MXPA98007990A - Low weight molecular polyethylenimine biologically compatible - Google Patents
Low weight molecular polyethylenimine biologically compatibleInfo
- Publication number
- MXPA98007990A MXPA98007990A MXPA/A/1998/007990A MX9807990A MXPA98007990A MX PA98007990 A MXPA98007990 A MX PA98007990A MX 9807990 A MX9807990 A MX 9807990A MX PA98007990 A MXPA98007990 A MX PA98007990A
- Authority
- MX
- Mexico
- Prior art keywords
- cell
- pei
- lmw
- molecular weight
- nucleic acid
- Prior art date
Links
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Abstract
The invention relates to low molecular weight polyethylenimines, to vectors for the introduction of nucleic acids into cells containing low molecular weight polyethylenimines, as well as to the preparation and use of the low molecular weight polyethylenimine and the vector. The invention relates to a vector for the introduction of a nucleic acid into a cell, which contains a low molecular weight polyethylenimine (LMW-PEI) and a nucleic acid, the LMW-PEI having a molecular weight of less than 50,000.
Description
Polyethylene plus low molecular weight biologically compatible
The invention relates to low molecular weight polyethylene, to vectors for the introduction of nucleic acids, into cells containing polyethylene glycosides of molecular weight as well as to the preparation and use of low molecular weight polyethyleneimine and the vector 10 An essential success in therapy in the therapeutic application of live DNA has not been established so far in clinical studies in humans The causes have to be sought, above all, in the poor efficacy of gene transfer, the limited expression ae genetic information lb dotton et al, Meth Enzymol 217 618-644 (1993)] and in the deficient biocompatibility [Choksakuln mitr et al, J Control Rei 34 233-241 (1995)] of the cationic support materials used Despite of which viral vectors, such as retroviruses "Miller Natare 357 455-460
,1992)] or adenoviruses [Mulligan, Science 260 926-932 (1993)] yielded very promising results m vitro, its application m vivo was limited, in particular by virtue of inflammatory and immunogenic properties as well as the risk of mutagenesis and integration into the genome itself
the cell [Crystal, Science 270 404-410 (1995)] As a possible alternative, non-viral vectors were offered, which are not only easier to manipulate than the vincos systems, but can also safely and efficiently introduce DNA into cells [Tomlmson and Rolland, J Contr Reí 39 357- 30 -372 (1996 Synthetic vectors, based on water-soluble cationic polymers such as poly-L-lysma 'PLL) [Wu and Wu, Biotherapy 3 87-95 (1991)] , DEAE-dextran [Gopal, Mol Cell Biol 5 1183-93 (1985)], Haensler and Szoka dendrimers
Biocon ugate Chem 4 ^ 72-379 (1993) J or cationic derivatives of methacrylic acid [Wolfert et al, Hum Gene Ther 7 2123-2133 (1996)] have evolved over time as an alternative to the classical form of transfection, "lipofection" with cationic lipids [Gao and Huang, Gene Therapy 2: dO-722 (1995)] and amphiphiles [Behr, Bioconjugate Chem. 5: 382-3B9 (1994)]. The decisive advantage of the "polifec-cyan" with cationic polymers lies in the infinite plurality of structural possibilities of variation that can influence in a desired way the physical-chemical and biological properties of the polymers and their plasmid-polymer complexes. By additional coupling of cell-specific ligands, such as transferrin [Wagner et al. , Proc. Nati Acad. Sci. 87: 3410-3414 (1990)], asialogiicoprotein [Wu and Wu, J. Biol. Chem. 262: 4429-4432 (1987)], as well as different antibodies [Trubetskoy et al., Bioconjugate Chem. 3: 323 -327 (1992)] and carbohydrates [Midoux et al., Nucleic Acid Research 21: 871-878 (1993)] the efficacy of these vectors could be considerably increased. Polyethylenimine (PEI), a cationic polymer with a three-dimensional branched structure, has led in a plurality of different adherent and suspension cell lines to transfection rates in part higher than average [Boussif et al., Gene Therapy 3: 1074-1080 (1996)]. For example, 3T3 fibroblasts could be transformed in vitro by 95%. The transfer of genes induced by PEI in vivo in the brain of mice determined a long-term expression of reporter genes and of the Bcl2 gene in neurons and glia cells that are in the same order of magnitude as in the transfer of adenovirus genes [Abdallah et al., Hum. Gene Ther. 7: 1947-1954 (1996)]. Polyethylenimine has extraordinary properties compared to other polyols known from the literature such as PLL [Zenke et al., Proc. Nati Acad. Sci. 87: 3655-3559 (1990)], methacrylate derivatives [Cherng et al., Pharm. Res. 13: 1038-1042 (1996)] or DEAE-dextran [Gopal, Mol. Cell. Biol. 5: 1183-93 (1985)]. By virtue of its reticulated structure > of the high charge density, It is able to condense and complex plasmids to a large extent. DNA can then be introduced into cells in the form of complexes of this type. The mechanisms of absorption if intracellular processing / lysotrope activity of PEI-plasmid complexes are not definitively clarified to date The decisive advantage of the PEI appears to be a pH-dependent modification of its structure that leads to a destabilization of endosomal-lysosomal compartments and thus, facilitates the release of complexes in the c toplasma In particular, amino functions with different pKa values of the molecule must be responsible for a buffer capacity accused of PEI ("proton sponge") that in the acidification of endosomes leads to a protonization of the polymers or swelling and, therefore, to the rupture of vesicle membranes The influx of protons induced by the endoemal ATPase apparently determines at the same time the passive influx of ammonium chlorides that, in the presence of PEI , leads to a massive increase in the total concentration of ions and, thus, to the osmotic binding of endosomes [Behr, Chimia 51 34-36 (1997)] Lysomotropic agents such as chloroquine, which are, for example, essential for transion of PLL, therefore have no influence on the transion rate of PEI's [Remy and Behr, J. Lip Res. 6 535- -544 (1996)] In WO 9602655 Al is described The use of high molecular weight polyethyleneimine with a molecular weight of 50 kDa and 800 kDa (molar mass 50 000 g / mol or 800 000 g / mol i for the transion of DNA in cells) The PEI commercially available, according to the manufacturer's data (for example Fluka, Neu Ulm) a molecular weight of 600-1000 kDa. Preparations of PEI of this type with a high molecular weight ("PEI of high molecular weight" HMW-PEI) already have a concentration of 0.01 mg / ml and after a brief incubation of 3 h, Clear cytotoxicity In addition, the polyethylene-amine structure can not be dissociated either enzymatically or hydrolytically and, therefore, is not biologically degradable. Furthermore, HMW-PEI, presumably, can not be secreted either through the faeces or through of the kidneys. As a consequence, the live administration of the HMW-PEI hitherto used, for example in the context of gene therapy, is fraught with considerable risks. The object of the invention is a polyethyleneimine with a molecular weight below 50,000 Da , preferably between 500 Da and 30,000 Da? "PEI of ba or molecular weight" LMW-PEI), the method (or procedure) for the preparation of this LMW-PEI and the use of LMW-PEI in complex with sequences of nucleotides or viral and non-viral nucleic acids for the introduction of nucleotide sequences into a cell, the administration of this cell to a mammal for the purpose of the prophylaxis or therapy of a disease and the administration of LMW-PEI in complex with a sequence of nucleotides to a mammal for the purpose of prophylaxis or therapy of a disease An object of the present invention is a vector containing a low molecular weight polyethyleneimine (LMW-PEI) and a nucleic acid (sequence nucleotide molecule), the LMW-PEI having a molecular weight below 50 000 Da In particular, the invention relates to vectors for the introduction of nucleic acid constructs in a cell, containing the complex vectors based on polyeti-lenimma with a molecular weight below 50,000 Da with nucleic acids, preferably non-viral or vinca nucleic acid constructs Preferably, the LMW-PEI has a molecular weight of 500 to 30,000 Da In a preferred embodiment of the invention, the LMW-PEI has a molecular weight of 1,000 to 5,000 Da. A molecular weight of approximately 2,000 Da is particularly preferred. The invention is a vector containing a polymer and a low molecular weight and a nucleic acid, the LMW-1 is prepared by polymerization of mono-methyl ethyleneimine in aqueous solution by the addition of hydrochloric acid, the aqueous solution being preferably to 3.1% up to 90% of monomeric ethylenimine and to C, 1% to 10% of concentrated hydrochloric acid (to 37%). The object of the invention is a vector containing a polyethylenimine of ba or molecular weight and a nucleic acid, not showing the LMW-PEI in swelling studies at different pH values in the range of pH 4 to pH 10 in phosphate buffer 0, 1 M no turbidity or precipitation. The object of the invention is a vector that contains a polyethyleneimine of molecular weight and a nucleic acid, reaching with this vector transfection rates greater than 1%, preferably transfection rates of 5% or more and, in particular embodiments, rates of transfection of 10% or more The nucleic acid can be, for example, a DNA or RNA. The nucleic acid can be an oligonucleotide or a nucleic acid construct. The nucleic acid is preferably a viral or non-viral nucleic acid construct. Preferably, the nucleic acid construct is a gene or a plasmid The nucleic acid construct can contain a transgene The nucleic acid construct can contain one or more effector genes An effector gene can encode, for example, a pharmacological active substance or its form of Prodrug and / or Encode an Enzyme The nucleic acid construct is preferably configured such that the gene (e.g., effector or transgene gene) is specifically expressed, e.g. in a virus-specific manner (e.g. , only in cells infected with viruses), in a specific manner of the cell (target), in a metabolically specific manner, in a specific way of the cell cycle, in a specific manner of the development or in a non-specific manner. In the simplest case, the nucleic acid contains a gene encoding the desired protein that contains sequences of specific promoters and eventually other regulatory sequences. To reinforce and / or prolong the expression of the gene, for example, viral promoter and / or enhancer sequences may be contained. Promoter and / or enhancer sequences of this type are represented explanatory way, for example, in Dillon, TiBTech 11, 167 (1993) Examples of these are the LTR sequences of Rous sarcoma virus and retrovirus, the promoter region and CMV virus enhancer, ITR sequences and / or p5, pl9 and p40 promoter sequences of AAV virus, ITR and / or adenovirus promoter sequences, ITR and / or vaccinia virus promoter sequences, sequences ITR and / or Herpes virus promoter, parvovirus promoter sequences and promoter sequences (regulatory region located above) of papillomavirus. The LMW-PEI is complexed with the nucleic acid by mixing the two starting substances. Preferably, a mixing ratio leading to complexes with a neutral or cationic charge should be chosen. Preferably, the vector is composed of complexes with more than 50% (weight percentage) of LMW-PEI. The vector preferably has a weight ratio of LMW-PEI to nucleic acid of 3.1 or greater, particularly preferably of 5 1 or greater or of 8 1 or greater An effector gene can be expressed as a fusion protein with a ligand, for example when the nucleic acid construct contains, together with the sequence of the effector gene, a sequence encoding a ligand. Very generally, the invention relates to a vector containing an LMW-PEI, a nucleic acid and, optionally, a ligand. Preferably, the individual components of the vector are covalently bound and / or by adsorption bonds. For example, the encoded protein and / or the LMW-PEI may be coupled to a ligand. In particular, the invention relates to that the low molecular weight polyethylene imine is coupled to a specific ligand of the cell (or specific to the target cell). Preferably, the ligand is a specific ligand of the cell. specific to the target cell. A specific ligand of the target cell can be bound to the outer membrane of a target cell, preferably of an animal or human target cell. A specific ligand of the target cell has a high specificity for the target cell. A vector containing a specific ligand of the target cell can be used for specific transfer of a nucleic acid to the target cell. The target cell can be, for example, an endothelial cell, a muscle cell, a macrophage, a lymphocyte, a glia cell, a blood-forming cell, a tumor cell, for example a leukemia cell, a virus-infected cell, an epithelial cell of the bronchi or a liver cell, for example a sinusoidal cell of the liver. A ligand that specifically binds to endothelial cells can be chosen, for example, from the group consisting of monoclonal antibodies or their fragments that are specific for endothelial cells, carrier glycosylated carriers in the terminal position, glycolipids or polysaccharides, cytokines, growth factors. , adhesion molecules or, in a particularly preferred embodiment, of envelope glycoproteins of viruses having a tropism for endothelial cells. A ligand that binds specifically to smooth muscle cells can be chosen, for example, from the group encompassing monoclonal antibodies or their fragments that are specific for actin, cell membrane receptors as well as growth factors or, in one embodiment particularly preferred, of glycoproteins from the coat of viruses that have a tropism for smooth muscle cells. A ligand that binds specifically to macrophages and / or lymphocytes can be chosen, for example, from the group encompassing monoclonal antibodies that are specific for membrane antigens on macrophages / or Imfocytes, intact immunoglobulins or Fe fragments of polyclonal or monoclonal antibodies that are specific for membrane antigens on macrophages and / or lymphocytes, cytokines, growth factors, peptides carrying terminal mañosa, lipid or polysaccharide proteins or, in a particularly preferred embodiment, of virus coat glycoproteins, in particular of the HEF protein of influenza C virus with mutation at the position of nucleotide 872 or HEA cleavage products of influenza C virus containing the catalytic triad Sepna 71, istidine 368 or 369 and aspartic acid 261 A ligand that binds specifically to glia cells can be chosen, for example, from the group encompassing antibody and fragments of antibodies that bind specifically to membrane structures of glial cells, adhesion molecules, peptides carrying mannose in terminal position, proteins, lipids or polysaccharides, growth factors or, in a particularly preferred embodiment, Virus coat glycoproteins having a tropism for glia cells A ligand that binds specifically to blood forming cells can be chosen, for example, from the group encompassing antibodies or antibody fragments that are specific for a cell factor receptor. stem, IL-1 'in particular the type of receptor I or II), IL-3, (in particular the type of receptor ao 3), IL-6 or GM-CSF, as well as intact immunoglobulins or Fe fragments presenting this specificity, and growth factors such as SCF, IL-1, IL-3, IL-6 or GM-CSF as well as their fragments, which bind to the respective receptors A ligand that binds specifically The leukemia cells can be chosen, for example, from the group encompassing antibodies, antibody fragments, immunoglobulins or Fe fragments that specifically bind to membrane structures on leukemia cells, such as CD13, CD14, CD15, CD33, CAMAL , sialosyl-Le, CD5, CDle, CD23, M38, IL-2 receptors, T cell receptors, CALLA or CD19, as well as growth factors or fragments or retmoids that are derived from the above. A ligand that binds specifically Virus-infected cells may be chosen, for example from the group comprising antibodies, fragments of antibodies, intact immunoglobulins or Fe fragments which are specific for a virus antigen which, after infection by the virus, is expressed on the cell membrane of the infected cell A ligand that can bind specifically to epithelial cells of the bronchi.
, sinusoidal cells of the liver or cells to the liver can be chosen, for example, from the group encompassing transferapne, asialoglycoproteins such as asialoorosomucoid, neoglyc-protein or galactose, insulin, peptides carrying terminally-engineered mannose proteins, lipids or polysaccharides, intact immunoglobulins or Fe fragments that bind specifically to the target cells and, in a particularly preferred embodiment, of virus coat glycoproteins that specifically bind to the target cells. Further detailed examples of ligands are disclosed, for example, in EP 0 790 312 and EP 0 846 7 * ^ 2 The object of the invention is furthermore a process for the preparation of a low molecular weight cationic polymer conjugate (LMW-PEI) based on polyethylene ma 'PEI) by means of pol imepzacion with opening of the ring of azipdma (ethylemmina monomerai) In this case, the preparation of etilemmina from ethanolamin a is preferably carried out according to the method of Wenker JACS 57 2328 (1935)) The boiling point is preferably at 55.0-56.0 ° C In the German patent application 665 791 (1938) the synthesis is described of PEI by the addition of catalysts such as acids or boron trifluoride to the liquid monomeric ethyleneamine According to the invention, in analogy to Dick et al, J Macromol Sci A4 • 1301-1314 (1970) erilr-nimma monome : to which poly rizn <Aqueous solution with the addition of hydrochloric acid. For the polymerization, an 0.1% to 90% solution of ethylenenunium imonomer) is made with stirring in distilled water and 0.1% to 10% concentrated hydrochloric acid (3%) is added as a catalyst. The polymerization was carried out for 1-30 days, preferably 4 days, at a temperature of 30-70 = C, preferably 50 ° C. The characterization of the polymers is carried out, for example, with 13 C-NMR spectroscopy, chromatography with size exclusion, light scattering and / or viscosimetry. The method for determining the molecular weight with the aid of the light scattering method is described basically in B. Vollmert 11962) "Grundriss der Makromolekularen Chemie", Sppnger Verlag, Berlin, pages 216-225. Preferably, the determination of the molecular weight by the light scattering method, in particular the laser light scattering method, for example using a light scattering photometer, for example the Wyatt Dawn DSP light scattering photometer, is carried out at 633 nm after direct injection on a K5 measuring card. The molecular weight can be determined, for example, with the aid of determined calibration constants in toluene and the known weight of the sample. With the procedure described, low molecular weight PEI (LMW-PEI) can be prepared with molecule sizes between 500 Da and 50,000 Da. With this, the molecular weight of the low molecular weight PEI (LMW-PEI) is clearly below that of the HMW-PEI and clearly below the threshold of the 50 kDa kidneys, so renal clearance should be guaranteed . Surprisingly, it was found that the LMW-PEI is clearly superior to the HMW-PEI in relation to efficacy as a vector for the introduction of nucleic acids or nucleic acid constructs into cells and their biological compatibility. LMW-PEI with molecular sizes between 1000 Da and 30,000 Da was shown to be optimally adequate. The LMW - PEI is able to bind DNA, condense and posit it. LMW-PEI with a molecular weight of, for example, about 2000 Da led, in complex with DNA containing a reporter gene [in the presence of serum], to 100-fold higher reporter gene expressions in mammalian cells [e.g. in fibroblasts of mouse (3T3) and human endothelial cells (ECV 304)] than the commercial high molecular weight (HMW) PEI. At the same time, the cytotoxicity of the LMW-PEI on fibroblasts was clearly reduced compared to the HMW-PEI. Accordingly, the object of the invention is polyethylene-mma with a molecalar weight of less than 50,000 Da, preferably between 500 Da and 30,000 Da (LMW-PEI) the method for the preparation of this LMW-PEI and the use of LMW-PEI in complex with viral and non-viral nucleotide sequences for the introduction of nucleotide sequences into a cell, the administration of this cell to a mammal for the purpose of prophylaxis or therapy of the disease and the administration of LMW-PEI in complex with a nucleotide sequence to a mammal for the purpose of prophylaxis or therapy of a disease. The object of the invention is a polyethyleneimine of low molecular weight with a molecular weight of less than 50,000 Da, preferably LMW-PEI which is prepared by the described process. The object of the invention is also the use of LMW-PEI with a molecular weight of less than 50,000 Da, preferably 1,000-30,000 Da, in particular about 2,000 Da. The LMW-PEI can be used, for example, for the introduction of a nucleic acid into a cell, for the preparation of a vector for the introduction of a nucleic acid into a cell, for the preparation of a medicament and / or in therapy gene The invention further provides a method for the preparation of a vector for the introduction of a nucleic acid into a cell. The vector can be prepared, for example by mixing a corresponding amount of LMW-PEI with a corresponding amount of nucleic acid. Preferably, the mixing of LMW-PEI and nucleic acid is carried out in an aqueous solution. In addition, the use of the vector is for example, the vector can be used for the introduction of a nucleic acid into a target cell or cell (transfection or polifection), for the preparation of a medicament and / or in gene therapy. Preferably, the invention relates to the use of the vector for introduction of constructions of non-viral nucleic acids or vincas in a cell and the administration of this cell (transfected) to a patient for the purpose of prophylaxis or therapy of a disease, the cell being able to be, for example, an endothelial cell, a lymphocyte, a macrophage, a liver cell, a fibroblast, a muscle cell or an epithelial cell, and this cell can be injected, for example, locally on the skin, subcutaneously, intramuscularly, in a wound, in a body cavity, in an organ or in a blood vessel. In another preferred embodiment, the invention relates to the use of the vector for the prophylaxis or therapy of a disease, the vector being injected, for example, locally on the skin, subcutaneously, intramuscularly, in a wound, in a body cavity, in an organ or in a blood vessel The LMW-PEI or a vector containing the LMW-PEI can be used, for example, for the introduction of a nucleic acid into a target cell / cell, the cell being / target cell an endothelial cell, a lymphocyte, a macrophage, a liver cell, a fibroblast, a muscle cell or an epithelial cell Further object of the invention is a method for the preparation of a transfected cell or target cell , incubating LMW-PEI and / or the vector with this cell. Preferentially-transfection is carried out in vitro. It is furthermore object of the invention a transfected cell or target cell containing the LMW-PEI and / or A vector according to the invention The invention also relates to the use of the transfected cell, for example as a medicament or for the preparation of a medicament and / or for gene therapy. It is furthermore object of the invention a medicament containing a LMW-PEI and / or a vector according to the invention and / or a transfected cell? object of the invention also a method for the preparation of a medicament, by mixing a nucleic acid with a LMW- PEI and, optionally other additives Since the LMW-PEI according to the invention is less strongly branched than the HMW-PEI and, therefore, contains more groups than the HMW-PEI, the LMW-PEI offers, in a much better than the HMW-PEI, the possibility of coupling to a cell-specific ligand Accordingly, the object of the invention is the coupling of the LMW-PEI to a specific ligand of the cell and the use of the coupling product in complex with a viral or non-viral nucleotide sequence for the introduction of the nucleotide sequence into a cell or for the administration of the complex to a mammal for the prophylaxis or therapy of a disease The possibilities of the preparation and coupling of specific ligands of the cell has already been described extensively in patent applications P 97101506 0 and DE 19649645 4. These patent applications are expressly referred to as Comple cs between LMW-PEI, optionally coupled with a specific ligand of the cell and an acid construction Viral or non-viral nucleic acids represent a vector for gene therapy. In a preferred embodiment, these vectors are administered to patients externally or mentally, locally, in a body cavity, in an organ, in the bloodstream, in the respiratory tract, in the tract <.jar-tL-ointest -.in.l, in the urogenital tract or intramuscularly or subcutaneously. By means of the vector according to the invention, an effector gene can be introduced non-specifically of the cell and / or specifically of the cell in a target cell, being in the case of the effector gene preferably a gene encoding a pharmacologically active substance or an enzyme, which dissociates an inactive precursor of an active substance into an active substance. The effector gene may be chosen such that the pharmacologically active substance or enzyme is expressed as a fusion protein with a ligand, and this ligand binds to the surface of cells, for example endothelial cells or proliferating tumor cells. The subject of the present invention are also yeast or mammalian cells in which, with the help of the LMW-PEI according to the invention, a nucleic acid construct has been introduced. In a particularly preferred embodiment, the nucleic acid constructs are incorporated, with the help of the LMW-PEI according to the invention, into cell lines which can then be used for transgene expression after transfection. . Accordingly, these cells can be used for the provision of a therapeutic and curative agent for patients. A preferred use of the LMW-PEI according to the invention in complex with a nucleic acid construct consists in the treatment of a disease, the provision of the therapeutic and curative agent encompassing the introduction of the nucleic acid construct into a cell target and its specific expression of the virus or of the target or specific cell in a metabolic or nonspecific form and specific to the cell cycle. The subject of the invention is, furthermore, the administration of mammalian cells in which a nucleic acid construct has been introduced with the help of the LMW-PEI according to the invention, for the preparation of a therapeutic agent and Healing for the treatment of a disease. For example, endothelial cells can be obtained from the blood, treated in vitro with the vector according to the invention f- injected into the patient, for example, intravenously.
Transfected m vitro cells of this type can also be administered to the patient in combination with a vector according to the invention. This combination presupposes that cells and vectors are administered or injected in each case at the same time or at different time intervals, in equal places or in different places.
Examples:
1) Methods
a) Preparation of low molecular weight polyethylenimine (LMW-PEI) LMW-PEI is obtained from aziridine by ring-opening polymerization in aqueous solution under acid catalysis. For this, for example, a solution of 10% ethyleneimine monomer in water (5 ml of ethyleneimine monomer + 45 ml of distilled water, solution with stirring) is stirred for 4 days at 50 ° C under the addition of 1% ( 0.5 ml) of concentrated hydrochloric acid (37%) as a catalyst, is concentrated by rotary evaporation and dried under vacuum at room temperature. The molecular weight determinations were carried out by laser light scattering measurement (Wyatt Dawn DSP light scattering photometer) at 633 nm after direct injection on a K5 measuring card. The molar masses are determined by virtue of the calibration constants determined in toluene and the known weight of the sample. The determination of molecular weight with the aid of light scattering analysis yielded 2000 Da. Comparatively, the commercially obtained PEI (firm Fluka, Neu Ulm) had a molecular weight, corresponding to the analysis by - Ib - light scattering, of 791 kDa (HMW-PEI). The prepared doe (LMW-PEI and HMW-PEI) were examined comparatively.
b) Preparation of the polynucleotide complexes The complexation of the plasmid DNA with the PEI's is carried out based on the method of Boussif et al. [Boussif et al., Proc. Nati Acad. Sci. 92: 7297-7301 (1995)]. 9 mg of the commercial solution of 50% HMW-PEI or 9 mg of LMW-PEI were dissolved in 9 ml of double-distilled water, adjusted to pH 7.4 with 1 N HCl and supplemented with water to a final volume of 10.0 ml. The finished solutions were filtered under sterile conditions (0.2 μm) and stored for a long time at 4 ° C. For the formation of the complex, 10 μg of plasmid and the different amounts of the PEI source solutions were each diluted in 150 mM NaCl to a final volume of 250 μl and mixed in the vortexing device. Table 1 gives a perspective on the employment and equivalent relationships of the complexes. After incubation for 10 minutes at room temperature, the polymer solutions were added dropwise in portions to the plasma solutions and mixed in the vortexing device. Before adding the complexes to the cell culture medium, they were incubated again for 10 min.
c) Analysis of the displacement in agarose The capacity of binding to the plasmid of the different PEId was controlled in the displacement test in agarose gel. For this, 1.35-27 μg of HMW-PEI and 2.7-90 μg of LMW-PEI in each case were complexed with 10 μg of plasmid • Table 1). Aliquots of 50 μl were incorporated into a gel of approximately 0.5 cm thickness based on 1% (w / v) agarose and were developed in Tris-EDTA buffer, pH 7.4, at 80 mV for 2 h . The location of the DNA was visualized at 254 nm after the reaction with ethidium bromide.
For the displacement of the plasmids from the complexes, in each case 10 g of the complex were added.
DNA, 50 or 100 μl of a dextran sulfate solution (PM
. COO, 10 mg / ml, Sigma, Deisenhofen) 30 min after complex formation.
d) Cell cultures L929 mouse fibroblasts were cultured under standard conditions customary to the person skilled in the art. These cells were plated at a density of 8000 cells / well in 96-well cell culture plates and cultured for 24 h before they were used for the toxicity experiments. The culture of 3T3 fibroblasts was carried out in the same way under standard conditions. ECV 304 (ATCC, Rockville, MD, USA) a spontaneously transformed and adherent human endothelial cell line, which was established from an apparently normal umbilical cord, was cultured in Dulbecco's modified agie (DMEM) medium (Gibco , Eggenstein) with 5% fetal calf serum (STF), 5% horse serum and 1% N-acetyl-L-alanyl-L-glutamine (all from Gibco, Eggenstein). Cells incubated at 37 ° C, at a relative air humidity of 95% and 5% CO 2, were passed twice a week after reaching confluence with trypsin / EGTA solution (2-trypsin source solution, 5%, 50 mM ethylene glycol tetraacetic acid solution, PBS pH 7.4, in the ratio 1: 1: 8). Since the cells did not separate individually from the bottom, but formed clusters of cells, 1/8 step was carried out in each case. Brain capillary endothelial cells were isolated and cultured according to the method of Bowman et al. ((1983) Ann Neurol 14: 396-402) and Mischek et al. [Mischek et al., Cell. Tiss. Res. 256: 221-226 (1989)]. For transfection assays they were seeded immediately after isolation in 6-well cell culture plates and cultured to a confluence of about 50%.
e) Cytotoxicity study The toxicity of the polymers was determined with the MTT test according to the method of Mosmann et al. [Mosmann, J. Immunol. Methods 65: 55-63 ¡1983)] in mouse fibroblasts L929. The polymer dilution series were prepared in DMEM with 10% STF and 2 mM glutamine and filtered under sterile conditions (0.2 μm, Schleicher &Schuell, Dassel). If necessary, the pH value and the osmolarity of the solutions were corrected. After a previous incubation of 24 h, the cells were mixed with the polymer solutions and incubated for 1, 3, 12 and 24 h. The viability of the cells was quantified by UV photometry by measuring the concentration of formazan. In a second series of experiments, the cells were generally treated for only 1 h with the polymers, washed and continued growing in cell culture medium free of PEI for up to 3, 12 and 24 h. The evaluation was carried out as described above.
f) Transfections The 3T3 mouse fibroblasts and? CV 304 cells seeded in 3 cm2 Petri dishes as well as the primary endothelial cells seeded in 6-well culture plates were washed immediately before the experiments with PBS pH 7.4 and they were re-supplied with medium supplemented with serum. The complexes of HMW-PEI and LMW-PE were added correspondingly to 3.33 μg of DNA per well or plate and incubated for 1 h at 37 ° C. The cells were continued to incubate for 60 hours and the luciferase or dgalactosidase activity was determined analogously to Chapter 9 and to the manufacturer's data. [Complex of the HMW- -PEI or LMW-PEI are the corresponding vectors, * contain HMW-PEI or LMW-PEI and nucleic acids, in this case the plasmid DNA)].
Example 2: Results
a) Physical-chemical properties of PEIs The behavior of polymers in relation to their reaction in the endosomal-lysosomal compartment was determined by swelling studies at different pV values. in the range of 4-10 in 0.1 M phosphate buffer. While the HMW-P? I dissolved clearly and without leaving a residue at pH 9 and 10, intense turbulence was observed from pH 8. This clouding was largely stable at pH 7 and 8. Sedimentation phenomena manifested only after several hours. On the contrary, at pH values in the acid interval, the formation of a sediment that could be easily resuspended was produced in the space of 30 in. The LMW - PEÍ did not show, under the same conditions, any turbidity or precipitation, but clearly passed to solution.
b) Cytotoxicity studies The toxicity of the PEI's was determined in vitro in L929 mouse fibroblasts that are recommended by different standard organizations as a standard cell culture model for the determination of the cytotoxicity and biocompatibility of polymers. In previous experiments, a direct and linear proportionality was found between the measured absorption of formazan formed and the number of cells in the range of 1 x 103 and 3 x 10 4 cells. 8,000 cells / well were mixed, after a growth phase of 24 h, with the polymer solutions and incubated for 1, 3, 12 and 24 h. The observed toxic effects of HMW-PEI and LMW-PEI were dependent on time and concentration in the range of 0-1.0 mg / ml over a period of time up to 24 h, showing the profiles of clear cytotoxicity differences for PEI of high and Da or molecular weight. Thus, the CIS0 in the case of HMW-PEI was between 0.06 mg / ml (incubation for 1 h) and 0.04 mg / ml (incu-nation for 24 h), whereas in the case of concentrations of IJMW -PEI between 0.1 and 1.0 mg / ml only after 12 hours of incubation became toxic, an IC5r could be determined only after a duration of the 24 h incubation and was at approximately 0.1 mg / ml .
c) Agarose gel displacement test To determine the optimal binding and quantitative ratio between the plasmid and PEI, a constant amount of plasmid (10 μg) with different concentrations of HMW-PEI and LMW-PEI was complexed according to the prescription. It was analyzed electrophoretically. Table 1 provides a perspective on the employment relationship of the investigated complexes, the volume of source solution used and the absolute amount of PEI.
a) Relationship of (mplec olume of Absolute quantity piásmidcv HMW PEÍ the solution of HMW-PEÍ [equivalents-opgen μl] "μgJ 1 + 1 3 1,35 1 + 6,67 20 3 1 + 10 30 13, 5 1 + 13.3 40 18 1-20 60 27
Re. JCióri: e rt, p Ico • of the number of rivers- 'p] r.3 -nidt /: MW - P? I I solution of MW-PEI [e i ivaj. -e e-a 1 igen μl] tμg] 1 + 3; 2, 7; -:; , < < .0 18 1 + 3"3 D 27 1 + 2, 7 40 36 1+? -, 33 *> 0 72 _ - (<, 6 100 yC
Table 1: Perspectiva ^ cc e the relations of employment ie compleios ií: l: z d "S pal .a eiecti otoresis y transfcción con a) HMW-
The localization of the plasmids and their complexes was visualized by staining with ethidium bromide. The DNA formed two fluorescent bands that correspond to the supercoiled form and the circular shape of the plasmid and that moved in the direction of the anode. HMW-PEI and LMW-PEI were not detectable with ethidium bromide. The complexing of DNA with HMW-PEI in the ratio l + l led to a partial, still incomplete, delay of the plasmid at the site of application. The low total charge and / or a larger diameter prevented the complex formed from migrating to the gel matrix. Complexes in the ratio 1 + 6 to 1 + 20 were not detectable, since they did not show any fluorescence, an indication about the exclusion of ethidium bromide from the plasmids caused by an effective condensation and the physical compression of its structure by the HMW-PEI. The anion / cation ratios are in this case in 1: 1.2 (1 + 6) to 1: 4 (1 + 20). Therefore, the complexes should have a positive total charge. 2.7 μg of LMW-PEI could bind and almost completely retard 10 μg of plasmid. However, the complex still had a negative total charge and was oriented towards the anode. Complete cytization and condensation to the DNA could be observed without However, only from 54 μg of LMW-PEI In order to verify the effect of condensation on the part of PEI of high and low molecular weight, the DNA of the finished complexes was displaced with an excess of dextran sulphate, which passes to being part of a competition reaction with cationic polymers Both in the case of HMW-PEÍ and also of LMW-PEI, the DNA could be released again from the complexes and migrated totally or partially into the gel matrix The intercalation of ethidium bromide was again possible and, therefore, the DNA was detectable by fluorescence
d) Efficiency of m vitro transfection The transfection efficiency of the PEI complexes was determined both with cell lines (3T3 mouse fibroblasts and ECV304 human endothelial cell line) as well as with primary cultures (pig brain capillary endothelial cells) As a reporter gene, the pGL3 control vector acquired commercially from Promega was used, which carries a luciferase gene under the control of an SV 40 promoter and enhancer. The employment ratios of the complexes with respect to the plasmid and the polymer corresponded to those that were used in the electrophoresis. Concentrations of 1.35 μg to 27 μg of HMW- -PEI / 10 μg of DNA were examined. The transfection maximum was shown at 18 ug of HMW-PEI. An additional increase in polymer concentration led only to a relatively small reduction of luciferase expression. In the case of LMW-PEI concentrations of 20-80 μg of LMW-PEI / 10 μg of DNA In contrast to HMW-PEI, it was possible to detect, with an increasing concentration of LMW-PEI, a constant increase in the efficiency of transfection in ECV cells. In the case of 80 μg of LMW-PEI / 10 μg of DNA an activity of the reporter gene was measured approximately 100 times higher than in the case of using the maximum effective dose of 18 μg of HMW-PEI / 10 ^ g of DNA. No regression of luciferase expression was observed as in the case of the high molecular weight PEI and also at the highest LMW-PEI concentrations. Tests with ECV cells and 3T3 cells gave identical results.
For comparative purposes, transfection studies with dgalactosidase as a reporter gene were also carried out in primary endothelial cell cultures with the maximum non-cytotoxic tolerable dosages (MTD) of HMW-PEI and LMW-PEI complexes. The in vitro BAT was 13.5 wg of HMW-PEI / 10 μg of DNA and 90 μg of LMW-PEIdO μg of DNA. Capillar endothelial cells cultured from brains of pigs, which were incubated with complexes based on 10 μg of DNA and 3.5 μg of HMW-PEI, could barely be transfected. Only 2-3 cells per culture cavity (well) showed the characteristic blue staining in the area of the cell nuclei. The success rate of the transfection was below 1% of the treated cells. Incubation with complexes based on 90 μg of LMW-PEI and 10 μg of DNA led, on the contrary, to a clear expression of the marker protein in the endothelial cells. The percentage ratio of cells stained blue per culture cavity in relation to the total number of cells, ie the success rate of the transfection, was between 5% and 10%. In no case could toxic effects of the polymer / DNA complexes in the cells be observed by optical microscopy.
Claims (1)
- CLAIMS 1 - . 1 - Vector for the introduction of a nucleic acid into a cell containing a low molecular weight polyethyleneimine dMW-PEI) and a nucleic acid, the LMW -? I having a molecular weight of less than 50,000 Da 2 - Vector according to Review 1, in which the LMW-PEI has a molecular weight of 500 to 30,000 Da 3 - Vector according to one or more of the revolutions 1/2, in which the LMW-PEI has a molecular weight of 1000 to 500C Da 4 Vector according to one or more of claims 1 and 3, in which the LMW-PEI has a molecular weight of about 2000 Da 5 -Vene according to one or more of claims 1 to 4, wherein the nucleic acid is a viral or non-viral nucleic acid construct. 6 - Vector according to one or more of claims 1 to 5, wherein the nucleic acid construct contains one 0 several effector genes. 7 - Vector according to one or more of claims 1 to 6, wherein at least one effector gene encodes a pharmacological active substance or its prodrug form 8 - Vector according to one or more of claims 1 to "*", wherein that at least one effector gene encodes an enzyme 9 -. 9 - Vector according to one or more of claims I to 8, wherein at least one effector gene is expressed as a fusion protein with a specific ligand of the 10-Vector cell according to one or more of the claims 1 to 9, in which the LMW-PEI is coupled to a specific ligand of the 11-Vector cell according to one or more of claims 1 to 10, wherein the specific ligand of the cell is bound to the outer membrane of the cell. a target cell 12 - Vector according to one or more of claims 1 to 11, in which the target cell is an endothelial cell, a muscle cell, a macrophage, an imfocyte, or a glia cell, a blood forming cell , a tumorai cell, a virus infected cell, an epithelial cell of the bronchi or a liver cell. 13. - Vector according to one or more of claims 1 to 12, wherein the weight ratio of LMW-PEI to nucleic acid is 3: 1 or greater. 14. Vector according to one or more of claims 1 to 13, wherein the weight ratio of LMW-PEI to nucleic acid is 8: 1 or greater. 15. Process for the preparation of a low molecular weight polyethylenimine (LMW-PEI) with a molecular weight of less than 50,000 Da, in which the monomeric ethyleneimide is polymerized in aqueous solution by the addition of hydrochloric acid. 16. Process according to claim 15, in which the aqueous solution is 0.1% to 90% of monomeric ethyleneimine and 0.1% to 10% of concentrated hydrochloric acid. 17. Process according to one or more of claims 15 and 16, wherein the polymerization is carried out at a reaction temperature of 30 ° C to 70 ° C. 18. - Method according to one or more of claims 15 to 17, wherein the reaction time is 1 to 30 days 19. Low molecular weight polyethyleneimine with a molecular weight less than 50,000 Da, prepared by a process according to one or more of claims 15 to 18. 20. Use of a low molecular weight polyethyleneimine with a molecular weight of less than 50,000 Da for the preparation of a vector according to one or more of claims 1 to 14. 21. Process for the preparation of a vector according to one or more of claims 1 to 14, wherein a corresponding amount of LMW-PEI is mixed with a corresponding amount of nucleic acid in an aqueous solution 22.- Use of a vector according to one or several of claims 1 to 14 for the introduction of a nucleic acid into a cell. 23 - Use of a vector according to claim 22, wherein the cell is an endothelial cell, a lmfocito, m macrophage, a cella of the liver, a fibroblast, a muscle cell or an epithelial cell 24 - Procedure for the preparation of a transfected cell, wherein a vector according to one or more of claims 1 to 14 is incubated in vitro with this cell 25 -. 25 - Transfected cell containing a vector according to one or more of claims 1 to 14. 26.- Use of a transfected cell according to claim 25 for the preparation of a medicament. 27. Use of a low molecular weight polyethyleneimine according to claim 19, for the preparation of a medicament 28. Use of a vector according to one or more of claims 1 to 14 for the preparation of a medicament. 29 - Use of a vector according to one or more of claims 1 to 14 for the preparation of a medicament for gene therapy. 30.- Procedure for the preparation of a drug, in which a nucleic acid is mixed with an LMW-PEI. 31. - Medicament containing a vector according to one of the bases of claims 1 to 14. 32 - Medicament containing an LMW-PEI according to claim 19. 33. - Drug containing a transfected cell according to claim 25.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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DE19743135.6 | 1997-09-30 |
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MXPA98007990A true MXPA98007990A (en) | 2000-07-01 |
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