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WO1999051629A2 - Nouveaux composes destines a la transfection d'adn - Google Patents

Nouveaux composes destines a la transfection d'adn Download PDF

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Publication number
WO1999051629A2
WO1999051629A2 PCT/EP1999/002361 EP9902361W WO9951629A2 WO 1999051629 A2 WO1999051629 A2 WO 1999051629A2 EP 9902361 W EP9902361 W EP 9902361W WO 9951629 A2 WO9951629 A2 WO 9951629A2
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leu
cell
vitro
vivo
lys
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PCT/EP1999/002361
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German (de)
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WO1999051629A3 (fr
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Eric Argirios Kitas
Ernst-Jürgen SCHLAEGER
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Roche Diagnostics Gmbh
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Priority to JP2000542350A priority Critical patent/JP2002510706A/ja
Priority to EP99919208A priority patent/EP1068225A2/fr
Priority to CA002327367A priority patent/CA2327367A1/fr
Priority to AU37065/99A priority patent/AU3706599A/en
Publication of WO1999051629A2 publication Critical patent/WO1999051629A2/fr
Publication of WO1999051629A3 publication Critical patent/WO1999051629A3/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

Definitions

  • the present invention is concerned with novel compounds especially useful for non- viral introduction of biologically active molecules such as DNA, RNA, peptides or proteins into eukaryotic cells.
  • Non-viral systems have been developed to carry DNA into cells, e.g., the transfection technique based on a cationic lipid, the dioleoyloxypropyl trimethylammonium (Feigner et al., Proc. Natl., Acad. Sci. USA, 1987, 84, 7413-7417) commercialized as LipofectinTM. Since the discovery of this transfection technique, many more cationic lipids have been synthesized and some are commercially available as transfecting reagent for laboratory use: DOGS (Trans- fectamTM), DOSPA (LipofectamineTM), DOTAP (DOTAPTM).
  • DOGS Trans- fectamTM
  • DOSPA LipofectamineTM
  • DOTAPTM DOTAP
  • Transfection of cells with oligonucleotides such as DNA can be used, for example to express in a host cell or organism, a protein which is not normally expressed by that cell or organism.
  • a DNA molecule called a plasmid may be introduced into a cell that does not normally contain the gene (s) encoded by that plasmid in order to express a marker gene product in that cell, or to express a protein of interest such as a recombinant protein which is later harvested from such cells (See Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor, 1989), ch. 1.).
  • the transfection of oligonucleotides into cells can also be used therapeutically.
  • antisense oligonucleotides once in the cell or cell nucleus, bind to target single-stranded nucleic acid molecules by Watson-Crick base pairing or to double stranded nucleic acids by Hoogsteen base pairing, and in doing so disrupt the function of the target by one of several mechanisms: by preventing the binding of factors required for normal transcription, splicing, or translation; by triggering the enzymatic degradation of mRNA by RNAse H, or by destroying the target via reactive groups attached directly to the antisense oligonucleotide. (See Zamecnic et al., Proc. Natl. Acad. Sci. -USA, 1978, 75, 280-284). Gene therapy or DNA based vaccination are other therapeutic applications.
  • Proteins and other macromolecules are transfected into cells for therapeutic and screening purposes. For example, immunization is enhanced by introducing an immunogenic protein into a cell, so that it is more efficiently processed and presented on the surface of the cells, thereby enhancing the immunogenic response.
  • Negatively charged macromolecules which act inside a cell are transported past the cell membrane into the cytoplasm where they exert their effect.
  • Factors which enhance or hinder transcription of DNA can be used in a screening test to verify the transcription of a gene of interest. These transcription assays are very well known for use in screening compounds to determine their effect against a particular macro- molecule, for example a cell receptor.
  • EP-A-784 984 and Legendre et al. describe conjugates of a lipid and a basic, membrane disturbing peptide that bind polynucleotides and anionic macromolecules can be used for transfection of cells.
  • the peptide portion of the conjugate consists of natural amino acids linked by a natural amide binding.
  • the present invention is directed to novel compounds which avoid disadvantages associated with known transfection agents.
  • the invention is concerned with novel compounds which are conjugates of lipids and a modified basic membrane disturbing peptide, characterized in that the peptides comprise a reversed amide backbone.
  • conjugates means compounds consisting of a lipid chemically bound to the peptide, e.g., via a disulfide bond formed between a sulfur atom present in or attached to the lipid and a sulfur atom present in or attached to the peptide; or an amide bond formed between the carboxyl group present in or attached to the lipid and an amino group of the peptide.
  • lipid as used herein comprises straight-chain, branched-chain, saturated or unsaturated aliphatic carboxylic acids and phospholipids.
  • aliphatic carboxylic acids are lauric acid, palmitic acid, stearic acid, oleic acid and (CH 3 (CH 2 ) n ) 2 CH COOH, where n is an integer from 3 to 19.
  • phospholipids are phosphatidylethanolamines such as dioleoylphosphatidylethanolamine.
  • basic peptides denotes peptides containing at least one basic amino acid.
  • basic amino acids are natural and unnatural diamino-monocarboxylic acids, such as alpha-, beta-diaminopropionic acid, alpha-, gamma-diaminobutyric acid, lysine, arginine, ornithine and p-aminophenylalanine, etc.
  • membrane disturbing peptides denotes cell-lytic or antibacterial peptides that perturb the barrier function of membranes (G. Saberwal and R. Nagaraj, BBA, 1994, 1197, 109-131).
  • Examples of basic, cell-lytic peptides are melittin, hemolysin, mastoparan, bombolitin, crabrolin and derivatives thereof.
  • Examples of basic antibacterial peptides are cecropins, magainins, gramicidin S and tyrocidine and derivatives thereof.
  • derivatives refers to peptides wherein one or more amino acid residues are missing, have been added or have been replaced by another amino acid residue without substantially changing the biological activity of the original peptide concerned, i.e. allow transfection of a macromolecule, preferably a polynucleotide. into a cell.
  • derivatives also refers to peptides wherein the terminal carboxyl group is esterified, particularly to form lower alkyl esters such as the methyl and ethyl ester; or converted into an amide, lower alkyl amide or di-lower alkyl amide or hydrazide.
  • derivatives also relates to peptides wherein the NH 2 -group of the N-terminus may be acylated to form an amide or a lower alkyl amide.
  • the NH2-group is acetylated.
  • lower denotes groups containing from 1-6 carbon atoms.
  • reversed amide backbone refers to retro-peptides which characteristically have the same composition as its parent peptide, but the sequence is reversed, i.e., n, ... 3, 2, 1 instead of 1, 2, 3, ... n when reading both in the N to C direction. Both have normal peptide bonds.
  • n n, ... 3, 2, 1 instead of 1, 2, 3, ... n when reading both in the N to C direction. Both have normal peptide bonds.
  • novel compounds of the present invention are compounds of formula
  • R and R are a hydrocarbyl moiety of a straight-chain or branched-chain, saturated
  • R is a basic, membran- edisturbing peptide with a reversed amide backbone
  • Y is C2-10 alkylene
  • X is -C(O)-NH- or
  • Particularly preferred compounds are those wherein R and R independently are an acyl moiety of a C 1 . 20 carboxylic acid.
  • C 1 2-20 denotes a number of carbon atoms of from 12 to 20.
  • the acyl moieties R 1 and R 2 can be a straight-chain or branched-chain, saturated or unsaturated moiety. Examples of such moieties are lauroyl, palmitoyl, stearoyl and oleoyl. In a preferred aspect, R 1 and R 2 are oleoyl.
  • Y is preferably ethylene, propylene or decamethylene.
  • X is preferably -S-S-.
  • R 3 is a basic, membrane disturbing peptide with a reversed amide backbone.
  • R 3 is Gln-Gln-Arg-Lys-Arg-Lys-Ile-T -Ser-Ile-Leu-Ala-Pro-Leu-Gly-Thr-Thr-Leu-Val-Lys- Leu-Val-Ala-Gly-Ile-NH-CH[CONH 2 ]-(CH 2 )-.
  • the peptide has a reversed amide backbone.
  • R 3 comprises at least 50%, more preferably 65%, and even more preferably 80% of D-amino acids or derivatives thereof. In the most preferred embodiment all amino acids are D-amino acids.
  • D-amino acid refers to naturally as well as non-naturally D- ⁇ - amino carbonic acids or derivatives thereof.
  • R 3 also comprises membrane-disturbing peptide sequences such as magainin, cecropin, defensins, etc., or chimers of such e.g., cecropin-melittin (Hancock & Lehrer TIBTECH, 1998, vol. 16, 82-88) synthesized as the retro-inverso peptides and derivatized to allow conjugation to lipids, analogous to the methods as described below, especially analogous in fashion to Example 1.
  • membrane-disturbing peptide sequences such as magainin, cecropin, defensins, etc., or chimers of such e.g., cecropin-melittin (Hancock & Lehrer TIBTECH, 1998, vol. 16, 82-88) synthesized as the retro-inverso peptides and derivatized to allow conjugation
  • R is D-Gln-D-Gln-D-Arg-D-Lys-D-Arg-D-Lys-D-Ile-D-
  • the most preferred compound is
  • a further embodiment of the present invention refers to the peptide portion of R 3 , especially to the intermediate peptide Gln-Gln-Arg-Lys-Arg-Lys-Ile-Trp-Ser-Ile-Leu-Ala-Pro-Leu-Gly- Thr-Thr-Leu-Val-Lys-Leu-Val-Ala-Gly-Ile-Cys-NH 2 or derivatives and/or salts and/or sol- vates thereof having a reversed amide backbone and consisting of at least 50%, more preferably 65%, and even more preferably 80% of D-amino acids or derivatives thereof. In the most preferred embodiment all amino acids are D-amino acids.
  • D-amino acid refers to naturally as well as non-naturally D- ⁇ -amino carbonic acids or derivatives thereof.
  • the peptide is D-Gln-D-Gln-D-Arg-D-Lys-D-Arg-D-Lys-D-Ile-D- Trp-D-Ser-D-Ile-D-Leu-D-Ala-D-Pro-D-Leu-Gly-D-Thr-D-Thr-D-Leu-D-Val-D-Lys-D-Leu- D-Val-D-Ala-Gly-D-Ile-D-Cys-NH 2 .
  • this invention relates to a process for preparing the novel compounds defined above, i.e., conjugates of lipids and basic, membrane disturbing peptides wherein the peptides comprise a reversed amide backbone, and compositions comprising at least one such compound, a polynucleotide or any other anionic macromolecule, and, optionally, a helper lipid and/or a short chain phospholipid, and/or a cationic lipid and optionally an additional known transfection reagent other than a conjugate of this invention (i.e. a compound of formula I or II).
  • this invention relates to compositions comprising conjugates of lipids and basic, membrane disturbing peptides and a helper lipid and/or a short chain phospholipid, and/or a cationic lipid or and optionally an additional known transfection reagent other than a conjugate of this invention, e.g. a compound of formula I.
  • the invention further relates to the use of the novel compounds as a carrier for transfecting a cell with a polynucleotide or any other anionic macromolecule.
  • the compounds provided by this invention can be prepared by reacting a peptide of the formula R 3 NH2 with a lipid of the formula
  • the peptides may be prepared by the solid-phase synthesis technique. In this technique, synthesis is occurring while the peptide is attached to a polymeric support, therefore allowing for the separation of product from byproduct by washing steps. The completion of the acylation reaction is ensured by using large excesses of soluble reagents. Synthesis involves the cova- lent anchorage of the first amino acid in the sequence to the solid support followed by the de- protection of the protected amino function for the subsequent coupling to the incoming amino acid derivative. After n cycles of deprotection and coupling, the peptide is released from the solid phase by a chemical cleavage reaction. In a preferred embodiment, D-amino acids are used.
  • the coupling of peptide of the formula R 3 NH2 with a lipid of the formula II can be accomplished by reacting the compounds wherein amino group other than the amino group to be reacted are protected in a suitable solvent in the presence of a condensation agent such as dicyclohexylcarbodiimide in analogy to methods known for producing peptide bonds.
  • the reaction of a peptide of the formula R 3 SH with a compound of formula R-SZ can be carried out in an appropriate solvent or solvent mixture which solubilizes both reactants.
  • the compound of formula R-SZ can be dissolved in an organic solvent, e.g., in chloroform.
  • the peptide R 3 SH is suitably dissolved in aqueous buffer solution, such as phosphate buffer, that contains an appropriate amount of an water-miscible organic solvent such as acetonitrile to accomplish the formation of a single phase reaction system.
  • R 1 and R 2 are oleolyl
  • Y is ethylene, propylene or decamethylene and the compound of the formula
  • R 1 and R 2 are oleolyl, Y is ethylene and Z is 2-pyridinethio are commercially available as N-Succinyl-PE, N-Glutaryl-PE , N-Dodecanyl-PE and N-PDP-PE from Avanti Polar Lipids, Alabaster, Alabama, USA.
  • anionic macromolecule can be transfected into a cell using a compound of formula I.
  • An anionic macromolecule is a macromolecule which contains at least one negative charge per molecule.
  • anionic macromolecules which can be transfected in accordance with this invention include polynucleotides , such as deoxyribonucleic acids (DNA) and ribo- nucleic acids (RNA); and proteins, such as ribonucleoproteins and proteins used for immunization, e.g. viral proteins.
  • DNA for use in the present invention are plasmids and genes, especially those for which gene therapy protocols have been launched such as cystic fibrosis transmembrane regulator (CFTR), adenosine deaminase (ADA), thymidine kinase (tk) and HLA B7; as well as reporter genes such as beta-galactosidase, luciferase, green fluorescence protein (gfp), chloramphenicol transferase and alpha- 1 antitrypsin.
  • Other examples of DNA are oligodeoxynucleotides and their analogues used as antisense, aptamer or 'triple-helix' agents.
  • RNA are ribozymes or oligoribonucleotide antisense molecules.
  • the nature of the cell which is to be transfected is not narrowly crucial.
  • the cell can be a prokaryotic or eukaryotic cell, a mammalian or a plant cell.
  • the cell is contacted with the anionic macromolecule in the presence of an appropriate amount of such compound.
  • the appropriate amount of the conjugate, e.g. a compound of formula I for a given amount of anionic macromolecule depends on their respective charges.
  • the +/- charge ratio between the conjugate and the molecule to be transfected generally varies between 0.1- 10, preferably between 0.5-5.
  • the value of "+/- charge ratio" is calculated by dividing the number of positively charged groups on the amino acids in the group R 3 by the number of negative charges of the molecule to be transfected.
  • number of negative charges means the number of negatively charged phosphates in the backbone.
  • the optimal ratio within these ranges depends on the cell to be transfected and is readily ascertained by one of skill in the art to which this invention pertains.
  • the amount of anionic macromolecules to the number of cells is such that the amount of anionic macromolecule for transfecting 10 4 cells is from 0.1 ng to 10 mg, preferably from 0.2 mg to 2 mg.
  • the anionic macromolecule is DNA
  • the preferred amount of DNA for transfecting 10 cells in vitro is from 0.1 mg to 10 mg.
  • the preferred amount of DNA is from 0.1 ⁇ g to 1 g.
  • the transfection is further carried out in the presence of a helper lipid and/or short chain phospholipid, and/or a cationic lipid or any other known transfection competent molecule other than a conjugate of this invention.
  • a helper lipid and/or short chain phospholipid and/or a cationic lipid or any other known transfection competent molecule other than a conjugate of this invention.
  • Any conventional helper lipid can be used.
  • Helper lipids are phospholipids which are known to increase delivery of macromolecules to cells when used together with known transfection competent molecules.
  • helper lipids are phospholipids, such as phosphatidylcholines or phosphatidyl- efhanolamines or mixtures thereof.
  • Preferred helper lipids are phosphatidylethanolamines, such as dioleoylphosphatidylethanolamine.
  • Short chain phospholipids are phospholipids containing fatty acid residues, which fatty acid residues contain from 6 to 12 carbon atoms in their backbone.
  • Examples of short chain phospholipids are phosphatidylcholines that carry two C6-12 fatty acid residues.
  • Preferred short chain phospholipids are dicapryl- and dicapryloyl phosphatidylcholine.
  • transfection competent molecules include cationic lipids as described by J.B. Behr in Bioconjugate Chem., 1994, 5,382089 and X. Gao and L. Huang in Gene Ther., 1995, 2, 710-722; polycations as described by A.V. Kabanov and V.A.: Kabanov in Bioconjugate Chem., 1995, 6, 7-20; peptides and polymers and other non-viral gene delivery systems as described by F.D. Ledley in Human Gene Therapy, 1995, 6, 1129-1144.
  • the helper lipid and/or short chain phospholipid and/or a cationic lipid and optionally another additional known transfection competent molecule other than a conjugate of this invention is suitably in the form of a liposome, micelles, organic or aqueous dispersions, or organic or aqueous solutions.
  • the optimal molar ratio between the compound of formula I and the helper lipid is 0.1 :50, preferably 1 :10.
  • the optimal molar ratio between helper lipid and short-chain phospholipid is 2:20.
  • the optimal molar ratio between the compound of formula I or II and additional transfection competent molecules is 0.1 : 10.
  • the present invention also comprises the use of a composition as defined above for transfecting an eukaryotic or prokaryotic cell in vivo or in vitro with an anionic macromolecule, preferably with a polynucleotide.
  • the invention also comprises the use of compounds of formula (I) as defined above for transfecting a eukaryotic or prokaryotic cell in vivo or in vitro with an anionic macromolecule, preferably with a polynucleotide.
  • a further embodiment of the present invention is a process for transfecting a cell in vivo or in vitro with an anionic macromolecule, preferably a polynucleotide, comprising contacting a cell in vivo or in vitro with the anionic macromolecule in the presence of a compound of formula (I) or with a composition as defined above.
  • an anionic macromolecule preferably a polynucleotide
  • the invention is directed to a process for introducing a biologically active anionic molecule into a cell in vivo or in vitro with an anionic macromolecule, comprising contacting a cell in vivo or in vitro with the anionic macromolecule in the presence of a composition or a compound as defined above.
  • the present invention is directed to the use of a compound or composition as defined above for introducing in vivo or in vitro a biologically active molecule into cells.
  • a conjugate of this invention e.g., a compound of formula I is added to the molecule to be transfected (e.g., plasmid DNA), suitably in an aqueous solution.
  • a helper lipid and, if desired, a short chain phospholipid and/or a cationic lipid and optionally another additional known transfection competent molecule other than a conjugate of this invention is then added, either in form of liposomes, micelles, or as an organic solution or aqueous dispersion.
  • the molecule to be transfected may be added to a composition comprising a compound in accordance with this invention, a helper lipid, and, if desired, a short chain phospholipid and/or a cationic lipid and optionally another additional known transfection competent molecule other than a conjugate of this invention.
  • the composition may be in solid, liquid, semisolid or aerosol form, suitably in form of liposomes, micelles, or as an organic solution or aqueous dispersion.
  • the composition can be administered by oral, parenteral (i.v., i.m., s.c, i.d., i.p.) transdermal, pulmonary, nasal, rectal, ocular, ventricular, vascular (catheter) and intratumoral route.
  • parenteral i.v., i.m., s.c, i.d., i.p.
  • transdermal pulmonary
  • nasal rectal
  • ocular ventricular
  • vascular vascular
  • the present invention also refers to a composition
  • a composition comprising at least one compound as defined above and a helper lipid and/or a short chain phospholipid and/or a cationic lipid or optionally another additional transfection competent molecule.
  • the composition may comprise an anionic macromolecule, preferably a polynucleotide.
  • These compositions may also comprise a polycationic polymer, preferably polyethyleneimine (PEI).
  • PEI polyethyleneimine
  • the components of these compositions may be in the form of an aqueous or organic solution, an aqueous or organic dispersion, or a liposome or a micelle.
  • polyethylenimine refers to a synthetic organic, generally branched macromolecule with a high cationic charge/density potential, preferably with a molecular weight of about 25 kDA.
  • the invention also relates to the use of a compound as above for transfecting a cell with an anionic macromolecule, preferably a polynucleotide.
  • the invention is concerned with a process for transfecting a cell with an anionic macromolecule comprising, contacting the cell with the anionic macromolecule in the presence of a compound as described above, so as to transfect the cell with the anionic macromolecule.
  • the invention also relates to a process for production of large quantities of recombinant proteins in PEI-mediated transfected cells.
  • high-level expression of both G protein coupled receptors an ligand-gated ion channels by the use of Semliki Forest virus (SFV) has been realized.
  • SFV Semliki Forest virus
  • B max values of more than 50 pmol receptor per mg protein and receptor densities of more than 3 x 10 6 receptors per cell have been achieved.
  • optimal conditions for mammalian serum-free suspension cultures have been obtained.
  • Adaptation of BHK, CHO and HEK293 cells to these conditions has allowed efficient infection with SFV vectors to produce large volumes of recombinant protein expressing cell cultures.
  • the growth temperature of the cell cultures can have a dramatic effect on the duration and levels of recombinant protein-expression.
  • Expression of recombinant luciferase is increased 5 to 10-fold by lowering the growth temperature of BHK and CHO cells from 37°C to 33°C.
  • the expression time is much longer at 33°C with still high expression 65 hours post-infection.
  • the effect of the temperature on the expression of two 7TM receptors, human neu- rokinin-1 receptor and rat metabotropic glutamate receptor-2 and the 5-HT 3 ligend-gated ion channel could be shown.
  • a similar effect as observed for luciferase has also been obtained for the receptors.
  • the receptor density is much higher in cells grown at 33°C compared to 37°C.
  • the expression time for receptors is usually restricted to 24 hours in SFV-infected cells, but can be prolonged to 65 hours when cells are cultured at 33°C. This improvement can greatly facilitate production of large quantities of recombinant proteins.
  • helper lipids in combination with PEI exhibit a high transient gene expression in HEK293 cells and other mammalian cell lines at low DNA concentration.
  • the fre- quently occurring inhibitory effect of conditioned medium which is a serious problem for scale up expression methods is reduced or even avoided.
  • Fmoc-amino acids (2.5 equiv.) were activated with an equivalent amount of TATU (L. A. Carpino J. Am. Chem. Soc. 1993, 115, 4397-4398) and DIPEA. Fmoc depro- tection was achieved with 20% piperidine in DMF.
  • the homogenous peptide obtained in paragraph a) above (24.2 mg, 7 mmole) was dissolved in a mixture of 2 ml of 100 mM ammonium acetate buffer, pH 6.5, and 2 ml of acetonitrile. To this solution there was added 7.4 mg of l,2-dioleoyl-sn-glycero-3-phosphoethanolamine- N-[3-(2-pyridyldithio)propionate] in 0.5 ml of chloroform. The mixture was stirred at room temperature for 1 hour and the organic solvent was removed by evaporation. The remaining solution was washed 3 times with ethyl acetate and the aqueous phase was lyophilized.
  • Example 1 1 mg of the compound obtained in Example 1 was dissolved in 500 ⁇ l acetonitrile and diluted in 500 ⁇ l sterile pure water (1 mg/ml) and stored at 4°C. 15 ⁇ l of a plasmid solution (1 mg/ml) and stored at 4°C. 15 ⁇ l of a plasmid solution encoding the soluble human tumor necrosis factor receptor p55 gene was transferred into 1.5 ml medium and mixed. Various amounts of the compound obtained in a) was added , mixed and after 10 min at room temperature the mixture was transferred to 15 ml HEK293(EBNA) cells in suspension.
  • Table 1 shows the transfection efficiency of the compound as described in Example 1 and the cell viability in serum-free suspension culture.
  • Example 1 1 mg of the compound obtained in Example 1 was dissolved in 500 ⁇ l acetonitrile and diluted in 500 ⁇ l sterile pure water (1 mg/ml) and stored at 4°C. 90 mg polyethyleneimine (PEI), with a molecular weight of 25 kDa, were dissolved in sterile pure water (0.9 mg/ml), neutralized with HC1 and sterile filtered and stored at room temperature.
  • PEI polyethyleneimine
  • 3 ⁇ l of a plasmid solution (1 mg/ml) encoding the soluble human tumor necrosis factor receptor p55 gene was diluted in 1.5 ml culture medium and mixed.
  • Various amounts of the compound (1 mg/ml) as described in Example 1 were added, mixed, followed by 8.3 ⁇ l PEI (0.9 mg/ml).
  • PEI 0.9 mg/ml
  • the released receptor protein was measured 72 hrs post-transfection in the culture medium and expressed as ng receptor per milliliter culture.
  • Table 2 shows the transfection efficiency of the compound described in Example 1 in combination with PEI by measuring the released receptor protein in serum free suspension culture.
  • Example 1 1 mg of the compound obtained in Example 1 was dissolved in 500 ⁇ l acetonitrile and diluted in 500 ⁇ l sterile pure water (1 mg/ml) and stored at 4°C. 90 mg polyethelenimine, with a molecular weight of 25 kDa, was dissolved in sterile pure water (0.9 mg/ml), neutralized with HCl and sterile filtered and stored at room temperature.
  • luciferase activity was measured the soluble cell extract after 24 hrs incubation and expressed as relative light unit (RLU) per milligram protein.
  • Table 3 shows the transfection efficiency of the compound described in Example 1 in combination with PEI by measuring the luciferase activity in serum free suspension culture.
  • Example 1 1 mg of the compound obtained in Example 1 was dissolved in 50 ⁇ l acetonitrile and diluted in 500 ⁇ l sterile pure water (1 mg/ml) and stored at 4°C. 90 mg polyethelenimine, with a molecular weight of 25 kDa, was dissolved in sterile pure water (0.9 mg/ml), neutralized with HCl and sterile filtered and stored at room temperature.
  • 3 ml of a plasmid solution (1 mg/ml) encoding the green fluorescence protein was diluted in 1.5 ml culture medium and mixed. 2.25 ⁇ l compound (1 mg/ml) was added, mixed, and followed by 8.3 ⁇ l PEI (0.9 mg/ml). After mixing and incubation for 10 min at room temperature the mixture was transferred into 15 ml of various cells. Cells which were grown in serum-free suspension culture were transferred into fresh medium before adding the com- plexes. The transfection efficiency was measured by counting the number of fluorescent cells after 24 hours incubation and expressed as percent transfected cells.
  • Table 4 shows the transfection efficiency of the compound described in Example 1 in combination with PEI by measuring the green fluorescence within various cell lines in serum-free suspension culture.
  • Example 1 1 mg of the compound obtained in Example 1 was dissolved in 500 ⁇ l acetonitrile and diluted in 500 ⁇ l sterile pure water (1 mg/ml) and stored at 4°C. 90 mg polyethylenimine, with a molecular weight of 25 kDa, was dissolved in sterile pure water (0.9 mg/ml), neutralized with HCl and sterile filtered and stored at room temperature.
  • a plasmid solution (1 mg/ml) encoding the luciferase gene was diluted in 1.5 ml culture medium and mixed. 2.25 ⁇ l compound (1 mg/ml) as described in Example 1 was added, mixed, and followed by 8.3 ⁇ l PEI (0.9 mg/ml). After mixing and incubation for 10 min at room temperature 0.1 ml of the mixture was transferred into a well containing 1 ml medium of a 12 well plate with various adherent cell lines. Cells which were grown in the presence of 10% serum were incubated during 4 hours post-transfection in serum-free medium. The luciferase activity was measured in the soluble cell extract after 24 hours incubation and expressed as relative light unit (RLU) per milligram protein.
  • RLU relative light unit
  • Table 5 shows the transfection efficiency of the compound described in Example 1 in combination with PEI by measuring the luciferase activity in various adherent cell lines.

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Abstract

L'invention concerne des conjugués de lipides et de peptides basiques modifiés perturbant la membrane, lesdits conjugués étant caractérisés en ce que les peptides comprennent un squelette amidé inversé. Elle se rapporte en particulier aux composés correspondant à la formule (I) dans laquelle R1 et R2 représentent un groupe fonctionnel hydrocarbyle d'un acide carboxylique saturé ou insaturé à chaîne droite ou ramifiée ou d'un groupe fonctionnel phospholipidique, R3 représentant un peptide basique modifié perturbant la membrane qui possède un squelette amidé inversé, Y représentant alkylène C¿2-10? et X représentant C(O)-NH- ou -S-S-. L'invention se rapporte également aux sels de ces composés.
PCT/EP1999/002361 1998-04-07 1999-04-07 Nouveaux composes destines a la transfection d'adn WO1999051629A2 (fr)

Priority Applications (4)

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JP2000542350A JP2002510706A (ja) 1998-04-07 1999-04-07 Ｄｎａトランスフェクションのための新規化合物
EP99919208A EP1068225A2 (fr) 1998-04-07 1999-04-07 Nouveaux composes destines a la transfection d'adn
CA002327367A CA2327367A1 (fr) 1998-04-07 1999-04-07 Nouveaux composes destines a la transfection d'adn
AU37065/99A AU3706599A (en) 1998-04-07 1999-04-07 New compounds for dna-transfection

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EP98106302 1998-04-07
EP98106302.7 1998-04-07
EP98124837 1998-12-30
EP98124837.0 1998-12-30

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000074646A2 (fr) * 1999-06-02 2000-12-14 Aventis Pharma Deutschland Gmbh Nouveau complexe vecteur liposomal et son utilisation en therapie genique
WO2001051092A2 (fr) * 2000-01-07 2001-07-19 University Of Washington Transport ameliore d'agents au moyen d'agents perturbateurs de membranes
WO2001068873A1 (fr) * 2000-03-15 2001-09-20 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, molecule humaine d'adhesion intercellulaire 12, et polynucleotide codant pour ce polypeptide
EP1182208A2 (fr) * 2000-07-28 2002-02-27 Roche Diagnostics GmbH Composition pour la transfection d'ADN
WO2002028438A1 (fr) * 2000-10-05 2002-04-11 King's College London Lipopeptides activateurs d'absorption pour composes bio-actifs

Families Citing this family (1)

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GB9915074D0 (en) * 1999-06-28 1999-08-25 Cortecs Plc Ligand-binding composition

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000074646A2 (fr) * 1999-06-02 2000-12-14 Aventis Pharma Deutschland Gmbh Nouveau complexe vecteur liposomal et son utilisation en therapie genique
WO2000074646A3 (fr) * 1999-06-02 2001-08-09 Aventis Pharma Gmbh Nouveau complexe vecteur liposomal et son utilisation en therapie genique
WO2001051092A2 (fr) * 2000-01-07 2001-07-19 University Of Washington Transport ameliore d'agents au moyen d'agents perturbateurs de membranes
WO2001051092A3 (fr) * 2000-01-07 2001-12-06 Univ Washington Transport ameliore d'agents au moyen d'agents perturbateurs de membranes
WO2001068873A1 (fr) * 2000-03-15 2001-09-20 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, molecule humaine d'adhesion intercellulaire 12, et polynucleotide codant pour ce polypeptide
EP1182208A2 (fr) * 2000-07-28 2002-02-27 Roche Diagnostics GmbH Composition pour la transfection d'ADN
EP1182208A3 (fr) * 2000-07-28 2002-05-08 Roche Diagnostics GmbH Composition pour la transfection d'ADN
WO2002028438A1 (fr) * 2000-10-05 2002-04-11 King's College London Lipopeptides activateurs d'absorption pour composes bio-actifs

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EP1068225A2 (fr) 2001-01-17
AU3706599A (en) 1999-10-25
JP2002510706A (ja) 2002-04-09
CA2327367A1 (fr) 1999-10-14
WO1999051629A3 (fr) 1999-12-23

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