 DNAxationic lipid ratio ( ⁇ g DNA: nmole cationic lipid), 0.25 mg/ml DNA, in a 2.5 mM histidine buffer, pH 5.0 and 5% w/v dextrose.
  • Non-lipid material (such as biological molecules being delivered to an animal or plant cell or target-specific moieties) can be conjugated to the lipid carriers through a linking group to one or more hydrophobic groups, e.g., using alkyl chains containing from about 12 to 20 carbon atoms, either prior or subsequent to vesicle formation.
  • Various linking groups can be used for joining the lipid chains to the compound. Functionalities of particular interest include thioethers, disulfides, carboxamides, alkylamines, ethers, and the like, used individually or in combination.
  • the particular manner of linking the compound to a lipid group is not a critical part of this invention, as the literature provides a great variety of such methods.
  • the active compounds to be bound to the lipid mixture are ligands or receptors capable of binding to a biological molecule of interest.
  • a ligand binding specifically to a receptor on a particular target cell type can be used to target delivery of the lipid carrier (with, e.g., the DNA or antibiotic of interest) to the desired target cells.
  • the active compound may be a peptide or other small molecule designed to regulate intracellular trafficking of the delivered substance, e.g., triggering endosomal release or transport into the nucleus using a nuclear localizing sequence.
  • the active compounds bound to the lipid mixture can vary widely, from small haptens (molecular weights of about 125 to 2000) to antigens (molecular weights ranging from around 6000 to 1 million).
  • proteinaceous ligands that bind to and are internalized by specific complementary binding partners on cell surfaces.
  • Illustrative active compounds include cytokines, interferons, hormones, antibodies to cell surface receptors or other molecules, and fragments of such compounds that retain the ability to bind to the same cell surface binding partners that bind the original (non-fragment) molecules.
  • the number of active compounds bound to a lipid carrier will vary with the size of the complex, the size of the compound, the binding affinity of the molecule to the target cell receptor or ligand, and the like.
  • the bound active molecules will be present in the lipid mixture in from about 0.001 to 10 mole percent, more usually from about 0.01 to 5 mole percent based on the percent of bound molecules to the total number of molecules available in the mixture for binding.
  • the lipid carrier compositions are particularly useful as carriers for use in vivo, particularly in vivo in humans. Particularly where repeat administration is necessary or desirable, the carriers should be screened for toxicity. Choice of neutral lipid can modulate toxicities observed with cationic lipids in different formulations, and thus each combination should be tested separately.
  • An animal such as a mouse, can be administered one or more doses of material containing between lOnmole and lO ⁇ mole of the lipid to be tested, typically complexed with the intended active component (such as DNA). At various times after administration the animals are monitored for evidence of toxicity, e.g. lethargy or inflammation. The animals are sacrificed and the liver examined for toxicity.
  • Total lipid may also be analyzed for the particular lipids or partial degradation products using, e.g., HPLC.
  • 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 PCT patent application WO 93/12756.
  • the various lipid-nucleic acid complexes are prepared by known methods, for example, as described in PCT application number WO 95/14381 and WO 96/40962. Precipitation of resultant lipid-DNA mixtures is determined by visual inspection. While precipitation does not preclude the use of such complexes for in vitro transfection purposes, precipitated complexes are not desirable for in vivo transfection.
  • the complexes can be stained with a dye that does not itself cause aggregation, but which will stain either the DNA or the lipid. For example, Sudan black (which stains lipid) can be used as an aid to examine the lipid-DNA mixture to determine if aggregation has occurred.
  • Particle size can be studied by methods known in the art including, for example, electron microscopy, laser light scattering, CoulterTM counting/sizing, and the like. Standard-size beads can be used for calibration to determine the size of liposomes or complexes that form.
  • DOPE cationic lipid
  • All compounds tested were phosphatidyl ethanolamine derivatives, which varied in chain length, saturation, and extent of branching.
  • the different neutral lipids were tested in combination with the cationic lipid BODAI (also known as DOTIM) or DOTAP.
  • BODAI and DOTAP were also used with cholesterol as the neutral lipid, which combinations were know to give high transfection efficiencies in vivo.
  • the lipid combinations were tested as carriers for gene transfer by intravenous delivery in ICR female mice (25 g), and expression was determined using the plasmid p4119 containing the CAT reporter gene under the control of the HCMV promoter.
  • the lipids were dissolved in a mixture of chloroform and methanol (1 :1).
  • Lipid films of cationic and neutral lipid at a 1 :1 molar ratio were formed with a rotary evaporator.
  • the films were hydrated with 5% dextrose in water (D5W) at room temperature and extruded through a series of membranes having pore sizes of 400nm, 200nm, and 50nm.
  • DNA-liposome complexes were prepared at a 1 : 10 DNAxationic lipid ratio (mg DNA: ⁇ mole cationic lipid) by adding the DNA, in a solution at 0.625 mg/ml concentration in D5W to the solution of liposomes, in an equal volume, with constant stirring, using a Hamilton Dilutor 540B (Hamilton, Reno, Nevada). BODAI: cholesterol was used at a 1 :6 DNAxationic lipid ratio. The DNA solution was 0.3125 mg/ml DNA in D5W. The resulting complexes were sized using a Submicron Particle Sizer 370 (Nicomp, Santa Barbara, California). Zeta potential was determined by a Zeta Plus, Zeta Potential Analyzer (Brookhaven Instruments Corp.).
  • mice were tested per group.
  • a dose of 62.5 ⁇ g p4119 plasmid DNA in 200 ⁇ l D5W was injected by tail vein per mouse.
  • the lung, heart , liver, and spleen were harvested after 24h and assayed for CAT activity.
  • Each organ was homogenized in 1.0ml of 5mM EDTA 0.25M Tris-HCl pH 7.8 containing 5ug/ml Aprotinin (Boehringer Mannheim, Indianapolis, IN), 5ug/ml Leupeptin (Boehringer Mannheim, Indianapolis, IN), and 5mM PMSF (Boehringer Mannheim, Indianapolis, IN), The resulting extracts were centrifuged and aliquots of the supernatant were removed for protein analysis, utilizing a bicinchoninic acid based reagent kit (Pierce, Rockford, IL). The remaining supernatant was heat treated at 65 C for 15min.
  • the CAT activity assay was performed using 5ul of heat treated supernatant, 25ul of 125ug/ml n-Butyryl CoA (Sigma, St. Louis, MO), 50ul of 5uCi/ml 14C- chloramphenicol (DuPont NEN, Boston, MA ), and 50ul of 0.25M Tris-HCl 5mM EDTA. Samples were incubated at 37 C for 2h. An addition of 3 OOul of mixed xylenes (Aldrich, Milwaukee, WI) was made followed by vortexing and centrifugation at 14K rpm for 5min.
  • the xylene layer was then transferred into 750ul of 0.25M Tris-HCl/5mM EDTA, vortexed, and centrifuged at 14K rpm for 5min.
  • the upper organic phase was then transferred into scintillation vials containing 5ml of Ready Safe Liquid Scintillation Cocktail (Beckman, Fullerton, CA). Samples were counted for 1 min each.
  • Figure 2 shows resulting expression levels in the lung
  • Figure 3 shows expression in the spleen
  • Figure 4 shows expression in the liver
  • Figure 5 shows expression in the heart.
  • the results show that in the lung, only formulations using either cholesterol (not shown) or DLPE as the neutral lipid showed significant expression levels. All other neutral lipids tested, including DOPE, showed very low expression levels in the lung. In the other organs tested, DLPE and cholesterol (not shown) also showed significant expression levels. In the liver, spleen and heart, diphytanoyl phosphatidylethanolamine also showed high expression levels.
  • Transfection efficiencies using DLPE as the neutral lipid were compared to those obtained using cholesterol as the neutral lipid, each in combination with various cationic lipids.
  • the cationic lipids used were BODAI, DOTAP, MBN222 (also called MBOP or MeBOP), MBN231 (DMBG, disclosed in co-owned and co-pending U.S. patent application Serial No. 08/825,854, Attorney Docket No. 97,171) and MBN233 (DOBG, disclosed in co-owned and co-pending U.S. patent application Serial No. 08/825,854, Attorney Docket No. 97,171).
  • the formulations were tested at the DNAxationic lipid ratios shown in Table 1.
  • BODAIxholesterol 1 :6 ⁇ g DNA: nmole cationic lipid ratio was used as a positive control.
  • the transfection efficiency is expressed as relative activity compared to the BODAIxholesterol control.
  • the plates were washed four times in 0.2% Tween-20 in PBS, and incubated for 1 hr at 37 °C with 50 ⁇ l sample (in 1:2 serial dilutions). The plates were washed four times in wash buffer, and incubated 45 min at 37 °C in 50 ⁇ l Digoxigenin labeled sheep anti-CAT antibody (Boehringer Mannheim) (1 : 100 in Blotto). Plates were washed again and incubated 45 min at 37 °C in 50 ⁇ l peroxidase- conjugated Fab fragment of sheep anti-DIG antibody (Boehringer Mannheim) (1:400 in Blotto).
  • Table 2 is a summary of transfections performed using DLPE as the neutral lipid, and shows comparisons of different cationic lipids and different DNA: cationic lipid ratios. Again, relative activity represents the ratio of transfection activity compared to the BODAIxholesterol (1 :6) positive control. High transfection rates were obtained with all cationic lipids tested, with best results obtained at DNA: cationic lipid ratios in the 1:7.5 to 1:10 range.
  • Cell lines were cultured in media from GIBCO, BRL (Gaithersburg, MD); along with additional supplements needed for optimal growth.
  • C57 MG cells grew in DMEM supplemented with 10% fetal bovine serum, and 2 mM L-glutamine.
  • CHO- Kl (ATCC CCL-61) cells' growth media consisted of F12 media supplemented with 10% fetal bovine serum.
  • COS-1 (ATCC CRL-1650) cells were grown in DMEM/F12 media supplemented with 10% fetal bovine serum, and 2 mM L-glutamine.
  • Complexing was performed by mixing 100 ⁇ l of a 0.625 mg/ml solution of the plasmid of interest (CAT, GFP or Rhodamine labeled plasmid) with an equal volume of the cationic liposome formulations at ratios of 1 :15, 1 :10, 1 :6, 1 : 1 and 3:1 (mg DNA/ ⁇ mol cationic lipid) for a final concentration of 0.3125 mg DNA/ ml complex. If the final net charge of the complex was positive, the appropriate volume of DNA was added to the liposome; the reverse order was employed if the final net charge of the complex was negative. Immediately after this addition, the mixture was rapidly hand pipetted -10 times up and down with a micropipet.
  • CAT 0.625 mg/ml solution of the plasmid of interest
  • Rhodamine labeled plasmid plasmid of interest
  • cells were harvested by gently washing the wells with 2 ml lx PBS, followed by the addition of 200 ⁇ l of 0.25% trypsin in 1 mM EDTA (GIBCO, BRL; Gaithersburg, MD). After collecting the liberated cells by the addition of 1 ml serum-containing media, cells were centrifuged at 4,000 rpm for 6 minutes at 4° C.
  • FACS buffer 5% FBS, O.lmM EDTA, 5 ⁇ g/ml propidium iodide (used in GFP assay only) in PBS
  • FACscan instrument see below
  • the supernatants containing the soluble protein fraction were transferred to storage tubes and stored at -70°C until assayed.
  • the total protein concentration of each sample was determined using a BCA microplate assay and BSA protein concentration standards (BCA Reagent and BSA standard: Pierce, Rockford, IL).
  • BCA Reagent and BSA standard Pierce, Rockford, IL.
  • the concentration of CAT protein in each sample was determined by analysis in a CAT- specific ELISA with a quantitation range between 15.6 and 250 pg/ml. CAT protein values are reported as ng of CAT enzyme per mg of total protein in the sample
  • Figure 6 shows the levels of DNA uptake by C57, CHO and COS cells of DNA/lipid complexes containing BODAI and one of cholesterol, DOPE, DLPE or DiPPE as the neutral lipid, at ratios of 1 : 15, 1:10 and 1 :6.
  • C57 cells did not take up DNA as well as CHO or COS cells.
  • BODAI/DiPPE uptake appears very low at the higher lipid DNA ratios.
  • the procedure may not completely distinguish between complex association with the cell and actual cellular uptake.
  • Figures 7 and 8 show GFP and CAT expression, respectively, with the same DNA/lipid formulations. Surprisingly, both GFP and CAT expression are significantly higher with formulations containing DiPPE. The effect was most pronounced in CHO cells, but was also seen in COS cells. In COS cells, the DLPE- containing formulations also expressed at high levels in the 1:6 and 1:10 ratios. In C57 cells, expression is similar among the different formulations although the DiPPE- containing formulations were generally taken up to a lesser extent.
  • DiPPE allows improved intracellular processing of the complexes after uptake.
  • the improved expression with DiPPE-containing formulations may be due to improved endosomal release, improved nuclear transport, and/or an improved rate of decomplexation from the DNA within the cell.
  • Tlic posilivc conlrol was Dodai/Chol (1:1).
  • Tlic relative activity is the ratio of the Iransfcclion efficiency of new fo ⁇ nulalion lo t e positive conlrol.

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Abstract

L'invention concerne des méthodes et des compositions pouvant être utilisées pour introduire des molécules polyanioniques, en particulier des acides nucléiques, dans des cellules mammaliennes, en utilisant certains phosphatidyl éthanolamines comme lipides assistants conjointement avec divers lipides cationiques. En particulier, on améliore la transfection de cellules mammaliennes induite par des lipides cationiques en utilisant des supports lipidiques comprenant 1,2-dilauroyl-sn-glycéro-3-phosphoéthanolamine (DLPE) ou 1,2-diphytanoyl-sn-glycéro-3-phosphoéthanolamine (DiPPE) et des lipides cationiques.
EP98915263A 1997-04-04 1998-04-03 Methodes ameliorees de transport utilisant des lipides cationiques et des lipides assistants Withdrawn EP0975332A1 (fr)

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US5650096A (en) * 1994-12-09 1997-07-22 Genzyme Corporation Cationic amphiphiles for intracellular delivery of therapeutic molecules
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