| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | October 16, 2001 |
| PATENT TITLE |
Poly (organo) phosphazenes for use in synthetic transfection systems |
| PATENT ABSTRACT |
Water soluble polymers or polymeric hydrogels are used to encapsulate antigen to form vaccines. The antigen is mixed with a polymer solution, microparticles are formed of the polymer and antigen, and, optionally, the polymer is crosslinked to form a stable microparticle. Preferred polymers are alginate and polyphospazenes, and mixtures thereof. Microparticles can be administered parenterally or mucosally. For oral delivery, the microparticles are preferably fifteen microns or less in diameter, and adhere to the mucosal lining of the gastointestinal tract, increasing uptake by the reticuloendothelium. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | May 13, 1998 |
| PATENT REFERENCES CITED |
Verma et al. Gene therapy-promises, problems and prospects. Nature vol. 389:239-242, Sep. 18, 1997.* Editorial, Gene therapy: Better vectors, less hype. Nature Biotechnology. vol. 15:815, Sep. 15, 1997.* Marshall, E. Gene therapy's growing pains, Science vol. 269:1050-1055, Aug. 25, 1995.* Orkin et al. Report and recommendations of the panel to assess the NIH investment in research on gene therapy, Dec. 7, 1995. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A synthetic transfection system comprising as a carrier a cationic, water-soluble or water-dispersible polyphosphazene and DNA to be transfected, wherein the DNA to be transfected is in the form of a plasmid, a vector, a gene construct or an oligonucleotide and wherein condensed particles comprising the polyphosphazene and DNA to be transfected are enclosed in a delivery vehicle, which is a liposome. 2. A synthetic transfection system comprising as a carrier a cationic, water-soluble or water-dispersible polyphosphazene and DNA to be transfected, wherein the polyphosphazene is coupled to a targeting moiety. 3. A method for introducing DNA into target cells which method comprises contacting target cells with a synthetic transfection system which comprises a polyphosphazene and said DNA, wherein the DNA to be transfected is in the form of a plasmid, a vector, a gene construct or an oligonucleotide wherein said synthetic transfection system comprises a liposome and wherein the DNA is introduced into said target cells. 4. A method for introducing DNA into target cells which method comprises contacting target cells with a synthetic transfection system which comprises a polyphosphazene and said DNA, wherein the polyphosphazene is coupled to a targeting moiety and wherein the DNA is introduced into said target cells. 5. The method of claim 4 wherein said targeting moiety is galactose or transferrin. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
The present invention relates to the field of synthetic transfection systems useful in the delivery of gene constructs or DNA fragments to cells, especially to cells in living organisms. More in particular, the invention relates to cationic polymers having broad possibilities to be modified or adapted in order to create a flexible DNA gene delivery system, which can be used in, e.g., gene therapy applications. Gene therapy is seen as a promising method to correct hereditary defects or to treat life threatening diseases such as cancer and AIDS. In gene therapy, nucleic acid fragments or gene constructs are brought into target cells. These nucleic acid fragments or gene constructs are preferably incorporated in plasmids or other vectors. If the reconstructed plasmids are applied to an organism per se, this generally leads to low expression of the introduced gene, if any. There are three main reasons for this low expression. First, the plasmids will hardly ever reach the cell population where they are intended to be incorporated, due to degradation and elimination processes. Second, if the plasmids do reach the target cells, they cannot simply pass the cellular membrane, because of the strongly polar nature and the size of the plasmids. Third, if a plasmid does invade a target cell, it normally will be enclosed in an endosome, which will convert into a lysosome. In the lysosome, the plasmid will be degraded so that the incorporated gene cannot be expressed. For the above reasons, in gene therapy plasmids comprising a desired gene construct are transported to and delivered in the target cells by means of carrier systems. In recent years, many efforts have been made in the research on potentially suitable transfection systems, both of viral and non-viral origin. These transfection systems should deliver the desired gene to the target cell and cause it to be expressed to a high degree. Viral vectors are very suitable, because by nature adapted, to introduce plasmids in target cells and to avoid endosome disruption, the degradation of the plasmids in endosomes or the transition of endosomes into lysosomes. However, viral vectors have a number of pronounced disadvantages. Viral vectors are able to effect integration of the introduced gene in the chromosomal DNA of the target cell. The site where this integration is effected cannot (yet) be predicted or controlled, which involves the risk of destroying essential genes or activation of, e.g., oncogenes. In addition, it is at present very difficult to provide for viral vectors on a commercial scale. Moreover, viral vectors generally trigger the immune system of a living organism, which will lead to immuno responses against the transfection system when used in vivo. Finally, viral vectors inherently set limits to the size of the gene construct to be introduced in the target cell. In order to overcome the intrinsic disadvantages of viral vectors, synthetic transfection systems should offer good perspectives. In this light, a review article of Kabanov et al. in Bioconjugate Chemistry vol. 6, no. 1 (1995), 7-20 is mentioned. This review article describes in general terms the principle of delivery of genetic material onto cells employing soluble in terpolyelectrolyte complexes (IPEC's) of nucleic acids with linear polycations. It is indicated that various polycations have been used to produce IPEC's. As explicit examples polyvinyl pyrimidinium salts, polypeptides such as polylysine conjugates and lipopolylysines, and spermines are mentioned. Explicit reference is made to research carried out by the group of E. Wagner, relating to gene delivery by means of plasmid-polylysine complexes (Curiel et al., Adenovirus Enhancement of Transferrin-Polylysine-Mediated Gene Delivery, Proc. Natl. Acad. Sci. 88 (1991) 8850-8854; Plank et al., Gene Transfer into Hepatocytes Using Asialloglycoprotein Receptor Mediated Endocytosis of DNA Complexed with an Artificial Tetra-Antennary Galactose Ligand Bioconj. Chem. 3 (1992) 533-539; Wagner et al., Influenza Virus Hemagglutin HA2 N-Terminal Fusogenic Peptides Augment Gene Transfer by Transferrin-Polylysine-DNA Compleses: Toward a Synthetic Virus-like Gene-Transfer Vehicle, Proc. Natl. Acad. Sci. 89 (1992) 7934-7938; and Curiel et al., Gene Transfer to Respiratory Epithelial Cells via the Receptor Mediated Endocytosis Pathway, Am. J. Respir. Cell Mol. Biol. 6 (1992) 247-252). The plasmid-polylysine complex investigated upon exposition to certain cell lines showed at least some expression of the gene. Further, it was found that the expression efficiency increased considerably due to the binding of transferrin to the plasmid-polylysine complex. Transferrin gives rise to close cell-complex contact; it binds the entire complex to the transferrin receptor of cells. Subsequently, at least part of the entire complex was found to be incorporated in the cell. However, the transfection efficiency of these polylysine based transfection systems as well as other known synthetic transfection systems is much lower than the efficiency of the known viral vectors. The aim of the present invention is to provide for an effective and efficient synthetic transfection or blocking system. Such a system should fulfil the following conditions. The synthetic carrier system used must be biocompatible and preferably biodegradable. In order to be able to bind and condense DNA, e.g. in the form of a plasmid, wherein a gene construct is incorporated, the carrier system should possess a positive charge at physiological pH. It has now been found that such a system can be based on polyphosphazenes which are at least partially substituted with cationic substituents. More in particular, the invention relates to water soluble or water dispersible poly(organo)-phosphazene based transfection systems, wherein organic cationic moieties are attached to the polyphosphazene backbone. The polyphosphazene polymer which forms the basis of the poly(organo)phospnazene systems of the present invention essentially comprises a backbone of --[--P(R).sub.2 =N--].sub.n -- units, wherein (R).sub.2 represents two groups, which may or may not be the same, coupled to the phosphorous atom. A restricted number of other units may, however, be present. It is already known from other technical fields that polyphosphazenes are biocompatible and biodegradable. Polyphosphazenes were studied in the art of biomedical and pharmaceutical applications. In this light, reference is made to, e.g., the PhD Thesis of J. Goedemoed titled "Polyphosphazene Drug Delivery Systems for Antitumor Treatment", University of Leiden (1990); to Crommen et al. Biodegradable Polymers I, Synthesis of Hydrolysis-Sensitive Poly(organo)phosphazenes, Biomaterials 11 (1992), 511-520; to Domb et al. in Polymer Advanced Technology vol. 3, no. 6 (1992) 279-292; to Calicetti et al. in Il Farmaco vol. 49, no. 1, (1994) 69-74; and Andriarov et al. in J. Control Release vol. 27, no. 1 (1983) 69-77. These other technical fields essentially relate to controlled and sustained release systems. In order to be able to bind to and condense with plasmids, the poly(organo)phosphazene used must--at physiological pH--at least contain 5% cationic radicals or groups coupled to the phosphorous atom in the --[--P(R).sub.2 =N--].sub.n -- units. Preferably, at least 40% and more preferably 50% cationic groups are coupled to the phosphorous atoms. In these cases, the polyphosphazene is able to bind DNA electrostatically and condense therewith. Moreover, degradation and elimination of the DNA in the systemic environment is avoided. In addition, it appeared that such polyphosphazene-DNA complexes are taken up in the target cells in a considerably higher amount as compared with the plasmids per se. Suitable cationic substituents are preferably derived from organic moieties possessing an amino group. Such moieties are positively charged at physiological pH. Examples of these organic moieties are amino C.sub.1-10 alcohols, and amino C.sub.1-10 alkoxy C.sub.1-10 alcohols, as well as their secondary, tertiary and quaternary derivatives. Especially, tertiary amines are preferred. Although it is important that the polymer is positively charged as a whole, it is essential that the phosporous atoms in the poly phosphazene backbone not entirely comprise cationic moieties. Polyphosphazenes which are entirely substituted with cationic substituents essentially do not give rise to expression of the transported gene constructs. More in particular, part of the phosphorous atoms, preferably at least 10%, should be coupled to other hydrophobic and hydrophilic moieties. Especially good results are obtained when at least 10% of the substituents coupled to the phosphorous atoms in the polymer backbone are derived from essentially electrically neutral organic hydrophilic substituents such as glycerol, methoxy ethoxy ethanol and polyethylene glycol. A possible explanation for these advantageous results is that the plasmid will be less tightly bound to the polymer, so that it can dissociate easier in the target cell. Further, it is noted that PEG avoids recognition by macrophages. Hydrophobic groups, such as benzyl alcohol and lauryl alcohol, probably assist in transfer of the membranes of target cells. The polyphosphazene used in accordance with the present invention is of a cationic nature, and it is water soluble or water dispersible. More in particular, very good results are obtained if one of the substituents R in the formula --[--P(R).sub.2 =N--].sub.n -- is a cationic group while the other substituent R may represent in 5-100% of the occurrences a cationic moiety and in 95-0% of the occurences an anionic or neutral moiety. The molecular weight of the polyphosphazene polymers used can be easily adjusted to the size of the plasmid to be transported. Normally, polyphosphazenes having a molecular weight of from 10,000 to 1,000,000 can suitably be used. The weight ratio of the polyphosphazene polymers to the DNA fragments is very critical. The transfection efficiency does increase with increasing polymer to plasmid ratios. Suitable results are obtained when using weight ratios of between 0.1 and 200, preferably this ratio is higher than 1 most preferably higher than 7. The length of the polyphosphazene polymers can be controlled by using and maintaining suitable reaction conditions in the polymerization process. The synthetic transfection system of the invention which comprises at least one cationic, water soluble or water dispersible polyphosphazene as a carrier further comprises a DNA fragment such as a vector, a plasmid, a gene construct or an oligonucleotide. Oligonucleotides can be used as a blocking structure in cells. It is noted that the condensed particles comprising the polyphosphazene and DNA fragments can be enclosed or incorporated in known drug delivery systems, e.g. in liposomes. Genes to be incorporated into vectors or vehicles to be used in the synthetic transfection system are among others documented in McKusick, V. A. Mendelian inheritance in man, catalogs of autosomal dominant, autosomal recessive, and X-linked pheno-types. Eighth edition. John Hopkins University Press (1988). Stanbury, J. B., Wyngaarden, J. B., Frederickson, D. S., Goldstein, J. L. and Brown, M. S. The metabolic basis of inherited disease. Fifth edition. McGraw-Hill (1983). These genes include but are not limited to: genes associated with diseases of the carbohydrate metabolism such as for: fructose-1-phosphate aldolase fructose-1,6-diphosphatase glucose-6-phosphatase lysosomal .alpha.-1,4-glucosidase amylo-1,6-glucosidase amylo-(1,4:1,6)-transglucosidase muscular phosphorylase liver phosphorylase muscular phosphofructokinase phosphorylase-b-kinase galactose-1-phosphate uridyl transferase galactokinase all enzymes of the pyruvate dehydrogenase complex pyruvate carboxylase 2-oxoglutarate glyoxylate carboligase D-glycerate dehydrogenase; genes associated with diseases of the amino acid metabolism such as for: phenylalanine hydroxylase dihydrobiopterin synthetase tyrosine aminotransferase tyrosinase histidase fumarylacetoacetase glutathione synthetase .gamma.-glutamylcysteine synthetase ornithine-.delta.-aminotransferase carbamoylphosphate synthetase ornithine carbamyltransferase argininosuccinate synthetase argininosuccinate lyase arginase L-lysine dehydrogenase L-lysine ketoglutarate reductase valine transaminase leucine isoleucine transaminase "branched chain" 2-keto acid decarboxylase isovaleryl CoA dehydrogenase acyl-CoA dehydrogenase 3-hydroxy-3-methylglutaryl CoA lyase acetoacetyl CoA 3-ketothiolase propionyl CoA carboxylase methylmalonyl CoA mutase ATP:cobalamrine adenosyltransferase dihydrofolate reductase methylene tetrahydrofolate reductase cystathionine .beta.-synthase sarcosine dehydrogenase complex proteins belonging to the glycine cleavage system .beta.-alanine transaminase serum carnosinase cerebral homocarnosinase; genes associated with diseases of fat and fatty acid metabolisms such as for: lipoprotein lipase apolipoprotein C-II apolipoprotein E other apolipoproteins lecithin cholesterol acyltransferase LDL receptor liver sterol hydroxylase "Phytanic acid" .alpha.-hydroxylase; genes associated with lysosomal defects such as for: lysosomal .alpha.-L-iduronidase lysosomal iduronate sulphatase lysosomal heparan N-sulphatase lysosomal N-acetyl-.alpha.-D-glucosaminidase lysosomal acetyl CoA: .alpha.-glucosaminide N-acetyltransferase lysosomal N-acetyl-.alpha.-D-glucosaminide 6-sulphatase lysosomal galactosamine 6-sulphate sulphatase lysosomal .beta.-galactosidase lysosomal arylsulphatase B lysosomal .beta.-glucuronidase N-acetylglucosaminylphosphotransferase lysosomal .alpha.-D-mannosidase lysosomal .alpha.-neuraminidase lysosomal aspartylglycosaminidase lysosomal .alpha.-L-fucosidase lysosomal acid lipase lysosomal acid ceramidase lysosomal sphingomyelinase lysosomal glucocerebrosidase lysosomal galactosylceramidase lysosomal arylsulphatase A .alpha.-galactosidase A lysosomal acid .beta.-galactosidase .alpha.-chain of the lysosomal hexosaminidase A; genes associated with diseases of the steroid metabolism such as for: 21-hydroxylase 11.beta.-hydroxylase androgen receptor steroid 5.alpha.-reductase steroid suiphatase; genes associated with diseases of the purine and pyrimidine metabolism such as for: phosphoribosylpyrophosphate synthetase hypoxanthine guanine phosphoribosyltransferase adenine phosphoribosyltransferase adenosine deaminase purine nucleoside phosphorylase AMP deaminase xanthine oxidase orotate phosphoribosyltransferase orotidine 5'-phosphate decarboxylase DNA repair enzymes; genes associated with diseases of the porphirine and haemal metabolism such as for: uroporphyrinogene III cosynthase ferrochelatase porphobilinogene deaminase coproporphyrinogene oxidase proporphyrinogene oxidase uroporphyrinoqene III synthase uroporphyrinogene decarboxylase bilirubine UDP-glucuronyltransferase catalase; genes associated with diseases of the connective tissue, muscles and bone such as for: lysyl hydroxylase procollagen peptidase .alpha.1-antitrypsine dystrophine alkaline phosphatase guanosine nucleotide regulatory protein of the adenyl cyclase complex; genes associated with diseases of the blood and blood-forming organs such as for: blood coagulation factor V blood coagulation factor VII blood coagulation factor VIII blood coagulation factor IX blood coagulation factor X blood coagulation factor XII blood coagulation factor XIII all other blood coagulation factors all genes associated with osteopetrosis such as for: "carbonic anhydrase II" thrombocytes membrane glycoprotein Ib thrombocytes membrane glycoprotein IIb-IIIa spectrin pyruvate kinase glucose-6-phosphate dehydrogenase NADH cytochrome b.sub.5 reductase .beta.-globin .alpha.-globin; genes associated with diseases of transport systems such as for: lactase sucrase-.alpha.-dextrinase 25-hydroxyvitamin D.sub.3 -1-hydroxylase cystic fibrosis transmembrane conductance regulator (CFTR); genes associated with congenital immunodeficiencies such as for: the proteins of the complement system including B, C1q, C1r, C2, C3, C4, C5, C7, C8 and C10 the inhibitor of C1, a component of the complement system the inactivator of C3b, a component of the complement system; the genes for X-bound immunodeficiencies such as for: one of the enzymes of the NADPH oxidase complex myeloperoxidase the syndrome of Wiscott Aldrich and Ataxia Telangiectasia; genes coding for hormones as well as the genes coding for their receptors such as for instance for: growth hormone. Further, these genes also include genes which (to date) have not been associated with a hereditary defect but with which gene therapy can be practised in some manner. These include: the gene for tyrosine hydroxylase; drug resistance genes such as for instance: the P-glycoprotein P170 (the so-called multi drug resistance gene mdr1) mdr 3 dihydrofolate reduitase (DHFR) and methotrtxate resistant isotypes thereof metallothioneine aldehyde dehydrogenase (ALDH) glutathione transferase; genes coding for all cytokins including for instance all interleukins and all interferons; genes coding for all growth factors; genes coding for all growth factor receptors; genes coding for all transplantation antigens such as for instance the major and minor histocompatibility antigens; genes capable of affording resistance against infectious organisms, such as for instance TAR decoys; genes of infectious organisms which can be used for vaccination purposes such as for instance the envelope gene of HIV; genes which can be used for negative selection such as for instance the thymidine kinase gene of the Herpes simplex virus against which selection can be effected with substrates such as for instance gancyclovir or acyclovir. Vectors to be used include viral and non-viral regulatory elements for expression and/or replication. These vectors are well known in the field. Suitable transfection systems are able to target a gene construct to the aimed cell population. The polyphosphazene based transfection system of the invention therefore at least comprises one group that is selectively targetted to target molecules associated with the surfuace of the target cells. Such targeting moieties or homing devices are known to the skilled person and comprise, e.g., tri and tetra antennary cluster glycosides, transferrin or other protein contructs, monoclonal antibodies against cell membrane proteins, ligands for cell surface associated receptors and binding fragments of derivatives of said targeting moieties etc. If, for instance, galactose moieties are coupled to the polyphosphazene system of the present invention the transported gene fragments are incorporated in hepatocytes through the galactose receptor of hepatocytes. Furthermore, the presence of recognizable structures covalently or non-covalently coupled to the polyphosphazene part of a polyphosphazene-DNA complex facilitates the incorporation of the gene construct in the target cell. Moreover, the transfection system can be adapted to allow the gene construct to leave endosomes in the cellular system. Thereto membrane destabilizing structures, in particular polypeptide fragments, are conjugated to the poly(organo)phosphazene systems. Such destabilizing structures should be able to disturb or destabilize the endosomal membrane systems. The plasmids incorporating a gene construct so reach the cytoplasm of the target cell, where the gene construct can be expressed. Examples of such membrane destabilizing structures which are suitably used in accordance with the present invention are fusogenic structures, e.g. certain peptides and (parts of) viral coating proteins, for instance peptides derived from hemagglutinin protein of the influenza virus (see in this respect, e.g., Plank et al. The Influence of Endosome-Disruptive Peptides on Gene Transfer Using Synthetic Virus-Like Gene Transfer Systems, J. Biol. Chem. 269 (1994), 12918-12924). Another compound that is useful in accordance with the present invention is chloroquine. It is noted that chloroquine is only used in in vitro applications, because it is toxic in vivo. Since the invention is directed to both in vivo and in vitro applications, this embodiment is within the scope of the invention. As said herein above polyphosphazenes are known per se. This also applies to methods of preparing these polymers. A preferred method to Drepare Dolvphosphazenes starts from hexachlorotriphosphazene. This starting compound is subjected to ring opening polymerization yielding poly dichlorophosphazene. Ring opening polymerization of hexachlorotriphosphazene in the bulk is for instance described in Kircheldorf (ed.) Handbook of Polymer Synthesis Part B, Chapter 17, Phosphorous-Containing Polymers (1991); while solution polymerization is described in Mujumdar et al. Solution Polymerization of Selected Polyphosphazenes, Macromol. Chem. 19, (1989) 2293-2302. Poly(organo)phosphazenes used in accordance with the present invention can be prepared from these poly dichloro phosphazenes by effecting nucleophilic substitution with organic compounds which are cationic at physiological pH or can easily be converted to such cationic substituents. Random copolymers can be synthesized by carrying out the substitution reaction in the simultaneous presence of two or more substituents. It is also possible to subject the starting polymer first to a reaction with a first substituent and subsequently with another substituent. Suitable methods of these nucleophilic substitutions are well within the scope of the person skilled in the art. Fusogenic peptides and targeting molecules can be bonded to the poly(organo)phosphazenes using well-known techniques, for instance using thiol groups introduced in the polymer and maleimide groups introduced in the peptide or targeting molecule. In a preferred embodiment, the polyphosphazene polymer is substituted with N,N-dimethylamino ethanol groups. These cationic groups are introduced by contacting polydichloro-phosphazenes with N,N-dimethylamino ethanol. In a further embodiment, the invention relates to a method for introducing DNA fragments in target cells, comprising contacting these DNA fragments with a polyphosphazene which is at least partially substituted with cationic substituents and subsequently contacting the obtained transfection system with target cells. Finally, the invention relates to the use of a polyphosphazene which is at least partially substituted with cationic substituents as transfection vehicle. The polyphosphazene system can be used both in in vivo and in in vitro applications. IN THE FIGURES FIG. 1 shows the number of transfected COS-7 cells and the relative cell viability dependent on the polymer/plasmid ratio; FIG. 2 shows the number of transfected OVCAR cells and the relative cell viability dependent on the polymer/plasmid ratio; FIG. 3 shows the number of transfected OVCAR cells and the relative cell viability dependent on the polymer/plasmid ratio; FIG. 4 shows the percentage of protonated amine side chains as function of the pH; FIG. 5 shows the degradation of N,N-dimethyl-aminoalcohol substituted polyphosphazene (DS 75%) at 37.degree. C., pH 7.2; and FIG. 6 shows the number of transfected OVCAR cells and the relative cell viability as a function of the polymer/plasmid ratio. |
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