WO2003087384A1 - Preparation and use of dna-polyelectrolyte nanoparticles for gene transfer - Google Patents

Abstract

The invention relates to a non-viral method of transfecting cell cultures, tissue and/or organ cultures and can also be used for in vivo gene transfer when observing particular basic conditions. The invention is suitable for medical and biological basic research, particularly regarding preliminary studies on gene therapy and in the pharmaceutical industry. The method for gene transfer into animal cells is characterized in that vector DNA in aqueous solution is packed into compact nanoparticles by successive addition of oppositely charged polyelectrolytes and that transfection of the cells is performed thereafter. Furthermore, the method is characterized in that the vector DNA - is made to interact with polycations and condensed in a first step, - is mixed with polyanions to coat a layer in a second step, and that these steps are repeated, if necessary. For in vivo gene transfer, the vector DNA is packed into polyelectrolyte nanoparticles (shells) which are mixed with e.g. Pluronic F127 gel and, following exposure of the tissues/organs/tumors to be transfected, applied thereon in liquid form.

Description

Preparation and Use of DNA-Polyelectrolyte Nanoparticles for Gene Transfer

The invention relates to a non-viral method of transfecting cell cultures, tissue and/or organ cultures and can also be used for in vivo gene transfer when observing particular basic conditions. The invention is suitable for medical and biological basic research, particularly regarding studies on gene therapy and tumor therapy and in the pharmaceutical industry. The invention relates also to a complex for a gene transfer into vascular tissues.

All methods of non-viral gene transfer are based on an interaction of the transgene DNA with suitable carrier molecules assuming multiple functions in the transfection process: transformation of the DNA into a condensed conformational state enabling reception by the recipient cell and protecting the DNA from nucleolytic degradation. Furthermore, lysosomolytic and/or membranolytic effects must be integrated so as to allow escaping from the endosomes/- lysosomes and incorporate a signal locating the nucleus. Commercial transfectants or those described in the literature meet these demands only in part. Three groups of such transfectants are known, including e.g. cationic liposomes, peptide- and/or protein-mediated systems, and polymer-mediated systems.

One major problem in the preparation of transfection-active DNA complexes based on such systems is their poor solubility in physiological saline solutions. This is a property governed by the DNA and is seen in charge-neutralized DNA (e.g. chromatin). Due to the risk of embolism and insufficient biodistribution, the use of the resulting aggregates in gene therapy is out of the question. On the other hand, a direct correlation between aggregate size and in vitro transfection efficiency has been demonstrated in virtually all of the systems. However, large aggregates frequently were found to be toxic to the cells.

Gene vectors should be economical and safe. High transfection activity is to be expected. At present, the art is far from accomplishing such properties. To date, it has not even been possible to find a satisfactory solution to the requirement of high in vitro or in vivo efficiency and optimal size of the transfecting complex.

One way of circumventing the aggregation tendency, which has been pursued in the literature, is to coat the transfecting complex with a neutral polymer, for which purpose polyethylene glycol (PEG) is used among other things, and which is covalently coupled to the DNA carrier. Trubetskoy et al. 1999 have prepared nanoparticles (shells) based on plasmid DNA complexed with natural or synthetic polyelectrolytes which resulted in aggregation-free particles under certain conditions. Disclosed is also the transfection with binary - e.g. DNApolycations - or ternary - e.g. DNA polycations/polyanions- complexes (WO 00 03694). The structural relationship between ternary complexes and shells are clarified not finally. High transfection rates were preferentially obtained with large particles (max. 600 nm) which showed aggregation tendency. Transfection data were disclosed only for lung or cell cultures. The lung is a traditional model for in vivo transfection. Unfortunately, results obtained in the model lung are not necessarily reproducible in other organs. Frequently, very good lung transfection methods/reagents have an adverse effect on transfection in other targets. Maximum transfection coincided with DNA-complexes (i) near electro neutrality, (ii) strong flocculation tendency, and (iii) DNA-complexes 500 nm in diameter. Additionally, Trubetskoy disclosed that large complexes transfect cells in culture better than small complexes. Best results are obtained by flocculated, neutral and large, non-stable complexes, because probably the non-stable complexes release contents more efficiently than stable complexes.

Thus, the technical problem underlying the present invention is to provide methods and a DNA-complex which promote the transfection, preferably in vivo transfection.

The present invention solves the problem by providing a method for a gene transfer into animal cells in vivo or ex vivo, characterized in that vector DNA in aqueous solution is packed into compact nanoparticles by successive addition of oppositely charged polyelectrolytes and transfection of the cells is performed thereafter whereby the nanoparticles have in a preferred embodiment an average particle size of less than 150 nm and a negative zeta potential. The polyelectrolytes in such weight ratios are mixed that specific particle size or surface charge will receive, whereby the selection of parameters or ratios may be based on routine experiments.

In a preferred embodiment DNA-polycation complex and/or DNA-polycation/polyanion complex are present in a physiological saline solution. The term physiological saline solution referred to herein is to be understood in a broad sense— i.e., it should include every isotonic solution being tolerated by the body. Examples in this respect are the physiological saline solutions in narrower sense, the so-called Ringer's solution (1000 ml contain 8.6 g sodium chloride, 0.3 g potassium chloride and 0.33 g calcium chloride), as well as blood substitute substances, i.e. infusion and standard injection solutions as are listed for example in the catalogue of pharmaceutical specialities put out by the members of the Bundesverbandes der pharmazentrischen Industrie e.V. (Federal Association of the pharmaceutical industry), with the title "Rote Liste" 1975 under the numbers 56001 to 56082; 56086 to 56267. Mixtures of these substances are also to be. understood.

Preferred, nanoparticles for gene transfer have an average particle size of less than 120 nm, more preferably 100 nm, and most preferably 90 - 50 nm. The small nanoparticles are very stable and efficient carrier for nucleic acids, e.g. condensed DNA. In a preferred embodiment more than 30%, more preferably 40%, and most preferably 50 % of target cells in a patient - e.g. in a tissue such as an artery wall - are transfected. The method provided in accordance with the present invention is preferably useful for transfection in vivo, whereby the process of vector-transfer in the living system is realized by endocytosis. The preferred nanoparticles for in vivo transfection-method have an average particle size of less than 150 - 50 nm this leads to receptor-specific endocytosis of the complex into the cell. The preferred nanoparticles for in vitro transfection-method have an average particle size of more than 300 nm this leads to phagocytosis of the complex into the cell. The polyelectrolytes are in such weight ratios mixed that the nanoparticles are preferentially flocculated for the in vitro method, but lower transfection rates are also obtained by using unfloc- culated soluble particles. The in vivo method requires unflocculated nanoparticles. In a preferred embodiment the ratio of DNA and polyelectrolyte is C_DNA <,~ Cp₀ι_ycati_on < C_Pθι_yani_θn for in vivo gene transfer.

Nanoparticle means in the context of the present invention a submicroscopic solid object, essentially of regular or semi-regular shape, that is less than one micrometer in its largest dimension and exhibits a liquid core and a semipermeable shell. Shell means an soluble polymeric electrostatic complex composed of internal core polymer(s) and external bath polymer(s) molecularly bonded, or gelled, in close proximity. Core polymer means an internal part of the microcapsule, nanoparticle, or polymeric film.

The term nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless specifically limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly provides the complementary sequence thereof, as well as the sequence explicitly indicated. The preferred nucleic acid for use in the invention is DNA. Coding sequences may preferably comprise cDNA. The term nucleic acid is used interchangeably with gene and cDNA. The term polynucleotide is used to mean a polymeric form of nucleotides of any length, which contain deoxyribonucleotides, ribonucleotides, and/or their analogs. The terms polynucleotide and nucleotide as used herein are used interchangeably. Polynucleotides can have any three-dimensional structure, and can perform any function, known or unknown. The term polynucleotide includes double-stranded, single-stranded, and triple-helical molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double stranded form.

The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any se- quence, nucleic acid probes, and primers. A polynucleotide can be comprised of modified nucleotides, such as methylated nucleotides and nucleotide analogs. Analogs of purines and pyrimidines are known in the art, and include, but are not limited to, aziridinylcytosine, 4- acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5- carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1- methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, pseudouracil, 5- pentynyluracil and 2,6-diaminopurine. The use of uracil as a substitute for thymine in a de- oxyribonucleic acid is also considered an analogous form of pyrimidine. If present, modification to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications included in this definition are, for example, caps, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phospho- nates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L- lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chela- tors (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkyla- tors, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars can be replaced by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or can be conjugated to solid supports. The 5^' and 3^' terminal hydroxy groups can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, but not limited to, 2^'-O-methyl-, 2^'-O-allyl, 2^'-fluoro- or 2^'-azido-ribose, carbocyclic sugar analogs, .alpha. -anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. As noted above, one or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (thioate), P(S)S (dithioate), (O)NR2 (amidate), P(O)R, P(O)OR^', CO or CH.sub.2 (formacetal), in which each R or R^' is independently H or substituted or unsubsti- tuted alkyl (1-20 C) optionally containing and ether (-O-) linkage, aryl, alkenyl, cycloalky, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. Although con- ventional sugars and bases will be used in applying the-method of the invention, substitution of analogous forms of sugars, purines and pyrimidines can be advantageous in designing a final product, as can alternative backbone structures like a poiyamide backbone. An an- tisense polynucleotide is a sequence complementary to all or part of a functional RNA or DNA. For example, antisense RNA is complementary to sequences of the mRNA copied from the gene. A fragment (also called a region) of a polynucleotide (i.e., a polynucleotide encoding a sarp) is a polynucleotide comprised of at least 9 contiguous nucleotides of the novel genes. Preferred fragments are comprised of a region encoding at least 5 contiguous amino acid residues, more preferably, at least 10 contiguous amino acid residues, and even more preferably at least 15 contiguous amino acid residues. The term recombinant polynucleotide intends a polynucleotide of genomic, cDNA, semisynthetic, or synthetic in origin which, by virtue of its origin or manipulation: is not associated with all or a portion of a polynucleotide with which it is associated in nature; is linked to a polynucleotide other than that to which it is linked in nature; or does not occur in nature.

In the context of the present invention, the terms condensed nucleic acid and partially condensed nucleic acid are used to refer to a nucleic acid that has been contacted with an organic or anorganic cation or polycation (e.g. polyamines, including spermine and spermidine, polyammonium molecules such as Polybrene, basic polyamino acids, and basic proteins). A polycation is e.g. a polymer containing a net positive charge, for example poly-L-lysine hy- drobromide. The polycation can contain monomer units that are charge positive, charge neutral, or charge negative, however, the net charge of the polymer must be positive. A polycation also can mean a non-polymeric molecule that contains two or more positive charges. A polyanion is a polymer containing a net negative charge, for example polyglutamic acid. The polyanion can contain monomer units that are charge negative, charge neutral, or charge positive, however, the net charge on the polymer must be negative. The term polycation refers also to a peptide or polypeptide (i.e., protein) sequence which contains an abundance of amino acid residues having positively charged (i.e., basic) side chains (e.g., arginine and lysine) such that the peptide has a positive charge and is capable of binding ionically to nucleic acids (which are negatively charged). A polyanion can also mean a non-polymeric molecule that contains two or more negative charges. The term polyion includes polycation, polyanion, zwitterionic polymers, and neutral polymers. A charged polymer is a polymer that contains residues, monomers, groups, or parts with a positive or negative charge and whose net charge can be neutral, positive, or negative. Condensed nucleic acids typically occupy a significantly smaller volume than noncondensed nucleic acids. It is recognized, however, that the degree of condensation may vary with local environment (e.g., lipid as opposed to aqueous environment). Due to the electric charge, a subset of these polycationic agents are capable of condensing the desired nucleic acids to a compact size to facilitate delivery. Typi- cally, condensation collapses polynucleotides or nucleic acids into macromolecular structures, commonly into a toroid form. The smaller size of condensed nucleic acids eases delivery by facilitating, for example, packaging nucleic acids into liposomes and/or reducing exposure to proteases and/or nucleases. The condensed nucleic acids exhibit different properties compared to relaxed nucleic acids, such as (1) a decrease in intercalation of ethidium bromide or other intercalating dye or (2) a reduced mobility ih gel electrophoresis. Thus, condensation can be measured by at least two different assays, an intercalating dye assay or a band shift assay. One type of intercalating dye assay uses ethidium bromide. In this assay, test nucleic acids, conveniently plasmid DNA, are mixed with polycationic agent in a ratio from about 1:1 to a 1 :50 weight/weight ration of plasmid to condensing agent. Following incubation, ethidium bromide is added to the reaction to a final concentration of 1 .mu.g/mL If a nucleic acid such as RNA is used as the test nucleic acid, acridine orange may be used as the intercalating dye. The reaction mixtures are transferred into UV transparent plastic tubes spotted with 1% agarose gel, or placed upon UV transparent plastic and illuminated with 260 nm light. The emission from the DNA-ethidium bromide complex is recorded on film by a camera equipped with an appropriate UV filter. The ability of an agent to condense DNA is inversely proportional to the intensity of the fluorescence in each reaction mixture. The more precise test is a band shift assay. Briefly, this assay is performed by incubating nucleic acids, either labeled or unlabeled, with various concentrations of candidate condensing agents. Test nucleic acids, conveniently plasmid DNA, and condensing agent are mixed at 1 :1 to 1 :50 w/w ratios. Following incubation, each sample is loaded on a 1% agarose gel and electrophoresed. the gel is then either stained with ethidium bromide or dried and autoradiographed. DNA condensation is determined by the inability to enter the gel compared to a non-condensed standard. Sufficient condensation is achieved when at least 90% of the DNA fails to enter the gel to any significant degree.

The invention is based on the object of devising initial steps in complying with the above- mentioned requirements by using a new principle in particle assembly. More specifically, it is intended to develop small, stable particles having high transfection efficiency. According to the invention the:

(i) in-vivo transfection-particle should be small (70 - 95 nm), stable and unflocculated - preferably with a negative surface charge - and

(ii) the in-vitro transfection-particle should be large (250 - 550 nm),or flocculated and non- stable, preferably with a neutral surface or positive surface charge. Therefore, a vector for gene therapy in the context of the invention should be small to permit uptake by endocytosis. In a preferred embodiment of the invention, the vector/particles should not include crosslinked material or surfactants (e.g. PEG). Receptor-specific ligands could be present to allow for cell and organ targeting. Furthermore, gene vectors should be stable, i.e., have no tendency of aggregating, and exhibit no interaction with serum proteins.

In a preferred embodiment of the invention the ζ- potential of the in vivo-particle is < -30 mV, and more preferably < —4-0 mV.

The term flocculation, as used herein means polymers, e.g. shells, aggregates or precipitates in situ, in solution e.g. at the critical solution temperature. The term flocculation, as used herein, is synonymous with the term coagulation. Flocculation refers to the settling of suspended solid particles from aqueous systems.

Soluble complexes observed in addition to the aggregates are transfection-inactive. These findings are substantiated by simple centrifugation of the complex at 4000 x g for 2 minutes. Exceptional are polyethyleneimine and dendrimer complexes which have a lower tendency of aggregating at high excess of positive charges and are transfection-active.

The invention is supported by own results from part of the inventors in the preparation of nanoparticles (shells) and is based on a stepwise adsorption of layers of oppositely charged polyelectrolytes on the surface of colloid particles, or, in this case, on the DNA condensed by transfectants. The surface charge of the particles changes with each coated layer. Using suitable polyelectrolytes, it is possible to achieve desirable coat properties such as selective permeability, controlled release, stability, and biocompatibility.

The basic idea of the invention is to coat the DNA/DNA carrier (transfectant) complexes with an additional, stabilizing polyelectrolyte layer so as to prevent aggregation in physiological media, i.e. to stabilize the particles. On the contrary, a common point of view has-been that large, non-stable nanoparticles - as vector - should possess higher transfection activity because they release contents more efficiently. The term vector is understood to generally refer to the non-viral vehicle by which the nucleotide sequence is introduced into the cell. It is not intended to be limited to any specific sequence. The vector could itself be the nucleotide sequence that activates the endogenous gene or could contain the sequence that activates the endogenous gene. Thus, the vector could be simply a linear or circular polynucleotide containing essentially only those sequences necessary for activation, or could be these sequences in a larger polynucleotide or other construct used to introduce the critical nucleotide sequences into a cell. It is also understood that the phrase vector construct or the term construct may be used interchangeably with the term vector herein. The term transfection has been used herein for non-viral-mediated introduction of a polynucleotide into a cell. However, it is to be understood that the specific use of this term has been applied to generally refer to the introduction of the polynucleotide into a cell and is also intended to refer to the introduction by other in- or ex-vivo methods described herein such as in-vivo-electroporation (eg. skin cells), liposome-mediated introduction, and the like (as well as according to its own specific meaning).

Several parameters are likely to be critical factors for an efficient in vivo gene transfection like: size of the plasmid, pH, osmolarity, salinity, and cell type. A large size plasmid (>10kb) is detrimental for an efficient transfection but larger plasmids sometimes may be transfected efficiently. The pH of the vehicle liquid seems an important parameter. Generally, a slightly basic/neutral pH (8-8,5/or 7,4) has a better chance to increase gene delivery. This may be reinforced by the suspension of the DNA in a basic/neutral buffer like Hepes, Bicarbonate or a NaCI-buffer. This is particularly recommended for injections into tumors since the center of tumors is often acidic and induces a DNA precipitation. Osmolarity is also an important factor to be checked. A highly concentrated DNA solution is hypertonic and may not be suitable for a good gene transfer. On the other hand, DNA solutions must not be too diluted to avoid large volumes and leakage after injection. A concentration of 0.05 - 5.0 mg/ml is preferred. Salts and buffers are also important for DNA storage and for DNA transfer. As seen above, the choice of a buffer may be critical for DNA transfer. Thereafter, the appropriate buffer can be determined by the investigator depending on the animal or human patient. Finally, cell lines grafted, in vivo may behave very differently depending on the tissue/organ they are derived from. This is even true between cell lines derived from the same cell type. Optimized conditions for one cell line may be quite different for another. This will also depend on the way the DNA is being injected. The selection of parameters: size of the particle, pH, osmolarity, salinity, and cell type or tissue target may be based on routine experiments to determine combinations/ratios of polyelectrolytes which result in efficient transfection.

Delivery of a nucleic acid means to transfer a nucleic acid from a container outside a mammal to near or within the outer cell membrane of a cell in the mammal. The term transfection may be used, in general, as a substitute for the term delivery, or, more specifically, the transfer of a nucleic acid from directly outside a cell membrane to within the cell membrane. The transferred (or transfected) nucleic acid may contain an expression cassette. If the nucleic acid is a primary RNA transcript that is processed into messenger RNA, a ribosome translates the messenger RNA to produce a protein within the cytoplasm. If the nucleic acid is a DNA, it enters the nucleus where it is transcribed into a messenger RNA that is transported into the cytoplasm where it is translated into a protein. Therefore if a nucleic acid expresses its cognate protein, then it must have entered a cell. A protein may subsequently be de- graded into polypeptides, which may be presented to the immune system. Polypeptide refers to a linear series of amino acid residues connected to one another by peptide bonds between the alpha-amino group and carboxyl group of contiguous amino acid residues. Protein refers herein to a linear series of greater than 2 amino acid residues connected one to another as in a polypeptide. A therapeutic effect of the protein in attenuating or preventing the disease state can be accomplished by the protein either staying within the cell, remaining attached to the cell in the membrane, or being secreted and dissociated from the cell where it can enter the general circulation and blood. Secreted proteins that can be therapeutic include hormones, cytokines, growth factors, clotting factors, anti-protease proteins (e.g., ai- phal-antitrypsin), angiogenic proteins (e.g., vascular endothelial growth factor, fibroblast growth factors), anti-angiogenic proteins (e.g., endostatin, angiostatin), and other proteins that are present in the blood. Proteins on the membrane can have a therapeutic effect by providing a receptor for the cell to take up a protein or lipoprotein (e.g., low density lipopro- tein receptor). Therapeutic proteins that stay within the cell (intracellular proteins) can be enzymes that clear a circulating toxic metabolite as in phenylketonuria. They can also cause a cancer cell to be less proliferative or cancerous (e.g., less metastatic), or interfere with the replication of a virus. Intracellular proteins can be part of the cytoskeleton (e.g., actin, dys- trophin, myosins, sarcoglycans, dystroglycans) and thus have a therapeutic effect in cardio- myopathies and musculoskeletal diseases (e.g., Duchenne muscular dystrophy, limb-girdle disease). Other therapeutic proteins of particular interest to treating heart disease include polypeptides affecting cardiac contractility (e.g., calcium and sodium channels), inhibitors of restenosis (e.g., nitric oxide synthetase), angiogenic factors, and anti-angiogenic factors. Biomolecule refers to peptides, polypeptides, proteins, enzymes, polynucleotides, oligonu- cleotides, viruses, antigens, carbohydrates (and conjugates), lipids, and saccharides. Enzymes are proteins evolved by the cells of living organisms for the specific function of catalyzing chemical reactions.

One aim of the invention is to find anionic polyelectrolytes that would stabilize the cationic DNA complexes against aggregation tendencies under physiological conditions and result in transfection-active complexes having a negative surface charge. This latter condition is required to exclude interactions with negative serum proteins, which would result in rapid loss of the complexes in vivo. On the other hand, binding to the negative cell surface is to be achieved via ligand-receptor interaction. In a preferred embodiment of the invention transfer RNA, DNA or oligoribonucleotides are used as polyanions. In other preferred embodiment of the invention antisense ribonucleotides or mixtures of antisense ribonucleotides and transfer RNA or polyvinyl sulfate and/or polystyrenesulfonate are used as polyanion. As used herein, the term polyanion refers to materials having more than one anionic group. For example, polyanion is used to refer to nucleic acids, both DNA and RNA which are present in their polyanionic form having more than one anionic phosphodiester group along the nucleic acid backbone. The term polyanion also refers to those pharmaceutical agents which have more than one anionic group at neutral pH. Such pharmaceutical agents include peptides having multiple carboxylic acid functionalities present (i.e., Glu, Asp). The term polyanion may also refer to the anionic portion of the polyampholyte and the term polycation may refer to the cationic portion of the polyampholyte, e.g. Poly-L-Lysine, succinic anhydride-PLL, polyme- thacrylic acid, polyaspartic acid, and preferred transfer RNA or polyvinyl sulfate. Representative examples of polyanions include vinyl type synthetic polyanions, such as a polyacrylic acid, a polymethacrylic acid, a polyvinyl sulfonic acid, a polyvinyl sulfuric acid, a polymaleic acid, polyfumaric acid and derivatives thereof: styrene type synthetic polyanions, such as a poly(styrenesulfonic acid) and a poly(styrenephosphoric acid); peptide type polyanions, such as a polyglutamic acid and a polyaspartic acid; nucleic acid type polyanions, such as a poly U and a poly A; synthetic polyanions, such as a polyphosphoric acid ester, a poly-. alpha. - methylstyrenesulfonic acid and copolymers of styrene and methacrylic acid; and polysaccha- ride type polyanions, such as heparin, dextran sulfate, chondroitin sulfate, alginic acid, pectin, hyaluronic acid and derivatives thereof. However, the polyanoin to be used in the present invention is by no means restricted to the above-mentioned specific examples.

In a preferred embodiment synthetic polycations, peptides, nucleoproteins and/or polyamino acids are used as polycations. Particularly preferred is polyethyleneimine (PEI) used as synthetic polycation. In another embodiment peptides predominantly comprised of lysine and/or arginine are used as polycations. In yet another preferred embodiment, the nucleoproteins H1 histone or HMG1 are used as polycations. In another aspect of the preferred embodiment, polylysine, oligolysine K-\ Q or K-jβ fusion peptides are used as polycations. Preferably, transfer RNA, DNA or oligoribonucleotides are used as polyanions. Particularly preferred antisense ribonucleotides or mixtures of antisense ribonucleotides and transfer RNA or other polyanions are used as polyanions.

In another embodiment, biocompatible anionic polymers are used as polyanions, which polymers prevent aggregation of the complexes under physiological saline conditions. Preferably, polyvinyl sulfate and/or polystyrenesulfonate are used as polyanion. In another aspect of the preferred embodiment, polylysine is used as polycation and transfer RNA as polyanion. In further aspect of the preferred embodiment, polyethyleneimine is used as polycation and polyvinyl sulfate as polyanion. Preferably, polyethyleneimine (PEI) is used as polycation and RNA as polyanion. In a further preferred embodiment PEI is used as polycation and polystyrenesulfonate sodium (PSSS) as polyanion. It is also preferred that that polyethyleneimine is used as polycation and polystyrenesulfonate sodium as polyanion. In a preferred embodiment, the vector DNA is coated with more than 2 polyelectrolyte layers. In another embodiment the polycations and/or polyanions comprising ligands.

In yet another preferred embodiment the following parameters are selected independently:

the particle surface charge by selecting suitable polyanions/polycations on the surface,

provision of ligands for cellular uptake by receptor-specific endocytosis and for nuclear transport by means of electrostatic binding of the ligands using short polar amino acid segments,

lysosomolytic activity of the nanoparticles by incorporating lysosomolytic polycations.

In another aspect of the preferred embodiment, integrin-specific peptides are used as exterior polyelectrolyte layer or as polycation. Preferably, the integrin-specific peptide is K-iβ-

GGCRGDMFGCA or an analogous peptide. In a preferred embodiment, polystyrenesulfonate sodium (PSSS) is used as polyanion in the chemical coupling of the ligands. In another aspect of the preferred embodiment, polyethyleneimine or dendrimers are used as polycations in mediating lysosomolytic activity. In further embodiment, the transfection is carried out in the presence of chloroquine or CaCl2-

The inventive method for the gene transfer into animal cells is characterized in that a) vector DNA for the preparation of transfecting complexes is made to interact with polycationic substances, such as peptides predominantly constituted of lysines and arginines, nucleoproteins such as H1 or HMG1 , synthetic polycations such as polyethyleneimine (PEI) and polyamino acids, e.g. polylysine (investigated as an example herein). This is done in an aqueous medium in order to prevent aggregation of the resulting complexes. In a second step b) these complexes, which generally have a positive surface charge, are coated with a negative layer by mixing with a polyanion. This layer, preferably constituted of transfer RNA or synthetic polyanions, prevents aggregation of the complexes when exposing them to physiological saline conditions. According to the invention, synthetic polyanions or biologically relevant oligoribonucleotides may also be used as polyanions. It should be noted that a number of other investigated polyanions fail to prevent the formation of aggregates (see above). To confirm that the complexes obtained actually comply with, the criteria of layer assembly (small size, low polydispersity, negative zeta potential), they are examined in physiological saline solution with respect to their size distribution, using dynamic light scattering, AFM or electron microscopy, and their zeta potential. The particles obtained comply with these criteria and are found to be active in transfection experiments. Due to their negative surface charge, which impedes strong interaction with the cell surface, the transfection efficiency is lower than that of DN A poly lysine complexes alone, which exhibit extensive aggregation. Thereby, however, expectations are raised that introduction of ligands for cell surface receptors might improve the efficiency of targeted gene transfer. A simple method of preparing shells provided with ligands is addition of cationic peptides having ligand sequences (e.g. K-iβ-cRGD) acting as third outer layer. It is also possible to admix an anionic peptide having ligand sequences (e.g. E^-cRGD) to the polyanions of the outer layer. One problem in doing so, however, is the risk of aggregation which must be prevented by careful titration of the added peptide component in well-defined concentration steps.

According to the invention, it is possible to further increase the transfection efficiency by the presence of. lysosomolytic substances such as chloroquine in the post-incubation medium. However, it is also possible to integrate a lysosomolytic component, e.g. the polycationic polyethyleneimine (PEI), in the particles. With this objective, polylysine can be replaced by polyethyleneimine (PEI). PEI/DNA complexes are capable of escaping from endosomes or lysosomes. One major advantage of the invention is the potential of providing a homogeneous, transfection-active particle population of small size (in the present example using polylysine of about 100 nm), which can be used as a model in investigations for in vivo gene transfer.

According to the invention, the method illustrated above can be used in cell transfection of various cultured cell lines. Gene transfer into tissue and organ cultures is also possible. Given sufficient stability of the particles, an in vivo gene transfer can be achieved; according to the invention, it is essential to use RNA and synthetic polyanions (polyvinyl sulfate, polystyrenesulfonate sodium) as polyanions in building up the shells. Similarly, this applies to other biocompatible polyanions which, according to the invention, meet the demand of absent aggregation in saline solution. Using the above-mentioned polyanions, transfection- active particles free of aggregation are obtained under physiological saline conditions for the first time.

More specifically, a preferred method of the invention proceeds e.g. as follows:

1. In vitro transfection a) The peptide K^<\ Q (oligolysine), K-iβ-cRGD (integrin-specific K-|6). H1 histone, polylysine or PEI are made to interact with vector DNA in an aqueous medium. 2 μg of DNA is used in a transfection batch including 2 x 10 cells. b) Subsequently, excess transfer RNA (type V from wheat germ, supplied by Sigma; DNA/polylysine/RNA = 1 :1.5:6) is added to the complexes and brought to physiological ionic strength by adding 5 M NaCI solution. Due to the risk of aggregation, the concentration conditions of the individual components are to be optimized. Thereafter, the shells are used for transfection. 2. In vivo gene transfer

One essential embodiment to the invention is the new variant of in vivo application to be protected by the claims: Shells are suspended in Pluronic F127 gel. This can be done by mixing with the gel at 4°C, because the gel is present in liquid form at this temperature. The loaded gel, which undergoes solidification at 37°C, is applied on the exposed organ, skin or tumor. The surgical wound is closed. The gel dissolves within an incubation period of up to 3 days, the shells are absorbed by the tissue, and the heterologous gene is expressed, which expression is investigated after a total of 7 days. Other modes of application, such as direct injection into tumors, were also successful.

In a preferred embodiment of the invention the method vector DNA is packed in nanoparticles and administered into living systems; e.g. for treating diseases such as cancers. The living system is e.g. a patient, an organ, a tumor, a vascular wall or a cell. In a preferred embodiment the nanoparticles are directly injected into the tissue, organs and/or tumors to be transfected; preferably, the nanoparticles are injected on the intravenous route or the nanoparticles are injected on the intraperitoneal route.

The term treating refers to:

(i) preventing a disease, disorder or condition from occurring in an animal that may be predisposed to the disease, disorder and/or condition, but has not yet been diagnosed as having it; (ii) inhibiting the disease, disorder or condition, i.e., arresting its development; and (iii) relieving the disease, disorder or condition, i.e., causing regression of the disease, disorder and/or condition.

Further still, the methods of the invention can be used in a preferred embodiment to treat cancer. Cancer in the context of the invention is characterized primarily by an increase in the number of abnormal cells derived from a given normal tissue. The disease process also involves invasion of adjacent tissues by these abnormal cells, and spread of these abnormal cells to regional lymph nodes and to distant sites (metastasis) via the circulatory system. Clinical data and molecular biologic studies indicate that cancer is a multistep process that begins with minor preneoplastic changes, which may under certain conditions progress to neoplasia. The term cancer is interpreted broadly. The compounds and methods of the present invention can be anti-cancer agents, which term also encompasses anti-tumor cell growth agents and anti-neoplastic agents. Cancer is defined herein as any cellular malignancy for which a loss of normal cellular controls results in unregulated growth, lack of differentiation, and increased ability to invade local tissues and metastasize. Cancer may develop in any tissue of any organ at any age. Cancer may be an inherited disorder or caused by environmental factors or infectious agents; it may also result from a combination of these. The differential expression of genes that regulate cell growth, migration, and other functions enables a cell to grow out of control and become cancerous. In many cases, the activation of oncogenes, which override the intrinsic cellular growth regulatory commands of a cell, as well as the inactivation of tumor suppressor genes, which normally hold tumor formation in check, renders tumor cells free of growth restraints. The identification and characterization of these differentially expressed genes in malignant tumors will facilitate the understanding of the basic nature of the malignancy and yield novel molecular markers useful in diagnosis and treatment. For the purposes of utilizing the present invention, the term cancer includes both neoplasms and premalignant cells. For example, the methods of the invention are useful for treating cancers and tumor cells in cancers such as ACTH-producing tumors, acute lymphocytic leukemia, acute nonlymphocytic leukemia, cancer of the adrenal cortex, bladder cancer, brain cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelocytic leukemia, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer, Ewing's sarcoma, gallbladder cancer, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma. Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer (small and/or non-small cell), malignant peritoneal effusion, malignant pleural effusion, melanoma, mesothelioma, multiple myeloma, neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer, ovary (germ cell) cancer, prostate cancer, pancreatic cancer, penile cancer, retinoblastoma, skin cancer, soft-tissue sarcoma, squamous cell carcinomas, stomach cancer, testicular cancer, thyroid cancer, trophoblastic neoplasms, uterine cancer, vaginal cancer, cancer of the vulva and Wilm's tumor. In a preferred embodiment of the invention the tumor is selected from the group consisting of fibrosarcoma, myxosarcoma, li- posarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelio- sarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, broncho- genie carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocy- toma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. Within such methods, pharmaceutical compositions and vaccines are typically administered to a patient. As used herein, a patient refers to any warm-blooded animal, preferably a human. A patient may or may not be afflicted with cancer. Accordingly, the above pharmaceutical compositions and vaccines may be used to prevent the development of a cancer or to treat a patient afflicted with a cancer. A cancer may be diagnosed using criteria generally accepted in the art, including the presence of a malignant tumor. Pharmaceutical compositions and vaccines may be administered either prior to or following surgical removal of primary tumors and/or treatment such as administration of radiotherapy or conventional chemo- therapeutic drugs.

The present invention also relates to a complex for a gene transfer into animal cells in vivo or ex vivo, comprising a vector DNA which is condensed and packed into compact nanoparticles, whereby the nanoparticles comprising polyanions selected from the group consisting of transfer RNA, DNA, oligoribonucleotides, polyvinyl sulfate and/or polystyrenesulfonate. Preferred complexes for in vivo transfection are unflocculated in physiological solution. Surprisingly, the particles showed a low polydispersity. The term in vivo administration refers to administration to a patient, for example a mammal, of a polynucleotide encoding a polypeptide for expression in the mammal. In particular, direct in vivo administration involves transfecting a mammal's cell with a coding sequence without removing the cell, from the mammal. Thus, direct in vivo administration may include direct injection of the DNA encoding the polypeptide of interest in the region afflicted by the metabolic disease, tumor or autoimmune disease, resulting in expression in the patient's cells. The term ex vivo administration refers to transfecting a cell, for example, a cell from a population of cells that are under autoimmune attack, after the cell is removed from the patient, for example a mammal. After transfection the cell is then replaced in the mammal. Ex vivo administration can be accomplished by removing cells from a mammal, optionally selecting for cells to transform, (e.g. stem cell) rendering the selected cells incapable of replication, transforming the selected cells with a polynucleotide encoding a gene for expression including also a regulatory region for facilitating the expression, and placing the transformed cells (e.g. stem cell) back into the patient for expression.

In a preferred embodiment of the invention, the nanoparticles have an average particle size of less than 150 nm, more preferably less than 100 nm, and most preferably less than 90, 80, 70 or 60 nm. The selection of ratios may be based on routine experiments to determine polyelectrolyte-combinations which result in desired size. According to the invention it has been surprisingly observed that the in-vivo-particle (that means: complexes for in vivo transfection) should be small (e.g. 70 - 95 nm), stable and unflocculated - preferably with a negative surface charge - and that the in-vitro-particle should be large (e.g. 150 - 550 nm), flocculated and non-stable, preferably with a positive surface charge. In a preferred embodiment of the invention, the ratio of DNA polyanion of the complex is 0,5 -6; more preferred the ratio of DNA/polyanion is 4 -5,5.

In an other preferred embodiment of the invention surface charge of the nanoparticles is essentially negative. More preferred the nitrogen/phosphate ratio of the complex is 4 - 12; most preferred the nitrogen/phosphate ratio is 8. The ratio of DNA/ polycation/ polyanion of the complex is e.g. 0,3-2,5: 0,3-2,5: 2-10 weight/weight/weight.

In an other embodiment of the invention the ratio of DNA/ polycation/ polyanion of the complex is 0,5-2: 0,5-2: 4-11 , weight/weight/weight, 0,5-2: 0,5-2: 4-8; more preferred, the ratio of DNA polycation/ polyanion of the complex is 0,7-1 ,7: 0,7-1 ,7: 5-7; most preferred, the ratio of DNA/ polycation/ polyanion is 1: 1,5 : 6 or 1: 1: 6. Surprisingly, ratios of DNA/ polycation/ polyanion 0,5-1 ,5: 0,5-1 ,5: 4-11 weight/weight/weight of the nanoparticles are most effective, thus rendering the in vivo transfection more economical. Therefore, the complex provided in accordance with the present invention is preferably useful for transfection in living systems; preferred for a cancer-, or an atherosclerosis-gene therapy. Surprisingly, we have found that these preferred complexes are (i) stable and (ii) smaller than 200, 150 or 95 nm and showed (iii) a low polydispersity, and (iv) a low aggregation tendency, (v) a spherical shape, (vi) negative surface charge, and (vii) colloidal stability in physiological salt and preferably protein environment. The combination of these characteristics is favourable for the in vivo gene therapy; preferably via receptor-specific endocytosis.

Preferred stable, non-flocculated and small complexes or nanoparticles for in vivo transfection are obtained by using:

- polylysine as polycation and transfer RNA as polyanion,

- polyethyleneimine as polycation and transfer RNA as polyanion

- polyethyleneimine as polycation and polyvinyl sulfate as polyanion, and

- polyethyleneimine as polycation and polystyrenesulfonate sodium as polyanion.

It was also found that the particles are particularly active in vitro if a lysosomolytical agent was present.

Many methods for introducing complexes into living systems (e.g. cells or tissues) are available and equally suitable for use in vivo, in vitro and ex vivo. For ex vivo therapy, complexes may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. For the purpose of gene therapy the development of efficient and safe gene delivery systems is necessary. Gene transfer research is basically divided into two lines of investigations, namely, viral and non-viral systems. The efficiency of viral systems is generally high, but there are many concerns about their safety due to risk of homologous recombination with subsequent induction of mutagenesis and cancerogenesis, as well as the limitation of repeated administration because of induction of immune response. Although the efficiency of viral systems is generally high, non-viral systems are also under consideration because of their higher safety. Despite progresses have recently been made, the efficiency of non-viral systems is not satisfactory in in vivo animal models and in clinical studies. Colloidal particles (polyplexes and lipoplexes) carrying DNA have been frequently used as non-viral gene vectors. However, their efficiency is still low and has to be improved. One reason for the low efficiency of gene delivery using the above vehicles was the finding that these systems showed the high tendency to aggregate in physiological salt solutions, with the consequence of the inability of cells to uptake the large aggregates in vivo. Other reasons were low dissemination of aggregates in tissues and induction of thrombosis. Only slowly the knowledge was accepted that this behavior originated from the colloidal properties of polycation/DNA complexes in physiological salt solutions and that physicochemical studies are necessary in order to develop complexes without aggregation and with neutral or negative surface charge. The latter is necessary to prevent the interaction with blood and extracellular matrix components. Therefore, colloidal stability, small particle size and negative or neutral surface charge is realized in the in vivo gene particles of the invention. The said gene vectors fulfill the requirements for in vivo therapy. Contrary to that, recent publications (e.g. WO 003694 (Trubestkoy et^'al.), and Trubestkoy et al. 1999) indicates that these requirements can be realized by coating of large and flocculated polycation/DNA complexes with negative polyelectrolytes. This strategy was originally based on studies of different authors concerning the development of polyelectrolyte nanoparticles by stepwise deposition of alternatively charged polyelectrolytes on a charged core. Polyelectrolyte nanoparticles or shells formed in this way are suitable to encapsulate different pharmacological agents, proteins etc. Highly efficient interaction between DNA and polycations results in formation of a condensed particle (core), which could be further condensed by association with the oppositely charged polyelectrolyte. Additional layers of polyelectrolytes with alternating charge can be applied as well. Moreover, additional charged layers might protect encapsulated plasmid DNA both from substitution by the charged plasma components from the DNA-polycation complex outside the cell, and from nuclease degradation inside of cell. It was found that not all of the polyanions are suitable in providing the energy for charge rearrangements required for shell formation without liberating the DNA. In this respect, the car- boxyl/backbone distance or the charge density of the polyanion is an important parameter. Short distance or high charge density results in dissociation of the binary complex between DNA and polylysine, and large distance leads to stable complexes. The present invention indicates that these requirements can be realized by coating of the polycation/DNA complexes with negative polyelectrolytes: natural polyanions as transfer RNA, as well as synthetic polyanions, e.g. polyvinylsulfate or polystyrenesulfonate, whereby compact particles with low aggregation capacity are obtained. The present invention also relates to a pharmaceutical composition comprising a therapeuti- cally effective amount of the said complex and one or more pharmaceutically acceptable adjuvant, excipient, carrier, buffer, diluent and/or customary pharmaceutical auxiliary. The term pharmaceutical agent or pharmaceutically active agent as used herein encompasses any substance that will produce a therapeutically beneficial pharmacological response when administered to a subject, including both humans and animals. More than one pharmaceutically active substance may be included, if desired, in a pharmaceutical composition used in the method of the present invention. Accordingly, it is presently preferred that the DNA or RNA molecule introduced as a pharmaceutical agent - e.g. a vaccine - to induce a therapeutic effect - e.g. a protective immune response - encodes not only the gene product (i.e., active agent) to be expressed, but also initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the vaccinated subject. The vaccine polynucleotide can optionally be included in a pharmaceutically acceptable carrier as described herein.

The pharmaceutically active agent can be employed in the present invention in various forms, such as molecular complexes or pharmaceutically acceptable salts. Representative examples of such salts are succinate, hydrochloride, hydrobromide, sulfate, phosphate, nitrate, borate, acetate, maleate, tartrate, salicylate, metal salts (e.g., alkali or alkaline earth), ammonium or amine salts (e.g., quaternary ammonium) and the like. Furthermore, derivatives of the active substances such as esters, amides, and ethers which have desirable retention and release characteristics but which are readily hydrolyzed in vivo by physiological pH or enzymes can also be employed.

As used herein, the term therapeutically effective amount or effective amount means that the amount of the biologically active or pharmaceutically active substance is of sufficient quantity and activity to induce a desired pharmacological effect. The amount of substance can vary greatly according to the effectiveness of a particular active substance, the age, weight, and response of the individual subject as well as the nature and severity of the subject's condition or symptoms. Accordingly, there is no upper or lower critical limitation upon the amount of the active agent introduced into the cells of the subject although it is generally a greater amount than would be delivered by passive absorption or diffusion, but should not be so large as to cause excessive adverse side effects to the cell or tissue containing such cell, such as cytotoxicity, or tissue damage. The amount required for transformation of cells will vary from cell type to cell type and from tissue to tissue and can readily be determined by those of ordinary skill in the art using the teachings herein. The required quantity to be employed in practice of invention methods can readily be determined by those skilled in the art. In one embodiment of the invention method, the amount of active agent such as a nucleic acid sequence encoding a gene product introduced into the cells is a transforming amount. A transforming amount is an amount of the active agent effective to modify a cell function, such as mitosis or gene expression, or to cause at least some expression of a gene product encoded by the nucleic acid sequence.

The term slow release polymer, such as a polyol, refers to a polyol which is biocompatible and which is capable of maintaining high pericellular concentrations of the expression vehicle. The polymer may be cationic, anionic, or non-ionic. In one embodiment, the polymer is a non-ionic polyol. In one embodiment, the non-ionic polyol is a polyoxyalkylene polymer, and more preferably a polyoxyalkylene block copolymer, wherein the polyoxyalkylene groups each have from 2 to 5 carbon atoms. The polyoxyalkylene polymer may have a molecular weight of from about 300 to about 40,000, preferably from about 900 to about 20,000. In a more preferred embodiment, the polyoxyalkylene block copolymer is a polyoxypropylene- polyoxyethylene block copolymer. In one embodiment, the polyoxypropylene component is present in an amount of from about 20 to about 90 wt. % of the copolymer, and the poly- oxyethylene component is present in an amount of from about 10 wt. % to about 80 wt. % of the copolymer. In a preferred embodiment, the polyoxypropylene is present in an amount of about 30 wt. % of the copolymer, and the polyoxyethylene is present in an amount of about 70 wt. % of the copolymer. An example of such a polyoxypropylene-polyoxyethylene block copolymer which may be employed in accordance with the present invention is a poly- oxypropylene-polyoxyethylene block copolymer having a molecular weight of about 12,500, and sold under the trade names Pluronic F127 or Poloxamer 407 by BASF Corporation, Chemical Division, Parsippany, NJ. It is to be understood, however, that the scope of the present invention is not to be limited to any particular polyol.

The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, in- tramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intra- nasal, enteral, topical, sublingual, or rectal means. In a preferred embodiment of the invention the complex of the invention can be administered in a pharmaceutically acceptable formulation. The present invention pertains to any pharmaceutically acceptable formulations, such as synthetic or natural polymers in the form of macromolecular complexes, nanocap- sules, microspheres, or beads, and lipid-based formulations including oil-in-water emulsions, micelles, mixed micelles, synthetic membrane vesicles, and reseated erythrocytes. In addition to the complex and the pharmaceutically acceptable polymer, the pharmaceutically acceptable formulation used in the method of the invention can comprise additional pharmaceutically acceptable carriers and/or excipients. As used herein, pharmaceutically accept- able carrier includes any and all solvents, dispersion media, coatings, antibacterial and anti fungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. For example, the carrier can be suitable for injection into the tumor or the artery wall. In another embodiment, the complex may be incorporated or impregnated into a bioab- sorbable matrix. In addition, the matrix may be comprised of the said polymer. A suitable biopolymer for the present invention can include also one or more maeromolecules selected from the group consisting of collagen, elastin, fibronectin, vitronectin, Iaminin, polyglycolic acid, hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparin sulfate, heparin, fibrin, cellulose, gelatin, polylysine, echinonectin, entactin, thrombospondin, uvomorulin, biglycan, decorin, and dextran. The formulation of these maeromolecules into a biopolymer is well known in the art. In constructing the matrix, it may be useful for the matrix to further include a substructure for purposes of administration and/or stability. Suitable substructures include freeze dried sponge, powders, films, flaked or broken films, aggregates, microspheres, fibers, fiber bundles, or a combination thereof. In addition, the matrix may be attached to a solid support for administration purposes. Suitable supports depend upon the specific use and can include a prosthetic device, a porous tissue culture insert, an implant, a suture, and the like. Therapeutic compositions of the present invention may include a physiologically tolerable carrier together with at least one species of complex of this invention as described herein, dispersed therein as an active ingredient. It is further contemplated that the complexes as described herein can be used therapeutically in a variety of applications. For example, as described above, a variety of useful compositions and formats, including bioab- sorbable materials or matrices may be used in conjunction with the complex of the present invention to coat the interior of tubes used to connect severed arteries; they may be added directly to suture materials or incorporated in bioabsorbable materials in and on sutures; further, they may be utilized on/in implants and prosthetic devices, either alone or in conjunction with other bioabsorbable and supporting materials. Thus in one embodiment, a complex of this invention can be incorporated into a bioabsorbable matrix, which matrix can be formulated into a variety of mediums, including a semi-solid gel, a liquid permeable but porous insoluble matrix, or a porous biopolymer as described further herein. A variety of useful compositions, including bioabsorbable materials (e.g., collagen qels) may be used in conjunction with a complex of the present invention in a variety of therapeutic applications.

A therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isoprdpylamine, trimethylamine, 2- ethylamino ethanol, histidine, procaine and the like. Physiologically tolerable carriers are well known in the art. Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate- buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, propylene glycol, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, organic esters such as ethyl oleate, and water-oil emulsions. A therapeutic composition contains a complex of the present invention, typically an amount of at least 0.1 weight percent of complex per weight of total therapeutic composition. A weight percent is a ratio by weight of complex to total composition. Thus, for example, 0.1 weight percent is 0.1 grams of complex per 100 grams of total composition.

Thus, the dosage ranges for the administration of a complex of the invention are those large enough to produce the desired effect in which the condition to be treated is ameliorated. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, and sex of the patient, and the extent of the disease in the patient, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication. The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. A therapeutic amount of a complex of this invention is an amount sufficient to produce the desired result, and can vary widely depending upon the disease condition and the potency of the therapeutic compound. The quantity to be administered depends on the subject to be treated, the capacity of the subject's system to utilize the active ingredient, and the degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the conditions of administration. Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent administration. A therapeutically effective amount of a complex of this invention is typically an amount such that when it is administered in a physiologically tolerable composition, it is sufficient to achieve a plasma or local concentration of from about 0.1 to 1 ,000 micromolar (uM), preferably about 1 to 100 uM. Alternatively, the dosage can be me- tered in terms of the body weight of the patient to be treated. In this case, a typical dosage of a therapeutic composition is formulated to deliver a pharmacologically active complex of this invention is amount of about 0.1 microgram (ug) to 100 ug per kilogram (kg) body weight, or more preferably about 1 to 50 ug/kg. Furthermore, certain utilities of the present invention involve local administration of a pharmacologically complex to a site of plaque or lesion (e.g. an aterial wall lesion or ateriosclerosis), and therefore is best expressed in unit dosage form. Such local administration is typically by topical or local administration of a liquid or gel composition containing about 1 to 1000 micrograms (ug) of complex per milliliter (ml) of composition, preferably about 5 to 500 ug/ml, and more preferably about 10 to 100 ug/ml. Thus a therapeutic composition can be administered via a solid, semi-solid (gel) or liquid composition, each providing particular advantages for the route of administration. A complex of the invention can be administered parenterally by injection or by gradual infusion over time. For example, a complex of the invention can be administered topically, locally, perilesionally, perineuronally, intracranially, intravenously, intrathecally, intramuscularly, subcutaneously, intracavity, transdermally, dermally, or via an implanted device, and they may also be delivered by peristaltic means. The complex of the present invention are typically administered as a pharmaceutical composition in the form of a solution, gel or suspension. However, therapeutic compositions of the present invention may also be formulated for therapeutic administration as a tablet, pill, capsule, aerosol, sustained release formulation or powder.

In another embodiment of the method of the invention, the pharmaceutically acceptable formulation provides sustained delivery, e.g., slow release of the complex of the invention to a subject for at least one, two, three, or four weeks after the pharmaceutically acceptable formulation is administered to the subject. As used herein, the term subject is intended to include animals, preferably mammals. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the primate is a human. Other examples of subjects include dogs, cats, goats, and cows. As used herein, the term sustained delivery is intended to include continual delivery of complex species in vivo over a period of time following administration, preferably at least several days, a week or several weeks. A therapeutically effective amount of slow release formulation may vary according to factors such as the disease state, age, and weight of the subject, and the ability of the complex species to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response.

The invention further contemplates a apparatus that comprises a bioabsorbable matrix and an effective amount of said complex or the pharamaceutical agent. The matrix can include a substructure comprising freeze dried sponge, powders, films, flaked or broken films, aggregates, microspheres, fibers, fiber bundles, or a combination, thereof. The solid support can be formulated into a prosthetic device, a porous tissue culture insert, an implant and a suture. The invention also relates to an arteriosclerosis/athrosclerosis-treating apparatus comprising a bioabsorbable matrix and an effective amount of a pharmaceutical composition of the invention. As used herein the term arteriosclerosis is a degeneration of the walls of the arteries due to the formation of foam cells and aortic streaks which narrow the arteries. This limits blood circulation and predisposes an individual to thrombosis. Atherosclerosis in the context of the invention is a cardiovascular condition occurring as a result of narrowing down of the arterial walls. The narrowing is due to the formation of plaques (raised patches) or streaks in the inner lining of the arteries. These plaques consist of foam cells of low-density lipoproteins, bxidized-LDL, decaying muscle cells, fibrous tissue, clumps of blood platelets, cholesterol, and sometimes calcium. They tend to form in regions of turbulent blood flow and are found most often in people with high concentrations of cholesterol in the bloodstream. The number and thickness of plaques increase with age, causing loss of the smooth lining of the blood vessels and encouraging the formation of thrombi (blood clots). As used herein the term athrosclerosis is a disease of the arteries in which fatty plaques develop on the inner walls, with eventual obstruction of blood flow. Both arteriosclerosis (calcium deposits) and athrosclerosis (fat deposits) involve a buildup on the inside of artery walls. As used herein the term cardiovascular disease is a disease of the blood vessels of the circulation system caused by abnormally high concentrations of lipids in the vessels. It is noted that term artery is presented only by way of example and that the present invention is applicable for any type of tubular organ. Therefore, the term artery is used herein in a very broad sense to encompass arteries, veins, internal organs and the like.

Without intending to be limiting, the invention will be illustrated with reference to the examples below.

Examples

1. Preparation and characterization of DNA-polyelectroiyte shells for the in vitro transfection of cell cultures

Vector DNA and cationic polymer are mixed at room temperature in excess polycation in water as described above. In this case, pCMV Luc and polylysine of m.w. 58,000 at a ratio of 1 :1.5 (w/w) based on 2 μg of DNA are used. To these complexes is added excess transfer RNA (type V or XI from Sigma) so as to make a DNA/polylysine/RNA mixing ratio of 1 :1.5:6 (g/g/g). The solution is brought to physiological saline concentration by adding 3 M NaCI. This batch is directly used for transfection purposes.

Similar formulations were prepared using polyethyleneimine (PEI) as polycation and polyvinyl sulfate (PVS) or polystyrenesulfonate sodium (PSSS) as polyanions.

Ten such batches are combined for physicochemical analysis. The shells having formed are examined for size and aggregation tendency by means of dynamic light scattering in a Zeta- sizer 3000 HS (Malvern Instr., Ltd, Maivern, England). Fig. 1 shows complex formation starting with the polylysine/DNA complex in water. Illustrated therein is the successive change of these complexes when adding the DNA and transferring into NaCI. Based on charge rearrangements, a relatively homogeneous complex size distribution is established.

Fig. 2 shows the zeta potential of the shells (DNA polylysine/RNA 1 :1.5:6, w/w/w), measured in the same instrument, compared to the polylysine/DNA (1:1.5, w/w) complex which constitutes the basis of the shells. As can be seen, the positive polylysine/DNA complex (+21.2 mV) is converted to the negatively charged shell (-37.1 mV) as expected.

In contrast to the polylysine/DNA complexes, these shells cannot be separated by centrifugation at 4000 x g for 2 minutes, or only in part. This simple test, demonstrating reduced aggregation tendency, can be used for initial identification of the particles. Fig. 3 shows such shells, imaged by atomic force microscopy.

2. In vitro transfection of ECV 304 cells with DNA-polyelectrolyte shells

The final shells produced as in 1., based on 2 μg of DNA (pCMV Luc), in 200 μl of working buffer (0.15 M NaCI, 10 mM Tris-HCI, pH 7.6) are filled up with 0.8 ml of ceil culture medium and added to the washed cells (2 x 10 cells). RPM I with 10% FCS is used as cell culture medium. The transfection mixture remains on the cells for 2-4 hours, the cells are washed with RPMI, fresh culture medium (RPMI, 10% FCS), optionally added with 0.1 mM chloro- quine (or 2 mM CaCl2), is added as post-transfection medium, and this is incubated for about 24 hours in a 5% CO2 atmosphere. Subsequently, the assay or, in the event of stable expression, the selection is started. In the post-transfection batch, the chloroquine remains on the cells for 24 hours. In this event, the toxicity of the batch is considerably lower than it would be if the DNA was present over the entire period of time. The results are illustrated in Fig. 4. While the transfection efficiency of the supernatant subsequent to centrifugation of the shells at 4000 x g for 2 minutes is lower than that without centrifugation, it is not zero as is the case with polylysine alone (the blank value is at 2000 RLU). Consequently, some of the particles cannot be removed by centrifugation, i.e., are small in size. The RLU values relate to RLU/20 μl cell lysate of 150 μl/well. To obtain the RLU values per mg of protein, the former have to be multiplied by a factor of 100. Hence, this type of shell affords 10 RLU/mg protein.

Shells of the type DNA(pCMV Luc)/PEI/PVS at a ratio of 1 :1 :6 (g/g/g) and DNA/PEI/PSSS at

7 8 a ratio of 1 :1 :11 furnish transfection rates of 10 -10 RLU/mg protein on ECV 304 cells. By using aggregated shells at smaller polyanion concentration, higher transfection rates can be obtained. 3. Direct injection of shells into tumors

Transplantation tumors were generated in nude mice using human tumor cells which were previously checked for highest in vitro transfection efficiency using shells. The best in vitro

Q transfection rates were observed in the range of 10 RLU/mg protein with HCT-116 (colon) and MCF-7 (mamma) cells. Works were performed on tumors generated with HCT-116. After 10 days, the tumors had a diameter of about 4 mm. DNA/PEI/PVS shells and DNA/PEI/PSSS shells with a weight ratio of 1:1:6 and 1 :1 :11 , respectively, based on merely 1.65 μg and 3.5 μg of DNA (pCMV Luc), respectively, were prepared (see application examples 1.). As to the PVS shells, DNA and PEI were in about equal volumes (12.5 μl) prior to mixing. As to the PSSS shells, to 6.3 μl of DNA 3.7 μl of PEI was added. Following mixing, the PEI/DNA complexes were allowed to stand for 10 minutes. Subsequently, PVS and PSSS, respectively, were added in such a way that both types of shells were in 50 μl of 20 mM HEPES after mixing the components. Thereafter, 8.8 μl of 1 M NaCI is added to make a final concentration of 0.15 M NaCI. The shells firstly are characterized by means of electron microscopy after 15 minutes, with a similar result as in 3. (Fig.5), and secondly injected directly into the tumors through the skin. The animals were sacrificed after 24 hours, the tumors were removed, and the homogenate was analyzed for luciferase activity using a luciferase reporter gene assay and a luminometer. The results were reproducible, indicating high efficiencies: (8.2±4.15 SD) x 10⁵ RLU/tumor for the PSSS shells and (2.3±1.5 SD) x 10⁵ RLU/tumor for the PVS shells. The PEI/DNA controls (1 :1.5 g/g) were positive: (5.5±6.2 SD) x 10 RLU/tumor. When assessing these results, however, one should keep in mind that these shells are particles having a negative surface charge, whereas PEI/DNA complexes have positive charge. This suggests varying mechanisms and possibly, varying transfected cell types in the tumor. Furthermore, reference is made to the favorable fact that nonspecific interactions with negatively charged serum components and with components of the extracellular matrix can be excluded when using shells.

4. Vascular gene delivery

In the example 4 our aim was to create on the shells-based technology nanoparticles which will exhibit colloidal stability and high transfection efficiency in vascular systems. Shells were prepared in two steps: First the polycationic core was produced by mixing plasmid DNA (pC3 ^β-gal DNA)with polyethylenimine (PEI) in HEPES buffer. A fixed nitrogen/phosphate ratio of N/P = 8 was used in all experiments. Covering of the core as the second step was performed with either natural polyanions such as transfer-RNA (tRNA) or synthetic polyanions, e.g. polyvinylsulfate (PVS). Optimal colloidal stability was empirically determined and achieved at the following ratio: DNA/PEI/polyanion 1 :1 :6 w/w/w. The resulting shells were characterized in HEPES buffer and physiological salt solution according to size/aggregation by PCS and surface charge by Zeta-potential (Table 1). Electron microscopy is shown in Fig. 6 D,E,F. Shells showed a spherical shape, negative surface charge and low aggregation tendency under physiological conditions.

For vascular gene delivery we devised an application method using Pluronic gel F127 as a biodegradable adhesive to keep shells in contact with the targeted vessel. Briefly, the carotid artery was injured using a balloon catheter, and 0.5 ml Pluronic gel (40%) in PBS containing either preformed shells or not covered PEI/DNA cores were placed around the injured artery segment. The shells contained 6.6 μg pcDNA3 β-gal plasmid or 3.3 μg per animal for tRNA- or PVS shells, respectively. After operation, the gel kept the shells in close contact with the vessel for about three days. The vessel segments were assayed for β-galactosidase gene expression after 4 days. Fig. 7 shows β-galactosidase expression in the rat carotid artery using pcDNA3 β-gal containing tRNA shells (Fig. 6A) or the corresponding PVS shells (Fig. 6B). Fig. 6C shows a PEI/DNA control, β-galactosidase was expressed in more than 50 % of the cells in the periadventitia and in 20% cells in the adventitia, but only in a few cells of the media. The DNA/PEI (core) control containing 6.6 μg DNA was virtually inactive.

Human uPA expression and cell accumulation in the developing neointima: Basing on the fact that urokinase plasminogen activator (uPA) stimulates the migration and proliferation of vascular SMC, the key processes in vessel wall remodeling after injury, we compared the effect of uPA gene delivery on accumulation of neointimal cells after balloon catheter injury with the effect of recombinant human uPA application. As we have shown previously, the recombinant uPA resulted in augmented neointimal cell accumulation after vessel injury. The tRNA shells containing 6.6 μg of the plasmid encoding human uPA (Hu-uPA) were applied periadventitially as described above. Contrails were pcDNA3 β-gal plasmid delivered in tRNA shells (irrelevant for this case), pure Pluronic F127 gel and Pluronic F127 containing 20 nmol/kg recombinant Hu-uPA. On the 7th day animals were sacrificed, and left and right carotids were subjected to histochemical analysis. Perivascular recombinant Hu-uPA application more than doubled the number of SMCs in the neointima compared to control pure gel (Table 2). The increases in neointima SMC numbers were also observed after Hu-uPA cDNA application but it was less pronounced than the effect of recombinant uPA (Fig. 7A, Table 2). Control application of pcDNA3 β-gal plasmid did not affect neointima cell numbers (Table 2). Human uPA immunoreactive peptides were not detected in the uninjured and injured rat carotid arteries after application of pure gel or pcDNA3 β-gal plasmid (Fig. 7C,D). The expression of Hu-uPA was detected only in injured left carotid artery after Hu-uPA cDNA application and localized predominantly to periadventitial, adventitial and neointimal cells (Fig.7A). Only unit cells in the media expressed Hu-uPA. Controls (Fig. 7 B,C,D) did not exhibit positive reaction with anti-Hu-uPA antibodies. Our aim in example 4 was the creation of a new, high effective in vivo non-viral gene delivery system. It is evident from an extensive body of literature that such a system must have certain properties, namely nanosize, colloidal stability in physiological salt and preferably protein environment, as well as the possibility to carry foreign DNA into the cells in vivo with their subsequent expression. Several attempts in this direction are reported by Trubetskoy et al. In their recent paper they used the principle of recharging of cationic DNA complexes with highly charged polyanions and obtained efficient transfection in vitro and in mice lungs. Different groups also designed shell-like particles using protective copolymers, but no in vivo application were demonstrated. Since our aim was to assemble a transfection system which could work without any additional enhancing agents, we used polyethylenimine (PEI) as DNA compacting agent. PEI is known as an effective DNA carrier, that due to endosomolytic properties can perform transfection without chloroquine or fusogenic peptides. N/P=8 was chosen because on the one hand it was demonstrated that transfection complexes with this ratio show high efficacy on a wide range of different cell lines and, on the other, being assembled in Hepes buffer, have small size, low polydispersity and positive charge (see Table

1 ).

Now, we focus on a variety of shells with different composition. Two different anionic polymers were tested: natural ones as transfer RNA type V (tRNA) from wheat germ and synthetic ones as polyvinylsulfate (PVS). Coating was performed by adding of different amounts of polyanions to the preformed PEI/DNA cores. The ratio polyanion/DNA w/w (R|) was step- wise varied from 0.5 till 6. The main selection criterium was the absence of aggregation of complexes after raising the salt concentration up to physiological. Rough turbidity experiments were performed by spectrophotometry by measuring the absorption of particle containing solutions at λ=400 nm (data not shown). It was empirically found out that the desired colloidal stability of the shells was achieved at a mass ratio polyanion/DNA of equal to 6. The properties of these complexes were more deeply investigated by electron microscopy, dynamic light scattering and measurement of the ζ potential (see Fig. 7 D,E and Table 1). The staining pattern of the particles with uranyl acetate and negative surface charge clearly showed that we really obtained an encapsulation of the PEI/DNA core by polyanions into a shell-like structure. The absence of aggregation and the negative surface charge (ζ potential) were good prerequisites for in vivo gene delivery. It is possible to presume no interaction of shells with plasma proteins and no sticking to the blood vessels. It is also worth to mention that we examined the particles with all the rows of ratios for transfection activity in vitro. Efficacy in vitro was diminishing with the reduction of aggregation tendency of the particles. At a mass ratio polyanion/DNA of 6, shells were nearly transfection-inactive (data not shown). For the vascular gene delivery we devised an application method at which Pluronic F127 gel was used as a biodegradable adhesive. This method allows to keep shells in contact with the targeted vessel up to 3 days and therefore gives an opportunity to apply low doses of foreign DNA. In our case it was possible to get high gene delivery efficiency even with 3.3 μg of plasmid DNA per animal.. Experiments with the β-gal reporter gene revealed that β- galactosidase was expressed in > 50 % of the cells in the periadventitia and in up to 20% cells in the adventitia. Expression was observed up to a depth of about 15 cell layers. Therefore, shells showed a high gene delivery efficiency and are able deeply to penetrate into tissues. The DNA/PEI (core) control containing 6.6 μg DNA was nearly inactive (Fig. 7A,B,C). We found β-galactosidase only in a few cells of media. May be it requires more DNA. In each case, we supposed that this difficulty can be overcome if the product encoded by the plasmid will be secreted by transfected cells and then distributed within the vessel wall.

It is well known that urokinase plasminogen activator (uPA), secreted by many type of cells, is capable of stimulating SMC migration and proliferation in vitro and in vivo and is expressed early after injury, when neointima is developing and medial smooth muscle cells are proliferating. In this example, we have shown that the application of RNA shells containing 6.6 μg of Hu-uPA encoding plasmid imitates the effect of recombinant uPA. to the injured vessel. Histochemical analysis revealed the distribution of transgene Hu-uPA expression allover the rat vessel wall. This evidently results in a significant increase in the accumulation of SMCs in the neointima as a result of increased cell migration and proliferation (Fig.1 ).

These results and data on direct injection of shells free of Pluronic F127 gel into transplantation tumors (not shown here) suggest that this system is suitable not only for gene delivery to blood vessels, but can be also efficiently applied for gene transfer to other organs and tissues. We therefore believe that this method represents a significant progress in the development of non-viral gene delivery systems for in vivo gene transfer and will continue to study it in more detail.

Table 1 Physical characterization of the shells.

Standard deviation given, at least 3 experiments Table 2 Neointimal cell number per vessel cross-section

Values are mean + SEM

*p<0.05 versus βGal; #p<0.05 versus Gel F127 Materials and methods

Shell assembly

The reporter plasmid pcDNA3 β-gal as well as the plasmid pcDNA3 Hu-uPA were grown in E. coli DH5, isolated and purified using standard methods (Qiagen Plasmid Maxi Kit, Hilden, Germany). Transfer RNA Type V from wheat germ, Polyethylenimine (PEI), Pluronic F127 were from Sigma, USA. Polyvinylsulfate (PVS) was from Wako Pure Chemical lnd. Ltd., China.

Shells were prepared in Hepes buffer (20mM, pH 7,4) by adding the polyanion to the preformed core - DNA/PEI complex (N/P=8). The following conditions were selected: DNA/PEI/polyanion (1 :1 :6 w/w/w).

Physicochemical measurements

Size and Zeta potential measurements were performed using a Malvern Zetasizer (Malvern lnd., England).

Animals

Male Wistar-Kyoto rats (4 to 5 months old) were obtained from a colony maintained at the Cardiology Research Center, Moscow, Russia. Their left common carotid artery was subjected to balloon catheter injury using surgical procedures approved by the Cardiology Research Center's Animal Experimentation Committee.

Surgery and gene delivery into the arterial wall

Rat carotid arteries were injured with an inflated balloon catheter as described previously. Briefly, after anesthetizing the rats with ketamine (100 mg/kg body wt, i.p., Pfaffen- Schwabenheim, Germany), a midline incision was made in the neck to expose the left external carotid artery. A 2 F Fogarty arterial embolectomy catheter (Baxter Healthcare, USA) was introduced into this artery through an arteriotomy and passed into the common carotid artery to the aortic arch. The balloon was inflated and then slowly rotated while pulling the catheter back towards the external carotid artery. This was repeated three times and then the external carotid artery was ligated. The contralateral right carotid artery as well as uninjured left carotid arteries from sham-operated rats served as controls. For periadventitial administration of shells, the arteries were gently dissected free of their surrounding connective tissue and then 0.5 ml Pluronic gel (40%) in PBS containing preformed shells or not covered cores was placed around the injured artery. Pluronic F127 (50%) gel was. prepared in PBS at 4°C and mixed with the shells (100 μi particle solution plus 400 μl of gel). After mixing salt concentration was adjusted to 0.15 M NaCI. The gel polymerized once loaded on the vessel segment of appr. 1.5 cm length. The incisions were closed and the animals were allowed to recover, β-galactosidase reporter gene and Hu-uPA gene activities were assessed in the vessel segment after 7 days.

Tissue collection and processing

The animals were deeply anesthetized with sodium pentobarbital (100mg/kg body wt), and Evans blue (60 μg/kg body wt; IV) was administered, so that removal of endothelium in the damaged vessels could be confirmed. Then the animals were perfused (120mm Hg) with saline solution, followed by 4% formaldehyde solution for 10 minutes. Left and right common carotid arteries were removed, cleaned from extraneous material and cut into three equal segments before embedding in paraffin or Tissue-Tek (Sakura Finetek, Netherlands). Cross- sections (7 μm for immunohistochemistry), were cut from each block, at 100 to 200 μm intervals.

Histochemistry and microscopy

Electron micrographs of shells were obtained with a Zeiss EM 910 electron microscope at an accelerating voltage of 80 kV after negative staining of the samples with 1 % uranyl acetate on formvar coated grids.

For the detection of β-galactosidase expression, the excised aorta segments were fixed in PFM, stained with β-gal assay, cut into slices of 7 μm in deeply frozen state, lightly stained with haematoxylin and analyzed by microscopy.

The immunohistochemical detection of Hu-uPA was performed on frozen sections (7 μm) according to the standard method used in our laboratory. The sections were fixed in 10 % solution of formalin in PBS for 2 minutes at room temperature, than they were fixed in 96% ethanol for 30 minutes at -20° C. The activity of endogenous peroxidase was blocked by incubation of sections in 3 % solution of hydrogen peroxide in PBS for 30 minutes at room temperature. The non-specific staining caused by secondary antibodies was blocked by incubation of sections in 10 % solution of normal horse serum in 1% BSA/PBS for 30 minutes at room temperature. The sections were immunoreacted with solution of mouse monoclonal antibodies (Ab) against Hu-uPA (Loxo, Dossenheim, Germany) in 1% BSA/PBS, concentration 6.7 μg/ml for 1 hour at room temperature. Bound primary Ab were detected with biotin- conjugated horse anti-mouse antibodies in 1% BSA/PBS (Vector Laboratories, Burlingame, Ca, USA), concentration 5.0 μg/ml and with Vectastain ABC Kit (Vector Laboratories, Burlingame, Ca, USA). The staining was visualized with the diaminobenzidine reaction (DAB chromogen system; Immunotech, Hamburg, Germany). Sections were lightly counterstained by Mayer's haematoxylin and analyzed by microscopy.

Total cell numbers in the neointima (i.e. hematoxylin stained nuclei in the neointima) were determined by counting (three sections per rat).

All results are mean + standard error of mean (S.E.M.). Comparisons between groups were performed using the one-way analysis of variance (ANOVA). A value of P<0.05 was considered statistically significant. All statistical analyses were performed using 'Jandel SigmaS- taf.

6. Incorporation of ligands into DNA-polyelecrolyte shells.

Shells containing peptidic nuclear localization signals were constructed as an example for the incorporation of receptor-specific ligands. Nuclear localization signals target specific factors of the nuclear transport maschinerie of the cell and facilitate the transfer of the DNA from the cytoplasm into the nucleus.

The following synthetic peptides contain 16 lysine residues for DNA binding fused to a nuclear localization signal:

NLS: CKKKKKKKKKKKKKKKKGGGPKKKRKVG SLN: CKKKKKKKKKKKKKKKKGGGGVKRKKKP VN: KKKKKKKKKKKKKKKKGGGCNEWTLELLEELKNEAVRHF

CYC: -1 VKLKVYPLKKKRKP '

KKKKKKKKKKKKKKKKC

The NLS contains a nuclear localization signal derived from the SV 40 T-large antigen. SLN is a nuclear transport deficient control peptide with an identical chemical composition of the NLS. VN contains a nuclear localization signal from the N-terminal HIV 1 Vpr and is known to follow a route into the nucleus different from the SV 40 T-large antigen derived signal. CYC represents an optimized version of the NLS and consists in a cyclic peptide containing the SV 40 T-large antigen derived nuclear localization signal and was fused to the oligolysine DNA-binding domain. These peptides were used as the DNA condensing agent in the shells.

2 μg plasmid DNA was pre-condensed with the peptide in water at a peptide to DNA charge ratio of 1.5 for the peptides NLS, SLN and CYC and of 0.8 for VN. PVS was then added to different PVS to DNA weight ratios, and 150 mM NaCI was subsequently added. Complex morphology was controlled using transmission electron microscopy following uranyl acetate staining. Colloidal stable particles were typically obtained with a PVS to DNA weight ratio between 0.6 and 0.875, depending on the peptide. At these ratios, shells displaying a negative Zeta-potential are generated. Particles were also characterized using dynamic light scattering for measuring the apparent the size and the ethidium bromide intercalation assay to evaluate the condensation rate of the encapsulated DNA.

Shells were used in transfection experiments of human colon carcinoma cells HCT116. Figure 8A shows the obtained expression levels of the luciferase reporter gene. The incorporation of the nuclear localization signal into the shells lead to a specific 10-fold increase of the gene expression as compared to the SNL control peptide. The incorporation of the peptide containing the optimized signal (CYC) lead to a further 100-fold increase of gene expression as compared to the linear NLS peptide. Figure 8B shows that colloidal stable nanoparticles of similar size and morphology were effectively used in the transfection experiments, demonstrating that the obtained enhancement of gene expression was due to the specific activity of the ligand.

In the mentioned transfection experiments, chloroquine and CaCI were also added to the transfection medium containing 10 % serum. Similar transfection results were also obtained without the presence of chloroquine, CaCI₂ or serum.

Figure 9A shows the apparent size of shells constructed with the peptides using increasing amounts of PVS. Aggregation was lost at PVS to DNA weight ratios between 0.25 and 0.4 depending on the peptide. Moreover, the shells used in the transfection experiments displayed an apparent size between 100-200 nm which is in a good agreement with the transmission electron microscopy observations. In Figure 9B, the polydispersity index obtained following dynamic light scattering experiments of the shells is shown. The results shows that polydispersity increases slightly when the PVS to DNA weight ratio becomes sufficient to overcome aggregation. With a further increase of PVS, the polydispersity decreases and reaches a relative minimum value which reflects a particular ratio in which the generated particles have a highly uniform size distribution. This was confirmed by transmission electron microscopy observations.

In order to examine the condensation rate of the DNA within the shells, the ethidium bromide intercalation assay was used (Figure 10) on complexes containing the NLS peptide. The fluorescence generated by naked DNA in water was set to 100% and represents complete decondensation of the DNA. After adding the peptide, fluorescence decreased to 6.5 % indicating a highly condensed state of the DNA. After the addition of PVS, fluorescence was recovered to 85 %, demonstrating an extensive dissociation of the complex. This was also confirmed by electron microscopy and dynamic light scattering. Finally, the addition of salt to a final concentration of 150 mM induced a significant recondensation of the DNA (55% of relative fluorescence). This condensation rate is also observed in a naked DNA solution containing 150 mM NaCI (Fig. 10). Therefore, the DNA molecules within the shells display a particular uncondensed structure, which may account to the colloidal stability of the particles as well as to a facilitated intracellular dissociation of the DNA.

Different particle formation procedures using the NLS were also applied. The resulting shells were characterized regarding their apparent size, their polydispersity and their capacity for ethidium bromide intercalation (Table 3). PVS was added to preformed DNA/peptide complexes in water (preparation 1 ) or in salt (preparation 3). Peptide was also added to a pre- incubated DNA/PVS-mixture in water (preparation 2) or salt (preparation 4). The results shows no significant differences concerning size, polydispersity and condensation rate between the preparations. This was also confirmed by electron micoscopy.

In addition to plasmid DNA bearing the luciferase reporter gene, shells with the same properties were also constructed with DNA containing the GFP reporter gene. Furthermore, peptides containing the RGD-binding motif for cell type specific targeting of the complex were also used to construct DNA-polyelectrolyte shells in a similar manner.

Equivalents: Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Legends to the Figures

Fig. 1 : Monitoring the preparation of DNA-polyelectrolyte shells by means of size determination using quasi-elastic light scattering. Illustrated therein are the scattering intensities as a function of the particle size (size distribution).

A: DNA/polylysine complexes (1 :1.5 w/w) in water; B: DNA/polylysine complexes (1 :1.5 w/w) in 0.15 M NaCI. The insert shows aggregates >1000 nm. C: DNA/polylysine/tRNA shells (1 :1.5:6 w/w/w) in water, centrifuged at 4000 x g for 4 minutes, as supernatant. D: DNA/polylysine/RNA shells (1 :1.5:6 w/w/w) in water, centrifuged at 4000 x g for 2 minutes (supernatant), subsequently transferred into 0.15 M NaCI. The measured batches relate to 10 μg of DNA.

Fig. 2: Zeta potential of DNA/polylysine complexes (1 :1.5 w/w) in water (A), and DNA/polylysine/tRNA shells (1:1.5:6 w/w/w) in water (B).

Fig. 3: Atomic force microscopy of DNA/polylysine/tRNA shells (1 :1.5:6 w/w/w) in TAE buffer (40 mM Tris acetate, 1 mM EDTA). Fig. 4: Transfection of ECV 304 cells with DNA/polylysine complexes (1 :1.5 w/w) and DNA/polylysine/tRNA complexes (1 :1.5:6 w/w/w), prepared using 2 commercially available transfer RNAs supplied by Sigma. The solution of the shells (0.2 ml) included 0.15 M NaCI. The shells in centrifuged form as supernatant (shaded) or without centrifugation (white) were added to 0.8 ml of transfection medium (RPMI) and added to the cells. 2 μg of pCMV Luc DNA, 2 x 10⁵ ECV 304 cells, 2 hours transfection in RPMI, post-incubation in RPMI, 0.1 mM chloroquine.

Fig. 5: Electron microscopy of DNA/PEI/PSSS shells (1:1:11 w/w/w), as used in direct tumor injection. 3.5 μl of pCMV Luc prepared as shells in 50 μl total volume under physiological saline conditions. 50 μl of shells/animal used for injection. Contrast staining with uranyl acetate.

Fig. 6: Expression of β-galactosidase in a rat carotid artery following in vivo gene transfer of shells applied in Pluronic F127 gel following exposure of the artery. (A) DNA/PEI/tRNA (1 :1 :1 w/w/w); (B) DNA PEI/PVS (1 :1 :6 w/w/w); (C) DNA/PEI (1 :1 w/w) control, r denotes the polyanion/DNA ratio (w/w). 3.3 μg of pcDNA β-Gal in RNA shells or 6.6 μg of DNA in PVS shells/0.1 ml total volume was employed, mixed with 0.4 ml of Pluronic F127 at 4°C and coated onto the artery. β-Galactosidase detection was performed after 7 days. (D-F) Electron micrographs of the respective shells. Bars on micrographs indicate 100 nm.

Fig. 7: Expression of Hu-uPA in rat carotid arteria after gene delivery by tRNA shells. tRNA shells containing Hu-uPA encoding cDNA (A), an isotypic control (recombinant Hu-uPA) (B) or an irrelevant β-galactosidase encoding plasmid (C) and empty Pluronic F127 gel (D) were transferred to rat carotid arteria as described in Materials and methods. The sections were immunoreacted with mouse monoclonal AB against Hu-uPA. Bound primary AB were detected with biotin-conjugated horse anti-mouse AB and stained as described in Materials and methods.

Fig. 8: Utilization of DNA/peptide/PVS complexes for transfection of cancer cells. A Luciferase expression following transfection of human colon carcinoma cells (HCT 116) with DNA/peptide/PVS complexes, prepared using synthetic cationic peptides containing oligolysine (16 residues) for DNA binding fused to a nuclear localization signal, denoted in the diagram. NLS, nuclear localization signal derived from the SV 40 T-large antigen. SLN, a control sequence of the same chemical composition of the NLS. CYC, an optimized nuclear localization sequence obtained through cyclization of a peptide containing the NLS. VN, a nuclear localization signal derived from the N-terminal region of the HIV 1-Vpr. The complexes contained PVS at the indicated DNA to PVS weight ratio. Note the sequence specific enhancement of gene expression using the NLS as compared to the SLN and a further dramatic increase of gene expression using the CYC-peptide. B. Electron micrographs showing typical DNA/peptide/PVS-complexes used in the transfection experiments. The incorporated peptide and the used PVS to DNA weight ratio are indicated.

Fig. 9: Dynamic light scattering of DNA/peptide/PVS-complexes in physiological salt solution at increasing PVS to DNA weight ratios. The incorporated peptide is indicated.

A. Apparent size. Note that aggregation was overcome at a PVS to DNA ratio between 0,25 and 0,4 depending on the used peptide. The apparent size of complexes which were used in transfection experiments were between 100-200 nm.

B. Relative polydispersity of the complexes at increasing PVS-concentrations as indicated by the polydispersity index. Note that the polydispersity increaes slightly when aggregation is lost. With a higher amount of PVS the polydispersity decreases and reaches a minimum which coincides with the particular PVS to DNA ratio used in transfection experiments.

Fig. 10: Ethidium bromide intercalation assay for monitoring of the DNA-condensation during the formation of DNA/peptide/PVS complexes containing the NLS-peptide and a PVS to DNA weight ratio of 0.875. Note that fluorescence was almost recovered after the addition of PVS to the preformed DNA/peptide-complex. The subsequent addition of salt induces a slight condensation of the DNA within the complex which was almost comparable to naked DNA in salt solution

Table 3. Characterization of DNA/peptide/PVS complexes in physiological salt solution according to four different complex formation procedures. 1 , 2: Complex formation in water and subsequent addition of 150 mM NaCI. 3, 4: Complex formation in 150 mM NaCI.

1 , 3: The PVS was added to the preformed DNA/peptide complex.

2, 4: The peptide was added to the pre-incubated DNA/PVS-mixture.

Note that no significant differences were found between the preparations in terms of apparent size and polydispersity of the resulting complexes and the condensation rate of the DNA. Table 3

Claims

Claims:

1. A method for a gene transfer into animal cells in vivo or ex vivo, characterized in that vector DNA in aqueous solution is packed into compact nanoparticles by successive addition of oppositely charged polyelectrolytes and transfection of the cells is performed thereafter.

2. The method according to claim 1 , characterized in that a DNA-polycation/polyanion complex is present in a physiological saline solution.

3. The method according to claim 1 or 2, characterized in that the weight ratio of DNA and polyelectrolyte is C_DNA <, ~ Cp₀ι_ycation < C_Pθ|y_aniθn.

4. The method according to any one claim 1 - 3, characterized in that the polyelectrolytes in such weight ratios are mixed that the nanoparticles have an average particle size of less than about 150 nm.

5. The method according to any one claim 1 - 4, characterized in that the nanoparticles have an average particle size of less than 100 nm.

6. The method according to any one of claims 1-5, characterized in that more than 30 % of the cells are transfected.

7. The method according to claim 6, characterized in that more than 50 % of the cells are transfected.

8. The method according to any one of claims 1-7, characterized in that the surface charge of the nanoparticles is essentially negative or neutral.

9. The method according to claim 8, characterized in that the ζ- potential is negative, prefereably < - 30 mV.

10. The method according to any one of claims 1-9, charaterized in that the nanoparticles are essentially unflocculated.

11. The method according to any one of claims 1 - 10, characterized in that the ratio of a DNA/polyanion is 0,5 -6.

12. The method according to claim 11 , characterized in that the ratio of a DNA/polyanion is 4 -5,5.

13. The method according to any one of claims 1-12, characterized in that the nitrogen/phosphate ratio of DNA/polycation is 4-12.

14. The method according to claim 13, characterized in that the nitrogen/phosphate ratio is 8.

15. The method according to any one of claims 1-14, characterized in that the ratio of DNA/ polycation/ polyanion is 0,5-2 : 0,5-2 : 4-11 weight/weight/weight.

16. The method according to claim 15, characterized in that the polycation is polylysine or PEI and the polyanion is transfer RNA or polyvinylsulfate.

17. The method according to claim 15, characterized in that the ratio of DNA/ polycation/ polyanion is 1 : 1 ,5 : 6 or 1 : 1 : 6 weight/weight/weight.

18. The method according to claim 17, characterized in that the polycation is polylysine or PEI and the polyanion is transfer RNA or polyvinalsulfate.

19. The method according to any one of claims 1-18, characterized in that the nanoparticles are transferred into physiological saline solution wherein transfection is performed.

20. The method according to any one claims 1-19, characterized in that the vector DNA is made to interact with polycations and condensed in a first step, is mixed with polyanions to coat a layer in a second step, and that these steps are repeated, if necessary.

21. The method according to any one of claims 1-20, characterized in that synthetic polycations, peptides, nucleoproteins and/or polyamino acids are used as polycations.

22. The method according to claim 21 , characterized in that polyethyleneimine (PEI) is used as synthetic polycation.

23. The method according to claim 21 , characterized in that peptides predominantly comprised of lysine and/or arginine are used as polycations.

24. The method according to claim 21 , characterized in that the nucleoproteins H1 histone or HMG1 are used as polycations.

25. The method according to claim 21 , characterized in that polylysine, oligolysine K^g or K-|6 fusion peptides are used as polycations.

26. The method according to any one of claims 1-25, characterized in that transfer RNA, DNA or oligoribonucleotides are used as polyanions.

27. The method according to any one of claims 1-26, characterized in that antisense ribonucleotides or mixtures of antisense ribonucleotides and transfer RNA or other polyanions are used as polyanions.

28. The method according to any one of claims 1-27, characterized in that biocompatible anionic polymers are used as polyanions, which polymers prevent aggregation of the complexes under physiological saline conditions.

29. The method according to any one of claims 1-28, characterized in that polyvinyl sulfate and/or polystyrenesulfonate are used as polyanion.

30. The method according to any one of claims 1-29, characterized in that polylysine is used as polycation and transfer RNA as polyanion.

31. The method according to any one of claims 1-30, characterized in that polyethyleneimine is used as polycation and polyvinyl sulfate as polyanion.

32. The method according to any one of claims 1-31 , characterized in that polyethyleneimine (PEI) is used as polycation and RNA as polyanion.

33. The method according to any one of claims 1-32, characterized in that PEI is used as polycation and polystyrenesulfonate sodium (PSSS) as polyanion.

34. The method according to any one of claims 1-33, characterized in that polyethyleneimine is used as polycation and polystyrenesulfonate sodium as polyanion.

35. The method according to any one of claims 1-34, characterized in that the vector DNA is coated with more than 2 polyelectrolyte layers.

36. The method according to any one of claims 1-35, characterized in that the polycations and/or polyanions comprising ligands.

37. The method according to any one of claims 1-36, characterized in that the following parameters are selected independently:

- the particle surface charge by selecting suitable ratios of polyanions/polycations on the surface,

- provision of ligands for cellular uptake by receptor-specific endocytosis by means of electrostatic binding of the ligands using short polar amino acid segments,

- provision of ligands for nuclear localization by receptor-specific endocytosis by means of electrostatic binding of the ligands using short polar amino acid segments,

- lysosomolytic activity of the nanoparticles by incorporating lysosomolytic polycations.

38. The method according to any one of claims 1-37, characterized in that integrin-specific peptides are used as exterior polyelectrolyte layer or as polycation.

39. The method according to claim 38, characterized in that the integrin-specific peptide is K-I6-GGCRGDMFGCA or an analogous peptide.

40. The method according to one of claims 36 to 39, characterized in that the ligang is a nuclear localization peptide NLS, preferably CKKKKKKKKKKKKKKKKGGGPKKKRKVG.

41. The method according to one of claims 36 to 40 37, characterized in that the ligand is nuclear localization peptide VN, preferably

KKKKKKKKKKKKKKKKGGGCNEWTLELLEELKNEAVRHF.

42.The method according to one of claims 36 to 41 , characterized in that the ligand is a cyclic nuclear localization peptide CYC

I VKLKVYPLKKKRKP '-

KKKKKKKKKKKKKKKKQ

43. The method according to any one of claims 36 - 42, characterized in that polystyrenesulfonate sodium (PSSS) is used as polyanion in the chemical coupling of the ligands.

44. The method according to any one of claims 1-42, characterized in that polyethyleneimine or dendrimers are used as polycations in mediating lysosomolytic activity.

45. The method to any one of claims 1-44 characterized in that the transfection is carried out in the presence of chloroquine or CaCl2-

46. The method to any one of claims 1-45, characterized in that vector DNA is packed into polyelectrolyte nanoparticles and administered into living systems.

47. The method according to claim 46, characterized in that the living systems is a tumor or a vascular wall.

48. The method according to claim 47, characterized in that the tumor is selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangio- endotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomy- osarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cysta- denocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepa- toma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, men- ingioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease.

49. The method according to claim 47, characterized in that the vascular wall is an artery wall.

50. The method according to any one of claims 46-49, characterized in that the nanoparticles are mixed with a cationic, anionic, and/or non-ionic gel and, following exposure of tissues, organs and/or tumors to be transfected, applied thereon in liquid form.

51. The method according to claim 50, characterized in that the gel is a Pluronic F127 gel or a Poloxamer 407 gel.

52. The method according to any one of claims 46-51 , characterized in that the nanoparticles are directly injected into the tissues, organs and/or the tumors to be transfected.

53. The method for according to any one of claims 46- 48, characterized in that the nanoparticles are injected on the intravenous route.

54. The method according to any one of claims 46-48, characterized in that the nanoparticles are injected on the intraperitoneal route.

55. A complex for a gene transfer into animal cells in vivo or ex vivo, comprising a vector DNA which is condensed and packed into compact nanoparticles, whereby the nanoparticles comprising polyanions selected from the group consisting of transfer RNA, DNA, oligoribonucleotides, polyvinyl sulphate and/or polystyrenesulphonate.

56. The complex according to claim 55, characterized in that polyelectrolytes in such weight ratios are mixed that complexes are unflocculated.

57. The complex according to claim 55 or 56, characterized in that the nanoparticles have an average particle size of less than 00 nm.

58. The complex according to claim 57, characterized in that the nanoparticles have an average particle size of less than 90 nm.

59. The complex according to any one of claims 55-58, characterized in that the surface charge of the nanoparticles is essentially negative.

60. The complex according to any one of claims 55-59, characterized in that the nitrogen/phosphate ratio is 4 - 12.

61. The complex according to claim 60, characterized in that the nitrogen/phosphate ratio is 8.

62. The complex according to any one of claims 55-61 , characterized in that the ratio of DNA/ polycation/ polyanion is 0,5-2 : 0,5-2 : 4-11 weight/weight/weight.

63. The complex according to claim 62, characterized in that the polycation is polylysine or PEI and the polyanion is transfer RNA or polyvinylsulfate.

64. The complex according to any one of claims 55-63, characterized in that the ratio of DNA/ polycation/ polyanion is 1 : 1 ,5: 6 or 1 : 1 : 6 weight/weight/weight.

65. The complex according to claim 62, characterized in that the polyanion is transfer RNA or polyvinalsulfate.

66. A pharmaceutical composition comprising a complex according to any one of claims 55- 65 and one or more pharmaceutically acceptable adjuvant, excipient, carrier, buffer, diluent and/or customary pharmaceutical auxiliary.

67. An arteriosclerosis/athrosclerosis-treating apparatus comprising a bioabsorbable matrix and an effective amount of a pharmaceutical composition of claim 66.