EP1208133A1 - Copolymeres pour le transport d'acide nucleique dans les cellules - Google Patents

Copolymeres pour le transport d'acide nucleique dans les cellules

Info

Publication number
EP1208133A1
EP1208133A1 EP00947874A EP00947874A EP1208133A1 EP 1208133 A1 EP1208133 A1 EP 1208133A1 EP 00947874 A EP00947874 A EP 00947874A EP 00947874 A EP00947874 A EP 00947874A EP 1208133 A1 EP1208133 A1 EP 1208133A1
Authority
EP
European Patent Office
Prior art keywords
dna
peptide
copolymer
complexes
pei
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00947874A
Other languages
German (de)
English (en)
Inventor
Christian Plank
Dirk Finsinger
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to EP00947874A priority Critical patent/EP1208133A1/fr
Publication of EP1208133A1 publication Critical patent/EP1208133A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33396—Polymers modified by chemical after-treatment with organic compounds containing nitrogen having oxygen in addition to nitrogen

Definitions

  • the invention relates to the field of gene transfer, in particular to non-viral vectors.
  • Organism to the target cell (the extracellular aspect) and 2) transfer of the agent to be transferred from the cell surface into the cytoplasm or the cell nucleus (the cellular aspect).
  • An essential prerequisite for receptor-mediated gene transfer is
  • DNA complexes With a suitable composition of the DNA complexes, specific uptake and efficient gene transfer in cells can be achieved by receptor-ligand interaction (Kircheis et al., 1997, Zanta et al., 1997). Complexes of DNA with cationic peptides are also particularly suitable for receptor-mediated gene transfer (Gottschalk et al., 1996; Wadhwa et al., 1997; Plank et al., 1999).
  • biodegradable synthetic polymers are used to package pharmaceuticals in a form that ensures increased residence time in the organism and leads to the desired bioavailability in the target organ ("controlled release").
  • the surface modification of colloidal particles with polyethylene glycol is designed in such a way that the undesired opsonization is suppressed.
  • biodegradable polymers for use in a large number of medical applications (Coombes et al., 1997).
  • the chemical bonds in the polymer backbone are varied. By suitable positioning of ester, amide, peptide or urethane bonds, the desired lability can be achieved in a physiological environment, the
  • Sensitivity to the attack of enzymes is specifically varied. Combinatorial synthesis principles have proven effective for the fast and efficient synthesis of biologically active substances (Balkenhohl et al., 1996). By systematically varying a few parameters, a large number of connections are obtained which have the desired basic structure (Brocchini et al., 1997). With a suitable, meaningful biological selection system, those who show the desired properties can be selected from this pool of compounds.
  • branched cationic peptides are suitable for efficient binding to DNA and for the formation of particulate DNA complexes (Plank et al., 1999). c) Polycation-DNA complexes go strong
  • the object of the present invention was to provide a new, improved non-viral gene transfer system based on nucleic acid-polycation complexes.
  • the starting point was the idea of enveloping the nucleic acid or nucleic acid complexes with a charged polymer which physically stabilizes the complexes and protects them from opsonization.
  • the present invention relates to a charged copolymer of the general formula I
  • R is an amphiphilic polymer or a homo- or heterobifunctional derivative thereof
  • X i) is an amino acid or an amino acid derivative, a peptide or a peptide derivative or a spermine or spermidine derivative;
  • Ci-Cß-alkyl is, where a is H or, optionally halogen- or dialkylamino-substituted, Ci-Cß-alkyl;
  • b, c and d are the same or different, optionally halogen- or dialkylamino-substituted Ci-C ⁇ -alkylene; or
  • a is H or, optionally halogen- or dialkylamino-substituted, Ci-C ⁇ -alkyl, and wherein b and c mean the same or different, optionally halogen- or dialkylamino-substituted Ci-C ⁇ -alkylene; or
  • W 1 Y 1 Z 1 is a substituted aromatic compound with three functional groups W 1 Y 1 Z 1 , where W, Y and Z have the meanings given below;
  • W, Y or Z are identical or different radicals CO, NH, 0 or S or a linker group capable of reacting with SH, OH, NH or NH 2 ;
  • n and n are independently 0, 1 or 2;
  • 1 is 1 to 5, preferably 1.
  • the unit XZ m -E n may be the same or different.
  • aromatic means a monocyclic or bicyclic aromatic hydrocarbon radical having 6 to 10 ring atoms which, in addition to the substituents mentioned at the outset, optionally independently with one or more further substituents, preferably with one, two or three substituents selected from the group Ci-C ⁇ - Alkyl, -0- (-C 6 alkyl), halogen - preferably fluorine, chlorine or bromine - cyano, nitro, amino, mono- (Ci-C ⁇ -alkyl) amino, di- (Ci-C ⁇ -alkyl) amino may be substituted.
  • the phenyl radical is preferred.
  • Aromatics in the sense of the present invention can also be used for a heteroraryl radical, i.e. : are a monocyclic or bicyclic aromatic hydrocarbon radical having 5 to 10 ring atoms, which contains one, two or three ring atoms selected from the group N, 0 or S independently of one another, the remaining ring atoms being C.
  • alkylamino or dialkylamino stands for an amino group which is substituted by one or two C 1 -C 6 -alkyl radicals, where - in the case of two alkyl radicals - the two alkyl groups can also form a ring.
  • C ⁇ _-Cg-alkyl generally represents a branched or unbranched hydrocarbon radical having 1 to 6 carbon atoms, which may optionally be substituted by one or more halogen atoms, preferably fluorine, which are identical to one another J or can be different.
  • halogen atoms preferably fluorine
  • lower alkyl radicals having 1 to 4 carbon atoms such as methyl, ethyl, propyl, isopropyl, n-butyl,
  • alkylene means a branched or unbranched double-bonded hydrocarbon bridge with 1 to 6 carbon atoms, which may optionally be substituted by one or more halogen atoms, preferably fluorine, which may be identical or different from one another.
  • amphiphilic polymer R is preferably a polyalkylene oxide, polyvinyl pyrollidone, polyacrylamide, iO
  • polyalkylene oxides examples include polyethylene glycols (PEG), polypropylene glycols, polyisopropylene glycols, polylbutylene glycols.
  • polyalkylene oxides in particular PEG.
  • the polyalkylene oxide can be present in the copolymer as such, or as a thio, carboxy or amino derivative.
  • the polymer R preferably has a molecular weight of 500-10,000, preferably 1000-10,000.
  • X is an amino acid
  • an amino acid with three functional groups can be used for the synthesis of the copolymer, two of these groups being capable of copolymerization with the polymer and one being able to couple the effector molecule E; in this case Z can be omitted.
  • the natural amino acids glutamic acid, aspartic acid, lysine, ornithine and tyrosine are preferred.
  • synthetic amino acids can be used instead of natural amino acids (e.g. corresponding spermine and spermidine derivatives).
  • linker groups are pyridylthiomercaptoalkylcarboxylates (see FIG. 1) or maleimidoalkane carboxylates.
  • X can also be a peptide (derivative).
  • E is coupled to it directly or via Z.
  • X is a positively or negatively charged peptide or peptide derivative or a spermine or spermidine derivative
  • the peptide in this case consists of a linear sequence of two or more identical or different natural or synthetic amino acids, the amino acids being chosen so that the peptide as a whole is either positively or negatively charged.
  • Suitable anionic peptide derivatives X have the general formula (peptide) n -B-spacer- (Xaa).
  • the peptide is a sequence of amino acids or amino acid derivatives with an overall negative charge.
  • the peptide preferably consists of three to 30 amino acids, preferably it consists exclusively » ⁇ from glutamic acid and / or aspartic acid residues.
  • n represents the number of branches depending on the functional groups contained in B.
  • B is a branching molecule, preferably lysine or a molecule of type X in the case of ii) to iv).
  • the spacer is a peptide consisting of 2 to 10 amino acids or an organic aminocarboxylic acid with 3 to 9 carbon atoms in the carboxylic acid skeleton, for example 6-aminohexanoic acid.
  • the spacer serves to spatially separate the charged effector molecule from the
  • Xaa is preferably a trifunctional amino acid, in particular glutamic acid or aspartic acid and can generally be a compound of type X, case i) - iv).
  • X may be a peptide derivative, the modification of the peptide being a charged moiety other than an amino acid; Examples of such groups are sulfonic acid groups or charged ones
  • Carbohydrate residues such as neuraminic acids, or sulfated glycosaminoglycans.
  • the modification of the peptide can be carried out according to standard methods, either directly in the course of the peptide synthesis or subsequently on the finished peptide.
  • the effector molecule E can be a polycationic or polyanionic peptide or peptide derivative or a spermine or spermidine derivative.
  • the peptide also represents a linear sequence of two or more identical or different natural or are synthetic amino acids, the amino acids being chosen so that the peptide as a whole is either positively or negatively charged.
  • the peptide can be branched. Examples of suitable branched cationic peptides have been described by Plank et al., 1999.
  • Suitable anionic molecules E have the general formula (peptide) n -B-spacer- (Xbb), where Xbb is preferably an amino acid with a reactive group which can be coupled to X directly or via Z.
  • the coupling of the effector peptide E to Z or directly to X takes place via a reactive group which is present in the peptide from the outset or is subsequently introduced, e.g. a thiol group (in a cysteine or through
  • the coupling can also take place via amino or carboxylic acid groups which are already present or have been introduced subsequently.
  • E can be a peptide derivative, the modification of the peptide consisting of a charged group other than an amino acid.
  • groups are sulfonic acid groups or charged ones
  • Carbohydrate residues such as neuraminic acids, or sulfated carbohydrate residues.
  • the coupling to X takes place directly or via Z.
  • the copolymer is modified with a cellular ligand for the target cell (receptor ligand L). In this case, the majority are
  • Linker positions Z are occupied by E; in between, a cellular ligand is coupled to individual positions of linker Z instead of cationic or anionic effector E. Alternatively, the ligand is coupled to individual positions of the effector molecule E.
  • the ratio E: L is preferably approximately 10: 1 to 4: 1.
  • the receptor ligand can be of biological origin (e.g. transferrin, antibodies, carbohydrate residues) or synthetic (e.g. RGD peptides, synthetic peptides, derivatives of synthetic
  • copolymers according to the invention can be prepared according to the following process scheme:
  • the copolymerization partner X or XZ m -E n is, if it is a peptide or peptide analog, after Standard methods, for example on the solid phase (solid phase peptide synthesis, SPPS) synthesized according to the Fmoc protocol (Fields et al., 1990).
  • the amino acid derivatives are activated with TBTU / HOBt or with HBTU / HOBt (Fields et al., 1991).
  • the following derivatives are used in their N-terminal Fmoc-protected form for the ionic amino acid positions:
  • Fmoc-K (Fmoc) -OH are used.
  • the peptides are cleaved from the resin using TFA / DCM.
  • the polymerization partner X is a peptide of the general structure (peptide) n -B-spacer- (Xaa) in the subsequent copolymerization, glutamic acid or aspartic acid is used at position Xaa, which is provided with a benzyl protective group at a carboxyl position. This is selectively removed by hydrogenolysis (Felix et al., 1978). The N-terminal amino acid positions of the peptide chain are occupied by Boc-protected amino acids in order to be able to carry out the deprotection after copolymerization of the peptide with PEG in one step. If the polymerization partner X is an amino acid derivative that contains a linker grouping (e.g.
  • Peptide chemistry can be obtained.
  • Mercaptopropionic acid is reacted with 2, 2 '-dithiodipyridine and purified by chromatography.
  • the reaction product is reacted with carboxyl-protected glutamic acid (O-t.butyl) with HOBt / EDC activation (see FIG. 1).
  • 6-Fn ⁇ oc-aminohexanoic acid is reacted analogously.
  • Carboxyl protecting groups are removed in TFA / DCM, the resulting glutamic acid derivative is purified using chromatographic methods.
  • Copolymers illustrates:
  • poly (PEG-0- OC-) matrix (“polyester”): the copolymerization of the ionic, partially side-chain-protected peptide dicarboxylic acids or
  • the p (PEG-peptide) copolymers are made according to established methods, e.g. with dicyclohexylcarbodiimide / DMAP, preferably in a strictly alternating sequence (Zalipsky et al., 1984;
  • the PEG macromonomer is put together 7 mixed with a side-chain protected peptide or glutamine or aspartic acid derivative in dichloromethane solution with DCC / DMAP. After the urea derivative formed has been separated off, the polymer can be obtained by precipitation with cold ether. The remaining side chain protecting groups can be split off with TFA in dichloromethane (the PEG ester bond is also stable under these conditions (Zalipsky et al., 1984)). The ionic polymer is obtained by precipitation and a final chromatography step. The conduct of the reaction enables the degree of polymerization and the amount of charge per PEG unit in the polymer to be checked.
  • polyamide poly (PEG -HN- 0C-) matrix
  • polyamide poly (PEG -HN- 0C-) matrix
  • Polymer matrix can be built up if, when using a copolymer-DNA complex in a gene therapy application, hydrolyzability that is too fast and therefore too high instability in systemic application is to be expected.
  • diamino-PEG derivatives are used instead of those of the PEG macromonomers which are copolymerized with the ionic peptides or glutamine or aspartic acid derivatives in analogy to the synthesis described above. In this synthesis, a hydrolysis-stable A skeleton is obtained.
  • Diamino-modified polyethylene glycols are commercially available as raw materials in defined molar mass ranges between 500 and 20,000 (eg Fluka).
  • the remaining acid-labile side chain protective groups of the peptide components are cleaved off, for example with TFA / DCM, and the polymers are purified by means of chromatographic methods.
  • Copolymers of glutamine or aspartic acid derivatives are reacted in a further step with anionic or cationic peptides which contain a suitable reactive group.
  • copolymers of 3- (2'-thio-pyridyl) mercaptopropionyl-glutamic acid are reacted with peptides which contain a free cysteine thiol group.
  • the Fmoc protective group is removed from copolymers which have arisen from 6-Fmoc-aminohexanoyl-glutamic acid under basic conditions.
  • the product is reacted with a carboxy-activated, protected peptide.
  • the peptide protective groups (t-Boc or Ot.butyl) are removed in DCM / TFA, the end product is purified by chromatography.
  • the amino group of Ahx (6-aminohexanoic acid) can be derivatized with bifunctional linkers and then reacted with a peptide.
  • the ligand L can be coupled directly by activating carboxyl groups on the effector E (preferably in the case of anionic copolymers) or on the ligand or by interposing bifunctional linkers such as succinimidylpyridyldithioproprionate (SPDP; Pierce or Sigma) and similar compounds.
  • SPDP succinimidylpyridyldithioproprionate
  • the purification of the reaction product can be carried out by gel filtration and ion exchange chromatography.
  • Type is the most selectable variable 13 and the molecular weight (degree of polymerization) of the polymer R, the identity of the polymerization partner XZ m -E n or the effector molecule E (for example a series of anionic peptides with an increasing number of glutamic acids) and the total degree of polymerization p.
  • a multi-parameter system is created which enables the rapid parallel construction of a homologous series of different copolymers and subsequently, after complexation with the nucleic acid, different non-viral vectors.
  • the synthesis concept is implemented on a cell culture plate scale (eg 96 wells per plate). For this purpose, the chemical synthesis is adapted to the required micro scale (reaction volumes in the range of 500 ⁇ l).
  • the copolymers are, for example, mixed with DNA complexes and then subjected to tests which permit an assessment of the polymer properties with regard to the intended application (for example gene transfer).
  • the same selection procedures can be used for copolymer-coated nanoparticles.
  • Screening and selection methods can serve, for example, complement activation tests in 96-well plate format (Plank et al., 1996), turbidometric measurements of the aggregation induced by serum albumin or salt in the same format or in vitro
  • Such examinations give e.g. Information about which copolymers from a combinatorial synthetic approach are suitable for modifying the surface of DNA complexes so that their solubility is sufficient for gene transfer applications in vivo, their interaction with blood and
  • Tissue components is restricted so that their residence time and duration of action in the blood circulation is increased sufficiently to enable receptor-mediated gene transfer in target cells.
  • copolymers according to the invention are preferably used for the transport of nucleic acids into higher eukaryotic cells.
  • the present invention thus relates to complexes containing one or more nucleic acid molecules and one or more charged copolymers of the general formula I.
  • the nucleic acid molecule is preferably condensed with an organic polycation or a cationic lipid.
  • the invention thus relates to complexes of nucleic acid and an organic polycation or a cationic lipid, which are characterized in that they have bound on their surface a charged copolymer of the general formula I via ionic interaction.
  • the nucleic acids to be transported into the cell can be DNAs or RNAs, with no restrictions on the nucleotide sequence and size.
  • the nucleic acid contained in the complexes according to the invention is primarily defined by the biological effect to be achieved in the cell, for example in the case of use in the context of gene therapy by the gene to be expressed or the gene segment, or by the intended substitution or repair of a gene defective gene or any target sequence (Yoon et al., 1996; Kren et al. 1998), or by the target sequence of a gene to be inhibited (eg in the case of the use of antisense oligoribonucleotides or ribozymes).
  • the nucleic acid to be transported into the cell is preferably plasmid DNA which contains a sequence coding for a therapeutically active protein.
  • the sequence codes, for example, for one or more cytokines, such as interleukin-2, IFN- ⁇ , IFn- ⁇ , TNF- ⁇ , or for a suicide gene that is used in combination with the substrate.
  • the complexes contain DNA coding for one or more Tumor antigens or fragments thereof, optionally in combination with DNA, coding for one or more cytokines.
  • therapeutically active nucleic acids are given in WO 93/07283.
  • the copolymer according to the invention has the property of sterically stabilizing the nucleic acid-polycation complex and reducing or suppressing its undesired interaction with components of body fluids (e.g. with serum proteins).
  • Suitable organic polycations for complexing nucleic acid for transport into eukaryotic cells are known; due to its interaction with the negatively charged nucleic acid, it is compacted and brought into a form suitable for absorption into the cells.
  • polycations that have been used for the receptor-mediated gene transfer such as homologous linear cationic polyamino acids (such as polylysine, polyarginine, polyornithine) or heterologous linear mixed cationic-neutral polyamino acids (consisting of two or more cationic and neutral amino acids), branched and linear cationic peptides (Plank et al., 1999; Wadhwa et al.
  • non-peptide polycations such as linear or branched polyethyleneimines, polypropyleneimines), dendrimers (spheroidal polycations with a well-defined diameter and a exact number of terminal amino groups can be synthesized; (Haensler and Szoka, 1993; Tang et al., 1996; WO 95/02397), cationic carbohydrates, e.g. chitosan (Erbacher et al. 1998).
  • the polycations can also be modified with lipids (Zhou et al. 1994; WO 97/25070).
  • cationic lipids (Lee et al. 1997), some of which are commercially available (e.g. lipofectamine, transfectam).
  • polycation is used below to represent both polycations and cationic lipids.
  • Polycations preferred in the context of the present invention are polyethyleneimines, polylysine and dendrimers, e.g. Polyamidoamine dendrimers ("PAMAM" dendrimers).
  • PAMAM Polyamidoamine dendrimers
  • the size or charge of the polycations can vary over a wide range; it is chosen so that the complex formed with nucleic acid does not dissociate at physiological salt concentration, which can be determined in a simple manner by the ethidium bromide displacement assay (Plank et al, 1999).
  • a defined amount of nucleic acid is incubated with increasing amounts of the selected polycation, the complex formed is applied to the cells to be transfected and the gene expression (generally using a reporter gene construct, e.g. luciferase) is measured using standard methods.
  • the nucleic acid complexes are built up via electrostatic interactions.
  • the DNA can be in excess in relation to the polycation, so that such complexes have a negative surface charge exhibit; conversely, when the polycation condensing the nucleic acid is in excess, the complexes have a positive surface charge.
  • the polycation is preferably present in excess in the context of the present invention.
  • the ratio of polycation: nucleic acid in the case of a positive charge excess is preferably set so that the zeta potential is approximately +20 to +50 mV, in the case of the use of certain polycations, e.g. Polylysine, it can be above that too.
  • the zeta potential is approximately -50 to -20mV.
  • the zeta potential is measured using established standard methods, e.g. by Erbacher et al. 1998.
  • the polycation is optionally conjugated to a cellular ligand or antibody; suitable ligands are described in WO 93/07283.
  • suitable ligands are described in WO 93/07283.
  • Tumor therapy prefers ligands or antibodies for tumor cell-associated receptors (e.g. CD87; uPA-R) that are able to increase the gene transfer in tumor cells.
  • tumor cell-associated receptors e.g. CD87; uPA-R
  • the nucleic acid In the preparation of the complexes, the nucleic acid, generally plasmid DNA, is incubated with the polycation (possibly derivatized with a receptor ligand), which is present in excess charge. Particles are formed, which are absorbed by receptor-mediated endocytosis Cells can be included.
  • the complexes are then incubated with a negatively charged copolymer according to the invention, preferably polyethylene glycol copolymer.
  • the effector E in the copolymer is preferably a polyanionic peptide.
  • the copolymer is first mixed with nucleic acid and then incubated with polycation, or, as a third variant, first mixed with polycation and then incubated with nucleic acid.
  • the nucleic acid is incubated with a polycation present in electrostatic deficit and then a cationic copolymer is added.
  • the order of the mixing steps can also be varied here, as described above for anionic copolymers.
  • the relative proportions of the individual components are chosen so that the resulting DNA complex has a weakly positive, neutral or weakly negative zeta potential (+10 mV to -10 mV).
  • positively charged copolymers they can be used as the sole nucleic acid binding and condensing polycationic molecules; the proportion of a polycation or cationic lipid can thus be omitted.
  • the relative proportions of the individual components are chosen so that the resulting DNA complex has a weakly positive, neutral or weakly negative zeta potential (+10 mV to -10 mV).
  • polycation and / or copolymer may have been modified with the same or different cellular ligands.
  • nucleic acid complexes according to the invention which are stabilized in size by the electrostatically bound copolymer of the general formula I and are thus protected against aggregation, have the advantage that they can be stored in solution for longer periods (weeks). In addition, they have the advantage that, due to the protective effect of the bound copolymer, there is less or no interaction with components of body fluids (e.g. with serum proteins).
  • the invention relates to a pharmaceutical composition containing a therapeutically active nucleic acid, the copolymer according to the invention and optionally an organic polycation or cationic lipid.
  • the pharmaceutical composition according to the invention is preferably in the form of a lyophilisate, optionally with the addition of sugar, such as sucrose or dextrose, in an amount which gives a physiological concentration in the ready-to-use solution.
  • the composition can also be in the form of a cryoconcentrate.
  • composition according to the invention can also be frozen (cryopreserved) or as a chilled solution.
  • the positively or negatively charged copolymers according to the invention serve to colloidal particles ("nanoparticles"), as they are for Application of classic drugs are developed to stabilize sterically and to reduce or suppress their undesirable interaction with components of body fluids (eg with serum proteins).
  • the copolymers according to the invention modified with receptor ligands can be used to provide such nanoparticles with receptor ligands on their surface in order to transfer drugs with increased specificity to target cells (“drug targeting”).
  • Fig. 1 Preparation of the copolymer frameworks from 3- (2'-thio-pyridyl) mercaptopropionyl-glutamic acid and 0, 0 'bis (2-aminoethyl) poly (ethylene glycol) 6000 or 0.0'-bis (2-aminoethyl ) poly (ethylene glycol) 3400
  • Fig. 2 Coupling of charged peptides to the copolymer backbone
  • Fig. 5 Erythrocyte lysis test
  • Fig. 9 Gene transfer in K562 cells with PEI (25 kD) DNA complexes in the presence and absence of the copolymer P3YE5C
  • Fig. 10 Transfection of the breast cancer cell line MDA-MB435S with polylysine-DNA complexes in the presence and absence of the envelope polymer P3INF7
  • Fig. 11 Lipofection in NIH3T3 cells in the presence and absence of the copolymer P3YE5C
  • Fig. 12 Transfection of HepG2 cells with
  • Fig. 13 Transfection of HepG2 cells with PEI-DNA complexes in the presence of different amounts of copolymer at different charge ratios
  • Fig. 14 Dependence of the transfection efficiency on the amount of copolymer, on the complex cleaning and on the presence of salt
  • Fig. 15 Intravenous gene transfer in vivo using DNA / polycation complexes with a copolymer shell ⁇ 5
  • Fig. 16 Gene transfer with copolymer-protected PEI-DNA
  • Fig. 17 Gene transfer with copolymer-protected PEI-DNA vectors in vivo - PYE5C-PEI-DNA in
  • Fig. 18 P6YE5C enhances the transport of DNA-PEI complexes into the tumor (tumor targeting)
  • Fig. 19 Interaction of PEI-DNA complexes and human serum components
  • X 3-mercaptopropionyl-glutamic acid, that is, an amino acid derivative according to case i), which was obtained by coupling the linker group 3- (2 '-Thiopyridyl) - mercaptopropionic acid to glutamic acid;
  • Ice cooling was carried out in succession in a 50 ml polypropylene tube (from Peske) one mmol of di-t-butyl ester of glutamic acid (Glu (OtBu) OtBu, Bachern), 1-hydroxybenzotriazole (Aldrich), N-ethyl-N'- ( dimethylaminopropyl) carbodiimide (Aldrich) and
  • Product 2b was dissolved in 3 ml of dimethylformamide (Fluka) and made up to 20 ml with dichloromethane. 5 ml of this solution (67.5 ⁇ mol) were sequentially treated with 506 mg diamino-PEG-6000 (84 ⁇ mol, corresponds to 1.25 equivalents; Fluka), 30 mg dicyclohexylcarbodiimide (135 ⁇ mol, 2 equivalents, 135 ⁇ l of a 1 M solution in DMF) and 2 mg
  • Product 4 was obtained using the same batch and purification as product 3.
  • the main fraction (54% of the product fractions) after gel filtration was a product which eluted with an apparent molecular weight of 22,800 Da (secondary fractions are a product with 64 kD, 14% of the Total, and a product with 46 kD, 32% of the total).
  • the peptides were prepared according to the FastMoc TM protocol on an Applied Biosystems 431A peptide synthesizer.
  • Peptide YE5C (sequence [Ac-YEEEEE] 2 -ahx-C) was used using 330 mg of cysteine-loaded chlortrityl resin (0.5 mmol / g; Bachern) with the protecting groups trityl- (Cys), di-Fmoc (Lys) and Ot - Butyl (Glu) manufactured. 1 mmol each of the protected amino acids was used. Double couplings were carried out continuously after the branch point (Lys).
  • the acetylation of the N-termini was carried out on the peptide-resin with 2 mmol acetic anhydride in 2 ml N-methylpyrrolidone in the presence of 2 mmol diisopropylethylamine.
  • the peptide was obtained as a crude product after cleavage from the resin (500 ul water, 500 ul thioanisole, 250 ul ethanedithiol in 10 ml trifluoroacetic acid) and precipitation with diethyl ether.
  • the crude product was dissolved in 100 mM HEPES pH 7.9 and purified by means of perfusion chromatography (Porös 20 HQ, Boehringer Mannheim, filled in a 4 x 100 mm PEEK column. 0-0.5 M NaCl in 8 min, flow rate 10 ml / min).
  • the extinction coefficient of the peptide in 50 mM sodium phosphate buffer in 6 M guanidinium hydrochloride at 280 nm is 2560 M "1 cm " 1 (Gill and von Hippel 1989).
  • Peptide INF7 (sequence GLFEAIEGFIENGWEGMIDGWYGC) was synthesized according to the same procedure on 500 mg chlortrityl resin (0.5 mmol / g) as described for YE5C and cleaved from the resin and with Diethyl ether like. The crude product was dried in vacuo. Aliquots of 20 mg each were dissolved in 500 ⁇ l of 1 M triethylammonium hydrogen carbonate buffer pH 8 and purified by gel filtration (Sephadex G-10 from Pharmacia filled into an HR 10/30 column from Pharmacia. Flow rate 1 ml / min.
  • Peptide SF029-ahx (sequence K 2 K-ahx-C) was synthesized in an analogous manner (500 mg Fmoc-Cys (Trt) - chlorotrityl resin from Bachern; 0.5 mmol / g) and purified by standard methods (Sephadex G10 with 0.1% TFA as eluent; reverse phase HPLC, 0.1% TFA - acetonitrile gradient).
  • the lysine at the branch point was alpha, epsilon-di-Fmoc-L-lysine, the following lysines were alpha-Fmoc-epsilon-Boc-L-lysine.
  • Peptide E4E (sequence [EEEE] 2 KGGE) was synthesized analogously. Batch size 0.25 mmol Fmoc-Glu (OBzl) - chlorotrityl resin. The resin was coated by suspending appropriate amounts
  • O-chlorotrityl chloride resin Alexis
  • FmocGlu OBzl
  • diisopropylethylamine After shaking for several hours, the mixture was filtered and washed several times with dimethylformamide, methanol, isopropanol, dichloromethane and diethyl ether.
  • a modified Fmoc protocol is used.
  • the N-terminal amino acid carries a Boc protective group in order to turn the solid phase synthesis into a fully protected base stable Peptide derivative of the sequence (E (Boc) [E (tBu)] 3 ) ⁇ KGGE (OBzl) OH (E4E PR0T ).
  • Glutamic acid was split off selectively with H 2 / palladium on activated carbon according to the standard procedure.
  • the peptide masses were determined by means of electrospray mass spectroscopy and the identity of the peptides was thus confirmed.
  • the available thiopyridyl binding sites are determined by adding a dilute solution of the polymer with 2-mercaptoethanol and then measuring the absorbance of the released 2-thiopyridone at a wavelength of 342 nm, the concentration of the free thiol functions of the cysteine-containing peptide
  • Ellman reagent determined at a wavelength of 412 nm according to Lambert-Beer. After the reaction, the completeness of which was determined by the absorption of the released thiopyridone at 342 nm, the mixture was concentrated and the product was fractionated by gel filtration (Superdex 75 material, Pharmacia).
  • Copolymer P6YE5C was prepared from fraction 3 (40,200 Da) of the product (3) and purified peptide. A product with an apparent molecular weight of 55,800 Da was obtained. The degree of polymerization is approx. 7.
  • the endosomolytic peptide INF7 was used, which is linked to the 3-mercapto-propionyl-glutamic acid group via a disulfide bridge of the cysteine thiol.
  • a) Copolymer P3INF7 was prepared from fraction 3 (22,800 Da) of product (4) and purified influenza peptide.
  • Copolymer P6INF7 was prepared from fraction 3 (40,200 Da) of product (3) and purified influenza peptide INF7.
  • lactosylated peptide SF029-ahx 9 parts of the branched peptide YE5C, which are linked to the 3-mercaptopropionyl-glutamic acid group via a disulfide bridge of the cysteine thiol, were used.
  • the reaction scheme for coupling the charged peptides to the copolymer structure according to 1.3 is shown in FIG. 2: peptides with free thiol groups are coupled to product 3 or 4, for example the peptide INF7 (left) or the peptide YE5C.
  • product 3 or 4 for example the peptide INF7 (left) or the peptide YE5C.
  • X is an amino acid derivative which was obtained by coupling Fmoc-6-aminohexanoic acid to glutamic acid.
  • Z can be omitted or a bifunctional linker such as SPDP or EMCS.
  • An effector E suitable for coupling to this polymer structure can be a peptide of the type E4E PR0T (Z omitted) or of the type YE5C, which reacts with the linker molecule Z (for example SPDP or EMCS) via the cysteine thiol.
  • Di-t-butyl-protected derivative (5) was dissolved in 30 ml dichloromethane / trifluoroacetic acid 2: 1 and stirred for one hour at room temperature. After the reaction was complete (reaction control with reversed phase HPLC), the solvent was reduced to about 5% of the initial volume and the product (6) was obtained by precipitation from diethyl ether. Final cleaning was carried out by RP-HPLC with an acetonitrile / water / 0.1% TFA gradient.
  • the copolymer can be conjugated to any peptide with a free C-terminus using standard peptide coupling chemistry.
  • trifluoroacetic acid with addition of up to 5% scavenger (preferably ethanedithiol, triethylsilane, thioanisole) was added, as described in the literature, and the mixture was stirred at room temperature for two hours.
  • scavenger preferably ethanedithiol, triethylsilane, thioanisole
  • the crude product was isolated by precipitation from diethyl ether.
  • the final purification was carried out as described above using gel filtration (Superdex75, Pharmacia).
  • Fig. 3 shows the reaction scheme:
  • Benzyl protective group on carboxylate 1 of the C-terminal glutamic acid of the fully protected peptide E4E PR0T is selectively cleaved with H 2 / palladium on activated carbon.
  • the product is activated with DCC with 0.0 'bis (2-aminoethyl) poly (ethylene glycol) 6000 or with 0.0'-
  • Polylysine (average chain length 170; Sigma) - DNA was prepared as a stock solution by adding 64 ⁇ g pCMVLuc (corresponding to pCMVL, described in WO 93/07283) in 800 ⁇ l HBS to 256 ⁇ g pL in 800 ⁇ l HBS and mixing by pipetting were, this corresponds to a calculated charge ratio of 6.3. From this suspension (hereinafter the DNA-polycation complexes are also referred to as “polyplexes”), 50 ⁇ l each in column 1 A-F were used as positive controls
  • PEI (25 kD, Aldrich) - DNA complexes were combined by combining equal volumes of a DNA solution (80 ⁇ g / ml in 20 mM HEPES pH 7.4) and a PEI solution (83.4 ⁇ g / ml in 20 mM HEPES pH 7.4 ) manufactured.
  • a DNA solution 80 ⁇ g / ml in 20 mM HEPES pH 7.4
  • a PEI solution 83.4 ⁇ g / ml in 20 mM HEPES pH 7.4
  • DNA complexes were centrifuged three times for 15 min at 350 xg in Centricon-100 filter tubes (Millipore) to remove excess, unbound PEI, filling to the original volume in each case with 20 mM HEPES pH 7.4 between centrifugations. After the last centrifugation step, a stock solution of DNA complex was obtained which corresponded to a DNA concentration of 300 ⁇ g / ml. 182 ⁇ l of this solution were diluted to 2520 ⁇ l with 20 mM HEPES pH 7.4. Each 610 ⁇ l of this solution (corresponding to 13.2 ⁇ g DNA) was pipetted into solutions of P6YE5C in 277.6 ⁇ l 20 mM HEPES pH 7.4 each.
  • the resulting solutions were brought to a salt concentration of 150 mM with 5 M NaCl.
  • 150 ⁇ l each of the resulting solutions were transferred in column 1, A to F, of a 96-well plate.
  • the dilution series in GVB 2+ buffer was carried out as described (Plank et al. 1996).
  • CH50 max denotes the CH50 value that is obtained with untreated human serum.
  • human serum was incubated with gene vectors.
  • CH50 values obtained with serum treated in this way are lower than CH50 max when the vectors activate the complement cascade.
  • the data are shown in percent of CHSO max .
  • Polylysine-DNA complexes have been observed, can be completely prevented by coating polymer P6INF7.
  • copolymer P6YE5C completely protects against complement activation even in small amounts added.
  • the test is used to examine the ability of peptides to lyse natural membranes depending on the pH.
  • the erythrocytes used in this example were obtained as follows: 10 ml of fresh blood was obtained from
  • the concentration of the erythrocytes was determined with an "extinction coefficient" of 2,394 x 10 8 ml / cells determined at 541 nm. To derive the extinction coefficient, the cell number was determined in an aliquot with a Neubauer chamber and the extinction of this solution was then determined after adding 1 ⁇ l of 1% Triton X-100 at 541 nm.
  • DOTAP / cholesterol-DNA complexes were prepared from DOTAP / cholesterol (1: 1 mol / mol) liposomes in 330 ⁇ l 20 mM HEPES pH 7.4 and DNA in the same volume at a charge ratio of 5.
  • the lipoplexes were with 0, 1, 2, 3 and 5 equivalents of the copolymer P3YE5C incubated in 330 ul buffer.
  • the final DNA concentration of the complex was 10 ⁇ g / ml.
  • the size of the DNA complexes was determined on the one hand by dynamic light scattering (Zetamaster 3000, Malvern Instruments) immediately after addition of polymer and then at different times over several hours, on the other hand by electron microscopy as in Erbacher et al., 1998, and Erbacher et al., 1999.
  • the particle size is 20 to 30 nm.
  • Copolymer-protected DNA complexes remain stable and do not aggregate, in contrast to unprotected PEI-DNA complexes, which precipitate immediately under these conditions (not shown).
  • the zeta potentials were determined on the same samples as in Example 6 using the Malvern instrument, the parameters refractive index, viscosity and dielectric constant being set to the values of deionized water, which can only be approximate.
  • FIG. 7A shows the zeta potential of PEI and DOTAP / cholesterol-DNA complexes as a function of the amount of copolymer P3YE5C added.
  • the zeta potential as a measure of the surface charge of the complexes decreases from strongly positive to neutral to slightly negative with increasing amount of added copolymer. This shows that the copolymer binds to the DNA complexes and balances or shields their electrostatic charge.
  • Figure 7B shows the zeta potential of purified PEI-DNA complexes.
  • Excess PEI was removed as described in Example 4B. Aliquots corresponding to a DNA amount of 40 ⁇ g each were taken from the PEI-DNA stock solution, which resulted from the purification, and made up to 666 ⁇ l each with 20 mM HEPES pH 7.4.
  • the resulting PEI-DNA suspensions were combined with solutions of P3YE5C in 333 ⁇ l 20 mM HEPES pH 7.4 and mixed by pipetting.
  • the solutions of P3YE5C contained polymer amounts corresponding to 1, 2, 3 and 5 charge equivalents relative to the amount of DNA used.
  • the zeta potentials were determined as described in Example 7A.
  • Adherent cells become 20,000 - 30,000 cells per day in flat-bottom plates the day before the transfection
  • the confluence should be about 70-80%).
  • Medium is aspirated before transfection. For transfection, 150 ⁇ l of medium are added to the cells and then 50 ⁇ l of DNA complexes are added.
  • composition of the DNA complexes preferably 1 ⁇ g DNA / well final concentration; Calculation for 1.2 times the amount; 20 ⁇ l volume per component (DNA, PEI, polymer). Finally 50 ⁇ l DNA complex are used for the transfection.
  • PEI Polyethyleneimine
  • Envelope polymer If you want e.g. for the specified amount of DNA and PEI shell polymer in an amount of
  • ⁇ l (envelope pol.) 1000 xÜ £ - ⁇ ⁇ x charge equ. ⁇ '330 c (envelope pol. [ ⁇ mol / ml])
  • DNA is vortexed to PEI. After 15 minutes, shell polymer is again added to the prefabricated PEI-DNA complex with vortexing. After a further 30 min, 50 ⁇ l DNA complexes are added to the cells, which are each in 150 ⁇ l medium.
  • the vessels used depend on the calculated total volume.
  • PEI is conveniently placed in a 14 ml polypropylene tube (e.g. Falcon 2059), the other two components in 6 ml tubes (e.g. Falcon 2063).
  • the components can also be mixed in a 96-well plate. If the final total volume of the DNA complexes is 1 - 1.5 ml, Eppendorf tubes are suitable. To mix, pipette up and down with the micropipette instead of vortexing.
  • Fig. 8 shows schematically the formulation of
  • the copolymer can be modified with receptor ligands, symbolized by asterisks (right).
  • the protein content of the lysates was determined using the Bio-Rad Protein Assay (Bio-Ra): 150 ⁇ l (or 155 ⁇ l) of dest were added to 10 ⁇ l (or 5 ⁇ l) of the lysate. Water and 40 ⁇ l of Bio-Rad protein assay dye concentrate are placed in a well of a transparent 96-well plate (type "flat botto", from Nunc, Denmark) and at 630 nm with the absorbance reader "Biolumin 690" and the computer program “Xperiment” (both from Molecular Dynamics, USA) measured the absorption.
  • Bio-Rad Protein Assay Bio-Ra
  • BSA Bovine serum albumin
  • K562 cells (ATCC CCL 243) were cultivated in RPMI-1640 medium with the addition of 10% FCS, 100 units / l penicillin, 100 ⁇ g / ml streptomycin and 2 mM glutamine at 37 ° C. in an atmosphere of 5% CO 2 , Desferoxamine was added to the cells to a final concentration of 10 ⁇ M the evening before the transfection. The medium was changed immediately before the transfection. 50,000 cells each in 160 ⁇ l medium were placed in the wells of a 96-well plate.
  • Transferrin-PEI (hTf-PEI 25 kD) was essentially as described by Kircheis et al. described (Kircheis et al., 1997) prepared by reductive amination. A product was obtained which coupled an average of 1.7 transferrin molecules per PEI molecule.
  • DNA complexes without hTf were produced with the equivalent amount of PEI (40 ⁇ g DNA + 42 ⁇ g PEI + shell polymer). 60 ⁇ l of the resulting mixtures (corresponding to an amount of 1 ⁇ g DNA / well) were in each
  • Tris pH 7.8; 0.1% Triton X-100 was added. After 15 minutes of incubation, the mixture was mixed by pipetting and 10 .mu.l samples were transferred to the black plate (Costar) for the luciferase test in 96-well plate format and 100 .mu.l luciferin substrate buffer were added. The resultant light emission was measured by means of a microplate scintillation & luminescence counter "Top Count" (Canberra-Packard, Dreieich).
  • the copolymer does not interfere with gene transfer and even increases it when a receptor ligand is in the DNA complex is present.
  • the expression of the reporter gene luciferase is shown based on the amount of total protein in the cell extract (mean values and standard deviations from triplicates).
  • MDA-MB435S cells (ATCC 45526; human breast cancer cell line) were in DMEM medium with the addition of 10% FCS, 100 units / ml penicillin, 100 ⁇ g / ml streptomycin and 2 mM glutamine at 37 ° C. in an atmosphere of 5% CO 2 cultivated. The evening before the transfection, the cells became 20,000 cells each
  • the DNA complexes were prepared as follows:
  • DNA complexes were mixed as indicated in the following table, with DNA first being added to polylysine and then again after 15 min to polymer P3INF7 or to the buffer. The experiments were carried out in triplicate. 60 ⁇ l DNA complexes were added to cells covered by 150 ⁇ l medium. After 4 h the medium was changed, after 24 h after washing with PBS and adding 100 ⁇ l lysis buffer, the luciferase test and the protein test were carried out as described in Example 9. Q
  • 10A shows the result of the gene transfer experiments with polylysine-DNA complexes in the human breast cancer cell line MDA-MB435S in the presence and absence of the copolymer P3INF7. No measurable reporter gene expression occurs in the absence of the copolymer. The pH-dependent membrane-destroying and thus endosomolytic activity of the copolymer results in efficient gene transfer. 5 nmol and 10 nmol P3INF7 refer to the amount of the polymer-bound peptide INF7 used.
  • NIH3T3 cells (ATCC CRL 1658) were cultivated in DMEM medium with the addition of 10% FCS, 100 units / ml penicillin, 100 ⁇ g / ml streptomycin and 2 mM glutamine at 37 ° C. in an atmosphere of 5% CO 2 .
  • DNA complexes were added to the cells which were in 800 ul fresh medium; this corresponds to 2 ⁇ g DNA per well.
  • the experiments were carried out in triplicate.
  • HepG2 cells (ATCC HB 8065) were cultivated in DMEM medium with the addition of 10% FCS, 100 units / ml penicillin, 100 ⁇ g / ml streptomycin and 2 mM glutamine at 37 ° C. in an atmosphere of 5% CO 2 .
  • DOTAP / cholesterol was done exactly as described above for NIH3T3 cells, this time using polymer P6YE5C. Furthermore 7 ⁇ g DNA were dissolved in 105 ⁇ l HEPES buffer to 7.3 ⁇ g PEI 25 kD in the same L ⁇
  • Figure 12 shows gene transfer in HepG2 cells in the presence and absence of the copolymer P6YE5C (designated P6 in the figure). Transfection by DOTAP / cholesterol DNA is not significantly inhibited. The transfection by PEI-DNA complexes is reduced (3 charge equivalents of the copolymer).
  • Example 14 The complexes were prepared in 20 mM HEPES pH 7.4, as indicated in Example 14, using unpurified complexes and adjusted to a concentration of 5% glucose before addition to the cells.
  • Fig. 14 shows the dependence of
  • mice 75 ul 50% glucose added. 100 ⁇ l of this solution was injected into the tail vein of mice (corresponding to a dose of 20 ⁇ g DNA per animal).
  • DOTAP cholesterol liposomes were produced according to standard instructions (Barron et al., 1998). In this case, liposomes with a molar DOTAP to cholesterol ratio of 1: 1 and a final concentration of DOTAP of 5 mM in 5% glucose. 130 ⁇ g DNA in 91.1 ⁇ l 20 mM HEPES pH 7.4 were added to 393.5 ⁇ l liposome suspension. After 15 minutes, 65 ul 50% glucose was added. 100 ⁇ l of this solution was injected into the tail vein of mice (corresponding to a dose of 20 ⁇ g DNA per animal).
  • DOTAP / cholesterol DNA (5: 1) with a copolymer shell: 393.9 ⁇ l liposome suspension were pipetted directly into a solution of 130 ⁇ g DNA in 65.3 ⁇ l water. After 15 min, 3 charge equivalents of P3YE5C in 216.9 ⁇ l HEPES buffer were added and after a further 30 min 75 ⁇ l 5% glucose. 115.5 ⁇ l of this solution were injected into the tail vein of mice (corresponding to a dose of 20 ⁇ g DNA per animal). G
  • the size of the complexes was determined by means of dynamic light scattering and was 20 to 30 nm.
  • mice were injected into the mice (age 5 weeks) intradermally on day 0, as described (Kruger et al. 1994). On day 21, the animals were injected via the tail vein with 200 ⁇ l DNA complex, each containing 50 ⁇ g DNA (p55pCMV-IVS-luc +, supplied by Dr. Andrew Baker, Bayer Corp., USA).
  • DNA complexes DNA (250 ⁇ g in 700 ⁇ l 20 mM HEPES pH7.4) was mixed with PEI (260.6 ⁇ g in 700 ⁇ l of the same buffer) by pipetting. After 15 minutes of incubation, the DNA complexes were pipetted into 400 ⁇ l of the shell polymer solution which contained 5 charge equivalents P3YE5C. After a further 15 min incubation, 200 ul 50% glucose (in water) was added. The complexes were purified from excess PEI using ultrafiltration as described in Example 4b. The vector suspension was finally made up to a volume of 1 ml at 20 mM ln
  • HEPES replenished.
  • the test animals were injected with 200 ⁇ l each via the tail vein. After 24 hours, the animals were sacrificed and reporter gene expression in the organs was determined: the animals were given general anesthesia by means of an intraperitoneal injection of 100 mg of ketamine and 5 mg of xylazine per kg of body weight.
  • the abdominal cavity was then opened and perfused with 20 ml of isotonic sodium chloride solution via the vena cava caudalis.
  • the organs were transferred to 2 ml screw cap tubes containing 500 ⁇ l lysis buffer (0.1% Triton X-100 in 250 mM Tris-HCl pH 7.8) and zirconium beads (2.5 mm diameter; Biospec Products, Bartlesville, USA).
  • the tissue samples were homogenized 2 x 20 sec using a mini bead bead heater (Biospec Products). 50 ⁇ l each
  • Tissue homogenates were used in the luciferase test (Promega Luciferase Assay Kit); the result is shown in Fig. 16.
  • DNA complexes 350 ⁇ g DNA in 420 ⁇ l 20 mM HEPES pH 7.4 were mixed with 364 ⁇ g PEI in the same volume of the same buffer. After 5 min. Incubation was either 420 ul 20 mM HEPES or 3 charge equivalents P6YE5C in 420 ul 20 mM HEPES pH 7.4 and mixed. Ultimately, 140 ul
  • Tissue homogenate was used. The result is shown in Fig. 17.
  • P6YE5C enhances the transport of DNA-PEI complexes into the tumor (tumor targeting)
  • tumors were set by placing lxlO 6 murine melanoma M3 cells (ATCC No. CCL 53.1) in 100 ⁇ l Ringer's solution subcutaneously in DBA / 2 mice.
  • Amount of P6YE5C ( ⁇ l) [1000 * ( ⁇ g of DNA) * charge equivalent of P6YE5C] /330*19.3; where the charge equivalent is the one defined above (3) and 19.3 is the concentration in mM of the solution of P6YE5C used.
  • glucose was added prior to administration of the complexes to provide an isotonic solution for in vivo injection.
  • mice were treated 20 days after tumor placement - the tumors had an average size of 15mm x 15mm at this point in time. 250 ⁇ l polyplexes per mouse were injected strictly iv (into the lateral tail vein). There were two groups consisting of 3 mice treated, the first group receiving unprotected DNA-PEI complexes and the second group of complexes encased with P6YE5C.
  • the animals were euthanized and the organs (liver, spleen, kidneys, heart and lungs) and the respective tumor were removed.
  • the further treatment of the organs and the measurement of the luciferase activity were carried out as described in the previous examples; the result of the tests is shown in Fig. 18.
  • Sensor chip is shown by an increasing measurement signal (resonance units, RU, y-axis).
  • the time axis over the entire experiment (x-axis) was 1200 seconds.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Polymers & Plastics (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Veterinary Medicine (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Nanotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Medicinal Preparation (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Other Resins Obtained By Reactions Not Involving Carbon-To-Carbon Unsaturated Bonds (AREA)

Abstract

La présente invention concerne des copolymères obtenus à partir d'un polymère amphiphile, de préférence du polyéthylène glycol, et d'une molécule effectrice chargée, en particulier un peptide ou dérivé peptidique. Elle concerne également des complexes d'acide nucléique dans lesquels l'acide nucléique est condensé avec un polycation et qui contiennent le polymère chargé lié à leur surface, utilisés en tant que vecteurs non viraux pour la thérapie génique.
EP00947874A 1999-06-25 2000-06-23 Copolymeres pour le transport d'acide nucleique dans les cellules Withdrawn EP1208133A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP00947874A EP1208133A1 (fr) 1999-06-25 2000-06-23 Copolymeres pour le transport d'acide nucleique dans les cellules

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP99112260A EP1063254A1 (fr) 1999-06-25 1999-06-25 Copolymères pour le transport d'acide nucléique dans la cellule
EP99112260 1999-06-25
PCT/EP2000/005869 WO2001000709A1 (fr) 1999-06-25 2000-06-23 Copolymeres pour le transport d'acide nucleique dans les cellules
EP00947874A EP1208133A1 (fr) 1999-06-25 2000-06-23 Copolymeres pour le transport d'acide nucleique dans les cellules

Publications (1)

Publication Number Publication Date
EP1208133A1 true EP1208133A1 (fr) 2002-05-29

Family

ID=8238431

Family Applications (2)

Application Number Title Priority Date Filing Date
EP99112260A Withdrawn EP1063254A1 (fr) 1999-06-25 1999-06-25 Copolymères pour le transport d'acide nucléique dans la cellule
EP00947874A Withdrawn EP1208133A1 (fr) 1999-06-25 2000-06-23 Copolymeres pour le transport d'acide nucleique dans les cellules

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP99112260A Withdrawn EP1063254A1 (fr) 1999-06-25 1999-06-25 Copolymères pour le transport d'acide nucléique dans la cellule

Country Status (6)

Country Link
EP (2) EP1063254A1 (fr)
JP (1) JP2003503569A (fr)
AU (1) AU6152000A (fr)
CA (1) CA2377211A1 (fr)
MX (1) MXPA01012802A (fr)
WO (1) WO2001000709A1 (fr)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2508015C (fr) 2002-12-30 2012-04-03 Nektar Therapeutics Al, Corporation Copolymeres sequences polypeptide-poly(ethylene glycol) a plusieurs bras en tant qu'excipients d'apport de medicaments
JP4535229B2 (ja) 2003-05-08 2010-09-01 国立大学法人 東京大学 ポリエチレングリコール−ポリカチオンブロック共重合体
JP2005080598A (ja) * 2003-09-10 2005-03-31 Japan Science & Technology Agency β−１，３−グルカン誘導体を用いるプラスミドＤＮＡの導入法
WO2005078084A1 (fr) * 2004-02-13 2005-08-25 Toudai Tlo, Ltd. Acide oligonucléique double brin portant un complexe de polyion, procédé pour produire celui-ci et composition pharmaceutique contenant celui-ci
JP4870941B2 (ja) * 2005-05-09 2012-02-08 公立大学法人大阪府立大学 高分子化合物
DE102005023993A1 (de) * 2005-05-20 2006-11-23 TransMIT Gesellschaft für Technologietransfer mbH Nicht virales Vektorsystem zum Transport von Nukleinsäure in die Lunge
KR101263057B1 (ko) 2006-01-19 2013-05-09 올엑셀, 인크. 자기 조립형 양쪽성 중합체를 이용한 약물의 가용화 및 표적화된 전달
MX2009005345A (es) * 2007-01-22 2009-06-12 Allexcell Inc Polimeros anfifilicos de autoensamblaje como agentes antivirales.
NZ581210A (en) * 2007-07-19 2012-04-27 Allexcel Inc Self-assembling amphiphilic biocompatible copolymers as anticancer agents
MA51127A (fr) 2017-12-07 2021-03-17 Adocia Solution injectable a ph 7 comprenant au moins une insuline basale dont le pi est compris entre 5,8 et 8,5 et un co-polyaminoacide porteur de charges carboxylates et de radicaux hydrophobes
TWI813604B (zh) 2017-12-07 2023-09-01 法商阿道洽公司 包含至少一具有pi從5.8至8.5之基礎胰島素以及一帶有羧酸鹽電荷及疏水基之共聚胺基酸的可注射ph7溶液

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5219564A (en) * 1990-07-06 1993-06-15 Enzon, Inc. Poly(alkylene oxide) amino acid copolymers and drug carriers and charged copolymers based thereon
DE69312951T2 (de) * 1992-10-14 1997-12-11 Nycomed Imaging As Chelatpolymere
WO1996021036A2 (fr) * 1994-12-30 1996-07-11 Chiron Viagene, Inc. Agents de concentration d'acide nucleique ayant une immunogenicite reduite
GB9600272D0 (en) * 1996-01-06 1996-03-06 Univ Nottingham Polymers
JP3808938B2 (ja) * 1996-07-16 2006-08-16 久光製薬株式会社 核酸運搬体
GB9623051D0 (en) * 1996-11-06 1997-01-08 Schacht Etienne H Delivery of DNA to target cells in biological systems
JP2002515418A (ja) * 1998-05-20 2002-05-28 エクスプレッション・ジェネティックス・インコーポレーテッド 肝細胞を標的とするポリエチレングリコール接合ポリ−ｌ−リシンのポリマー遺伝子キャリヤー

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0100709A1 *

Also Published As

Publication number Publication date
AU6152000A (en) 2001-01-31
CA2377211A1 (fr) 2001-01-04
JP2003503569A (ja) 2003-01-28
MXPA01012802A (es) 2003-06-24
WO2001000709A1 (fr) 2001-01-04
EP1063254A1 (fr) 2000-12-27

Similar Documents

Publication Publication Date Title
EP1198489B1 (fr) Combinaisons pour l'introduction d'acides nucleiques dans des cellules
DE69725878T2 (de) Zusammensetzungen zur polynukleotidabgabe
DE69433519T2 (de) Sich selbst zusammensetzendes polynukleotid-abgabesystem, das dendrimer-polykationen enthält
Remy et al. Gene transfer with lipospermines and polyethylenimines
US5990089A (en) Self-assembling polynucleotide delivery system comprising dendrimer polycations
Behr Synthetic gene transfer vectors II: back to the future.
DE69713381T2 (de) Nukleinsäurekomplex mit kationischen polymer nützlich für gentherapie
DE69635609T2 (de) Nukleinsäure enthaltende zusammensetzung, herstellung und verwendung
Yao et al. The gene transfection efficiency of a folate–PEI600–cyclodextrin nanopolymer
He et al. Combinatorial optimization of sequence-defined oligo (ethanamino) amides for folate receptor-targeted pDNA and siRNA delivery
Wagner Polymers for nucleic acid transfer—an overview
ES2265569T3 (es) Composicion farmaceutica que mejora la transferencia de genes in vivo.
DE69822473T2 (de) Lipid-polyamid-konjugate und zusammensetzungen zur verabreichung von nucleinsäuremolekülen
JP2007521247A (ja) 生体適合性−遺伝子送達剤としての新規カチオン性リポポリマー
Bono et al. Design and synthesis of biologically active cationic amphiphiles built on the calix [4] arene scaffold
Berger et al. Optimizing pDNA lipo-polyplexes: a balancing act between stability and cargo release
DE69804463T2 (de) Kationische polymere, diese kationische polymere enthaltende komplexe und wenigstens eine negative ladung enthaltende therapeutisch wirksame mittel, insbesondere nukleinsäuren und ihre verwendung in gentherapie
EP0905254A2 (fr) Vecteur d'ADN associés avec des polyéthylèneimines à bas poids moléculaire, biocompatibles
EP1208133A1 (fr) Copolymeres pour le transport d'acide nucleique dans les cellules
DE69928679T2 (de) Kationische dendrimer-verbindungen und deren verwendung als oligonucleotid/polynucleotid-träger
EP2741783B1 (fr) Nanocomplexes polyanioniques pour applications thérapeutiques
Tarwadi et al. Preparation and in vitro evaluation of novel lipopeptide transfection agents for efficient gene delivery
DE69709891T2 (de) Chitosan enthaltende zusammensetzung
Sun et al. New amphiphilic N-phosphoryl oligopeptides designed for gene delivery
OSKOUEI et al. Modified polyethylenimine: Self assemble nanoparticle forming polymer for pDNA delivery

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20020125

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

17Q First examination report despatched

Effective date: 20030411

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20050804