HK1020753A - Biologically tolerated low molecular weight polyethylenimines - Google Patents

Description

Biologically tolerated low molecular weight polyethylenimines

The invention relates to low molecular weight polyethylenimine (polyethylenimine), a vector containing the low molecular weight polyethylenimine for inserting nucleic acids into cells, and a preparation method and application of the low molecular weight polyethylenimine and the vector.

In the context of clinical studies in humans, in vivo administration of DNA has not to date achieved any significant successful therapeutic effect. This is due, inter alia, to the low efficiency of gene transfer and the limitation of the expression of the genetic information [ Cotton et al, enzymology (meth. enzymol.)217:618-644(1993) ] and the lack of biocompatibility of the cationic carrier substances used [ Choksaksauronimitter et al, J.Control. Rel.) -34: 233-241(1995) ]. Although viral vectors such as retroviruses [ Miller, Nature 357:455-460(1992) ] or adenoviruses [ Mullgan, Science 260:926-932(1993) ] have shown promising results in vitro assays, their use in vivo has been limited due to their inflammatory and immunogenic properties, as well as their mutagenesis and integration into the cellular genome [ Crystal, Science 270:404-410(1995) ]. Non-viral vectors, which are not only easier to manipulate than viral systems, but also allow DNA to be pooled into cells in a reliable and efficient manner, offer another possible option [ Tomlinson and Rolland, J.Contr.Rel.). 39:359-372(1996) ].

Over time, synthetic vectors based on water-soluble cationic polymers such as polymerized L-lysine (PLL) [ Wu and Wu, biotherapeutic (Biotherapy)3:87-95(1991) ], diethylaminoethyl-dextran (DEAE-dextran) [ Gopal, biological cell biology (mol. cell. biol.) (5:1183-93(1985) ], dendrimers [ Haensler and Szoka, Bioconjugate chemistry (Bioconjugate Chem.). 4:372-379(1993) ] or cationic methacrylate derivatives [ Wolfert et al, human gene therapy (hum. Gene. Ther.) ] 7:2123-2133(1996) ] have been developed instead of the traditional lipofection approach, i.e.the possible structural changes of the cationic polymers used in transfection methods of Gao and amphiphilic Huang, Gene therapy 2:710-722(1995) ] and of the Bioconjugate substances [ Behr, biochemical-389. 382) ],1994, the physicochemical and biological properties of the polymers and their plasmid/polymer complexes are influenced in an ideal manner. It has been possible to significantly increase the efficacy of these vectors by additional coupled cell-specific ligands such as transferrin [ Wagner et al, Proc. Natl. Acad. Sci.87:3410-3414(1990) ], asialoglycoprotein [ Wu and Wu, J. biochem. 262:4429-4432(1987) ], various antibodies [ Trubetskoy et al, bioconjugate chemistry 3:323-327(1992) ] and carbohydrates [ Midoux et al, Nucl. acids Res. 21:871-878(1993) ].

In many different adhesion and suspension cell lines, the cationic polymer Polyethylenimine (PEI) with a three-dimensional branching structure has in some cases led to transfection rates that are much higher than the average [ Boussif et al, Gene therapy 3: 1074-. For example, 3T3 fibroblasts can be transformed by up to 95% in vivo. PEI-mediated gene transfer in vivo in the mouse brain resulted in long-term expression of reporter and Bcl2 genes in neurons and glial cells, the same size as in adenovirus gene transfer [ Abdallah et al, human Gene therapy 7:1947-1954(1996) ].

PEI has outstanding properties compared with other literature-described polymeric cations, such as PLL [ Zenke et al, Proc. Natl. Acad. Sci.87:3655- & 3659(1990) ], methacrylic acid derivatives [ Cherng et al, Pharmacology 13:1038- & 1042(1996) ] or diethylaminoethyl-dextran (DEAE-dextran) [ Gopal, molecular cytobiology 5:1183-93(1985) ]. Due to its cross-linked structure and high charge density, it is capable of highly agglutinating and complexing plasmids. The DNA can then be pooled into cells in the form of these complexes. The mechanisms of uptake, intracellular processes and denaturation of the PEI/plasmid complex have not been finally elucidated to date.

The main advantage of PEI is the pH-dependent change in its structure, which leads to instability of the endosomal/lysosomal compartment and thus facilitates the release of the complex into the cytoplasm. In particular, the different pKa values of the amino functions of the molecule, give PEI a significant buffering capacity ("proton sponge") and, when the endosomes are acidified, lead to protonation of the polymer and the resulting swelling, which breaks the vesicle membrane. Proton influx mediated by endosomal ATPase may at this point result in passive injection of anion chloride, which, when PEI is present, may lead to a significant increase in total ion concentration and osmotic swelling of the endosomes [ Behr, Chimia 51:34-36(1997) ]. For this reason, lysosomal agents, such as chloroquine, which are necessary for transfection of Polylysine (PLL) do not affect the rate of PEI transfection [ Remy and Behr, J. Lipid Res. 6:535-544(1996) ].

WO9602655A1 describes the use of high molecular weight PEI having molecular weights of 5 and 80 kilodaltons (Da) (molar masses of 50,000 and 800,000g/mol, respectively) for intracellular transfection of DNA.

Commercial PEI has a molecular weight of 60-100 kilodaltons, according to the information provided by the manufacturer (e.g.Fluka, Neu Ulm). Such high molecular weight PEI preparations (HMW PEI) started at a concentration of 0.01mg/ml and showed significant cytotoxicity after a short incubation period of 3 hours. Further, the PEI structure is not broken down by enzymatic and hydrolytic degradation and thus is not biodegradable. Furthermore, HMW-PEI may not be excreted via feces and kidney.

Thus HMW PEI, which is still used today for in vivo administration, e.g. for gene therapy, is at considerable risk.

The present invention relates to PEI having a molecular weight of less than 50,000 daltons, preferably PEI (low molecular weight PEI: LMW PEI) in the range of 500Da to 30,000 Da; also relates to a method for preparing the LMW PEI; and the use of LMW PEI to form complexes with viral and non-viral nucleotide sequences or nucleic acids for inserting the nucleotide sequences into cells; to a method of administering such a cell to a mammal for the purpose of preventing or treating a disease; also relates to a method for administering LMW PEI to a mammal in a complex with a nucleotide sequence for the purpose of preventing or treating a disease.

The present invention relates to a vector comprising a Low Molecular Weight Polyethylenimine (LMWPEI) and a nucleic acid (nucleotide sequence), the LMW PEI having a molecular weight of less than 50,000 daltons. The invention relates in particular to a vector for inserting a nucleic acid construct into a cell, which vector comprises a complex of PEI having a molecular weight of less than 50,000Da and a nucleic acid, preferably a non-viral or viral nucleic acid construct.

LMW PEI preferably has a molecular weight of 500 to 30,000 daltons. In a preferred embodiment of the invention, the molecular weight of the LWM PEI is between 1000 and 5000 daltons. Especially preferred are those having a molecular weight of about 2000 daltons.

The present invention relates to a support comprising low molecular weight PEI and nucleic acid, LMW PEI polymer prepared by polymerization of monomeric aziridine in an aqueous solution, preferably at a concentration of 0.1% to 90%, with the addition of concentrated hydrochloric acid (37%) preferably at a concentration of 0.1% to 10%, by adding hydrochloric acid.

The present invention relates to a vector comprising low molecular weight PEI and a nucleic acid, the LMW PEI not exhibiting any turbidity or precipitation when subjected to swelling studies in phosphate buffers of different pH in the range of 0.1M pH4 to pH 10.

The present invention relates to a vector comprising low molecular weight PEI and a nucleic acid, wherein the vector is used at a transfection rate of greater than 1%, preferably at a transfection rate of 5% or greater, and in particular embodiments at a transfection rate of 10% or greater.

The nucleic acid may be DNA or RNA. The nucleic acid may be an oligonucleotide or a nucleic acid structure. The nucleic acid is preferably a viral or non-viral nucleic acid construct. The nucleic acid construct is preferably a gene or a plasmid. The nucleic acid construct may contain a transgene. The nucleic acid construct may contain one or more effector genes. The effector gene may encode a pharmaceutically active compound or a prodrug form thereof, and/or encode an enzyme. The nucleic acid construct is preferably configured to allow specific expression of a gene (e.g., an effector gene or transgene), e.g., virus-specific (i.e., e.g., only in virus-infected cells), (target) cell-specific, specific with respect to metabolism, cell cycle-specific, specific with respect to evolution; or in a form that allows non-specific expression of the gene. In the simplest case, the nucleic acid comprises a gene which codes for the desired protein, and the gene comprises a specific promoter sequence and, where appropriate, regulatory sequences. For example, viral promoter sequences and/or enhancer sequences may be present for enhancing and/or amplifying gene expression. For example, Dillon, TiBTech11,167(1993) reviews promoter sequences and/or enhancer sequences having this property. Examples of such sequences are the Long Terminal Repeat (LTR) of Rous sarcoma virus and the Long Terminal Repeat (LTR) of retrovirus, the promoter and enhancer region of Cytomegalovirus (CMV), the terminal inverted repeat (ITR) sequence and/or the promoter sequence p5, p19, p40 of adeno-associated virus (AAV), the ITR and/or promoter sequence of adenovirus, the ITR and/or promoter sequence of vaccinia virus, the ITR and/or promoter sequence of herpes virus, the promoter sequence of parvovirus and the promoter sequence of papilloma virus (upstream regulatory region).

LMW PEI forms a complex with nucleic acid as two starting materials by mixing. Preferably a mixing ratio which results in the formation of a complex with a neutral or positive charge. Preferably the support consists of a complex containing more than 50% by weight of LMW PEI. Preference is given to vectors having a weight ratio of LMW PEI to nucleic acid of 3:1 or more, particularly preferably a weight ratio of 5:1 or more, or 8: 1 or more.

The effector gene may be expressed with a ligand as a fusion protein marker, for example, when the nucleic acid construct contains a sequence encoding the ligand in addition to the effector gene sequence.

The present invention relates generally to a vector comprising a LMW PEI, a nucleic acid and, where appropriate, a ligand. The individual components of the support are preferably linked by covalent and/or adsorptive bonds. Such as the encoded protein and/or LMW PEI may be coupled to a ligand. The invention particularly relates to a vector wherein LMW PEI is coupled to a cell-specific (or target cell-specific) ligand.

Preferably a cell-specific or target cell-specific ligand. The target cell-specific ligand may bind to the outer membrane of a target cell, preferably an animal or human target cell. The target cell-specific ligand is highly specific for the target cell. Vectors containing target cell-specific ligands can be used for target cell-specific transfer of nucleic acids. For example, the target cell may be an endothelial cell, a muscle cell, a macrophage, a lymphocyte, a (neural) glial cell, a hematopoietic cell, a tumor cell, such as a leukemia cell, a virally infected cell, a bronchial epithelial cell, or a liver cell, such as a sinusoidal cell of the liver.

A ligand which binds specifically to endothelial cells may be selected, for example, from monoclonal antibodies or fragments thereof specific for endothelial cells, glycoproteins, glycolipids or polysaccharides which bear mannose at the end, cytokines, growth factors, adsorption molecules or, particularly preferably, glycoproteins from viral envelopes which have an endothelial tropism. A ligand which binds specifically to smooth muscle cells may be selected from the group consisting of monoclonal antibodies or fragments thereof specific for actin, cell membrane receptors and growth factors, or, particularly preferably, glycoproteins from viral envelopes having a tropism for smooth muscle cells. A ligand which binds specifically to macrophages and/or lymphocytes may be selected from monoclonal antibodies specific for membrane antigens on macrophages and/or lymphocytes, intact immunoglobulins, or Fc fragments of monoclonal or polyclonal antibodies specific for membrane antigens on macrophages and/or lymphocytes, cytokines, growth factors, peptides carrying mannose at the terminal, proteins, lipids or polysaccharides, or, particularly preferably, glycoproteins from the envelope of the virus, in particular the influenza C virus HEF protein with a mutation at nucleotide 872 or the influenza C virus HEF cleavage product containing the catalytic triads serine 71, histidine 368 or 369 and aspartic acid 261. A ligand which binds specifically to glial cells may be selected from antibodies or antibody fragments which bind specifically to structural bonds in the glial cell membrane, adsorbed molecules, peptides carrying mannose at their termini, proteins, lipids or polysaccharides, growth factors, or, particularly preferably, glycoproteins from viral envelopes with glial cell tropism. A ligand which binds specifically to hematopoietic cells may be selected from antibodies or antibody fragments specific for stem cell factor receptors, IL-1 (especially class I or II receptors), IL-3 (especially alpha or beta receptors), IL-6 or granulocyte macrophage colony stimulating factor (GM-CSF), intact immunoglobulins or crystallizable fragments (FC fragments) which exhibit this specificity, and growth factors such as stem cell growth factor (SCF), IL-1, IL-3, IL-6 or granulocyte macrophage colony stimulating factor and fragments thereof which bind to the relevant receptors. A ligand which binds specifically to leukemia cells may be selected from antibodies, antibody fragments, immunoglobulins or Fc fragments which bind specifically to cell membrane structures of leukemia cells, such as cluster of differentiation 13(CD13), CD14, CD15, CD33, Cell Adhesion Molecules (CAMAL), sialosyl-Le, CD5, CD1e, CD23, M38, IL-2 receptor, T cell receptor, CALLA or CD19, and may also be a growth factor or a fragment derived therefrom, or a retinoid. A ligand that specifically binds to a viral infectious cell may be selected from an antibody, an antibody fragment, an intact immunoglobulin or an Fc fragment specific for a viral antigen, which is expressed on the infected cell membrane following viral infection. A ligand which binds specifically to bronchial epithelial cells may be selected from transferrin, asialoglycoproteins such as asialoglycoproteins, neoglycoproteins or galactose, insulin, peptides bearing mannose at the terminal, proteins, lipids or polysaccharides, intact immunoglobulins or Fc fragments which bind specifically to target cells, or particularly preferably glycoproteins from viral envelopes which bind specifically to target cells. Other detailed examples of ligands are disclosed in european patents 0790312 and 0846772.

The invention further relates to a process for the preparation of low molecular weight, cationic conjugated polymers based on polyethylenimines (LMW PEI) by ring-opening polymerization of aziridines (monomeric aziridines).

In this application, aziridine is preferably prepared from ethanolamine by the method of Wenker (JACS 57:2328 (1935)). The boiling point is preferably in the range 55.0 ℃ to 56.0 ℃.

German patent application 665,791(1938) discloses a process for the synthesis of PEI by adding a catalyst such as an acid or boron trifluoride to a monomeric aziridine liquid. According to the invention, the polymerization is carried out by adding hydrochloric acid to an aqueous solution of monomeric aziridine, in analogy with the method disclosed by Dick et al in J.Macro science A4:1301-1314 (1970).

For the polymerization, a 0.1% to 90% aziridine (monomer) solution was prepared in distilled water with stirring and 0.1 to 10% concentrated hydrochloric acid (37%) was added as catalyst. The polymerization is carried out for 1 to 30 days, preferably 4 days, at a temperature of from 30 ℃ to 70 ℃ and preferably 50 ℃.

The polymer is characterized by 13C-NMR spectroscopy, particle size separation chromatography, light scattering and/or viscometry. Vollmert (1962) "Grundris derMakromolekularen Chemie", Springer Verlag, Berlin, Pages216-225 illustrate the determination of the molecular weight by light scattering. The molecular weight is preferably determined by light scattering, particularly preferably by laser light scattering using a spectrophotometer, e.g.by direct injection of the sample into a K5 cell and determination at 633nm using a Wyatt Dawn DSP spectrophotometer. The molecular weight can be determined using standard constants determined in toluene and known initial sample weights.

The method can be used to prepare LMW PEI having a molecular weight of from 500 daltons to 50,000 daltons. The molecular weight of LMW PEI is therefore significantly lower than HMW PEI and significantly lower than the 5 ten thousand Da renal threshold, which means that renal excretion of LMW PEI can be ensured.

Unexpectedly, LMW PEI is significantly superior to HMW PEI in terms of its efficacy as a vector for inserting nucleic acids or nucleic acid structures into cells and its biocompatibility. LMW PEI having a molecular size of 1000Da to 30,000Da is most suitable. LMW PEI can bind DNA, coagulate DNA, and enhance the positive charge properties of DNA. LMW PEI (in serum) with a molecular weight of approximately 2000 daltons, when complexed with DNA containing the reporter gene, resulted in different levels of expression of the reporter gene in mammalian cells (e.g., in mouse fibroblasts (3T3) and human endothelial cells (ECV 304)), which was 100-fold more effective than commercially available High Molecular Weight (HMW) PEI. Meanwhile, LMW PEI is significantly less cytotoxic to fibroblasts than HMW PEI.

The invention therefore relates to polyethylenimines having a molecular weight of less than 50,000 dalton, preferably those having a molecular weight of 500-30,000 dalton (LMW PEI), to a process for the preparation of such LMW PEI and to the use of complexes of LMW PEI with viral and non-viral nucleotide sequences for inserting nucleotide sequences into cells, to a method of administration of such cells for the purpose of preventing or treating diseases in mammals, and to a method of administration of LMW PEI complexed with nucleotide sequences for the purpose of preventing or treating diseases in mammals.

The present invention relates to LMW PEI having a molecular weight of less than 50,000 daltons, preferably LMW PEI prepared by the methods disclosed herein.

The invention also relates to the use of LMW PEI having a molecular weight of less than 50,000 Dalton, preferably 1000-30,000 Dalton, particularly preferably 2000 Dalton. The LMW PEI can be used for inserting a nucleic acid into a cell, for preparing a vector for inserting a nucleic acid into a cell, or for preparing a medicament and/or for gene therapy.

The invention further relates to a method for preparing a vector for inserting a nucleic acid into a cell. This vector can be prepared, for example, by mixing an appropriate amount of LMW PEI with an appropriate amount of nucleic acid. The LMW PEI is preferably mixed with the nucleic acid in an aqueous solution.

The invention further relates to the use of such a vector. Such vectors can be used, for example, for inserting nucleic acids into cells or target cells (transfection or polymerization transfection), or for preparing medicaments and/or for gene therapy. The invention preferably relates to the use of the vector for inserting a non-viral or viral nucleic acid construct into a cell, and to the use of the transfected cell, which may be an endothelial, lymphocyte, macrophage, hepatocyte, fibroblast, muscle or epithelial cell, for the purpose of preventing or treating a disease in a patient, for local injection into the skin, subcutaneous, intramuscular, wound, body cavity, organ or blood vessel. In another preferred embodiment, the invention relates to the use of the carrier in the prevention or treatment of a disease, for example, the carrier may be injected topically into the skin, subcutaneously, intramuscularly, in a wound, in a body cavity, in an organ or a blood vessel.

LMW PEI or a vector containing LMW PEI may be used to insert a nucleic acid into a cell/target cell, which may be an endothelial cell, a lymphocyte, a macrophage, a hepatocyte, a fibroblast, a muscle cell, or an epithelial cell.

The invention further relates to a method for preparing transfected or target cells by incubating these cells with LMW PEI and/or a vector. Transfection is preferably carried out in vitro. The invention also relates to transfected or target cells containing LMW PEI and/or a vector of the invention. The invention further relates to the use of the transfected cells as a medicament or for the preparation of a medicament and/or for gene therapy.

The invention further relates to a medicament comprising a LMW PEI and/or a vector of the invention and/or a transfected cell. The invention also relates to a method for preparing a medicament by mixing a nucleic acid with LMW PEI, if appropriate with further additives.

Since the LMW PEID of the present invention is significantly less structurally branched than HMW PEI and therefore contains more amino groups than HMW PEI, there is a greater chance of coupling LMWPEI to cell-specific ligands than HMW PEI. The invention thus relates to the coupling of LMW PEI with a ligand which is cell-specific and to the use of this coupling product to form complexes with viral or non-viral sequences, to insert nucleotide sequences into cells or to the use of such complexes for the pharmaceutical treatment of mammals for the purpose of preventing or treating diseases. The possibility of preparing and coupling cell-specific ligands has been disclosed in detail in patent applications EP97101506.0 and DE 19649645.4. These patent applications are incorporated herein by reference.

Complexes of LMW PEI, coupled with cell-specific ligands as appropriate, with viral or non-viral nucleic acid constructs constitute a vector for gene therapy. In a preferred embodiment, the carriers are administered to the patient topically, internally, to a body cavity, tissue, blood circulation, respiratory, gastrointestinal or urinary tract, or intramuscularly, or subcutaneously.

The effector genes, which are preferably genes which code for pharmaceutically active compounds or for enzymes which cleave inactive precursors of an active compound into active compounds, can be integrated into the target cells by the vectors of the invention in a non-cell-specific or cell-specific manner. The effector gene may be selected such that the pharmaceutically active compound or enzyme is expressed as a fusion protein marker with a ligand that binds to the surface of a cell, such as a proliferating endothelial cell or tumor cell.

The invention also relates to yeast or mammalian cells into which a nucleic acid construct has been inserted from the LMW PEI of the invention. In a particularly preferred embodiment, the LMW PEI of the invention is used to introduce nucleic acid constructs into cell lines that, after transfection, can be used to express transgenes. These cells can therefore be used for the preparation of a medicament for a patient. The preferred use of the LMW EPI of the invention forming a complex with a nucleic acid structure is to prepare a medicament for the treatment of diseases, which comprises the insertion of the nucleic acid structure in a target cell and the expression of this structure in a virus-specific or target cell-specific or metabolism-specific or non-specific manner and in a cell cycle-specific manner.

The invention further relates to a method for administering mammalian cells, wherein the LMW PEI of the invention is used to insert a nucleic acid construct into a cell for the preparation of a medicament for the treatment of a disease. For example, endothelial cells can be isolated from blood, treated in vitro with a vector of the present invention, and then introduced into the body by intravenous infusion.

These cells, which are transfected in vitro, may also be administered to a patient in combination with the vectors of the present invention. Such combinations refer to administration of the cells and the carrier, either simultaneously or at different times, separately, at the same or different sites or by injection.

Example (b): 1) method a) preparation of a low molecular weight polyethylenimine (LMW PEI)

LMW PEI is obtained by ring-opening polymerization of aziridine in an aqueous solution using an acid catalyst. For this, 1% (0.5ml) of concentrated hydrochloric acid (37%) catalyst was added to a 10% aziridine monomer aqueous solution (5ml aziridine monomer +45ml distilled water, dissolved with stirring), stirred at 50 ℃ for 4 days, then rotary evaporated and dried under vacuum at room temperature. LMW PEI was directly injected into a K5 measuring cell and the molecular weight was measured at 633nm by laser dispersion measurement (Wyatt Dawn DSP spectrophotometer). The gram molecular weight was calculated from the standard constant determined in toluene and the known initial sample weight.

The molecular weight was 2000 daltons as determined by spectrophotometry. The molecular weight of commercially available PEI (Fluka, Neu Ulm) determined by spectroscopic analysis was 791kDa (HMW PEI).

Both LMW PEI and HMW PEI samples were subjected to a control test. b) Preparation of Polynucleotide complexes

The DNA plasmid was complexed with PEI as described by Boussif et al (Boussif et al, Proc. Natl. Acad. Sci.92:7297-7301 (1995)). 9mg of 50% commercially available HMW PEI solution or 9mg of LMW PEI dissolved in 9ml of double distilled water the pH was adjusted to 7.4 with 1N HCl and then dissolved in water to a total volume of 10.0 ml. The resulting solution was sterile filtered (0.2 μm) and stored at 4 ℃ for a prolonged period of time.

For the preparation of complexes, 10. mu.g plasmid and various amounts of PEI stock solutions were diluted to 250. mu.l with 150mM NaCl and mixed in a vortex apparatus. The mixing ratio and the equivalent ratio for preparing the complex are given in table 1. After incubation at room temperature for 10 minutes, the polymer solution was added dropwise in portions to the plasmid solution and mixed in a vortexer. After incubation of the complex for an additional 10 minutes, it is added to the cell culture medium. c) Agarose migration assay

The ability of different PEI to bind to the plasmid was examined in an agarose migration assay. The procedure was to complex 1.35-27. mu.g of HMW PEI and 2.7-90. mu.g of LMW PEI with 10. mu.g of plasmid, respectively (Table 1). A50. mu.l aliquot was loaded on a 1% (w/v) agarose gel having a thickness of about 0.5cm and developed at 80mV in Tris (hydroxymethyl) aminomethane-ethylenediaminetetraacetic acid (Tris-EDTA) buffer, pH7.4, for 2 hours. After reaction with ethidium bromide, the position of the DNA was observed at 254 nm.

To visualize the plasmid from the complex, 50. mu.l or 100. mu.l dextran sulfate solution (Mw5000,10mg/ml, Sigma, Deisenhofen) was added per 10. mu.g DNA complex 30 minutes after the complex was formed. d) Cell culture

L929 mouse fibroblast cultures were performed under standard conditions well known to those skilled in the art. These cells were spread in a 96-well cell culture dish at a cell density of 8000 cells/well, and subjected to cytotoxicity test 24 hours after culture.

3T3 fibroblasts were cultured in the same manner under standard conditions.

Spontaneously transformed extracellular virus (ECV)304(ATCC, Rockville, MD USA), attached to a human endothelial cell line defined by a dominant normal control code, was cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Eggenstein) containing 5% Fetal Calf Serum (FCS), 5% horse serum and 1% N-acetyl-L-alanyl-L-glutamine (aminoamide) (all from Gibco, Eggenstein).

After confluency was achieved by incubating the cells at 37 ℃ under 95% relative humidity and 5% carbon dioxide, the cells were passaged twice a week with trypsin/ethylene glycol bis (2-aminoethylether) tetraacetic acid (EGTA) solution (2.5% trypsin stock, 50mM ethylene glycol tetraacetic acid solution, pH7.4 phosphate buffered saline, 1: 8). Since the cells were shed from the medium in the form of clusters of cells rather than individually, 1/8 passages were performed separately.

Brain capillary endothelial cells were isolated and cultured according to the methods of Bowman et al (1983) Ann. neuron. 14: 392-402) and Mischek et al (Mischek et al, cell tissue research (cell. Tiss. Res.)256:221-226 (1989). For transfection experiments, they were spread in 6-well cell culture dishes immediately after isolation until approximately 50% confluency. e) Cytotoxicity Studies

The cytotoxicity of the polymers was determined on L929 mouse fibroblasts using the MTT assay according to the method of Mosmann et al [ Mosmann, J. Immunol., 65:55-63(1983) ]. Serial dilutions of the polymer were prepared in minimal essential medium (DMEM) with 10% FCS and 2mM glutamine, and filter sterilized (0.2 μm, Schleicher & Schuell, Dassel). The pH and osmotic pressure of the solution were adjusted as needed. After 24 hours of pre-incubation of the cells, the polymer solution was mixed with the cells and incubated for 1, 3, 12 and 24 hours. The formazan * (formazan) concentration was measured by uv spectrophotometry to quantify cell viability.

In the second set of experiments, cells were treated only gently with polymer for 1 hour, then washed and cultured in PEI-free cell culture medium for 3, 12, 24 hours. Quantitative determination was carried out as described above. f) Transfection

Will be spread at 3cm²ECV304 cells and 3T3 mouse fibroblasts in the dish, and primary endothelial cells spread in a 6-well dish, were washed with ph7.4 phosphate buffer immediately before the experiment, and provided with fresh serum supplement medium. HMW PEI and LMW PEI complexes corresponding to 3.33. mu.g DNA were added to each well or each dish and incubated at 37 ℃ for 1 hour. The cells were then incubated for 60 hours and luciferase and beta-galactosidase activities were determined analogously to the method of section 9 and according to the manufacturer's instructions. [ HMW PEI and LMW PEI complexes are corresponding vectors containing HMW PEI and LMW PEI, respectively, and a nucleic acid, here a DNA plasmid]. Example 2: results a) physicochemical Properties of PEI

Swelling studies with 0.1M phosphate buffer solutions at various pH values in the range of pH4-10 demonstrated the reactivity of the polymer in the endosome/lysosome compartment. When HMW PEI dissolves at pH9 and pH10, a clear residue-free solution is formed, with a high degree of haze being seen at and below pH 8. This haze is essentially stable at pH7 and 8. Sedimentation occurs only after a few hours. In contrast, in the acidic range of pH, a sediment that was easily resuspended formed within 30 minutes. Under the same conditions, LMW PEI did not produce any turbidity or precipitation, but formed a clear solution. b) Cytotoxicity Studies

In vitro PEI cytotoxicity assays were performed on L929 mouse fibroblasts, which are recommended by various standardization bodies to be standard cell culture models for determining cytotoxicity and biocompatibility of polymers. Preliminary experiments determined the absorption of formazan * (formazan) formed and was found to be at 1X 10³-3×10⁴Direct, linear proportionality between cell numbers within a range. After the 24 hour growth phase, 8000 cells/well were mixed with the polymer solution and incubated for 1, 3, 12 and 24 hours. In the range of 0-1.0mg/ml, it is observedThe toxic effects of HMW PEI and LMW PEI of (g) are time and concentration dependent over 24 hours, and the cytotoxicity curves for high and low molecular weight PEI show significant differences. Thus, for HMW PEI, IC50 was between 0.06mg/ml (1 hour incubation) and 0.04mg/ml (24 hour incubation), whereas LMW PEI at a concentration between 0.1 and 1.0mg/ml was toxic only after 12 hours incubation and IC50 values were only determined when LMW PEI at a concentration of about 0.1mg/ml was incubated for 24 hours. c) Agarose gel migration assay

To determine the optimal binding and quantitative ratio between plasmid and PEI, a quantity of plasmid (10. mu.g) was complexed with various concentrations of HMW PEI and LMW PEI, followed by electrophoretic analysis. The mixing ratios of the complexes, the stock solutions used and the absolute amounts of PEI determined are given in Table 1.

TABLE 1a)

plasmid/HMW PEI mixing ratio [ equivalents]1+11+6.671+101+13.331+20

Volume of stock solution [ mu.l]Absolute amount of HMW PEI [ μ g]3 1.3520 930 13.540 1860 27

b)

plasmid/LMW PEI mixing ratio [ equivalents]	Volume of stock solution [ mu.l]	Absolute amount of LMW PEI [ μ g]
			1+2	3	2.7
1+13.33	20	18
			1+20	30	27
1+26.67	40	36
			1+40	60	54
1+53.33	80	72
			1+66.66	100	90

Table 1: complex mixture scale for electrophoresis and transfection containing a) HMW PEI and b) LMW PEI

The position of the plasmid and its complex is shown by ethidium bromide staining. The DNA forms two fluorescent bands corresponding to the supercoiled circular form of the plasmid and migrating in the direction of the anode. HMW PEI and LMW PEI could not be detected with ethidium bromide.

Complexation of DNA and HMW PEI in a 1+1 ratio results in partial, but not total, hindrance of plasmid migration at the point of loading. Decreasing the total loading and/or increasing the loading diameter prevents migration of the formed complex in the gel matrix.

Complexes in the ratio 1+6 to 1+20 cannot be detected because they do not show any fluorescence; this suggests that ethidium bromide is rejected by the plasmid because its structure is fully condensed and physically compressed by the HMW PEI. For these complexes, the negative/positive charge ratio is from 1: 1.2(1+6) to 1: 4(1+ 20). The complex therefore has a positive charge overall.

2.7. mu.g of LMW PEI almost completely bound and blocked 10. mu.g of plasmid. However, this complex is overall negatively charged and migrates toward the anode. However, complete cationic and DNA aggregation was observed only at LMW PEI. gtoreq.54. mu.g.

To verify the coagulation effect of high and low molecular weight PEI, excess dextran sulfate was used to replace DNA from the complex that had formed and the dextran sulfate was competitively reacted with the cationic polymer. For both HMW PEI and LMW PEI, the DNA may be released from the complex again and all or a portion of the DNA may migrate into the gel matrix. Ethidium bromide can re-enter the DNA structure and thus the DNA can be detected by fluorescence. d) In vitro transfection efficiency

The transfection efficiency of the PEI complexes was determined with cell lines (3T3 mouse fibroblasts and the human endothelial cell line ECV304) and primary cultures (porcine cerebral capillary endothelial cells). Promega commercially available pGL3 control vector, which carries the luciferase gene under the control of SV40 promoter and enhancer, can be used as a reporter gene. The ratio of the plasmid and the polymer mixed in the complex was the same as that of the complex subjected to electrophoresis.

The concentration of HMW PEI was determined at 1.35. mu.g to 27. mu.g/10. mu.g DNA. Maximal transfection was obtained at 18. mu.g HMW PEI. Further increasing the polymer concentration resulted in only a minor decrease in luciferase expression.

For LMW PEI, the concentration of 20-80. mu.g LMW PEI/10. mu.g DNA was determined. In contrast to the case of HMW PEI, increased stability of transfection efficiency was measured in ECV cells when LMW PEI concentration was increased. At 80. mu.g LMW PEI/10. mu.g DNA, the reporter activity was determined to be about 100-fold greater than the activity determined with 18. mu.g HMW PEI/10. mu.g DNA, which is the maximum activity shown. No decrease in luciferase expression was observed with the HMW PEI, as observed with the highest concentration of LMW PEI. Experiments with 3T3 and ECV cells gave the same results.

For comparison, transfection studies with β -galactosidase as a reporter gene were also performed on endothelial primary cell cultures and the highest tolerated non-cytotoxic dose (MTD) of HMW PEI and LMW PEI complexes were used. The in vitro MTD values were 13.5. mu.g HMW PEI/10. mu.g DNA and 90. mu.g LMW PEI/10. mu.g DNA. It was almost impossible to transfect cultured porcine cerebral capillary endothelial cells cultured with a complex comprising 10. mu.g DNA and 13.5. mu.g HMW PEI. Only 2-3 cells per well showed a specific blue reaction at the nuclear site. Only less than 10% of the treated cells were successfully transfected. In contrast, incubation with complexes containing 90 μ g hmw PEI and 10 μ g dna resulted in significant expression of the marker protein in endothelial cells. The percentage of blue-stained cells to total cells per well, i.e., transfection success, was between 5% and 10%. No toxic effect of the polymer/DNA complex on the cells was observed with light microscopy.

Claims (33)

1. A vector for inserting a nucleic acid into a cell, comprising a low molecular weight polyethylenimine (LMW PEI) and a nucleic acid, wherein the LMW PEI has a molecular weight of less than 50,000 daltons.

2. The vector according to claim 1, characterized in that the LMW PEI has a molecular weight of 500 to 30,000 Dalton.

3. The vector according to one or more of claims 1 and 2, characterized in that the LMW PEI has a molecular weight of 1000 to 5,000 daltons.

4. The vector according to one or more of claims 1 to 3, characterized in that the LMW PEI has a molecular weight of about 2000 daltons.

5. Vector according to one or more of claims 1 to 4, characterized in that the nucleic acid is a viral or non-viral nucleic acid structure.

6. Vector according to one or more of claims 1 to 5, characterized in that the nucleic acid structure contains one or more effector genes.

7. The vector according to one or more of claims 1 to 6, wherein at least one effector gene is encoded by a pharmaceutically active compound or a prodrug thereof.

8. The vector according to one or more of claims 1 to 7, characterized in that at least one effector gene is encoded by an enzyme.

9. The vector according to one or more of claims 1 to 8, characterized in that at least one effector gene is expressed as a fusion protein marker together with a cell-specific ligand.

10. The vector according to one or more of claims 1 to 9, wherein the LMW PEI is coupled to a cell-specific ligand.

11. The vector according to one or more of claims 1 to 10, wherein the cell-specific ligand binds to the outer membrane of the target cell.

12. The vector according to one or more of claims 1 to 11, wherein the target cell is an endothelial cell, a muscle cell, a macrophage, a lymphocyte, a glial cell, a hematopoietic cell, a tumor cell, a virally infected cell, a bronchial epithelial cell or a liver cell.

13. The 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 more.

14. The 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 more.

15. A process for preparing low molecular weight polyethylenimines having a molecular weight of less than 50,000 daltons, characterized in that the process comprises polymerizing monomeric aziridine in aqueous solution by addition of hydrochloric acid.

16. The method of claim 15, wherein the concentration of monomeric aziridine in the aqueous solution is from 0.1% to 90% and the concentration of concentrated hydrochloric acid is from 0.1% to 10%.

17. The process according to one or more of claims 15 and 16, characterized in that the polymerization is carried out at a temperature of between 30 ℃ and 70 ℃.

18. The process according to one or more of claims 15 to 17, characterized in that the reaction time is 1-30 days.

19. A low molecular weight polyethylenimine having a molecular weight of less than 50,000 dalton, characterised in that it is prepared by a process according to one or more of claims 15-18.

20. Use of low molecular weight polyethylenimine having a molecular weight of less than 50,000 dalton, characterized in that it is used for the preparation of a vector according to one or more of claims 1 to 14.

21. A method for preparing a vector according to one or more of claims 1 to 14, characterized in that it comprises mixing an amount of LMW PEI with an amount of nucleic acid in an aqueous solution.

22. Use of a vector according to one or more of claims 1 to 14 for inserting a nucleic acid into a cell.

23. Use of a vector according to claim 22, wherein the cell is an endothelial cell, a lymphocyte, a macrophage, a hepatocyte, a fibroblast, a muscle cell or an epithelial cell.

24. A method for preparing a transfected cell, characterized in that it comprises incubating a vector according to one or more of claims 1 to 14 with this cell in vitro.

25. A transfected cell comprising 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 polyethylenimine 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. A method of preparing a medicament, comprising mixing a nucleic acid with a LMW PEI.

31. A medicament, characterized by comprising a vector according to one or more of claims 1 to 14.

32. A medicament comprising a LMWPEI as claimed in claim 19.

33. A medicament comprising a transfected cell according to claim 25.