AU1820999A - Grafted copolymers as gene carriers - Google Patents
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Description
WO 99/29839 PCT/US98/26451 GRAFTED COPOLYMERS AS GENE CARRIERS BACKGROUND OF THE INVENTION This invention relates to a gene carrier for use in connection with in vivo 5 applications. More particularly, the invention relates to a non-toxic composition and a method for effectively delivering a selected nucleic acid into a host cell by forming an electrostatic complex of the nucleic acid with a cationic polymer. Graft copolymers of poly-L-lysine (PLL) and a polyoxyalkyl glycol are particularly effective. 10 Genes are very attractive candidates for therapeutic use in a variety of disease states due to their ability to produce bioactive proteins using the biosynthetic machinery provided by host cells. M.S. Wadhwa et al., 6 Bioconjugate Chemistry 283 (1995). There are many established protocols for transferring genes into cells, including calcium phosphate precipitation, electroporation, particle bombardment, 15 liposomal delivery, viral-vector delivery, and receptor-mediated gene delivery. Id. Although all of these methods can be used for delivering genes into cultured mammalian cells, there are many difficulties in introducing genes into target cells in vivo. In particular, calcium phosphate and polycation precipitation are two techniques that are probably the most widespread in laboratory practices 20 (PROFECTION Mammalian Transfection Systems, Technical Manual 2 (Promega Corp., 1990)); however, they are characterized by relatively low transfection efficiency. A.V. Kabanov & V.A. Kabanov, 6 Bioconjugate Chemistry 7 (1995). These two techniques appear ineffective for introducing RNA molecules into cells and cannot be used for transfection in vivo. Id. 25 Transfection methods using retroviral or adenoviral vectors, E. Gilboa et al., 4 Biotechniques 504 (1986); M.A. Rosenfeld et al., 252 Science 431 (1991), overcome some of these limitations. Retroviral vectors, in particular, have been successfully used for introducing exogenous genes into the genomes of actively dividing cells such that stable transformants are obtained. D.G. Miller et al., 10 Mol. 30 Cell Biol. 4239 (1990). However, the method ofusing retroviral vectors for inserting genes into the host cell's genome depends on the viral infection pathway. Applying the retroviral method in human gene therapy raises serious concerns about possible recombination with endogenous viruses, oncogenic effects, and immunologic tlBARTITilTF 1FqHFrT (RIII F 26 WO 99/29839 PCT/US98/26451 2 reactions. A.V. Kabanov & V.A. Kabanov, 6 Bioconjugate Chemistry 7 (1995); H.M. Temin, 1 Human Gene Therapy 111 (1990). Such concerns have discouraged the use of iral vectors for human gene therapy. D.G. Miller et al., 10 Mol. Cell Biol. 4239 (1990). 5 On the other hand, non-viral gene delivery systems, such as cationic liposomes, H.M. Temin, 1 Human Gene Therapy 111 (1990), or PLL, G.Y. Wu & C.H. Wu, 263 J. Biol. Chem. 14621 (1988); E. Wagner et al., 87 Proc. Nat'l Acad. Sci. USA 3410 (1990); H. Farhood et al., 1111 Biochem. Biophys. Acta 239 (1992), have their own drawbacks. Even though they seem to be safe for human clinical use, 10 typical non-viral systems provide low transfection efficiencies or cause precipitation ofthe nucleic acids. N.H. Caplen et al., 1 Nature Medicine 1 (1995). At present, the LIPOFECTIN (trademark of GIBCO/BRL) protocol seems to be most reliable in this category, J.H. Felgner et al., Enhanced Gene Delivery and Mechanism Studies with a Novel Series of Cationic Lipid Formulations, 269 J. Biol. Chem. 2550 (1994), but 15 it bears the disadvantage of high cytotoxicity. H. Farhood et al., 1111 Biochim. Biophys. Acta 239 (1992). In view of the foregoing, it will be appreciated that development of a gene delivery system that is both safe and efficient would be a significant advancement in the art. 20 BRIEF SUMMARY OF THE INVENTION It is an object of the present invention to provide a composition and a method for delivering nucleic acids into cells. It is also an object of the present invention to provide a composition and a 25 method for gene delivery that are safe and efficient. It is another object of the invention to provide an efficient, non-viral composition and a method of use thereof for delivering exogenous DNA or RNA to a target cell. It is still another object of the invention to provide a method of delivering a 30 gene into a target cell in vitro. It is yet another object of the invention to provide a method of delivering a gene into a target cell in vivo.
WO 99/29839 PCT/US98/26451 3 These and other objects can be addressed by providing a composition for delivery of a selected nucleic acid into a target cell, wherein the composition is configured for forming an electrostatic complex with the selected nucleic acid, comprising a biocompatible graft copolymer of a cationic first polymer and an 5 amphiphilic second polymer. Preferably, the first polymer is a member selected from the group consisting ofpoly(L-lysine), derivatives thereof and mixtures thereof, and more preferably is poly(L-lysine). Preferably, the second polymer is a polyoxyalkyl glycol, such as those selected from the group consisting of polyethylene glycol homopolymers, polypropylene glycol homopolymers, alpha-substituted 10 poly(oxyalkyl) glycols, poly(oxyalkyl) glycol copolymers and block copolymers, and activated derivatives thereof. Polyethylene glycol is particularly preferred. In a preferred embodiment of the invention, the graft copolymer comprises polyethylene glycol grafted to an E-amino group ofpoly(L-lysine). The graft copolymer preferably comprises about 5 mole% to about 25 mole% of polyethylene glycol and, more 15 preferably, about 10 mole% of polyethylene glycol. In another aspect of the invention, a composition for delivery of a selected nucleic acid into a host cell comprises an electrostatic complex of a selected nucleic acid and a biocompatible graft copolymer comprising a cationic first polymer and an amphiphilic second polymer. In a preferred embodiment ofthe invention, the nucleic 20 acid and the graft copolymer are present in a weight ratio of about 0.3 to 10. Preferably, the composition further comprises an effective amount of an anti endosome functional agent, such as chloroquine. Such effective amount of chloroquine is preferably about 25-250 MM and more preferably about 75-150 4M. In still another aspect of the invention, a method of transforming a host cell 25 with a selected nucleic acid comprises contacting the host cell with an effective amount of an electrostatic complex comprising the selected nucleic acid and a biocompatible graft copolymer, wherein the biocompatible graft compolymer comprises a cationic first polymer and an amphiphilic second polymer; such that the host cell internalizes the selected nucleic acid. 30 In yet another aspect of the invention, a method of using a composition for delivering a selected nucleic acid to an individual comprises administering an effective amount of an electrostatic complex comprising the selected nucleic acid and a WO 99/29839 PCT/US98/26451 4 biocompatible graft copolymer, comprising a cationic first polymer and an amphiphilic second polymer, such that the complex is systemically circulated and contacts a host cell such that the host cell internalizes the selected nucleic acid. 5 BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS FIG. 1 is a schematic representation of complex formation between plasmid DNA and an illustrative gene delivery composition according to the present invention. 10 FIG. 2 shows an illustrative synthesis scheme of a grafted copolymer, PEG-g PLL, according to the present invention. FIG. 3 shows data from a fluorescence quenching assay: (0) PLL control; (0>) 5 mole% PEG-g-PLL; (o) 10 mole% PEG-g-PLL; (A) 25 mole% PEG-g-PLL. FIG. 4 shows size (diameter) determinations by dynamic laser light scattering 15 ofpSV-p-gal (DNA), a complex ofpSV-p-gal and PLL (PLL), a complex ofpSV-p gal and 5 mole% PEG-g-PLL (5 mol% PEG), a complex ofpSV-p-gal and 10 mole% PEG-g-PLL (10 mol% PEG), and a complex of pSV-P-gal and 25 mole% PEG-g PLL (25 mol% PEG). FIG. 5 shows transfection efficiency for transfection ofhumanliver carcinoma 20 (HepG2) cells with pSV-p-gal using LIPOFECTIN reagent (Lipofectin), a PLL complex (PLL), a 5 mole% PEG-g-PLL complex (5 mol% PEG), a 10 mole% PEG g-PLL complex (10 mol% PEG), and a 25 mole% PEG-g-PLL complex (25 mol% PEG). FIG. 6 shows the transfection efficiency of human liver carcinoma (HepG2) 25 cells as a function of the weight ratio of DNA to PEG-g-PLL. FIG. 7 shows cell viability of human liver carcinoma HepG2 cells after transfection with pSV-p-gal using a control (media), LIPOFECTIN reagent (Lipofectin), PLL (PLL), a 5 mole% PEG-g-PLL complex (5 mol% PEG), a 10 mole% PEG-g-PLL complex (10 mol% PEG), and 25 mole% PEG-g-PLL (25 mol% 30 PEG).
WO 99/29839 PCT/US98/26451 5 FIG. 8 shows transfection efficiency of human liver carcinoma HepG2 cells with pSV-P-gal using a 10 mole% PEG-g-PLL complex as determined 24, 48, 72, and 96 hours after transfection. FIG. 9 shows the effect of chloroquine concentration on transfection of 5 human liver carcinoma HepG2 cells with pSV-p-gal using a 10 mole% PEG-g-PLL complex. DETAILED DESCRIPTION Before the present compositions and methods of use thereof for gene delivery 10 are disclosed and described, it is to be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat. It is also to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting since the 15 scope of the present invention will be limited only by the appended claims and equivalents thereof. It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an anti-endosome 20 function agent" includes a mixture of two or more of such agents, reference to "an amphiphilic polymer" includes reference to one or more of such polymers, and reference to "a cationic polymer" includes reference to a mixture of two or more of such cationic polymers. In describing and claiming the present invention, the following terminology 25 will be used in accordance with the definitions set out below. As used herein, "PEG-g-PLL" means a grafted copolymer wherein PEG or another poly(oxyalkyl)glycol is conjugated to an E-amino group of a lysine residue of PLL. As used herein, "x mol% PEG-g-PLL," where x is a number between 1 and 30 100, refers to PEG-g-PLL having x mole% of PEG. For example, 5 mol% PEG-g PLL is PEG-g-PLL containing 5 mole% of PEG.
WO 99/29839 PCT/US98/26451 6 As used herein, "poly(oxyalkyl)glycol" refers to polyether glycol polymers that when grafted to PLL render the resulting composition non-toxic and water soluble. Each monomer portion of the polymer contains a carbon chain having up to about 5 carbon atoms. Preferred poly(oxyalkyl) glycols are selected from the group 5 consisting of polyethylene glycol (PEG) homopolymers, polypropylene glycol homopolymers, alpha-substituted poly(oxyalkyl) glycols (such as methoxypolyethylene glycols or other suitable alkyl-substituted derivatives such as those containing C 1
-C
4 alkyl groups), poly(oxyalkyl) glycol copolymers and block copolymers, and activated derivatives thereof The poly(oxyalkyl) glycols used in the 10 present invention preferably have a molecular weight of about 200 to about 50,000, and more preferably about 200 to about 20,000. An especially preferred poly(oxyalkyl) glycol is polyethylene glycol (PEG). PEG is preferred because it is inexpensive, approved by the U.S. Food and Drug Administration for administration to humans, and is resistant to eliciting an antibody response. 15 As used herein, "PLL" refers to poly(L-lysine), derivatives thereof, and mixtures thereof The PLL preferably has a molecular weight in the range of about 200 to 50,000 and more preferable in the range of about 500 to 30,000. As used herein, "effective amount" means an amount of a nucleic acid that is nontoxic but sufficient to provide the selected local or systemic effect and 20 performance at a reasonable benefit/risk ratio attending any medical treatment. As used herein, "administering" and similar terms mean delivering the complex formed by admixing the nucleic acid to be delivered with a gene carrier composition according to the present invention to the individual being treated such that the complex is capable of being circulated systemically to the parts of the body 25 where the complex can contact the target cells. Thus, the composition is preferably administered to the individual by systemic administration, typically by subcutaneous, intramuscular, or intravenous administration, or intraperitoneal administration. Injectables for such use can be prepared in conventional forms, either as a liquid solution or suspension or in a solid form suitable for preparation as a solution or 30 suspension in a liquid prior to injection, or as an emulsion. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like; and if WO 99/29839 PCT/US98/26451 7 desired, minor amounts of auxiliary substances such as wetting or emulsifying agents, buffers, and the like can be added. Delivery of a nucleic acid, i.e. DNA and/or RNA, can be used to achieve expression of a polypeptide or to inhibit expression of a polypeptide through the use 5 of an "antisense" nucleic acid, especially antisense RNA. As used herein, "polypeptide" means peptides of any length and includes proteins. The term "polypeptide" is used herein without any particular intended size limitation, unless a particular size is otherwise stated. Typical ofpolypeptides that can be expressed are those selected from the group consisting of oxytocin, vasopressin, 10 adrenocorticotrophic hormone, epidermal growth factor, prolactin, luteinizing hormone releasing hormone, growth hormone, growth hormone releasing factor, insulin-like growth factors, insulin, erythropoietin, obesity protein such as leptin, somatostatin, glucagon, glucagon-like insulinotropic factors, parathyroid hormone, interferon, gastrin, interleukin-2 and other interleukins and lymphokines, tetragastrin, 15 pentagastrin, urogastrin, secretin, calcitonin, enkephalins, endorphins, angiotensins, renin, bradykinin, bacitracins, polymixins, colistins, tyrocidin, gramicidins, and synthetic analogues, modifications, and pharmcologically active fragments thereof, monoclonal antibodies, and vaccines. This group is not to be considered limiting; the only limitation to the peptide or protein drug that may be expressed is functionality. 20 Delivery of DNA and/or RNA is useful in gene therapy, vaccination, and any therapeutic situation in which a nucleic acid or a polypeptide should be administered in vivo. E.g., U.S. Patent No. 5,580,859, hereby incorporated by reference. When the nucleic acid is DNA, it can be a DNA sequence that is itself non replicating, but is inserted into a plasmid wherein the plasmid further comprises a 25 replicator. The DNA may also contain a transcriptional promoter, such as the CMV IEP promoter, which is functional in humans. The DNA can also encode a polymerase for transcribing the DNA. Many expression vectors for expression of a cloned gene in a mammal are known in the art, and many such expression vectors are commercially available, for example, pEUK-C1 (Clontech, Palo Alto, Calif.). A gene 30 of interest can be inserted into such an expression vector according to recombinant DNA technology well known in the art. E.g., J. Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., 1989), hereby incorporated by reference.
WO 99/29839 PCT/US98/26451 8 The method can be used for treating a disease associated with a deficiency or absence or mutation of a specific polypeptide. In accordance with another aspect of the invention, the method provides for immunizing an individual, wherein such individual can be a human or an animal, comprising delivering a DNA and/or RNA 5 to the individual wherein the DNA and/or RNA codes for an immunogenic translation product that elicits an immune response against the immunogen. The method can be used to elicit a humoral immune response, a cellular immune response, or a mixture thereof. An illustrative method of forming the gene carriers according to the present 10 invention is accomplished by grafting polymer monomethoxy polyethylene glycol (mPEG) to the e-amino group of lysine of PLL. PEG, a straight-chain amphiphilic polymer has been used to modify several enzymes giving them longer half-lives in vivo. F.F. Davis et al., in 4 Enzyme Engineering 169 (1978); A. Abuchowski et al., 7 Cancer Biochem. Biophys.175 (1984). PEG has also been used to modify 15 interleukin-2 to give it increased solubility and increased half-life in vivo. N.V. Katre et al., 84 Proc. Nat'l. Acad. Sci. USA 1487 (1987). The PEG-grafted PLL (PEG-g PLL) provides a solubility increase when it forms an electrostatic complex with genes to be delivered (FIG. 1) because of the solubilizing effect of the PEG chains. A.V. Kabanov et al., 6 Bioconjugate Chemistry 639 (1995). The solubility increase 20 induced by the added PEG chains favorably affects the transfection efficiency as compared to a plasmid DNA/PLL complex without increasing the cytotoxicity of PLL. Rather, the presence of the PEG chains also acts to reduce the cytotoxicity of the PLL base and improve transfection duration. The electrostatic complex is formed by the affinity of the positively-charged polymer (e.g. PLL) and the negatively 25 charged nucleic acid. Example 1 Graft copolymers of PEG-g-PLL having PEG contents of 5 mol %, 10 mol % and 25 mol % were synthesized according to the procedure outlined in FIG. 2. 30 Thionyl chloride (SOC1 2 ), triethylamine (TEA), dimethyl sulfoxide (DMSO), and methoxy polyethylene glycol (mPEGOCH 2
CH
2 0H; MW=550) were purchased from WO 99/29839 PCT/US98/26451 9 Aldrich (Milwaukee, Wisconsin). PLL-hydrobromide (Repeating unit=120; MW=25,000) was purchased from Sigma (St. Louis, Missouri). For example, synthesis of 5 mol % PEG-g-PLL was performed as follows: Methoxy PEGOCH 2
CH
2
CO
2 H was synthesized by alkylation ofmPEGOCH 2 CH20H 5 with ethyl bromoacetate. Thionyl chloride (1.5 ml) was added to a round bottom flask containing 6 mg of mPEGOCH 2
CH
2
CO
2 H and refluxed for 40 min, followed by evaporation of thionyl chloride under vacuum. The resulting product was dissolved in 100 A of DMSO, and added to 1 ml of DMSO solution containing 25 mg of PLL-hydrobromide and 160 l of TEA while stirring at room temperature. 10 After stirring for 30 min., 4 ml of deionized water was added and the reaction mixture was adjusted to pH 1 with 6 N HC1. Finally, the product was dialyzed against water and lyophilized. The content of PEG was determined by 1 H-NMR using D 2 0 as a solvent (not shown). A peak at about 3.5 ppm signified the presence of mPEG in the product. 15 Example 2 The plasmid pSV-p-gal (Promega Corp., Madison, Wisconsin; EMBL accession No. X65335) is a positive control vector for monitoring transfection efficiencies of mammalian cells. The pSV-p-gal plasmid contains a SV40 early 20 promoter and enhancer sequence, transcription start sites, E. coli lacZ coding region encoding p-galactosidase, and SV40 small T antigen polyadenylation signals. SV40 early promoter and enhancer drive the transcription of the lacZ gene. Equal amounts of plasmid DNA and ethidium bromide (1 Atg each) were placed in 1 ml of buffer containing 20 mM Hepes (N-[2-hydroxyethyl]piperazine-N' 25 [2-ethanesulfonic acid]) and 0.15 M NaC1, (pH 7.4). Various amounts of PEG-g PLL prepared according to the procedure of Example 1 were then added to the plasmid DNA-ethidium bromide mixture. Fluorescence intensity of this mixture was measured with a spectrofluorometer (PC1, ISS Co., USA)(Xex=516 nm, Xem=590 nm). FIG. 3 shows the results from this fluorescence quenching assay. These data 30 indicate that PEG-g-PLL condenses up to 55% of the plasmid DNA. Almost no difference was observed in the plasmid DNA condensation ability of PLL compared to its PEG-modified derivatives.
WO 99/29839 PCT/US98/26451 10 Example 3 In this example, gel retardation assays were carried out to further determine whether PEG-g-PLL formed complexes with DNA. Mixtures of pSV-P-gal and PEG-g-PLL prepared as in Example 2 were fractionated by electrophoresis in a 1% 5 agarose gel. After fractionation, the gel was stained with ethidium bromide (0.5 Mg/ml) and illuminated on a UV illuminator. The movement of free plasmid DNA (not shown) was retarded as the amount of PEG-g-PLL for complex formation increased. These results indicate that PEG-g-PLL formed a stabale complex with plasmid DNA. Complete retardation was achieved at and above a 1:1 weight ratio 10 of plasmid DNA:PEG-g-PLL. Example 4 In this example, the sizes of plasmid DNA/PEG-g-PLL complexes prepared according to the procedure of Example 2 were measured by dynamic laser light 15 scattering (Brookhaven BI-DS) at a 900 angle using a 1:3 weight ratio of plasmid DNA to PEG-g-PLL. Data were analyzed by a cumulative analysis. FIG. 4 shows the size ofthe plasmid alone and the size of complexes with various compounds. The complexes have an average diameter of about 300 nm. 20 Example 5 In this example, the efficacy of compositions according to the present invention for mediating in vitro transfection of mammalian cells was demonstrated. HepG2 cells (human liver carcinoma cells) were obtained from the American Type Culture Collection (Rockville, MD; ATCC accession No. 8065-HB). Transfection 25 was performed using PEG-g-PLL in 96-well plates seeded at a cell density of 20 x 104 cells/m. The plasmid pSV-3-gal DNA/PEG-g-PLL complex was prepared by mixing 1 4g of plasmid DNA and 3 ag ofPEG-g-PLL in 100 Ml of serum-free MEM medium and incubating it for 30 min at room temperature, followed by the addition of 10% (v/v) fetal bovine serum (Hyclone Laboratories, Logan, Utah) and 100 4M 30 chloroquine (Sigma). Chloroquine, a cell permeant base, was used to partially neutralize acidic compartments of the cells and prevent the fusion ofendosomes with lysosomes. P. Midoux et al., 21 Nucleic Acids Research 871-78 (1993). The WO 99/29839 PCT/US98/26451 11 medium in each well of the 96-well plate was replaced by the transfection mixture. Cells were incubated for 4 hr in a tissue culture incubator (Napco Co.) at 37oC in 5%
CO
2 . Transfection mixtures were removed and fresh growth medium containing 10% fetal bovine serum was added to each well. Cells were incubated for an additional 5 44 hr in a tissue culture incubator at 37oC in 5% CO 2 . In situ X-gal (5-chloro-4-bromo-3-indolyl-p-D-galactopyranoside) staining of transfected cells was used for the detection of expressed P-galactosidase. J.R. Sanes et al., 5 EMBO J. 3133 (1986). Cells in each well of the 96-well plate were washed twice with IX phosphate-buffered saline (PBS) and fixed by 100 Al of 0.25% 10 (v/v) glutaraldehyde for 15 min at room temperature. Glutaraldehyde solution was then removed and cells were rinsed gently 3 times with IX PBS. Next, 60 A of X gal solution (1 mg/ml X-gal, 2 mM MgC12, 5 mM K 4 Fe(CN) 6 and 5 mM K 3 Fe(CN) 6 ) was added, followed by incubation for 16 hr in a tissue culture incubator at 37oC. X-gal solution was the removed and cells were covered with 1X PBS. Cells 15 expressing the P-galactosidase enzyme from transfection of pSV-p3-gal plasmid were stained blue by X-gal and could be seen and counted under a microscope. As shown in FIG. 5, transfection efficiency increased by up to 30-fold as the degree of PEG-modification increased. Of the three degrees of substitution tested, 10 mol % PEG-g-PLL showed the best transfection efficiency. The 25 mol% PEG 20 g-PLL showed significantly lower transfection efficiency. Thus, the optimal range ofPEG-modification in PLL seems to be around 10 mol %. A commercially available transfection agent, LIPOFECTIN reagent (GIBCO BRL), showed slightly higher transfection efficiency than 10 mol % PEG-g-PLL in HepG2 cells. LIPOFECTIN reagent is a 1:1 (w/w) liposome formulation of the cationic lipid 25 N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA), and dioleoyl phosphotidylethanolamine (DOPE) in membrane filtered water. Example 6 In this example, different complexes were made with a constant amount of 30 plasmid DNA (10 /pg/ml) and increasing amounts of 10 mol% PEG-g-PLL (1-50 4g/ml). These complexes were then used to transfect HepG2 cells according to the procedure of Example 5. As shown in FIG. 6, transfection efficiency increased as the WO 99/29839 PCT/US98/26451 12 weight ratio of PEG-g-PLL to plasmid DNA increased to 3:1. At higher ratios of PEG-g-PLL to plasmid DNA, transfection efficiency decreased. Example 7 5 In this example, the cytotoxicity of PEG-g-PLL was determined. The percentage of living cells was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay of T. Mosman, 65 J. Immunol. Methods 55 (1983), hereby incorporated by reference. At the end of the transfection experiment described in Example 5, the 10 transfection mixture was replaced by fresh growth medium containing 26 ul of 2 mg/ml MTT solution. Plates were incubated for an additional 4 hr at 37 oC in a tissue culture incubator, then MTT-containing medium was removed by aspiration, and 150 jl of DMSO was added to dissolve the formazan crystals formed by living cells. Absorbance was measured at 570 nm using a microplate reader (Model EL311, Bio 15 Tek instrument Co.), and the percentage of living cells was calculated from the following equation: Cell viability (%) = 570(sample) x 100
A
5 7 0 (control) As shown in FIG. 7, Both 5 mol %- and 10 mol %-modified PEG-g-PLL showed very mild cytotoxicity on HepG2 cells, whereas LIPOFECTIN reagent or PLL showed moderate to high cytotoxicity. Even though 25 mol %-substituted 20 PEG-g-PLL resulted in a lower cell viability than 5 mol %- or 10 mol %-substituted PEG-g-PLL, its cytotoxicity was still lower than either LIPOFECTIN or PLL. Therefore, the compositions of the present invention are improvements over known substances with respect to cytotoxicity. 25 Example 8 In this example, the procedure ofExample 7 was followed except that the cell viability assay was performed at 24, 48, 72, or 96 hours after transfection. Generally, a transfection assay is performed 48 hrs after transfection, but almost the same WO 99/29839 PCT/US98/26451 13 transfection efficiency was obtained when the assay was performed after only a 24 hr incubation (FIG. 8). In the case ofLIPOFECTIN reagent, P-galactosidase activity after a 24 hr incubation was half the P-galactosidase activity after a 48 hr incubation (data not shown). Further, the cells transfected by plasmid pSV-p-gal DNA/PEG-g 5 PLL mixture maintained its gene expression level up to 96 hr (FIG. 8). Example 9 In this example, the role of chloroquine in transfection efficiency was determined. The procedure of Example 5 was followed except that chloroquine 10 concentration was varied from 0-100 uM. FIG. 9 shows that chloroquine was not essential for successfully transfecting cells, however, chloroquine played an important role in increasing transfection efficiency. As the concentration of chloroquine increased up to 100 MM, the transfection efficiency increased up to 30-fold. 15 Example 10 In this example, a method of delivering a gene in vivo to an individual is illustrated. A nucleic acid encoding the leptin obesity protein, such as human leptin or a rat leptin cDNA, C. Guoxun et al., Disappearance of Body Fat in Normal Rats Induced by Adenovirus-mediated Leptin, 93 Proc. Nat'l Acad. Sci. USA 14795-99 20 (1996), or a mouse leptin cDNA, P. Muzzin et al., Correction of Obesity and Diabetes in Genetically Cloned Mice by Leptin Gene Therapy, 93 Proc. Nat'l Acad. Sci USA 14804-14808, both ofwhich are hereby incorporated by reference, is known in the art. The mammalian expression vector, pEUK-C 1 (Clontech, Palo Alto, Calif.) is designed for transient expression of cloned genes. This vector is a 4.9 kb plasmid 25 comprising a pBR322 origin of replication and an ampicillin resistance marker for propagation in bacteria, and also comprising the SV40 origin of replication, SV40 late promoter, and SV40 late polyadenylation signal for replication and expression of a selected gene in a mammalian cell. Located between the SV40 late promoter and SV40 late polyadenylation signal is a multiple cloning site (MCS) of unique XhoI, 30 XbaI, SmnaI, SacI, and BamHI restriction sites. DNA fragments cloned into the MCS are transcribed as RNA from the SV40 late promoter and are translated from the first ATG codon in the cloned fragments. Transcripts of cloned DNA are spliced and WO 99/29839 PCT/US98/26451 14 polyadenylated using the SV40 VPI processing signals. The leptin gene is cloned into the MCS ofpEUK-C1 using techniques well known in the art, e.g. J. Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., 1989). The resulting plasmid is delivered to a human or animal after incorporation into a complex 5 according to the present invention illustrated in Example 2. An effective amount of the resulting complex is systemically administered to an individual such that complex enters the bloodstream and contacts target cells. The target cells that are contacted by the complex take up the complex, thus internalizing the leptin DNA. The leptin DNA is then expressed in the recipient cell, resulting in 10 a positive effect in treatment for obesity or diabetes. The compositions of the present invention offer improved transfection ability than that obtained with PLL. The present compositions also demonstrate low cytotoxicity, early gene expression, and maintenance of the early gene expression 15 level up to 96 hrs. These characteristics are advantages over prior art compounds such as LIPOFECTIN reagent.
Claims (47)
1. A composition for delivery of a selected nucleic acid into a target cell, wherein said composition is configured for forming an electrostatic complex with said 5 selected nucleic acid, comprising a biocompatible graft copolymer comprising a cationic first polymer and an amphiphilic second polymer.
2. The composition of claim 1 wherein said first polymer is a member selected from the group consisting of poly(L-lysine), derivatives thereof, and 10 mixtures thereof.
3. The composition of claim 2 wherein said first polymer is poly(L lysine). 15
4. The composition of claim 3 wherein said poly(L-lysine) has a molecular weight of about 200 to 50,000.
5. The composition of claim 1 wherein said second polymer is a polyoxyalkyl glycol. 20
6. The composition of claim 5 wherein said polyoxyalkyl glycol is a member selected from the group consisting of polyethylene glycol homopolymers, polypropylene glycol homopolymers, alpha-substituted poly(oxyalkyl) glycols, poly(oxyalkyl) glycol copolymers and block copolymers, and activated derivatives 25 thereof.
7. The composition of claim 6 wherein said polyoxyalkyl glycol has a molecular weight of about 200 to about 50,000. 30
8. The composition of claim 7 wherein said polyoxyalkyl glycol has a molecular weight of about 200 to about 20,000. WO 99/29839 PCT/US98/26451 16
9. The composition of claim 6 wherein said polyoxyalkyl glycol is polyethylene glycol.
10. The composition of claim 1 wherein said graft copolymer comprises 5 polyethylene glycol grafted to an E-amino group of poly(L-lysine).
11. The composition of claim 10 wherein said graft copolymer comprises about 5 mole% to about 25 mole% of polyethylene glycol. 10
12. The composition of claim 11 wherein said graft copolymer comprises about 10 mole% of polyethylene glycol.
13. A composition for delivery of a selected nucleic acid into a host cell comprising an electrostatic complex of the selected nucleic acid and a biocompatible 15 graft copolymer comprising a cationic first polymer and an amphiphilic second polymer.
14. The composition of claim 13 wherein said first polymer is a member selected from the group consisting of poly(L-lysine), derivatives thereof, and 20 mixtures thereof.
15. The composition of claim 14 wherein said first polymer is poly(L lysine). 25
16. The composition of claim 15 wherein said poly(L-lysine) has a molecular weight of about 200 to 50,000.
17. The composition of claim 13 wherein said second polymer is a polyoxyalkyl glycol. 30
18. The composition of claim 17 wherein said polyoxyalkyl glycol is a member selected from the group consisting of polyethylene glycol homopolymers, WO 99/29839 PCT/US98/26451 17 polypropylene glycol homopolymers, alpha-substituted poly(oxyalkyl) glycols, poly(oxyalkyl) glycol copolymers and block copolymers, and activated derivatives thereof. 5
19. The composition of claim 18 wherein said polyoxyalkyl glycol has a molecular weight of about 200 to about 50,000.
20. The composition of claim 19 wherein said polyoxyalkyl glycol has a molecular weight of about 200 to about 20,000. 10
21. The composition of claim 18 wherein said polyoxyalkyl glycol is polyethylene glycol.
22. The composition of claim 13 wherein said graft copolymer comprises 15 polyethylene glycol grafted to an e-amino group of poly(L-lysine).
23. The composition of claim 22 wherein said graft copolymer comprises about 5 mole% to about 25 mole% of polyethylene glycol. 20
24. The composition of claim 23 wherein said graft copolymer comprises about 10 mole% of polyethylene glycol.
25. The composition of claim 13 wherein said nucleic acid and said graft copolymer are present in a weight ratio of about 0.3 to 10. 25
26. The composition of claim 13 further comprising an effective amount of an anti-endosome function agent.
27. The composition of claim 26 wherein said anti-endosome function 30 agent comprises an effective amount of chloroquine. WO 99/29839 PCT/US98/26451 18
28. The composition of claim 27 wherein said effective amount of chloroquine is about 25-250 4M.
29. The composition of claim 28 wherein said effective amount of 5 chloroquine is about 75-150 AM.
30. A method of transforming a host cell with a selected nucleic acid comprising contacting the host cell with an effective amount of an electrostatic complex comprising the selected nucleic acid and a biocompatible graft copolymer, 10 wherein the biocompatible graft copolymer comprises a cationic first polymer and an amphiphilic second polymer, such that said host cell internalizes said selected nucleic acid.
31. The method of claim 30 wherein said first polymer is a member 15 selected from the group consisting of poly(L-lysine), derivatives thereof, and mixtures thereof.
32. The method of claim 31 wherein said first polymer is poly(L-lysine). 20
33. The method of claim 32 wherein said poly(L-lysine) has a molecular weight of about 200 to 50,000.
34. The method of claim 30 wherein said second polymer is a polyoxyalkyl glycol. 25
35. The method of claim 34 wherein said polyoxyalkyl glycol is a member selected from the group consisting of polyethylene glycol homopolymers, polypropylene glycol homopolymers, alpha-substituted poly(oxyalkyl) glycols, poly(oxyalkyl) glycol copolymers and block copolymers, and activated derivatives 30 thereof. WO 99/29839 PCT/US98/26451 19
36. The method of claim 35 wherein said polyoxyalkyl glycol has a molecular weight of about 200 to about 50,000.
37. The method of claim 36 wherein said polyoxyalkyl glycol has a 5 molecular weight of about 200 to about 20,000.
38. The method of claim 35 wherein said polyoxyalkyl glycol is polyethylene glycol. 10
39. The method of claim 30 wherein said graft copolymer comprises polyethylene glycol grafted to an E-amino group of poly(L-lysine).
40. The method ofclaim 39 wherein said graft copolymer comprises about 5 mole% to about 25 mole% of polyethylene glycol. 15
41. The method ofclaim40 wherein said graft copolymer comprises about 10 mole% of polyethylene glycol.
42. The method of claim 30 wherein said nucleic acid and said graft 20 copolymer are present in a weight ratio of about 0.3 to 10.
43. The method of claim 30 further comprising an effective amount of an anti-endosome function agent. 25
44. The method of claim 43 wherein said anti-endosome function agent comprises an effective amount of chloroquine.
45. The method of claim 44 wherein said effective amount ofchloroquine is about 25-250 4M. 30
46. The method of claim 45 wherein said effective amount ofchloroquine is about 75-150 /M. WO 99/29839 PCT/US98/26451 20
47. A method ofusing a composition for delivering a selected nucleic acid to an individual comprising administering an effective amount of an electrostatic complex comprising the selected nucleic acid and a biocompatible graft copolymer, comprising a cationic first polymer and an amphiphilic second polymer, such that the 5 complex is systemically circulated and contacts a host cell such that the host cell internalizes the selected nucleic acid.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US6935197P | 1997-12-12 | 1997-12-12 | |
US60069351 | 1997-12-12 | ||
PCT/US1998/026451 WO1999029839A1 (en) | 1997-12-12 | 1998-12-11 | Grafted copolymers as gene carriers |
Publications (1)
Publication Number | Publication Date |
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AU1820999A true AU1820999A (en) | 1999-06-28 |
Family
ID=22088387
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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AU18209/99A Abandoned AU1820999A (en) | 1997-12-12 | 1998-12-11 | Grafted copolymers as gene carriers |
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EP (1) | EP1032659A1 (en) |
JP (1) | JP2001526181A (en) |
KR (1) | KR20010032879A (en) |
AR (1) | AR013017A1 (en) |
AU (1) | AU1820999A (en) |
BR (1) | BR9814273A (en) |
IL (1) | IL136679A0 (en) |
WO (1) | WO1999029839A1 (en) |
ZA (1) | ZA9811378B (en) |
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AU2001238485A1 (en) * | 2000-02-18 | 2001-08-27 | The Immune Response Corporation | Methods and compositions for gene delivery |
EP1421217A2 (en) * | 2001-08-27 | 2004-05-26 | Zeptosens AG | Surface for the immobilisation of nucleic acids |
CN100434520C (en) * | 2006-03-07 | 2008-11-19 | 湖北大学 | Polypeptide type transfection reagent |
CN102462846B (en) * | 2010-11-08 | 2014-07-09 | 复旦大学 | Chlorotoxin-modified glioma targeting gene delivery compound and preparation method thereof |
KR101357899B1 (en) * | 2011-02-25 | 2014-02-03 | 서울대학교산학협력단 | Galactosylated polyethylene glycol-chitosan-graft-spermine copolymer as a hepatocyte targeting gene carrier and gene therapy using the same |
ES2635087T3 (en) | 2011-03-03 | 2017-10-02 | Chugai Seiyaku Kabushiki Kaisha | Hyaluronic acid derivative modified with amino carboxylic acid |
KR101486989B1 (en) | 2013-10-15 | 2015-02-03 | 부산대학교 산학협력단 | Copolymer and gene transfer carrier comprising the same |
JP6564369B2 (en) | 2013-12-09 | 2019-08-21 | デュレクト コーポレイション | Pharmaceutically active agent conjugates, polymer conjugates, and compositions and methods involving them |
CN103755953B (en) * | 2013-12-12 | 2015-11-18 | 深圳先进技术研究院 | A kind of intelligent polycation nano-carrier, its preparation method and application thereof |
CN106554499B (en) * | 2015-09-25 | 2018-12-14 | 南京理工大学 | A kind of poly- (beta-amino ester) quasi polymer genophore and its synthetic method and application containing disulfide bond |
CN106978444B (en) * | 2016-01-15 | 2021-12-17 | 江苏命码生物科技有限公司 | Method for introducing nucleic acid into cell |
CN111393552B (en) * | 2020-03-22 | 2022-07-15 | 福建医科大学 | Preparation and application of polyvinyl amine-arginine grafted derivative as gene vector |
Family Cites Families (1)
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AU6254494A (en) * | 1993-02-16 | 1994-09-14 | Virginia Tech Intellectual Properties, Inc. | Polyelectrolyte dna conjugation and genetic transformation of an animal |
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1998
- 1998-12-11 IL IL13667998A patent/IL136679A0/en unknown
- 1998-12-11 JP JP2000524412A patent/JP2001526181A/en active Pending
- 1998-12-11 AR ARP980106331A patent/AR013017A1/en not_active Application Discontinuation
- 1998-12-11 EP EP98963119A patent/EP1032659A1/en not_active Withdrawn
- 1998-12-11 ZA ZA9811378A patent/ZA9811378B/en unknown
- 1998-12-11 WO PCT/US1998/026451 patent/WO1999029839A1/en not_active Application Discontinuation
- 1998-12-11 BR BR9814273-9A patent/BR9814273A/en not_active Application Discontinuation
- 1998-12-11 KR KR1020007006209A patent/KR20010032879A/en active Search and Examination
- 1998-12-11 AU AU18209/99A patent/AU1820999A/en not_active Abandoned
Also Published As
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BR9814273A (en) | 2000-10-03 |
IL136679A0 (en) | 2001-06-14 |
ZA9811378B (en) | 1999-08-05 |
EP1032659A1 (en) | 2000-09-06 |
JP2001526181A (en) | 2001-12-18 |
AR013017A1 (en) | 2000-11-22 |
KR20010032879A (en) | 2001-04-25 |
WO1999029839A1 (en) | 1999-06-17 |
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