text:
Polymeric gene delivery system
A means for obtaining efficient introduction of exogenous genes into a patient, with long term expression of the gene, is disclosed. The gene, under control of an appropriate promoter for expression in a particular cell type, is encapsulated or dispersed with a biocompatible, preferably biodegradable polymeric matrix, where the gene is able to diffuse out of the matrix over an extended period of time, for example, a period of three to twelve months or longer. The matrix is preferably in the form of a microparticle such as a microsphere (where the gene is dispersed throughout a solid polymeric matrix) or microcapsule (gene is stored in the core of a polymeric shell), a film, an implant, or a coating on a device such as a stent. The size and composition of the polymeric device is selected to result in favorable release kinetics in tissue. The size is also selected according to the method of delivery which is to be used, typically injection or administration of a suspension by aerosol into the nasal and/or pulmonary areas. The matrix composition can be selected to not only have favorable degradation rates, but to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when administered to a mucosal surface.
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This is a continuation, of application Ser. No. 08/467,811, filed Jun. 6, 1995, which is a division of application Ser. no. 08/213,668, filed Mar. 15, 1994.BACKGROUND OF THE INVENTION
The present invention is generally in the area of drug delivery devices and is specifically in the area of polymeric drug delivery devices.
Gene therapy is generally defined as the introduction and expression of an exogenous gene in an animal to supplement or replace a defective or missing gene. Examples that have received a great deal of recent attention include the genes missing in cystic fibrosis and severe combined immunodeficiency. Although tremendous progress has been made in the area of gene therapy, obtaining long term expression of the desired proteins remains elusive.
In the majority of cases, a retroviral vector is used to introduce the gene to be expressed into appropriate cells. Gene transfer is most commonly achieved through a cell-mediated ex vivo therapy in which cells from the blood or tissue are genetically modified in the laboratory and subsequently returned to the patient. The clinical studies by Steven Rosenberg, et al., "Immunotherapy of patients with metastatic melanoma using tumor-infiltrating lymphocytes and IL-2", Preliminary report, New England J. Med., 319 (1988) 1676-1680, using in vitro-activated LAK and TIL for tumor destruction illustrates this approach. In other cases, the vector carrying the gene to be expressed is introduced into the patient, for example, by inhalation into the lungs in the case of cystic fibrosis. Transfected cells have also been implanted, alone or encapsulated within a protective membrane that protects the cells from the inflammatory response of the body while at the same time allowing the gene product to diffuse out of the membrane. There have also been reports of the direct injection of an exogenous gene in combination with an appropriate promoter, into tissue, with some transient expression being noted.
Viral vectors have been widely used in gene transfer, due to the relatively high efficiency of transfection and potential long term effect through the actual integration into the host's genome. However, there are still concerns about the risks involved in the use of viruses. Activation of proto-oncogenes and reversion to wild-type viruses from replication incompetent viruses are some important potential hazards of viral delivery of genes.
Since the discovery that naked DNA is taken up by muscle cells and transiently expressed in vivo, and subsequent reports, by Wolff, Jon A et al., "Direct gene transfer into mouse muscle in vivo," Science, 247, 1465-1468, 1990; and Acsadi et al., "Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs," Nature, 352, 815-818, 1991, there has been increasing interest in using non-viral vehicles for in vivo transfections.
Plasmid DNA, which can function episomally, has been used with liposome encapsulation, CaPO4 precipitation and electroporation as an alternative to viral transfections. Recent clinical trials with liposome encapsulated DNA in treating melanoma illustrates this approach to gene therapy, as reported by Nabel, J. G., et al., "Direct gene transfer with DNA-liposome complexes in melanoma: Expression, biological activity and lack of toxicity in humans", Proc. Nat. Acad. Sci. U.S.A., 90 (1993) 11307-11311. A foreign gene coding for HLA-B was introduced into subcutaneous sites of melanoma tumors. Expression of the new gene and the absence of an anti-DNA host response was confirmed. Wolff, Jon A, "Persistence of plasmid DNA and expression in rat brain cells in vivo," Experimental Neurology, 115, 400-413, 1992, also reported expression of plasmid DNA. Thus, direct gene transfer offers the potential to introduce DNA encoding proteins to treat human diseases.
The mechanisms for cellular uptake of exogenous DNA and subsequent expression are not clear but gene transfer with naked DNA is associated with several characteristics. Unlike in the case of oligonucleotides, which are typically a maximum of twenty to thirty nucleotides in length, genes encoding most molecules of therapeutic interest are quite large, and therefore considerably more difficult to introduce into cells other than through retroviral vector, or in vitro, by chemical manipulation, so that the efficiency of transfer is low. In most reported cases to date, only transient expression of up to a few weeks or months has been observed. Although low level expression and short term expression are two important drawbacks with direct DNA transfer, transfections with naked DNA have several advantages over viral transfers. Most importantly, concerns related to the immunogenicity and transforming capability of viruses are avoided. In addition, naked DNA is easy to produce in large quantities, is inexpensive, and can be injected at high concentration into localized tissue sites allowing gene expression in situ without extensive ex vivo therapy.
The following additional articles review the current state of gene therapy and the problems associated therewith: Blau, Helen M, "Muscling in on gene therapy," Nature, 364, 673-675, 1993; Cohen, Jon, "Naked DNA points way to vaccines," Science, 259, 1691-1692, 1993; Dagani, Ron, "Gene therapy advance, anti-HIV antibodies work inside cells," C&EN, 3-4, 1993; Felgner, Philip L, "Lipofectamine reagent: A new, higher efficiency polycationic liposome transfection reagent," Focus/Gibco, 15, 73-78, 1993; Liu, Margaret A et al., "Heterologous protection against influenza by injection of DNA encoding a viral protein," Science, 259, 1745-1749, 1993; Marx, Jean, "A first step toward gene therapy for hemophilia B," Science, 262, 29-30, 1993; Mulligan, Richard C, "The basic science of gene therapy," Science, 260, 926-931, 1993; Nicolau, Claude et al., "In vivo expression of rat insulin after intravenous administration of the liposome-entrapped gene for rat insulin I," Proc. Natl. Acad. Sci. USA, 80, 1068-1072, 1983; Partridge, Terence A, "Muscle transfection made easy," Nature, 352, 757-758, 1991; Wilson, James M, "Vehicles for gene therapy," Nature, 365, 691-692, 1993; Wivel, et al., "Germ-line gene modification and disease prevention: Some medical and ethical perspectives," Science, 262, 533-538, 1993; and Woo, Savio L C et al., "In vivo gene therapy of hemophilia B: sustained partial correction in Factor IX-deficient dogs," Science, 262, 117-119, 1993.
Gene therapy is one of the most promising areas of research today. It would therefore be extremely useful if one had an efficient way to introduce genes into cells which yielded long term expression.
It is therefore an object of the present invention to provide a means for efficient transfer of exogenous genes to cells in a patient.
It is a further object of the present invention to provide a means for long term expression of exogenous genes in patients.
It is a further object of the present invention to provide a means for increasing or decreasing the inflammatory response to implanted polymeric devices.
It is a still further object of the present invention to provide a method for immunization of individuals over a more prolonged period of time than is achieved by a single or multiple immunization protocol.
It is another object of the present invention to provide a method for targeting of gene delivery either to tissue cells or to inflammatory type cells.SUMMARY OF THE INVENTION
A means for obtaining efficient introduction of exogenous genes into a patient, with long term expression of the gene, is disclosed. The gene, under control of an appropriate promoter for expression in a particular cell type, is encapsulated or dispersed with a biocompatible, preferably biodegradable polymeric matrix, where the gene is able to diffuse out of the matrix over an extended period of time, for example, a period of three to twelve months or longer. The matrix is preferably in the form of a microparticle such as a microsphere where the gene is dispersed throughout a solid polymeric matrix) or microcapsule (gene is stored in the core of a polymeric shell), although other forms including films, coatings, gels, implants, and stents can also be used. The size and composition of the polymeric device is selected to result in favorable release kinetics in tissue. The size is also selected according to the method of delivery which is to be used, typically injection into a tissue or administration of a suspension by aerosol into the nasal and/or pulmonary areas. The matrix composition can be selected to not only have favorable degradation rates, but to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when administered to a mucosal surface, or selected not to degrade but to release by diffusion over an extended period.
Examples demonstrate the effectiveness of the system in animals.DETAILED DESCRIPTION OF THE INVENTION
Gene transfer is achieved using a polymeric delivery system which releases entrapped genes, usually in combination with an appropriate promoter for expression of the gene, into surrounding tissue. Efficacy of transfer is achieved by: a) releasing the gene for prolonged period of time, b) minimizing diffusion of the gene out of the delivery system (due to its size) so that release is predominantly degradation dependent, and c) improving the transient time of expression and the low infection seen by direct gene therapy. In case of non-erodible polymers, the device is formulated so that the gene is released via diffusion. This is achieved by creating porous systems or adding soluble bulking agents that create pores as they leach out of the system.
The Polymeric Matrices
Selection of Polymer
Both non-biodegradable and biodegradable matrices can be used for delivery of genes, although biodegradable matrices are preferred. These may be natural or synthetic polymers, although synthetic polymers are preferred due to the better characterization of degradation and release profiles. The polymer is selected based on the period over which release is desired, generally in the range of at least three months to twelve months, although longer periods may be desirable. In some cases linear release may be most useful, although in others a pulse release or "bulk release" may provided more effective results. The polymer may be in the form of a hydrogel (typically in absorbing up to about 90% by weight of water), and can optionally be crosslinked with multivalent ions or polymers.
High molecular weight genes can be delivered partially by diffusion but mainly by degradation of the polymeric system. In this case, biodegradable polymers, bioerodible hydrogels, and protein delivery systems are particularly preferred. Representative synthetic polymers are: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellullose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone.
Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.
Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-co-caprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
Bioadhesive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
Selection of Matrix Form and Size
In the preferred embodiment, the polymeric matrix is a microparticle between nanometers and one millimeter in diameter, more preferably between 0.5 and 100 microns for administration via injection or inhalation (aerosol).
The microparticles can be microspheres, where gene is dispersed within a solid polymeric matrix, or microcapsules, where the core is of a different material than the polymeric shell, and the gene is dispersed or suspended in the core, which may be liquid or solid in nature. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably.
Alternatively, the polymer may be cast as a thin slab or film, ranging from nanometers to four centimeters, a powder produced by grinding or other standard techniques, or even a gel such as a hydrogel. The polymer can also be in the form of a coating or part of a stent or catheter, vascular graft, or other prosthetic device.
Methods for Making the Matrix
The matrices can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art.
Microsphere Preparation
Bioerodible microspheres can be prepared using any of the methods developed for making microspheres for drug delivery, for example, as described by Mathiowitz and Langer, J. Controlled Release 5,13-22 (1987); Mathiowitz, et al., Reactive Polymers 6, 275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci. 35, 755-774 (1988), the teachings of which are incorporated herein. The selection of the method depends on the polymer selection, the size, external morphology, and crystallinity that is desired, as described, for example, by Mathiowitz, et al., Scanning Microscopy 4,329-340 (1990); Mathiowitz, et al., J. Appl. Polymer Sci. 45, 125-134 (1992); and Benita, et al., J. Pharm. Sci. 73, 1721-1724 (1984), the teachings of which are incorporated herein.
In solvent evaporation, described for example, in Mathiowitz, et al., (1990), Benita, and U.S. Pat. No. 4,272,398 to Jaffe, the polymer is dissolved in a volatile organic solvent. The DNA, either in soluble form or dispersed as fine particles, is added to the polymer solution, and the mixture is suspended in an aqueous phase that contains a surface active agent such as poly(vinyl alcohol). The resulting emulsion is stirred until most of the organic solvent evaporates, leaving solid microspheres.
In general, the polymer can be dissolved in methylene chloride. Several different polymer concentrations can be used, for example, between 0.05 and 0.20 g/ml. After loading the solution with DNA, the solution is suspended in 200 ml of vigorously stirring distilled water containing 1% (w/v) poly(vinyl alcohol) (Sigma Chemical Co., St. Louis, Mo.). After four hours of stirring, the organic 'solvent will have evaporated from the polymer, and the resulting microspheres will be washed with water and dried overnight in a lyophilizer.
Microspheres with different sizes (1-1000 microns) and morphologies can be obtained by this method which is useful for relatively stable polymers such as polyesters and polystyrene. However, labile polymers such as polyanhydrides may degrade due to exposure to water. For these polymers, hot melt encapsulation and solvent removal may be preferred.
In hot melt encapsulation, the polymer is first melted and then mixed with the solid particles of DNA, preferably sieved to less than 50 &mgr;m. The mixture is suspended in a non-miscible solvent such as silicon oil and, with continuous stirring, heated to 5° C. above the melting point of the polymer. Once the emulsion is stabilized, it is cooled until the polymer particles solidify. The resulting microspheres are washed by decantation with petroleum ether to give a free-flowing powder. Microspheres with diameters between one and 1000 microns can be obtained with this method. The external surface of spheres prepared with this technique are usually smooth and dense. This procedure is useful with water labile polymers, but is limited to use with polymers with molecular weights between 1000 and 50000.
Solvent removal was primarily designed for use with polyanhydrides. In this method, the drug is dispersed or dissolved in a solution of a selected polymer in a volatile organic solvent like methylene chloride. The mixture is then suspended in oil, such as silicon oil, by stirring, to form an emulsion. Within 24 hours, the solvent diffuses into the oil phase and the emulsion droplets harden into solid polymer microspheres. Unlike solvent evaporation, this method can be used to make microspheres from polymers with high melting points and a wide range of molecular weights. Microspheres having a diameter between one and 300 microns can be obtained with this procedure. The external morphology of the spheres is highly dependent on the type of polymer used.
In spray drying, the polymer is dissolved in methylene chloride (0.04 g/ml). A known amount of active drug is suspended (if insoluble) or co-dissolved (if soluble) in the polymer solution. The solution or the dispersion is then spray-dried. Typical process parameters for a mini-spray drier are as follows: polymer concentration=0.04 g/ml, inlet temperature=24° C., outlet temperature=13 to 15° C., aspirator setting=15, pump setting=10 ml/min, spray flow=600 NLh−1, and nozzle diameter=0.5 mm. Microspheres ranging in diameter between one and ten microns can be obtained with a morphology which depends on the selection of polymer.
Double walled microspheres can be prepared according to U.S. Pat. No. 4,861,627 to Mathiowitz.
Hydrogel microspheres made of gel-type polymers such as alginate or polyphosphazines or other dicarboxylic polymers can be prepared by dissolving the polymer in an aqueous solution, suspending the material to be incorporated into the mixture, and extruding the polymer mixture through a microdroplet forming device, equipped with a nitrogen gas jet. The resulting microspheres fall into a slowly stirring, ionic hardening bath, as described, for example, by Salib, et al., Pharmazeutische Industrie 40-11A, 1230 (1978), the teachings of which are incorporated herein. The advantage of this system is the ability to further modify the surface of the microspheres by coating them with polycationic polymers such as polylysine, after fabrication, for example, as described by Lim, et al., J. Pharm. Sci. 70, 351-354 (1981). For example, in the case of alginate, a hydrogel can be formed by ionically crosslinking the alginate with calcium ions, then crosslinking the outer surface of the microparticle with a polycation such as polylysine, after fabrication. The microsphere particle size will be controlled using various size extruders, polymer flow rates and gas flow rates.
Chitosan microspheres can be prepared by dissolving the polymer in acidic solution and crosslinking with tripolyphosphate. For example, carboxymethylcellulose (CMC) microsphere are prepared by dissolving the polymer in an acid solution and precipitating the microspheres with lead ions. Alginate/polyethylene imide (PEI) can be prepared to reduce the amount of carboxyl groups on the alginate microcapsules. Table 1 summarizes various hydrogels, concentrations, ionic baths, and stirring rates used to manufacture them.TABLE 1 Preparation of Hydrogel Matrices Hydrogel dissolving bath ionic bath stirring Hydrogel concen. pH Temp ° C. concen. (w/v) rate chitosan 1.0% 5.0 23° C. 3% tripoly- 170 rpm phosphate alginate 2.0% 7.4 50° C. 1.3% calcium 160 rpm chloride alginate/ 2.0%/ 7.4 50° C. 1.3% calcium 160 rpm PEI 6.0% 7.4 50° C. chloride Carboxy 2.0% 7.4 50° C. 10.0% lead 100 rpm methyl nitrate cellulose
Other device forms
Other delivery systems including films, coatings, pellets, slabs, and devices can be fabricated using solvent or melt casting, and extrusion, as well as standard methods for making composites. The polymer can be produced by first mixing monomers and DNA as described by Sawhney, et al., and polymerizing the monomers with UV light. The polymerization can be carried out in vitro as well as in vivo. Thus, any biocompatible glue could be also used to incorporate the DNA.
Loading of Gene
The range of loading of the gene to be delivered is typically between about 0.01% and 90%, depending on the form and size of the gene to be delivered and the target tissue.
Selection of Genes to be Incorporated
Any genes that would be useful in replacing or supplementing a desired function, or achieving a desired effect such as the inhibition of tumor growth, could be introduced using the matrices described herein. As used herein, a "gene" is an isolated nucleic acid molecule of greater than thirty nucleotides, preferably one hundred nucleotides or more, in length.
Examples of genes which replace or supplement function include the genes encoding missing enzymes such as adenosine deaminase (ADA) which has been used in clinical trials to treat ADA deficiency and cofactors such as insulin and coagulation factor VIII.
Genes which effect regulation can also be administered, alone or in combination with a gene supplementing or replacing a specific function. For example, a gene encoding a protein which suppresses expression of a particular protein-encoding gene, or vice versa, which induces expresses of a protein-encoding gene, can be administered in the matrix..
Examples of genes which are useful in stimulation of the immune response include viral antigens and tumor antigens, as well as cytokines (tumor necrosis factor) and inducers of cytokines (endotoxin), and various pharmacological agents.
The chronic immune response to the polymeric matrix is mediated by the action of a variety of growth factors including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factors (FGFS), transforming growth factors (TGF-&agr; and TGF-&bgr;, interleukin-1 (IL-1), and tumor necrosis factor (TNF). Inhibitors of these inflammatory mediators in combination with a gene to be delivered other than the immune inhibitor would be effective in decreasing the normal inflammatory response directed toward the polymeric matrix. By inhibiting the amount of encapsulation of the matrix, the effective release would be further extended. Examples of materials which could inhibit encapsulation include antisense mRNA to suppress fibrin or collagen formation, inhibitors of EGF, PDGF, FGFs, TGF-&agr;, TGF-&bgr;, IL-1 and TNF and anti-inflammatory agents such as corticosteroids and cyclosporin.
Genes can be obtained using literature references or from commercial suppliers. They can be synthesized using solid phase synthesis if relatively small, or obtained in expression vectors, for example, as deposited with the American Type Culture Collection, Rockville, Md.
Selection of vectors to be introduced in combination with the gene.
As used herein, vectors are agents that transport the gene into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. Promoters can be general promoters, yielding expression in a variety of mammalian cells, or cell specific, or even nuclear versus cytoplasmic specific. These are known to those skilled in the art and can be constructed using standard molecular biology protocols. Although as demonstrated by the examples, the genes will diffuse out of the polymeric matrix into the surrounding cells where they are expressed, in a preferred embodiment, the genes are delivered in combination with a vector to further enhance uptake and expression. Vectors are divided into two classes:
a) Biological agents derived from viral, bacterial or other sources.
b) Chemical/ physical methods that increase the potential for gene uptake, directly introduce the gene into the nucleus or target the gene to a cell receptor.
Biological Vectors
Viral vectors have higher transaction (ability to introduce genes) abilities than do most chemical or physical methods to introduce genes into cells.
Retroviral vectors are the vectors most commonly used in clinical trials, since they carry a larger genetic payload than other viral vectors. However, they are not useful in non-proliferating cells.
Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation. However, many people may have pre-existing antibodies negating effectiveness and they are difficult to produce in quantity.
Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature. However, they cannot be transmitted from host to host and there are some safety issues since they can enter other cells.
Plasmids are not integrated into the genome and their life span is from few weeks to several months, so they are typically very safe. However, they have lower expression levels than retroviruses and since cells have the ability to identify and eventually shut down foreign gene expression, the continuous release of DNA from the polymer to the target cells substantially increases the duration of functional expression while maintaining the benefit of the safety associated with non-viral transfections.
Chemical/physical vectors
Other methods to directly introduce genes into cells or exploit receptors on the surface of cells include the use of liposomes and lipids, ligands for specific cell surface receptors, cell receptors, and calcium phosphate and other chemical mediators, microinjections directly to single cells, electroporation and homologous recombination. The chemical/physical methods have a number of problems, however, and will typically not be used with the polymeric matrices described herein. For example, chemicals mediators are impractical for in vivo use: when calcium phosphate is used there appears to be very low transduction rate, when sodium butyrate is used the inserted gene is highly unstable and when glycerol is used inserted gene is rapidly lost.
Pharmaceutical Compositions
The microparticles can be suspended in any appropriate pharmaceutical carrier, such as saline, for administration to a patient. In the most preferred embodiment, the microparticles will be stored in dry or lyophilized form until immediately before administration. They will then be suspended in sufficient solution for administration.
In some cases, it may be desirable to administer the microparticles in combination with an adjuvant to enhance the inflammatory response against the polymer and thereby increase the likelihood of phagocytosis by macrophages and other hematopoietic cells, with subsequent expression of the gene specifically within these cells, or, in the case where the microparticles contain an anti-cancer agent, to enhance the inflammatory reaction against the tumor cells in combination with the effect of the anti-cancer agent.
The polymeric microparticles can be administered by injection, infusion, implantation, orally (not preferred), or administration to a mucosal surface, for example, the nasal-pharyngeal region and/or lungs using an aerosol, or in a cream, ointment, spray, or other topical carrier, for example, to rectal or vaginal areas. The other devices are preferably administered by implantation in the area where release is desired.
The materials can also be incorporated into an appropriate vehicle for transdermal delivery as well as stents. Appropriate vehicles include ointments, lotions, patches, and other standard delivery means.
Targeting of cell populations through polymer material characteristics.
Studies with plasmid release using PLA/PCL biodegradable polymers indicate that the majority of transfected cells, assessed with the &bgr;-galactosidase reporter gene, are inflammatory cells involved in the "foreign body" response. In general, non-degrading polymers evoke a stronger inflammatory response when compared to non-biodegrading polymers. A strong foreign body response results in a thick layer of macrophages, fibroblasts, and lymphocytes around the implant. Because the polymer release device relies on diffusion for movement of its particles, a strong inflammatory response will limit the effective distance of diffusion. Accordingly, biodegrading polymers can be used to target inflammatory cells due to the inability of the plasmid DNA (pDNA) to migrate across the reactive tissue layer to the site specific tissue. A more biocompatible material which induces a weaker response from the host will result in a thinner layer of inflammatory cells, enabling the released pDNA to migrate across the inflammatory cells to the indigenous cells to be transfected.
Incorporation of Antiinflammatories and Immune Enhancers; Treatment of Cancers
In recent years, considerable attention has been focused on the use of gene therapy to treat various diseases including cancer. Generally, gene therapy for cancer therapeutics either targets the cells of the immune system to enhance their ability to kill malignant cells or directly targets the cancer cells to regulate their proliferation or enhance some cellular function which will result in a stronger activation of the immune response.
Most types of cancer are characterized by frequent relapses during the course of treatment and the continued non-specific and/or specific activation of the immune system resulting from gene therapy is crucial. Second, cell targeting is a major limitation of current vectors and implantation of a controlled release device directly inside a tumor where the DNA is released locally is one alternative to ex vivo therapy or the development of effective ligand specific vectors. As indicated by the prevalence of ex vivo therapy, targeting hematopoietic cells is especially difficult. The histological results from the implant site in the studies described in the examples below, reveal a substantial inflammatory response surrounding the intramuscular implant. The well known "foreign body" host response can be used to an advantage as this migration of lymphocytes and antigen presenting cells raises the possibility of directing the transfection to these specific cell populations.
Tumors elicit both the humoral and cell-mediated immune response, and lymphocytes, particularly cytotoxic T cells and NK cells, as well as macrophages, are known to play a crucial role in tumor elimination. Gene therapy for cancer treatment either targets these cells or the malignant cells themselves. An implant releasing naked DNA for long term functional gene transfer which can target inflammatory cells and/or tumor cells could significantly improve cancer therapy.
The approaches used include upregulation of class I MHC expression, transduction of antigen presenting cells with tumor-specific antigens, cytokine immunotherapy, transfection of tumor cells with tumor suppressor genes and anti-sense therapy.
The malignant transformation of cells is often characterized by a reduction of class I MHC expression leading to a severe depression of the CTL-mediated immune response. An increase in class I MHC expression on tumor cells could facilitate the activation of the immune system against these altered self-cells. Transfection of genes for cytokines such as tumor necrosis factor (TNF) into tumor cells or tumor suppressor genes such as p53 can be used to limit the ability of tumor cells to multiply. Anti-sense therapy targets cell proliferation or the production of necessary proteins such as tumor angiogenesis factor (TAF) by complementary RNA hybridization to block transcription of specific genes.
The immune system can be activated and induced to attack specific cells using cytokines such as Proleukin or monoclonal antibodies. For example, cancer cells proliferate in part due to a decreased immune response against the transformed cells. The matrices described herein provide a means to allow recognition and provocation of a response to cancer cells. For example, genes coding for antigens, such as the aberrant epithelial mucin of breast cancer, and monoclonal antibodies directed against tumor antigens have been shown to have potential in stimulating immune destruction of malignant cells. These genes, alone or in combination with monoclonal antibodies, can be delivered to the tumor sites in the polymeric matrices to achieve inhibition of the tumor cells.
Cancer cells can also be treated by introducing chemotherapy drug resistant genes into healthy cells to protect them against the toxicity of drug therapy, or by the insertion of appropriate vectors containing cytotoxic genes or blocking genes into a tumor mass to eliminate cancer cells. In a preferred embodiment, the immune system is specifically stimulated against antigens or proteins on the surface of the cancer cells.
These approaches can be used in vitro and in vivo. In vitro, the cells can be removed from a patient, the gene inserted into the cell and the cells reintroduced into the patient. In vivo, the gene can be directly introduced into the body either systematically or in localized sites.
Another approach is to use suicide genes that cause cell death when they are activated or when their product is combined with a pharmaceutical. The primary limitation of the method is the fact that the gene should be targeted to the cancer cell and not to normal cell. Current approach to overcome the problem is direct injection of the vectors into a localized area where normal cells do not proliferate. This would be greatly facilitated using the polymeric devices described herein. The advantages of polymeric devices in this setting include continuous and protracted release of the incorporated pharmaceutical. This increases the liklihood that the intended purposes, for example, treatment of cancerous cells, will be achieved.EXAMPLES
The method and materials of the present invention will be further understood by reference to the following non-limiting examples.Example 1
Expression of Linear and Supercoiled Plasmid DNA Encapsulated in Polymeric Implants in Muscle Tissue of Rats
The study described in this example confirms the feasibility of in vivo transfections using biodegradable polyester blends to release linear or supercoiled plasmid DNA. Although only short term expression was studied in this study, polymer devices releasing drugs offer the potential for sustained long term delivery of naked DNA.
Marker genes are used to study the movement of engineered cells containing exogenous genes, as well as the vectors and genes introduced with the vectors, to insure that the genes remain where they are introduced. Almost all of the initial research into gene therapy is with marker genes. Preferred marker genes are those whose product is innocuous and which can be readily detected by simple laboratory tools. An appropriate marker gene is &bgr;-galactosidase (&bgr;-gal), since expression is readily detected by addition of X-gal, a substrate which yields a blue color when the active enzyme is present.
Encapsulation of linear and supercoiled &bgr;-gal coding DNA in a PLA blend
1 g PLA (300K) and 2 g polyactic acid (PLA) (2K) was dissolved in 10 ml of methylene chloride and 5 drops of sorbitan trioleate (SPAN™) 85. The mixture was divided into two aliquots of 5 ml and 100 ul of either circular or linear DNA (between 1 and 2 mg/ml diluted 1:5 in buffer) was introduced into the aliquots. Each mixture was mixed well and aliquoted into glass vials (1 ml/vial). Between 20 &mgr;g and 40 &mgr;g of &bgr;-gal plasmid DNA was encapsulated in each glass vial. The glass vials were left in the -refrigerator for four days to evaporate the methylene chloride and then lyophilized.
Implantation of DNA/PLA pellets
Each sample was first sterilized with ethanol for 5 min and then washed with PBS-penicillin/streptomycin for 5 min. Surgery was done on Sprague Dawley rats. Linear DNA was implanted into the left leg and supercoiled DNA implanted into the right. Implants were inserted into incised muscle—either in the vastus or the hamstring. The muscle was sutured back together and then the skin was sutured closed. Rats were sacrificed for analysis at two weeks.
Rats were perfused with Phosphate Buffered Saline (PBS) with 2500 units of heparin followed by 3% paraformaldehyde and 0.2% glutaraldehyde in PBS. The tissue was post-fixed with 3% paraformaldehyde followed by 15% sucrose/PBS. Excised muscles were cut with a cryostat and stained with X-gal.
Histology of the implant sites revealed a substantial inflammatory response around the film at two weeks and two months. The bulk of the &bgr;-gal positive staining was localized to this area with few muscle cells exhibiting positive staining. The cells present around the implant probably consists of phagocytic cells, lymphocytes and fibroblasts. As expected, transfection was more efficient with supercoiled DNA.Example 2
In vitro Transfection with pRSV &bgr;-gal
NIH3T3 fibroblasts were plated onto a 6 well tissue culture dish with 1 ml of D-MEM (10% Fetal calf serum with penicillin/streptomycin). 24 hours after plating, the cells were transfected with pRSV &bgr;-gal control plasmids as per Promega Profection Mammalian Transfection system.Plate 1: 10 &mgr;l pRSV-Z (3.4 &mgr;g) Calcium Phosphate Precipitated Plate 2: 30 &mgr;l pRSV-Z (10.2 &mgr;g) Calcium Phosphate Precipitated Plate 3: 10 &mgr;l pRSV-Z (3.4 &mgr;g) Naked DNA Plate 4: 30 &mgr;l pRSV-Z (10.2 &mgr;g) Naked DNA Plate 5: DNA/PLA Plate 6: Control
Plate 5 with the PLA pellet was placed into the well with 4 ml of media to counter the effect of the decrease in pH. After 24 hours, the DNA/PLA pellet was removed and the media left unchanged. At 48 hours, the cells were fixed and stained with X-Gal (1 ml/plate) overnight.
The efficiency of transfection was very low. All plates except the control well had a handfull of blue staining cells. There was no observable differences in the number of blue cells among the 5 plates. It was interesting to note that the plate with the DNA/PLA had similar levels of staining as the other plates even after the fact that half the cells had died and detached due to the PLA degradation.Example 3
Duration of Expression with pSV &bgr;-gal DNA Encapsulated into PLA Blends
In vitro release of plasmid DNA
pSV &bgr;-gal was amplified in HB101 and purified with Qiagen's Mega Prep. 500 &mgr;l of plasmid in Tris-EDTA buffer (67.5 &mgr;g) was lyophilized and resuspended into 100 &mgr;l of sterile dH2O and incorporated into PLA. 0.05 g PLA (2K) and 0.05 g (300K) was dissolved in 1 ml of methylene chloride and 1 drop of SPAN™ 85. After the polymer was in solution, 100 &mgr;l of plasmid (67.5 &mgr;g) was added to the mixture and vortexed for 15 sec. The resulting film was left in a refrigerator overnight and subsequently lyophilized overnight.
This film was incubated with 1.0 ml of TE buffer at 37° C. under gentle agitation and sample supernatants tested at 24 hours and at 4 days for the presence of released DNA. DNA was assayed by agarose gel electrophoresis on the supernatants.
The results based on the gel of the supernatant after 24 hours of incubation show that a substantial amount of plasmid was released. After 4 days, the results indicate that there was a first phase of release due to the diffusion of plasmid molecules which are close to the surface of the device followed by a slower release at 4 days due to the low degradation rate of the polymer which was too low to be measured.
In vivo transfection levels
3 mg PLA (2K) and 1 mg PLA (1000K) were dissolved in methylene chloride (0.25 ml). 1 drop of SPAN™ 85 and 20 &mgr;l of plasmid (20 &mgr;g) was added to the solution and homogenized for 1 minute. This solution was air dried in a glass vial for 3 hours in a sterile hood. The brittle film was ground into fine granules and pressed into a pellet form. Three of these DNA containing pellets were made as well as three control pellets without DNA. All pellets were lyophilized overnight to extract residual solvents.
Three rats received DNA/PLA in their left hamstring and control/PLA in their right hamstring. Pellets were inserted into incised hamstrings and the muscles closed with 6-0 Vicryl. Three rats received an injection of pSV &bgr;-gal plasmids (20 &mgr;g in 100 &mgr;l of TE buffer) over a minute long period in their left leg and 100 &mgr;l of plain TE buffer in their right leg as controls. The site of injection was marked with suture.Rat ID Left Right Implant Duration R112 DNA/PLA Control/DNA 1 week R110 DNA/PLA Control/DNA 5 weeks R111 DNA/PLA Control/DNA 10 weeks R115 DNA/buffer Control/buffer 1 week R114 DNA/buffer Control/buffer 5 weeks
Rats were perfused with PBS/heparin, followed by 4% paraformaldehyde, and post-fixed in 4% paraformaldehyde followed by 15% and 25% sucrose/PBS. Excised muscles were cut with a cryostat and stained with X-Gal.
In vitro release studies indicate that plasmid DNA can be incorporated into polymers without degradation through manufacturing processes and released in functional form for possible uptake by surrounding cells.
In vivo studies reveal that with a 20 &mgr;g loading of DNA into the polymer, there is substantial transfection of inflammatory cells at 1 and 5 weeks as confirmed by X-gal staining and immunoblotting. At 10 weeks, there was no difference in staining intensity between the control PLA and DNA/PLA. This is believed to be due to the result of the low loading (20 &mgr;g) of the polymer such that after one week the release rate was below half maximal levels. Investigators using direct injection use doses in the 100 &mgr;g range to see their effects. A higher initial loading, which will lead to continued release of higher amounts of DNA from polymers, should prolong transfection durations. Rats injected with 20 &mgr;g of DNA in solution showed no transfection at 1 and 5 weeks.Example 4
Comparison of Plasmid DNA Release From Biodegradable and Non-degrading Polymers
Release of plasmids from biodegradable and non-degradable polymer was compared to test the feasibility of targeting either inflammatory cells or tissue specific cells by selection of polymer material. Plasmid DNA was incorporated into a non-degradable elastomer, ethylene vinyl acetate copolymer (EVAc) and implanted into the same site in different animals as PLA/PCL implants. EVAc is a very biocompatible polymer which can be manufactured into a microporous structure through which DNA can diffuse into the surrounding tissue.
Encapsulation of pRSV &bgr;-gal into Polymers.
pRSV &bgr;-gal in HB101 was purchased from the ATCC (American Type Culture Collection, Rockville, Md.). The plasmids were grown and purified with Promega's Maxi Prep. 1 ml of a 0.1% solution of ELVAX40 (Dupont) in methylene chloride was vortexed with 645.2 &mgr;l of pRSV &bgr;-gal (200 &mgr;g), frozen in liquid nitrogen and lyophilized. The resulting mixture was extruded at 55° C. into a rod shaped form.
PLA (2K) and polycaprolactone (PCL) (112K) were dissolved in methylene chloride in a 3:1 ratio and 80 mg of the polymer vortexed with 322.6 &mgr;l of pRSV &bgr;-gal (100 &mgr;g). The mixture was left in the refrigerator for 2 days and lyophilized.
Implantation of the Polymers.
The EVAc/DNA and PLA/DNA were implanted into rat hamstrings along with their control on opposite sides and sacrificed at 2 weeks.
Histological staining with X-gal reveals positive staining of muscle cells as well as inflammatory cells in close proximity to the EVAc polymeric implant at two weeks post-implantation. In comparison, the PLA/PCL implant reveals positive staining of mostly inflammatory cells only, in accordance with the earlier data regarding biodegradable polymers.
Thus the selection of a biodegradable or non-degradable polymer implant can be used to target delivery to inflammatory cells or tissue cells (for example, muscle). Comparison of PLA/PCL and the EVAc implants illustrates the different transfected cell populations. Specifically, the PLA/PCL implant results in almost exclusive transfection of inflammatory cells while the EVAc implant results in a large number of transfected muscle cells.
Modifications and variations of the method and compositions of the present invention will be obvious to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the following claims.
1. A method of delivering naked DNA to a tissue site of a mammalian subject, said method comprising implanting directly, into said tissue site in said subject, a composition comprising:
- (a) a preparation of microparticles between 1 and 300 &mgr;m in diameter, each of which preparation of microparticles comprises a synthetic, biocompatible, non-biodegradable polymeric matrix; and
- (b) an effective amount of naked DNA contained within said matrix, wherein said amount of naked DNA is greater than 20 &mgr;g, and wherein the DNA contains a gene operably linked to a promoter, the nucleotide sequence of said gene being greater than thirty nucleotides in length;
2. The method of claim 1, wherein approximately 0.1-90% by weight of naked DNA is loaded into the polymeric matrix.
3. The method of claim 1, wherein the microparticles are approximately between 1 and 100 &mgr;m in diameter.
4. The method of claim 1, wherein the microparticles are approximately between 1 and 10 &mgr;m in diameter.
5. The method of claim 1, wherein the naked DNA is greater than one hundred nucleotides in length.
6. The method of claim 1, wherein the naked DNA comprises supercoiled DNA.
7. A composition for delivery of naked DNA into a cell comprising:
- (a) a preparation of microparticles between approximately 1 and 300 &mgr;m in diameter, each of which preparation of microparticles comprises a synthetic polymeric matrix; and
- (b) an effective amount of naked DNA contained within said matrix, wherein said amount of naked DNA is greater than 20 &mgr;g, and wherein the DNA contains a gene operably linked to a promoter, the nucleotide sequence of said gene being greater than thirty nucleotides in length;
8. The composition of claim 7, wherein the microparticles are between approximately 1 and 100 &mgr;m in diameter.
9. The composition of claim 7, wherein the microparticles are between approximately 1 and 10 &mgr;m in diameter.
10. The composition of claim 7, wherein the polymeric matrix is biodegradable.
11. The composition of claim 7, wherein the polymeric matrix is non-biodegradable.
12. The composition of claim 7, wherein approximately 0.1-90% by weight of the naked DNA is loaded into the polymeric matrix.
13. The composition of claim 7, wherein the naked DNA is greater than one hundred nucleotides in length.
14. The composition of claim 7, wherein the naked DNA comprises supercoiled DNA.
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Type: Grant
Filed: Nov 25, 1997
Date of Patent: Jul 17, 2001
Assignee: Neurotech S.A. (Evry)
Inventors: Edith Mathiowitz (Brookline, MA), Yong Shik Jong (Seoul), Kim Boekelheide (Wakefield, RI)
Primary Examiner: Dave Trong Nguyen
Attorney, Agent or Law Firms: Mintz, Levin, Cohn, Ferris, Glovsky and Popeo, P.C., Ivor R. Elrifi, Esquire
Application Number: 08/978,522
International Classification: A61K/4800; A61K/914; A61K/922; C12N/1500;


text:
Quilted Portrait: Porcupine
Quilted Portrait: Porcupine
This card is a reproduction of an original acrylic ink and pen drawing.
The card is 5"x7" and comes with a matching envelope. Blank inside so that it can be used for any occasion.
Each card is carefully packed in a clear archival sleeve and shipped with a rigid backing.
© 2016 Polanshek of the Hills [Jess Polanshek]
Add To Cart

subset: commoncrawl


text:
Frozen II
Frozen II is funny, exciting, sad, romantic, and silly.
Frozen II is funny, exciting, sad, romantic, and silly.
It doesn't say much about abusive charlatans, their enablers, and their victims, that we don't already know.
Roger Ebert on James Ivory's "Howards End".
"The Ballad of Narayama" is a Japanese film of great beauty and elegant artifice, telling a story of startling cruelty. What a space it opens…
An article about today's noon premiere of a new movie about architect Benjamin Marshall at the Gene Siskel Film Center.
An article about the screening of Horace Jenkins' "Cane River" on Friday, November 1st, at the Academy Film Archive in Los Angeles.
An article about today's noon premiere of a new movie about architect Benjamin Marshall at the Gene Siskel Film Center.
A video essay celebrating Antonia Bird's maligned masterpiece, Ravenous.
A celebration of Yasujiro Ozu, as written by a Far Flung Correspondent from Egypt.
A Far Flung Correspondent weighs in on the MCU controversy.
An early review of Clint Eastwood's Richard Jewell out of AFI Fest.
A review of the new Hulu show imported from Channel 4, The Accident.
American enfant terrible Harmony Korine's characters are all so self-absorbed that they never quite realize just how freakish they are. The sideshow characters in "Spring Breakers" are prettier than Korine's usual protagonists, especially when compared to the Halloween-mask-clad stars of his last film, "Trash Humpers." But that difference only accentuates the joke that Korine ("Gummo," "Mister Lonely") leans too hard on throughout "Spring Breakers": here are attractive monsters. Their primary aspirations in life are to doff their clothes, take drugs and generally push themselves to new levels of MTV-approved Bacchanalian debauchery.
So, Korine's latest is a characteristically serious film about inherently ridiculous characters, but it's too montonous to be really thoughtful or funny. Korine genuinely loves the amoral posturing of his bikini-clad, thrill-seeking co-ed protagonists, but eventually it all feels like too much of the same thing. "Spring Breakers" has moments as provocatively clever as Korine's better works, but its mainly attractive for its abundant superficial pleasures.
Four nubile college students dream of bass-heavy pop music, day-glo bikinis, and booze-soaked simulations of sex. They want to be objectified, to put themselves in positions where they're assumed to be naive and insanely over-sexed eye candy. Spring break is, as Faith (Selena Gomez) puts it, the girls' chance to see things that they've never seen before, an understatement given how lost in the Floridian funhouse they eventually get.
The girls save up money, but discover that they don't have even half of what they need. So, out of sheer boredom and desperation, and armed with balaclavas, hammers, and (realistic-looking) toy guns, they rob a nearby diner. This is the first transgressive act that the quartet commits, and therefore the most shocking. Maybe the these girls-behaving-badly aren't as phony as they seem. They tease each other and the people around them but only because they ironically treat the real danger that they routinely put themselves in like, as one girl puts it, a video game. While easily ignoring the fratboys who routinely hit them up for sex, the girls become prey for more serious thugs, like James Franco's Alien, a gun-toting rapper who gives voice to the group's mantra: "Spring break forever."
At what point does simulated/learned behavior end, and innate personality take over? That's the most exciting thing about "Spring Breakers" — not just the role-reversals, but the characters' uneasy attempts at becoming the people they're posing as.
Still, even that main theme gets annoying after a while because Korine doesn't develop it; he just repeats it. The click of a lighter as it heats up a bong, amplified to the point where it sounds like a gun being cocked, makes for an effective transition between scenes — but it loses impact after three, four, then five times. By that point it's apparent that the noise is just a meaningless gimmick.
Likewise, the members of Korine's quartet are pretty much interchangeable. Faith, The Virginal One, is the only one who really stands out. Faith is inevitably the first to bridle at Korine's image of spring break as a lascivious day-glo gauntlet of throbbing body parts. "Spring Breakers" doesn't follow a conventional plot, but each new encounter tests the girls' collective resolve until they eventually become supporting characters in their own story. At that point, Alien becomes the film's main subject since he assumes he's an idol to the girls. But as a grill-sporting hustler who crows about the bundles of cash, weed, and guns he owns, Alien's just an intermediary step between the girls and Archie (Gucci Mane), the drug dealer/pimp who taught Alien how to be a real "gangsta" (ie: to treat Brian DePalma's "Scarface" as both a masterpiece and a way of life).
By refusing to out-and-out say that Alien's role model is just as inauthentic as he is, Korine proves that his film's ideas about authenticity are mostly reliant on titillating suggestion. It doesn't really matter how Archie learned to act phony because by the time he shows up in "Spring Breakers," Korine's pretty much made his skimpy point. There are effectively disturbing — part-funny, part-scary — scenes throughout the film, as when Alien heartily fellates two guns leveled at his head, or when Alien and the girls perform Britney Spear's "Everytime" in a musical montage that has them singing on the beach, and robbing people in a video game arcade at the same time. But that's all the movie has to reveal: a food-chain of fakers.
A Far Flung Correspondent weighs in on the MCU controversy.
A review of the new Star Wars spin-off, The Mandalorian.
This message came to me from a reader named Peter Svensland. He and a fr...
Our staff choices for the best films from 2010 through 2019.


text:
wu-tang clan
ODB changes name to Dirt McGirt ODB
- 2003-04-10 15:38:56
© The Wu-Tang Corp.
- 2003-04-10
Ol' Dirt Dog has done it again.
, the charismatic and often troubled member of
Wu Tang Clan
has changed his name again – this time to Dirt McGirt ODB. The rapper has assumed a variety of monikers including the infamous Big Baby Jesus to Ason to God Unique to Joe Bananas to Osiris.
"He's changed his name to Dirt McGirt ODB. He just feels that he's coming out and its time to start over and take the world by storm," Dirty's manager
Jared Weisfeld
told AllHipHop.com. "He's going to be Ol' Dirty Businessman now. you are going to see more of ODB than ever."
Dirty was recently released from an upstate prison and now reportedly resides in a psychiatric facility, but Weisfeld wouldn't confirm where the rapper was staying.
With the recent announcement of a new clothing line (Dirt McGirt) and accompanying underwear line (Ol' Dirty Drawers), Weisfeld maintained that his client has altered his state of mind as well as his name.
"Dirty is ready to come back, he's matured a lot. He's looking forward. He's never not been fit to be in the public," he maintained. "He is not schizophrenic. He's excited and wants to get it on."
"He's had a lot of time to think in prison. He wants to do everything from movies to clothing. He wants to do everything," he maintained.
Finally, budding journalists would be able to talk to the newly appointed Dirt McGirt emptying their pockets.
"He's not going charge like he used to [for interviews]"
Weisfeld insisted that Dirty would be back in free world before July, refuted previous notions, but wouldn't be able to confirm the rapper's actual date of release from the NY State facility where he currently resides
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Proof, that the Wu-Tang Clan ain't nothing to mess with
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text:
Identifying asset performance
SKF can offer a wide range of services and product solutions for condition monitoring. Or if it is preferred, SKF can provide these services by remote monitoring diagnosis. This means fast ramp up and limited own investments in special skills and technology to reach world class condition monitoring operations with always up to date technology and competency, provided as a contracted service.


text:
Finding the Best Lee's Summit Plumber for Your Emergency Needs
Plumbers 911 Has Trained and Efficient Technicians Available
Sometimes life throws you a curve ball. Life seems to be going along smoothly, when all of a sudden you hear a loud bang and the sound of rushing water in the middle of the night. Things are going to get complicated real fast. In order to deal with a plumbing emergency and prevent further damage, you need someone who can get there fast and get the job done right. The only real question is, how will you find the right plumbing contractor to help get your life back to normal?
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The first step is choosing only to work with independent, local Lee's Summit contractors who have spent years developing a reputation for excellence in the trade. Plumbers 911 is not a huge, impersonal chain or franchise operation. We don't spray our name on the side of the service truck that will arrive at your door.
The second step is hiring only highly-trained and reliable union plumbers. We've found that MO union technicians represent the highest standards in the industry. All of our techs are fully licensed, bonded, and insured. They are the best in the business, having completed at least 10,000 hours of training in order to receive their certifications. In addition, they agree to a comprehensive background screening as well as follow-up drug testing.
Complete 24/7 Emergency Plumbing Services in Lee's Summit, MO
Because our techs are the best in the business, we are able to provide a wide variety of services.
Emergency Plumbing: Let's go back to our original scenario. Let's say your pipes burst or your water heater starts leaking all over your basement. Because we work with technicians in your community, we are usually just around the corner when you call.
That means that our response times beat anyone else in the area. We are available and equipped 24/7 to handle any pipe, heater or toilet problems.
Leak Detection: On average, American households pay about 14% too much on their monthly water bills because of leaking faucets and other plumbing fixtures.
That's a shame because so much of it is completely avoidable. Our servicemen can come out to find and repair those leaks regardless of their location.
We'll check the following for drips and leaks:
• Shower heads
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• Faucets
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• Outside meter lines
• Garden hose bibs
• Pipe joints and connections
Toilet and Appliance Repair and Installation: Another great way to save money on your utility bills is to replace old and inefficient toilets and appliances with new energy and water efficient models with the EPA's WaterSense certification. Even a simple job like replacing an old showerhead can add up to big savings in the long term.
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New Construction: We are also fully capable of helping you build a home or commercial building from scratch. We can help you select and install water and energy efficient fixtures and appliances that will not only look good, but save you money as well. From laying the sewer mains for a new subdivision to adjusting the smallest faucet handle, we will make sure that every detail is perfect.
Hot Water Heater Installation: A hot water heater is an essential appliance in any building, and you want it to work right every time. Because of the size and weight of many hot water heaters, it is not a good DIY job for beginners. In addition, there are a lot of technological advancements to choose from nowadays:
• Conventional Storage Water Heaters: These are the traditional models with a huge metal water tank with a heating element in the base.
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• Heat Pump Water Heaters: These units are basically a refrigerator in reverse, moving heat from outside the home to heat the water.
• Solar Water Heaters: An excellent choice for saving energy.
Commercial Plumbing: When a plumbing emergency happens in a home, the structural and property damage can be devastating. However, if the same emergency happens to a business, the potential for lost profit makes things even worse. We'll do everything in our power to get your doors open again so that you can get back to business. We are also equipped for large scale projects such as backflow prevention, hydrojetting, sewer cleaning, gas system installation and sump pump repairs. We can also install and maintain water sub-metering systems for multi-family rental complexes.
Septic System Installation: A well-maintained septic system can be an environmentally sound method of disposing of waste water. A poorly-maintained one can be a nightmare, leading to backed-up sewage and waste-filled lawns. Our experts can inspect and repair every element of your septic system, keeping it problem-free for years.
Contact Us Immediately to Schedule a Service
Plumbers 911 is your source for the best plumbing contractors in Lee's Summit, Missouri.
We have the experience and equipment to handle your repair or installation job, regardless of the size. Call us today at (816) 326-8643 to be connected to our reliable and competent plumbing techs. We'll do a great job for you at a reasonable price, day or night.


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The Student Success Center provides many programs and services to help you build your confidence for learning. Our aim is to assist you to achieve your educational goals. If you want a GED/HSE, or want to improve your language, writing, reading, math, or study skills to prepare you for your certificate or degree, we're here to serve you.
The Center is located on the 2nd floor of the Gerald R. Meyerhoeffer Building. Many of the classes and services are also available in off-campus areas.


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In Store Banker - Sherman Oaks Ralphs
US Bank
In Store Banker - Sherman Oaks Ralphs
US Bank
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At U.S. Bank, we're passionate about helping customers and the communities where we live and work. The fifth-largest bank in the United States, we're one of the country's most respected, innovative and successful financial institutions. U.S. Bank is an equal opportunity employer committed to creating a diverse workforce. We consider all qualified applicants without regard to race, religion, color, sex, national origin, age, sexual orientation, gender identity, disability or veteran status, among other factors.
The In-Store Banker at our U.S. Bank In-Store branches breaks the mold of what it means to be a banker. Focusing on sales and service, In-Store Bankers open accounts, handle teller transactions, inform customers of other products and services that meet their needs, and solve customer questions and concerns with warmth and a collaborative spirit.
In-Store Bankers build relationships with customers based on trust, recommending financial solutions based on each customer's unique goals and needs. In-Store Bankers may be required to travel locally, serving customers throughout their community at various In-Store branches.
At U.S. Bank, you'll get the support and tools you need to meet your goals and build a meaningful career. We reward top performance and ethical team players. Eligibility for incentives is based on sales referrals, branch growth and/or customer satisfaction. In-Store Bankers have flexible schedules that may include weekends (depending on branch location).
We're looking for people who want more than just a job - who want to make a difference in the communities where we live and work. Apply today and explore what's possible with a career at U.S. Bank.
This position requires National Mortgage Licensing System (NMLS) registration under the terms of the S.A.F.E. Act of 2008 and Regulation Z. You will be subject to the required registration process, which includes a criminal background and credit check. Failure to meet or maintain any of the NMLS registration requirements, including maintaining a satisfactory criminal and credit record, may result in a rescission of your offer or termination of employment.
Basic Qualifications
- High school diploma or equivalent
- Minimum one year of experience in sales and/or cash handling activities, or commensurate training
Preferred Skills/Experience
- Ability to sell bank products, process transactions, and solve customer service issues
- Basic knowledge of all retail products and services, applicable bank and branch policies, procedures and support systems
- Effective interpersonal/customer service skills
- Well-developed selling and referral skills
- Strong mathematical, problem-solving and negotiation skills
- Strong verbal and written communication skills
- Ability to analyze customer credit data and other related financial information
- Strong interpersonal and relationship building skills
- Previous experience in a financial sales representative oriented role
Internal Number: 180004951
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At U.S. Bank, you'll get the support and tools you need to meet your goals and build a meaningful career. We reward top performance and ethical team players. Eligibility for incentives is based on sales referrals, branch growth and/or customer satisfaction
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This Week at Physics
|<< Spring 2013
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Event Number 3053
Tuesday, September 24th, 2013
- Chaos & Complex Systems Seminar
- Hydrogen energy levels in n dimensions via group theory
- Time: 12:05 pm
- Place: 4274 Chamberlin (refreshments will be served)
- Speaker: Adam Bincer, UW Department of Physics
- Abstract: My talk is a generalization to n dimensions of a brilliant group-theoretical treatment of the hydrogen energy levels by Pauli in 1926. To start I define the concept of a group and discuss cyclic groups as a simple example. Then the rotation group in two dimensions is introduced to get us started on Lie groups. From there it is just a short hop to rotations in n dimensions. Along the way I introduce the idea of matrices - hopefully all this mathematics will be introduced gently enough so as not to turn the non mathematicians in the audience completely off. I then turn to the non-relativistic hydrogen atom in n dimensions and show how it is governed by the rotation group in (n+1) dimensions. I conclude with some anecdotes about Pauli.
- Host: Clint Sprott
- Add this event to your calendar
- Hydrogen energy levels in n dimensions via group theory


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Dr. Stanley Toussaint, Sr. Professor Emeritus
Dr. Toussaint has faithfully taught New Testament studies at Dallas Seminary since 1960 and has pastored for more than 20 years. Dr. Toussaint aptly blends the mix of scholarship with a pastoral heart for God and God's people. Known as one of the world's leading authorities on the New Testament, Dr. Toussaint challenges us to deepen our walk with God.


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Weapons Policy
The Cooper Union prohibits the possession or use of any weapon or simulated weapon on campus by anyone except law enforcement officers or those who have received prior written authorization from the Director of Public Safety. The term "weapon" includes an instrument used to inflict physical harm, that is intended to inflict harm, or that could reasonably cause fear of infliction of harm, and any item that may be deemed a weapon under applicable law, including but not limited to: a pistol, revolver, shotgun, rifle, stun gun, BB or pellet gun, taser, electronic dart gun, and other instrument that launches a projectile by pressure resulting from combustion of propellant material, including a weapon related to or using air, sound, flare, hunting or springs; bombs, grenades, mines, explosives, or incendiary devices which can include "ignition devices" and aerosols; daggers, stilettos, swords, and knives including switchblade and gravity knives; and including parts, components, spare parts, or ammunition relating to the above.
A disarmed weapon is a weapon unless it is a simulated weapon. A simulated weapon means a facsimile of any category of weapon, as defined in this policy that resembles the actual weapon but is not capable of use as a weapon. The term "possession" includes, but is not limited to, storage of a weapon in the dormitory, in lockers, desks, briefcases, laboratories, studios, or a weapon carried (conspicuous or concealed) on the person. Hazardous materials that are not approved for use in a laboratory or studio or are approved for use, but are intentionally or recklessly used or possessed may be classified as a weapon.
The Cooper Union Weapons Policy is in addition to, and may be more restrictive than, weapons regulations that may be imposed by Federal, State, and New York City law. With respect to Cooper Union personnel (faculty, staff and student workers), possession of weapons on campus is subject to disciplinary action, up to and including termination of employment—consistent with any applicable employment manuals or union contracts.
With respect to students, possession of weapons on campus or while attending or participating in a Cooper Union-sponsored activity off campus is subject to disciplinary action up to and including dismissal, consistent with procedures in the Campus Safety, Security and Fire Report and Cooper Union's Code of Conduct.
All requests to use a simulated weapon in conjunction with a Cooper Union related event (e.g., play, film production, or art exhibition) should be submitted in writing to the Director of Public Safety for approval. If approved, the Director of Public Safety will issue written permission for the use of the simulated weapon and specify its restrictions. The restrictions will include the name of the individual who may possess the simulated weapon, the time and manner it may be used, the manner in which it will be transported to and from the location, and the location the simulated weapon is to be stored when not in use. Under no circumstances may a simulated weapon be stored in any Cooper Union owned, leased, or controlled facilities other than an approved safety storage area. The individual to whom written permission has been granted to possess a simulated weapon must maintain the custody of the simulated weapon at all times and may not transfer custody of the simulated weapon to any other person not specified in the written permission. The individual who has been granted permission to possess a simulated weapon may not do so while under the influence of alcohol or any substance that impairs judgment, and may not engage in any reckless behavior while in possession of the simulated weapon. There is no exception to the prohibition of having a simulated weapon in The Residence Hall at any time or for any purpose. Failure to comply with any and all restrictions will result in permission being revoked and possible disciplinary action. The Director of Public Safety will not approve requests for the use of any weapon as defined in this policy for any purpose that violates any Federal, State or New York City Law.
To Report Weapons on Campus or a Threat of Violence.
Cooper Union believes that in order to achieve a safe and secure campus a collaborative effort by all members of the campus community is required. All students, faculty, and staff who have knowledge of weapons on campus have an obligation under this policy to report that information immediately to the Director of Public Safety. Anyone with information regarding the possession or storage of weapons on campus that does not pose an immediate threat should report it to The Office of Public Safety at 212-353-4375. Any individual who believes there is an imminent threat of violence on campus should call the police by dialing 9-911 from any campus phone or 911 from a cell phone. Reports can be made anonymously by emailing email@example.com


text: Feel free to contact us if you have any questions or comments. We try to respond to all inquiries by the next business day. In Addition, Mr. Khanna hosts biweekly Free Community Conference Calls open to everyone. We are located at: Washington, DC Metro Office - 5225 N. Wilson Blvd, Arlington VA 22205. Ph: 703-908-4800 Staunton, VA Office - 6 Byers Street, Staunton, VA 24401, Phone: 540-886-6321.


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Join us at The Boom Boom Room for the most entertaining Saturday evening you can have in downtown St. Louis. The Boom Boom Revue is the perfect burlesque show just for you. Dancing, comedy, specialty cirque acts and more await you in our intimate venue. It's so much fun come and see what audiences have been enjoying for the past few years. We offer a full bar, and appetizers are available for purchase.
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Earlier Event: January 26THE BOOM BOOM REVUE SATURDAY DINNER SHOW
Later Event: January 27BE OUR GUEST SUNDAY BURLESQUE BRUNCH


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Lindsay Lohan Films Commercial While Under House Arrest [VIDEO]
Lindasy Lohan has apparently decided to get creative while under house arrest.
The troubled actress, who is currently confined to her home for allegedly stealing a $2,500 necklace, recently filmed a commercial of sorts for a new auctioning website called Beezid.com.
According to BuzzFeed, the 24-year-old 'Mean Girls' star was paid $25,000 to film the commercial, plus an additional $10,000 in purchasing credits on the site. Not bad, Linds.
This is just the latest stunt Lohan has pulled since she began her house arrest on May 26. Earlier this month, she threw a barbeque party on the rooftop of her Venice, California home.


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Wake up and get your down dog on! Set the tone for the weekend with this all levels vinyasa class, sequenced to meet everyone's needs. Begin with sun salutations and end in a sweet savasana, all the while flowing from pose to pose in between. Learn something new about yourself and walk out feeling rejuvenated, energized and blessed.
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Earlier Event: March 23Community Class
Later Event: March 25Vinyasa Flow


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Born in Tompkinsville on November 15, 1925, Joe Downing was one of eight children born to Aldridge Clifton and Katie Burton Goodman Downing. He grew up in Horse Cave, where he attended elementary and high school, graduating in 1943 as valedictorian of his class. Downing spent his youth observing and exploring the caves and landscape around him, unknowingly laying the groundwork for his amazingly creative works of art. Downing's work has long been known for his unique combination of media and technique, demonstrating a dedication to his personal artistic vision which he proudly acknowledged was rooted in his Kentucky home place.
Following his 18th birthday in November, 1943, Downing was inducted into the United States Army, where he served as an artillery observer in Europe during World War II. He was assigned to a unit that landed at Normandy soon after D-Day and the unit was engaged in a distinguished record of service leading to the end of World War II. Downing, himself, earned the Bronze Star with the citation for courage in action. While in Europe, Downing caught a brief glimpse of Paris, the city that would become his home in 1950 and where he maintained an apartment and studio until his death.
Following his return to the United States, Downing enrolled at Western Kentucky University for a portion of the 1945-46 school year, a time when he began to recognize his enthusiasm for art with the encouragement of Ivan Wilson. Following his parents' wishes, Downing turned his attention to preparation for a career in optometry when he enrolled in and graduated from the Northern Illinois College of Optometry in Chicago. During that time, however, Downing also took classes at the Chicago Art Institute. In 1950, following his graduation, Downing went to Paris for what was to be a three-month stint, only to find that he could not bring himself to leave. In 1952, Downing had his first one-man show, attended by Pablo Picasso, whose simple words of "well done" were encouragement enough to spur Downing on.
Downing's work is owned by such museums as the Paris Museum of Modern Art, the Smithsonian Institution and the Museum of Modern Art in New York City, as well as the Kentucky Museum in Bowling Green, the Speed Museum in Louisville and the Owensboro Museum of Fine Art. Collectors around the world own his work, and he had showings across the globe, including Tokyo and much of Europe, as well as several shows over the years in Kentucky. Downing's natural creativity led him to write several books of poetry and, most recently, a book about growing up in Horse Cave.
Horse Cave native and internationally renowned artist Joseph Dudley Downing died December 29, 2007, at the age of 82. Downing passed away in the village of Menerbes in the Provence region of France, a place where he kept a home for over 40 years.
"Time and again, on canvas and tile, wood and leather, paper and linen. Downing rejoices in the very act of seeing: the visual possession and transformation of life. In the end, what strikes one most in this long song of praise to sight is its gathering freshness. Innocence, curiosity and desire haunt the paintings as they did Downing's mind as he daydreamed in the grottoes of Horse Cave. The source has widened but never grown tainted. This is the world of a man whom the dreams of childhood have never failed."
—excerpt from The Achievement of Joe Downing by Michael Peppiatt | Art International | Volume XXVI/4 September-October 1983
"Uncle Dudley's life was passion fully formed from an early age, yet was always in metamorphosis. For creativity is a constant state of change, and if Uncle Dudley's existence demonstrated any pure concept, it was that openness to change, and the ability to embrace and learn from the struggles it might bring, will keep you young until at least the age of 82. There was no one who more epitomized what he, himself, laughingly referred to as 'pathological optimism'."
—Elizabeth Downing | niece of Joe Dudley Downing
—Elizabeth Downing | neice of Joe "Dudley" Downing
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A Literary and Domestic Incident in the Desert West
Aliens. Spacealiens. No such thing. How theyd fought it out, cosmic mysteries reduced to raw shouting in the uncaring morning. Her leaving in wrath and now returning, straight into the bedroom without him. Rustling and thumps.
She was in the doorway. She had stripped in there and dressed her body for the boudoir, soft ladyclothes baring flesh and something more, down at the forking of her legs. Hard nickelplate steel, the Smith & Wesson. Her hand was on it.
Whos crazy.
He watched drymouthed. The gunsight vanished up into the red dark inmost space of life. A child had come from that place, seed of a man before him, its birthing route retraced now by the instrument of death. The Smith & Wesson slid up and back in a mirthless parody of union. Warmth ebbed from his own flesh.
Whos crazy. You or me.
The gun now lost in the depths. No. Not lost. A swift turn of her wrist and there was the whole of it, winking back into the airy world and rising to fix on him. The shining shaft foreshortened into the lightless eye of an ancient uncaring deity, hollow gaze falling on his own fearstruck eyes.
Whos crazy.
It was her voice that spoke but it seemed to rise from the void of the gunbarrel. His stuporous amazement broke and he made a lunge, wrested away the wet and befouled sidearm from her grip. He lurched toward the bathroom. The Smith & Wesson plashed in the toilet water and lay there, cold and still like a homunculitic babe unwombed premature, lost to god and man alike.
She started for the bathroom herself. It would not do. He scrabbled in the water, pulled the silver gun up streaming. It dripped back across the floor to the door and out, and he flung it down into the garbagecan. There the law could find it.
[Novelist's Ex in Bizarre Handgun Threat Arrest: Cormac McCarthy's former wife pulled weapon from her vagina.]


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A proud and historic port city
In the south-eastern corner of Spain, Almeria is more than the gateway to the arid heart of Andalusia. Shaped by the legacy of the Moors (and today by intensive agriculture) it's a city of fascinating contrasts – from spaghetti westerns to amazing marine wildlife.
Almer�a's bustling local markets sell everything from bric-a-brac to seasonal specialities, and make a great introduction to the daily life of the city. Start at the Mercado Central de Abastos (Circunvalaci�n Del Mercado), a covered market that specialises in takeaway and bargain outlets, before strolling down to San Jos� and browsing the selection of hippy-style craft stalls. On Thursdays, a big street market in Carboneras sells handicrafts, ceramics and fresh produce from the surrounding area. Calle de las Tiendas and Calle Real both have an eclectic mix of local boutiques and big-name brands, while Paseo de Almer�a draws a loyal fashionable crowd.
Food & Drink
Mediterranean and Arabic influences combine to create the Almer�a region's distinctive and delicious cuisine. The best place to sample local specialities is Casa Puga (Jovellanos 7), an ancient taverna that has it roots in the 1900s and now counts among the city's brightest gastronomic stars. La Encina Plaza Vieja (Calle Mar�n) does a combination of scrumptious tapas and traditional rustic dishes. The freshest and the best seafood is served at glitzy Club de Mar (Playa de Almadrabillas), although El Bello Rinc�n (Carretera Nacional-340) combines an imaginative fish-focused menu with far-reaching sea views. At Casa Sevilla (Calle Rueda L�pez), creative dishes interchange effortlessly with more traditional fare.
Procession of the Virgin of the Sea<
Honouring the patron saint of Almer�a, 'la Virgen del Mar', this lively fiesta sees an ancient statue of the Virgin Mary returned to the beach where it was originally discovered. After the procession, locals gather on the beach to enjoy bonfires, music and food.
Noche de San Juan
Part Christian celebration, part Pagan ritual, this midsummer festival is an excuse for the residents of Almer�a to let their hair down. Expect to see the burning of papier m�ch� figures, followed by street bonfires, music and traditional verbena dancing.
Almer�a Fair
This summer celebration combines the Spanish traditions of bullfighting and flamenco dancing with a thoroughly modern knees-up. The last Saturday of the fair is a public holiday, and a spectacular floral offering is made to the Virgin Mary while costumed crowds look on.
Almer�a Western Film Festival
The first European festival dedicated exclusively to Western films, this new event is set to become a staple of the Almer�a calendar. It takes place in the desert of Tabernas and features a combination of classic films and new screenings.
International Festival of Spanish Keyboard Music
A series of concerts, workshops and fringe events make up this niche festival, which aims to appeal both to musical academics and to members of the public. Those keen to learn a new skill can also take part in courses run by nationally acclaimed artists.
The Moorish heritage of Almer�a is reflected in the exotic opulence of the city's best hotels. Plaza Vieja Hotel & Lounge (Plaza de la Constituci�n) is overlooked by the Alcazaba Fortress and channels the Moroccan style of this famous monument in its d�cor. For visitors who prefer to be based closer to the sea, VITA Gran Hotel (Avenida Reina Regente) offers a more contemporary brand of luxury – its balcony rooms all have ocean views and access to an outdoor pool. The best budget accommodation tends to be in family-run pensions such as Pension Americano (Avenida de la Estacion), a basic but homely collection of rooms close to the city centre.


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