GENE DELIVERY SYSTEM

Abstract: A gene delivery system is made of enzymatically degradable polymeric cation and nucleic acid (DNA or RNA) nanospheres optionally with a linking moiety or a targeting ligand attached to the surface. The delivery system can be made by a simple method of coacervation. Targeting ligands can be attached to the nanosphere directly or via a linking moiety. The linkage design allows the attachment of any molecule onto the nanosphere surface including antibodies, cell adhesion molecules, hormones and other cell-specific ligands.

Description: GENE DELIVERY SYSTEM This application is a continuation-in part of Serial No. 08/657,913 filed June 7, 1994, and Serial No. 60/021,408 filed July 9, 1996. BACKGROUND OF THE INVENTION A variety of techniques nave been used to introduce foreign genes into cells. Physical methods include co-precipitation with calcium phosphate, electroporation, and particle bombardment. While these direct transfer techniques are adequate in vitro, they are impractical in vivo. Promising in vivo gene therapy relies on a carrier such as viral vectors or lipoβomes for delivery. There are still lingering safety concerns for viral vectors. Another limitation is the size of the DNA sequences, usually limited to 7-8 kb, that can be incorporated into the viral vector. Lipoβomeβ, on the other band, nave low loading level in general. In both cases, there is the issue of cell or tissue specificity for these gene delivery systems. Controlled drug delivery has significantly improved the success of many drug therapies (Langer, R., 1990, New methods of drug delivery, Science, 249:1527-33; Poznansky, et al., 1984, Biological approaches to the controlled delivery of drugs: a critical review, Pharmacol. Rev., 36:277-336). A major goal of drug delivery is to localize the drug to the target site. These targeted delivery systems often take the form of injectables composed of lipoβomeβ (Gregoriaαis, G., 1988, Lipoβomeβ as Drug Carriers, New York: Wiley; Litzinger, et al., 1992, Phosphatidylethanolamine lipoβomes: drug delivery, gene transfer and immunodiagnoβtic applications, Biochimica et Biopnysica →A ta., 1113:201-27) and microsphereβ made of proteins (Cummings, et al., 1991, Covalent coupling of doxorubicin in protein microβpheres is a major determinant of tumor drug deposition, Biochem. Pharm., 41:1849-54; Verrijik, et al., 1991, Polymer-coated albumin microspheres aβ carriers for intravaβcular tumor targeting of cisplatin, Cancer Chemotner. ana Pharm. , 29:117-21; Tabata, et al., 1988, Potentiation of antitumor activity of macrophageβ by recombinant interferon alpha A/D contained in gelatin microspheres, Jpn. J. Cancer Res., 79:636-646), polysaccharides (Rongved, et al., 1991, Croββed-linked, degradable starch microspheres as carriers of paramagnetic resonance imaging: synthesis, degradation, and relaxation properties, Carbohydrate Res., 145:83-92; Carter, et al., 1991, The combination of degradable starch microβpheres and angiotensin II in the manipulation of drug delivery in an animal model of colorectal metastasis, British J. Cancer, 65:37-9), and synthetic polymers (Davis, et al., 1984, Microβpheres and Drug Therapy, Amsterdam; Eldridge, et al., 1991, Biodegradable microβpheres as a vaccine delivery system, Molec. Immunology, 28:287-94; Pappo, et al., 1991, Monoclonal antibody-directed targeting of fluorescent polystyrene microβpheres to Peyer'β patch M cells, Immunology, 73:277- 80). Polymeric systems βhare some of the advantages of liposomal systems such as altered pharmacokineticβ and biodistribution. While liposomes might have better prospects of biocompatibility and potential for fusion with cells, polymeric microβphereβ have more controllable release kinetics, better stability in storage, and higher drug-loading levels for some classes of compounds. There is a need in the art for a DNA delivery system which can provide controlled release, iβ simple to make, is stable, is cost effective, has a high DNA loading level, and is relatively non-immunogenic. SUMMARY OF THE INVENTION It is an object of the invention to provide polymeric particles for delivery of DNA to cells. It is an object of the invention to provide a method of making polymeric particles for delivery of DNA to cells. It is another object of the invention to provide a method of delivering DNA to cells using polymeric particles. Theβe and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment a nanosphere for gene delivery is provided. The nanosphere comprises a polymeric cation and DNA, and optionally a linking molecule or a targeting ligand is attached to the surface of said nanosphere. In another embodiment of the invention a method of forming nanospheres for gene delivery is provided. The method compriβeβ the steps of: forming nanospheres by coacervation of DNA and a polymeric cation; and optionally adhering a linking molecule or a targeting ligand to the surface of the nanospheres. In yet another embodiment of the invention a method for introducing genes into cells is provided. The method compriβeβ incubating cells to be transfected with solid nanospheres comprising a polymeric cation and DNA Optionally a targeting ligand is attached to the nanoβphere's surface. The targeting ligand binds to the surface of the cells to be transfected. Thus the present invention provides the art with an attractive DNA delivery system which is simple to prepare, is cost effective, has controlled release ability, is storage stable, and is biocompatible. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Schematic diagram showing the synthesis of gelatin-DNA coacervateβ. Figure 2. Gel electrophoresiβ of cDNA before and after encapsulation. (βtd= standard; S up = supernatant, Pellet = nanospheres pelleted by centrifugation) . Figure 3. Controlled release of intact LAMP-1 cDNA was demonstrated in vitro. The nanospheres were cross-linked with glutaraldehyde at various glutaraldehyde concentrations then degraded with trypsin. Figure 3A shows the time course of DNA release at various glutaraldehyde-cross- linking levels. Figure 3B shows (on gelβ and densitometer tracing) the DNA which waβ released from the nanospheres at various times and at various levels of glutaraldehyde-cross-linking. Figure 4. Fluorescent images of U937 cellβ transfected by controls and LAMP- 1 cDNA-loaded nanospheres (at day 3 poβt-transfection). Figure 4A: anti-DC44 nanospheres without cDNA; Figure 4B: calcium phosphate tranβfection; Figure 4C: LAMP-1 nanospheres without antibody; Figure 4D: LAMP-1 nanospheres coated with anti-CD-44 mAB. LAMP-1 expression is manifested as granules (in lysoβomes) in the cells. Figure 5. Flow cytometric analysis of the transfection efficiency of U937 cells by antilymphocyte function associated antigen- 1 coated nanospheres and controls. The actual mean fluorescence intensity (MFI) is shown in the insert. Msp= microspheres, MRK= a mismatched anti-P- glycoprotein antibody, PLM = anti-LFA antibody. Figure 6. Temporal expression of LAMP-1 in 293s cells transfected by anti- CD44 coated nanospheres. Figure 7. Preparation of DNA-chitoβan nanoβphereβ by complex coacervation. Nanospheres form as a result of Na^S0 4 induced desolvation of the polyelectrolyte complexes. Procedures or features that may help tran-βfection efficiency, but are not necessary for transfection include: the coencapβulation of lysoβomolytic agentβ or therapeutic agentβ into the nanoβphereβ during the complex coacervation; varying the cross- linking to affect the stability of nanospheres; conjugation of ligands to the surface of the nanoβphereβ to enhance receptor-mediated endocytosis. Figure 8. The chemistry of coupling of mannose-6-phσsphate to the surface of a DNA-nanoβphere using carbodiimideβ. Figure 9- The chemistry of coupling of folate to the surface of a DNA- nanoβphere using carbodiimideβ. Figure 10. The chemistry of coupling of tranβferrin to the surface of a DNA- nanosphere using carbodiimideβ. Figure 11. The persistence of luciferaβe expression in HEK293 cells after transfection with DNA-chitoβan nanoβphereβ is demonβtrated. 8 X 10 HEK293 cells were seeded into each well plate 24 hourβ before transfection, nanospheres containing 1 μg of DNA were incubated with the cells in each well for 4 hourβ. The cells were further incubated with fresh medium. After 5 days, the cells were passaged at 1:5 dilution every 3-7 days (indicated by an asterisk in the figure). Luciferaβe activities were measured at different time points. Figure 12. Expreββion of green fluorescence protein in HEK293 cells after transfection with chitosan nanospheres containing pDI-neoGFP plaβmid. 8 X 10 HEK293 cells were seeded into each well of 12 well plate 24 hours before transfection, nanospheres containing 1 mg of DNA were incubated with the cells in each well for 4 hours. The cells were further incubated with fresh medium for 3 days, trypsinized and analyzed on a FACScan analyzer. The percentage of positive cells (transfection percentage) versus the amount of nanospheres is shown. Figure 13. Transfection of HEK293 cells with mannoβe-6-phoβphate or folate conjugated nanospheres. 8 x cells were seeded into each well of a 12 well plate 24 hours before transfection. nanospheres containing 2 μg of DNA were incubated with the cells in each well for 4 hours. The cells were further incubated with fresh medium for 3 days, and luciferase expression levels were measured using a Promega luciferaβe assay system. Figure 14. Three-phase diagram for pRE-luciferase pcDNA-chitoβan coacervation at 50 mM Na 2 S0 4 and 55° C. The dark shaded area shows the concentration at which the nanosphere phase can be observed. Precipitation or aggregation occurs within the light area and there is no - O - phaβe separation in the white area. The conditions used to prepare nanoβphereβ are shown by the arrow. The final composition of the nanospheres are 34.65 ± .0.95 % by weight of plasmid DNA and 65.35 ± 0.95% by weight of chitosan; their encapsulation efficiencies are 98 ±2.0%and 92.7 ± 3.7%, reβpectively. Figure 15. Size distribution of chitoβan-baβed nanospheres is shown as determined by Dynamic Light Scattering and Photon Correlation Spectroβcopy. The βize of the nanospheres clustered tightly between 200 and 300 nm. Figure 16. Nanopshere formulation protectβ plasmid DNA from DNaβe I digestion. Naked DNA and nanoβphereβ were both incubated with different concentrations of DNasel for 15 min. At 37° C. The reaction was stopped with iodoacetic acid, a DNaβe I inhibitor. The nanoβphereβ were then digested with chitoβanase and lysozyme for 2 hours. All samples were run on an 0.08% polyacrylamide gel and βtained with ethidium bromide. Lane 1: molecular weight markers; lane 2: plasmid; lane 3: nanospheres; lane 4: digested nanospheres; lane 5: plasmid + DNAse (0.1 μg); lane 6: nanoβphereβ + DNAse (0.1 μg); lane 7: nanoβphereβ + DNaβe + digestion; lane 8: nanopsheres + 0.5 μg DNaβe; lane 9: nanoβpheres + 0.5 μg DNase + digestion. Figure 17. Cytotσxicity of polycationic gene carriers. HEK 293 cells (4 X 10 / well) wee plated in 96-well plates. Different concentrations of chitosan, poly-L-lysine and Lipofectamine were added 24 hrβ. later. After 48 hrβ cytotσxicity was determined by an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; thiaolylblue)reaction assay according to a standard protocol. Diamonds: chitosan; squares: poly-L-lysine; triangles: lipofectamine. Figure 18. Green fluorescence protein expression in HEK 293 cells 2 days after transfection. Transfections were carried out in a 24 well plate (5 x 10* cells/well) using different amounts of nanoβphereβ. Transfected cells were sorted after 2 days using FACβcan. Ncβp 0.1 = nanospheres iwth 0.1 μg DNA, Ncβp 0.5 = nanoβphereβ with 0.5 μg of DNA, etc. Seep + Chlq 1 = nanospheres with chloroquine and 1 μg of DNA Figure 19. Uptake of nanoβphereβ in HEK 293 cellβ. Nanoβphereβ were made with the luciferase plasmid and βtained with TO-PRO (Molecular Probeβ Inc.), a cell-impermeable DNA-binding dye. Cells were washed, trypsinized at different time points arter transfection and analyzed by FACscan. Cellβ transfected at 4° C were used as controls to account for particles bound to cell surface. Figure 20. Storage stability of DNA-chitoβan nanospheres and freeze- dried nanospheres. Nanospheres were made according to methods described below. PEG- conjugated nanosphere were freeze dried directly after being spun down to βucroβe gradients (50%). Transfection of HEK 293 cells was performed according to the same method as before. Figure 21- Chemical scheme for attaching ligands to the surface of the DNA nanoβphereβ. Figure 22. Expression of β-gal in muscle of BALB/c mice transfected with pcBLacZ gene delivered in different forms. Levels were determined at days 7 and 21 post-tranefection, and the endogenous background galactosidase activity was subtracted. Values are average (n=6) + SD. _ _ DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS It is a discovery of the present invention that nucleic acid molecules of various chain lengths can complex with polymeric cations in aqueous conditions to form solid nanoβphereβ ranging from βubmicron to microns in size. These nucleic acid-loaded nanospheres can efficiently transfect cellβ. According to the present invention, a polymeric cation having a similar charge density to gelatin, iβ used to complex with nucleic acids to form nanoβphereβ. Proteins such as tubulin, actin, cytochrσme C, human serum albumin, and histoneβ may be used as the polymeric cation. Polymeric amino acids, such as polyarginine and polylysine, may also be used, although natural proteins are preferred. Alternatively, carbohydrates such aβ chitosan, prσteσglycan, methylcelluloβe, amylose, and starch can be used aβ the polymeric cation. Typically the polymeric cation has a molecular weight of between 5,000-1,000,000. Chitosan may be particularly useful as the polymeric cation of the present invention. Desirably sodium sulfate iβ used to induce the coacervation of polymeric cation and nucleic acids. Ethanol can also be used at a concentration of about 40 to 60% to induce coacervation. Targeting ligands, if desired, can be directly bound to the surface of the nanosphere or can be indirectly attached using a "bridge" or "spacer". Because of the amino groups on some polysaccharideβ and on proteins (aβ provided by arginine and lysine groups of the proteins), the surface of the nanoβphereβ can be easily derivatized for the direct coupling of targeting moieties. For example, carbo-diimides can be vised aβ a derivatizing agent. Alternatively, spacers (linking molecules and derivatizing moieties on targeting ligands) such as avidin-biotin can be used to indirectly couple targeting ligands to the nanoβphereβ. Biotinylated antibodies and/or other biotinylated ligands can be coupled to the avidin-coated nanosphere surface efficiently because of the high affinity of biotin M '1 ) for avidin (Hazuda, et al., 1990, Processing of precursor interleuhin 1 beta and inflammatory disease,/. Biol. Chem., 265:6318- 22; Wilchek, et al., 1990, Introduction to avidin-biotin technology, Methods In Bnzymology, 184:5-13). Orientation-βelective attachment of IgGβ can be achieved by biotinylating the antibody at the oligoβaccharide groups found on the F c portion (O'Shannessy, et al., 1984, A novel procedure for labeling immunoglobulinβ by conjugation to oligosaccharides moieties, Immunol. Lett., 8:273-277). This design helps to preserve the total number of available binding sites and renders the attached antibodies less immunogenic to F c receptor-bearing cells such aβ macrophages. Spacers other than the avidin-biotin bridge can also be used, aβ are known in the art. For example, Staphylococcal protein A can be coated on the nanospheres for binding the F c portions of immi oglobulin molecules to the nanospheres. Croβs-linking can be used to stabilize nanoβphereβ. This iβ particularly useful for protein-based nanospheres, such aβ those made of gelatin. The rate of nanosphere degradation and nucleic acid release can be designed a priori by varying the extent of α-oββ-unking. Increased cross-linking yields increased stability of the nanosphere. The loading level of nucleic acid can be as high aβ 30% (w/w), with an encapsulation efficiency of >95%. Cross-linking of linking molecules or targeting ligands to the nanosphere is used to promote the stability of the nanosphere as well as to covalently affix the linking molecule or targeting ligand to the nanosphere. The degree of cross-linking directly affects the rate of nucleic acids release from the microspheres. Croβs-linking can be accomplished using glutaraldehyde, carbodiimideβ such as EDC (l-ethyl-3-(3- dimethyla-minσproρyl)-carbodiinιide, DCC (N,N'-dicyclohejcylcarbodϋmide), carboxylβ (peptide bond) linkage, bis (sulfoβuccinimidyl) βuberate, dimethylsuberimidate, etc. Targeting ligands according to the present invention are any molecules which bind to specific types of cells in the body. These may be any type of molecule for which a cellular receptor exists. Preferably the cellular receptors are expressed on specific cell types only. Examples of targeting ligands which may be used are hormones, antibodies, cell-adhesion molecules, saccharides, drugs, and neurotransmitters . The nanoβphereβ of the present invention have good loading properties. Typically, following the method of the present invention, nanoβphereβ having at least 5% (w/w) nucleic acids can be achieved. Preferably the loading is greater than 10 or 15% nucleic acids. Often nanoβphereβ of greater than 20 or 30%, but less than 40 or 50% nucleic acids can be achieved. Typically loading efficiencies of nucleic acids into nanoβphereβ of greater than 95% can be achieved. The method of the present invention involves the coacervation of polymeric cations and nucleic acids. Because this process depends on the interaction of the positively charged polymeric cations and the negatively charged nucleic acids it can be considered aβ a complex coacervation proceβs. However, sodium sulfate (or ethanol) induces the coacervation reaction by inducing a phase transition, and therefore it could also be considered aβ a simple coacervation reaction. Nucleic acids are present in the coacervation mixture at a concentration of between 1 ng/ml to 500 μg/ml. Desirably the nucleic acids are at least about 2-3 kb in length, and may range up to 10, 20, or 50 kb. Sodium sulfate iβ present at between 5 and 100 mM. Gelatin or other polymeric cation is present at between about .01 and 7%, preferably between 2 and 5% (w/v), in the coacervation mixture. Typically, about ten-fold higher amounts of amino acid-based polymers are required than carbohydrate based polymers. Thus gelatin is typically used at 1-5%, while chitosan is typically used at 0.01 to 0.05%. Uβing chitosan, suitable concentrations of nucleic acids are between 0.002% and 0.008%. An attractive nanosphere delivery system requires a delicate balance among factors such as the simplicity of preparation, cost effectiveness, nucleic acids loading level, controlled release ability, storage βtability, and immunogenicity of the components. he gene delivery system described here may offer advantages compared to other particulate delivery systems, including the liposomal system. The problems of instability, low loading level, and controlled release ability are better resolved with the polymeric nanosphere systems. Gelatin has received increasing biologic use ranging from surgical tissue adheβive (Weinβchelbaum, et al., 1992, Surgical treatment of acute type A dissecting aneurysm with preservation of the native aortic valve and use of biologic glue. Follow-up to 6 years, /. → orac. Caraioυasc. Surg. , 130:369-74) to quantitative immunohistochemical assays (Izumi, et al., 1990, Novel gelatin particle agglutination test for serodiagnoβiβ of leproβy in the field, /. Clinical Microbiol. , 28:525-9) and aβ drug delivery vehicle (Tabata, et al., 1991, Effectβ of recombinant alpha-interferon-gelatin conjugate on in vivo murine tumor cell growth, Cancer Res., 51:5532-8), due to its biocompatibility and enzymatic degradability in vivo. Compared to other synthetic polymeric systems, such aβ the extensively studied polylactic/polyglycolic copolymers, the mild conditions of nanosphere formulation are appealing. Unlike the solvent evaporation and hot-melt techniques used to formulate synthetic polymeric nanospheres, complex coacervation requires neither contact with organic solvents nor heat. It is also particularly suitable for encapsulating bio- macromoleculeβ βuch aβ nucleic acids not only through passive solvent capturing but also by direct charge-charge interactions. Unlike viral vectors, which cannot deliver genes larger than 10 kb, the nanosphere delivery system of the present invention doeβ not have such size limitations. Nucleic acid molecules of greater than about 2 kb can be used, and nucleic acid molecules even from 10 to 50 kb may be used. In general, the range of possible targets iβ dependent on the route of injection, e.g., intravenous or intraarterial, subcutaneous, intra-peritoneal, intrathecal, etc. For systemic injections, the specificity of this delivery system iβ affected by the accessibility of the target to blood borne nanoβphereβ, which in turn, is affected by the βize range of the particles. Size of the particles iβ affected by temperature, component concentration, and pH in the coacervation mixture. The particles can also be size- fractionated, e.g., by sucrose gradient ultracentrifugation. Particles with size less than 150 nanometers can acceββ the interstitial space by traversing through the fenestrations that line most blood vessels walls. Under such circumstances, the range of cellβ that can be targeted is extensive. An abbreviated list of cells that can be targeted includes the parenchymal cells of the liver sinusoids, the nbroblaβts of the connective tissues, the cellβ in the Islets of Langerhanβ in the pancreas, the cardiac myocytes, the Chief and parietal cellβ of the intestine, osteocyteβ and chondrocyteβ in the bone, keratinocyteβ, nerve cells of the peripheral nervous system, epithelial cellβ of the kidney and lung, Sertoli cells of the testis, etc. The targets for particles with sizes greater than 0.2 microns will be confined largely to the vascular compartment. Here, the targetable cell types include e ythrocytes, leukocytes (i.e. monocyteβ, macrσphageβ, B and T lymphocytes, neutrophils, natural kdler cellβ, progenitor cellβ, mast cells, eoβinophilβ), platelets, and endothelial cellβ. For subcutaneous injections, the targetable cells include all cellβ that resides in the connective tissue (e.g., fibroblaβtβ, mast cells, etc.), angerhans cells, keratinocyteβ, and muscle cells. For intrathecal injections, the targetable cells include neurons, glial cells, aβtrocytes, and blood-brain barrier endothelial cells. For intraperitoneal injection, the targetable cells include the macrophageβ and neutrophils. Carbohydrate-based nanoβphereβ such as those made using chitosan offer many advantages. Cross-linking is not required. In addition, a targeting ligand is not required, although it can be used. Although applicants do not wiβh to be bound by any particular theory, it iβ poβtulated that cells have a receptor to which chitosan binds. The concentration of DNA required in the coacervation mix using chitosan is about half of that required using gelatin. In addition, the concentration of chitosan required iβ about one tenth that required for making gelatin nanospheres. Chitosan based nanoβphereβ can be freeze dried and are storage stable. Because chitoβan-baβed nanoβphereβ need not be cross-linked or reacted with targeting ligands, they can be readily used after formation without the need for extensive post-synthesis purifications. Elimination of this post-βyntheβis processing permits the direct use of freshly made coacervateβ to tranβfect cells. An additional contraβt between chitoβan- and gelatin-based nanospheres iβ the effect of chloroquine on transfection efficiencies. In the gelatin syβtem, chloroquine improves the transfection efficiency. However, in the chitosan system, chloroquine does not improve the transfection efficiency. The size of the nanospheres is believed to be critical for efficient cellular delivery. Typically the nanoβphereβ are less than 4, 3, 2, or even 1 μm. Most preferably the solid particles are between 200 and 300 run. -Examples: Example 1 Matrix Materials: Gelatin (60 bloom, type A from porcine skin), chondroitin 4-βulfate, glutaraldehyde (25%, grade 1), l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide hydrochloride (EDC hydrochloride), and ultra -pure sucrose were purchased from Sigma Chemical Co. (St. Louis, MO). Biotin LC hydrazide, and NeutrAvidin, and Coomassie protein aββay reagents were from Pierce (Rockford, IL). Centricon microconcentratorβ were from Amicon (Beverly, MA). Monoclonal antibodies: mAb PLM-2, a BALB/c mouse anti-porcine LFA-1 (IgG j i,) which also cross reacts with murine LFA-1, was isolated and purified as previously described (Hildreth, et al., 1989, Monoclonal antibodies against porcine LFA-1: species croββ-reactivity and functional effects of b-βubunit-βpecific antibodies, Molec. Immunol , 26:883-895). IB-4B, a rat anti-mouse LAMP-1 ascite fluid and a mouse anti-human CD44 mAb were isolated as previously described (de Wet, et al., 1987, Firefly lucif erase gene: structure and expression in mammalian cells, Mol. & Cell. Bio!., 7:725-37). CHA is a IgG x that does not recognize any known in vivo mouse epitσpeβ (Hybritech Inc., San Diego, CA). Affinity-purified FITC and Texas Red-labeled polyclonal anti-rat IgGβ were obtained from Sigma. Gene : Two geneβ were uβed to demonstrate the feasibility of this delivery system. The LAMP-a cDNA is a 6.4 kb circular βupercoiled plasmid cDNA with a mouse LAMP-1 gene (2.4 kb) inserted into an Invitrogen plasmid cDNA with a CMV promoter (Guaamieri, et al., 1993,/. Bid. Chem., 268:1941). Detection of LAMP- 1 expression was done by staining cellβ with anti-LAMP-1 mAb and with secondary anti-IgG mAb conjugated with Texas Red. The gene coding for luciferaβe enzyme is widely used in cell biology for the study of gene expression because of the high sensitivity of the aβsay, its simplicity, and low cost. In addition, the enzyme is a good reporter of gene expression because it is a cytosolic protein that does not require post- translational processing for enzymatic activity (de et, et al., 1987, Firefly luciferaβe gene: structure and expression in mammalian cellβ, Mol. & Cell. Biol., 7:725-37; Wood, et al., 1989, Introduction to beetle luci erases and their applications, /. of Biolumin. & Chemilum. , 4:289-301). The presence of luciferaβe can be readily detected by an enzymatic reaction that involve the oxidation of beetle luciferin with concomitant production of a photon (in the form of chemilumineβcence.) The aββay waβ carried out using an aββay kit purchased from Promega Corp. (Madison, l). Synthesis of microspheres: A detailed schematic diagram for the synthesis of the gelatin-DNA coacervates iβ shown in Figure 1. All concentrations described are final concentrations in the reaction mixture set at 67°C unless otherwise stated. Gelatin/plaβmid DNA nanospheres coated with avidin were synthesized by first preparing a 3.5 mg ml βolution of plaβmid DNA encoding a lysosomal associated membrane protein-1 (LAMP-1) (6.7 Kb, circular supercoiled) in 42 mM sodium sulfate (Na j SO . Coacervation waβ initiated by the addition of gelatin (5%) to the DNA/Na 2 0 4 βolution at equal volume while vortexing at high speed for 1 minute. Coencapβulation of drugβ and other agentβ can be achieved by adding directly to the DNA/Na^O j βolution before initiating coacervation with gelatin. Avidin (5 mg/ml) waβ added to the micrσβphere suspension at a final concentration of 75 ug avidin/ml microsphere solution. The microspheres mixture waβ layered onto a layer of 70% sucrose (w/v) and centrifuged at 6,000 x g for 4 minutes (Brinkman Instruments Inc., Westbury, NY, model L8-75). Micrcβphere fractions recovered from the sucrose layer was diluted 5-fold with water then cross-linked with glutaraldehyde (12.5 mM final concentration) for 10 minutes at room temperature. Unreacted glutaraldehyde was quenched by adding ethanolamine (1 M) for 10 minutes. The microβpheres were dialyzed by sucrose centrifugation as described above. -Attachment of biotinylated m→Abs to avi din-coated microspheres: 30 ug of antibodieβ (biotinylated according to established procedures (O'Shannesβy, et al., 1984, A novel procedure for labeling immunoglobulinβ by conjugation to oligosaccharides moieties, Immunol. Lett. , 8:273-277)) waβ added to 1 ml of avidin- coated microβphereβ suspension (11 mg/ml) for 1 hour with gentle agitation. Unbound mAb waβ removed from microβphereβ by dialysis. Characterization of miσrosphere ana binding performance: DNA loaded microspheres exhibited polymorphic colloid shape with a polydiβperβed particle size of less than 3 microns aβ determined by light microscopy. Purified microβphereβ were stable for at least one month without appreciable degradation. The loading level for LAMP-1 plasmid DNA was 20% (w/w). The encapsulation efficiency waβ typically >95%. The mobility of the free LAMP-1 DNA and the releaβed DNA (from the microβphere) in 1% agaroβe gel electrophoreβiβ were identical (Fig. 2), suggesting that the encapsulated DNA was releaβed in itβ original form. Release rate of the cDNA from the microβphereβ waβ dependent on the cross-linking density and on the enzyme level (Figure 4). Sustained releaβe of up to weeks can be readily obtained. We tested the ability of the LAMP-1 DNA loaded microβphereβ to bind and βubβequently tranβfect a human hiβtiocytic lymphoma cell line (U937) in tiββue culture. When coated with either anti-LFA or anti-CD44 monoclonal antibody (both protein targets were expreββed in high amount of U937 cell βurface), expresβion LAMP-1 protein waβ detected by day 3 (Fig. 4A, fluorescent granules) when stained with antibodies recognizing LAMP-1. The staining pattern of U937 cells incubated with LAMP-1 microspheres was identical to the calcium phosphate method of transfection (Figure 4B). Microβphereβ that were either coated with avidin or non- specific CHA mAb showed no granular staining patterns, and were identical to untreated cellβ (Fig. 4C). Using flow cytometry, we showed that the expreββion of LAMP-1 waβ detected in up to 5% of U937 cellβ in culture (Figure 5). None of the controls ~ blank microβpheres, microβpheres with cDNA but no antibodies, microβphereβ with cDNA and coated with a mismatched anti-P-glycoprotein antibody (MRK-Mβp.), and free cDNA at a concentration βix time higher than entrapped in the microspheres — showed any evidence of transfection. The efnciency of transfection appears to be doβe- reβpσnβive. In general, the transfection efnciency of this particular gene and cell type - ) - is comparable between the proposed microspheric delivery system (1-10%) and the calcium phosphate precipitation method (2-15%). Figure 6 demonstrates the concept in a different cell type using a different monoclonal antibody. Again, free cDNA could not tranβfect the cellβ. Eventually the LAMP-1 expression disappeared after several passages. Positive results were also obtained for the luciferaβe reporter gene system. Transfection waβ clearly detected by measurement of luciferase enzymatic activity, in 293s cellβ incubated with luciferaβe gene-loaded microβphereβ. Example 2 The general concept of synthesizing the DNA nanoβphereβ is shown in Figure 7. DNA-gelatin nanoβphereβ were synthesized by vortexing 100 ml of a solution containing gelatin (5% w/v in distilled water) and 4 mM chloroquine with 100 ml of a βolution containing 0.2 mg/ml plasmid DNA and 4.3 mM sodium sulfate (N - j SO^) at 55 °C for one minute. Nanoβphereβ without chloroquine were synthesized similarly except with a higher Na^O j concentration (45 mM). The unreacted components were removed by centrifuging the mixture in a 100 ml sucrose layer (55% w/w) at 40,000 x g for 7 minutes. The sucrose fraction containing the nanospheres waβ diluted with water to 200 ml, then croββlinked and conjugated with tranβferrin by adding the nanosphere and tranβferrin βolution (20 mg) to 22 ml of a 0.2 M MES buffer βolution, pH 4.5, containing 0.1 mg/'ml of (l-ethyl-3-[3-di-methylaminopropyll-carbodiimide hydrochloride) (EDC). The reaction waβ carried out in room temperature for 30 minutes, then quenched by addition of glycine reaching a 0.2 M final concentration. Croβslinking was also achieved by reaction with various concentrations of glutaraldehyde for 10 min. DNA-chitoβan nanoβphereβ were prepared similarly with the exception that the chitosan solution diluted to 0.02% (w/v, P H5.5 in 5 mM NaAc-HOAc). Example 3 The general concept of synthesizing DNA nanospheres iβ shown in Figure 7. Microparticular coacervates of chitosan or gelatin-DNA complexes formed as a result of Na j SO^-induced deβolvation of the local water environment of the polyelectrolytes. Synthesis of the nanospheres waβ optimized with respect to βalt concentration, pH, temperature, and reactant concentrationβ, aβ shown in the three- phase diagram of Figure 14. At 55 °C, pH 5.5, and a βodium sulfate concentration of 50 mM, phaβe βeparation of partictdate chitoβan-DNA complexes occurred at the concentration range of 0.005 to 0.02 % (w/v) for chitoβan, and 0.004 to 0.008 % for the DNA. Aβ βhown by TEM and SEM in Figure 15, nanoβphereβ in the range of 200-320 nm were formed. The solid nanoβphereβ protect the DNA from enzymatic degradation (Figure 16). Stability studies of the DNA-chitoβan nanoβphereβ were conducted in 10% fetal bovine serum (FBS), where the nucleaβe decomposed the DNA. Gel electrophoretic mobility analysis indicated naked DNA incubated for 15 minutes in the serum βhowed clear indication (Lane 5), whereas the DNA in the nanosphere remained intact (Lanes 6 and 7). Even at a 5-fold higher nucleaβe concentration of 0.5 ug/ml, there was still no βign of degradation (Lanes 8 and 9)- The cytot wάcity of chitoβan was compared to other polycationic gene carriers (Figure 17). Chitoβan was nontσxic at least up to a concentration of 200 ug/ml. The DNA-chitoβan nanospheres containing the GFP DNA transfected the 293 cells in a dose-responsive manner (Figure 18). Uptake of the DNA-chitoβan nanospheres was rapid aβ demonstrated by FACScan analysis (Figure 19). Almoβt 100% of cells have taken up the nanospheres in 2 hourβ of incubation, displaying βaturable kinetics. To allow lyophilization without aggregation, nanospheres were derivatized according to the chemical scheme βhown in Figure 21. One mL of DNA-chitoβan nanoβphereβ suspension containing 50 mg of pcDNA waβ mixed with 5 mL of DSS solution (10 mM), 10 mL of a-malemidyl-w-N-hydroxysuccinimidyl polyethylene glycol) βolution (NHS-PEG-MAL, MΨ 2000, 5 mM), 20 mL of βuccinimidyl succinamide of methoxy polyethylene glycol) βolution (SSA-PEG, MW 5000, 10 mM) and 100 mL of PBS buffer under vortexing. The mixture was stirred at room temperature for 30 min before 50 mL of 1 M glycine was added to quench the reaction, followed by adding 250 mg of transferrin modified with βulfhydryl group (free βulfhydryl groups were introduced into tranβferrin by 2-iminothiolane reaction, modification degree waβ 3~*6 per tranβferrin). After reaction for 2 hourβ at room temperature, the mixture was ultracentrifuged at 50,000g for 20 min in a 55% sucrose gradient. The nanoβphereβ iβolated were lyophilized directly. Conjugation of PEG5000 to the βurface of the nanoβphereβ minimized any aggregation in βolution. T-liese nanoβphereβ could also be lyophilized without any anti-caking agent and easily reβuβpended. Transfection emciency of the nanospheres waβ not affected by this PEG derivatization (Figure 20). Neither waβ it affected by βtorage in βucroβe βolution or in the lyophilized state. Comparison of in vivo transfection efnciency waβ conducted by injecting gelatin nanoβphereβ containing the LacZ plaβmid (1 mg total DNA), DNA-Lipofectamine complex (1 mg total DNA), naked plaβmid DNA (1 mg), or 5 x 1010 viral particles of AAV-LacZ suspended in 20 ml into the exposed tibialiβ anterior muscle bundles of six-week old BALB/c mice. One and three weeks later, the muscle waβ iβolated, homogenized, and the reporter gene expreββion determined by an aββay kit CGalacto-Laght 1 ) supplied by Tropix (Bedford, MA). Injection of gelatin nanospheres containing 1 mg of the LacZ gene into the tibialiβ muscle bundle of mice produced β-gal expression for at least 21 days (Figure 22). The level for an equivalent dose of naked DNA was 10-30 fold lower at day 7, and declined to background level by day 21. In contrast to their relative performance in vitro, the Lipofectamine complexes were not as efficient aβ the nanospheres. The expression level at day 7 waβ even lower than that of naked DNA. By gross observation, there waβ acute inflammatory response in the muscle tissue treated by the Lipofectamine complexes, which might account for the poor result. The AAV vector waβ the moβt efncient, eliciting a β-gal expression of 50-100 timeβ higher than that of the nanoβphereβ at day 7, and the level increased 6-12 fold further at day 21, probably due to viral replication. Nanospheres synthesized by salt-induced complex coacervation of cDNA and polycations such aβ gelatin and chitosan are efficient gene delivery vehicles. DNA-nanoβphereβ in the βize range of 200-750 nm could tranβfect a variety of cell lineβ. Although the transfection efnciency of the nanoβphereβ waβ typically lower than that of Lipofectamine and calcium phosphate controls in cell culture, the β-gal expreββion in muscle of BALB/c mice waβ higher and more sustained than that achieved by naked DNA and Lipofectamine complexes. This gene delivery system has several attractive features: 1) ligands can be conjugated to the nanosphere to βtimulate receptor-mediated endocytoβiβ; 2) lysosomolytic agentβ can be incorporated to reduce degradation of the DNA in the endoβomal and lyβoβomal compartments; 3) other bioactive agentβ or multiple plaβmidβ can be co-encapβulated; 4) bioavailabihty of the DNA can be improved because of protection from serum nucleaβe degradation by the matrix; 5) the nanosphere is stable in plasma electrolytes, and can be lyophilized for storage without loss of bioactivity.

Claims: CLAIMS I. A βolid nanosphere of leββ than 3 μm for gene delivery to cellβ, compriβing a polymeric cation and a polyanion, wherein the polyanion consists of nucleic acidβ, wherein the polymeric cation iβ not polylyβine. 2. The nanosphere of claim 1 wherein the polymeric cation is a carbohydrate. 3. The βolid nanosphere of claim 2 wherein the polymeric cation is chitoβan. 4. The nanosphere of claim 1 wherein said nanosphere comprises greater than 5% (w/w) nucleic acidβ. 5. The nanosphere of claim 1 wherein said nanosphere comprises greater than 20% (w/w) nucleic acidβ. 6. The nanosphere of claim 1 wherein said nucleic acids comprise a gene of 2-20 kb. 7. A method of forming βolid nanospheres for gene delivery to specific target cells, compriβing the βtep of: forming nanoβphereβ of leββ than 3 μ-m by coacervation of nucleic acids and a polymeric cation which iβ not polylyβine. 8. The method of claim 7 wherein the polymeric cation is a carbohydrate. 9. The method of claim 8 wherein the coacervation is performed in the presence of βodium sulfate. 10. The method of claim 8 wherein the polymeric cation iβ chitoβan. II. The method of claim 8 wherein the polymeric cation iβ preβent at a concentration of about 0.01-7% in the βtep of coacervation. 12- The method of claim 8 wherein the nucleic acidβ are preβent in a concentration of 1 ng/ml to 500 μg/ml in the step of coacervation. 13. The method of claim 8 wherein the concentration of sodium sulfate is between about 5 and 100 mM in the βtep of coacervation. 14. A method for introducing genes into cellβ, compriβing the steps of: incubating (a) cellβ to be tranβfected with (b) βolid nanoβphereβ of less than 3 μ-m compriβing polymeric cationic molecules and nucleic acid molecules, whereby the cells are tranβfected with the nucleic acid molecules, wherein the polymeric cationic molecules are not polylysine. 15. The method of claim 14 wherein the polymeric cationic molecules jure carbohydrates. 16. The method of claim 15 wherein the polymeric cationic molecules are chitoβan. 17. The method of claim 15 wherein the nucleic acid iβ DNA. 18. The method of claim 15 wherein the nucleic acid iβ RNA. 19- The method of claim 14 wherein the cellβ are in culture. 20. The method of claim 14 wherein the cellβ are in an animal.

Inventors: AUGUST THOMAS, LEONG KAM W, MAO HAI-QUAN, ROY KRISHNENDU, TRUONG THOMAS, AUGUST, THOMAS, LEONG, KAM, W., MAO, HAI-QUAN, ROY, KRISHNENDU, TRUONG, THOMAS
Applicants: UNIV JOHNS HOPKINS, THE JOHNS HOPKINS UNIVERSITY
Application year: 1997
Main classification: A61K47
IPC codes: A61K9, A61K47, C12N15