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WO2001008709A1 - Traitement par ultrason des tumeurs - Google Patents

Traitement par ultrason des tumeurs Download PDF

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Publication number
WO2001008709A1
WO2001008709A1 PCT/US2000/020631 US0020631W WO0108709A1 WO 2001008709 A1 WO2001008709 A1 WO 2001008709A1 US 0020631 W US0020631 W US 0020631W WO 0108709 A1 WO0108709 A1 WO 0108709A1
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WIPO (PCT)
Prior art keywords
tumor
nucleic acid
sonoporation
plasmid
expression
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PCT/US2000/020631
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English (en)
Inventor
Khursheed Anwer
Sean Sullivan
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Valentis, Inc.
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Publication date
Application filed by Valentis, Inc. filed Critical Valentis, Inc.
Priority to AU63890/00A priority Critical patent/AU6389000A/en
Publication of WO2001008709A1 publication Critical patent/WO2001008709A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/208—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to products and methods useful for delivering one or more nucleic acid molecules to a tumor and sonoporating the tumor.
  • nucleic acids in vivo have been pursued by a variety of methods. These include lipofectin/liposome fusion: Feigner et al, Proc. Natl. Acad. Sci., Volume 84, pp. 7413 - 7417 (1987); and transferrin:transferrin receptor delivery of nucleic acid to cells: Wagner et al., Proc. Natl. Acad. Set, Volume 87, pp. 3410 - 3414 (1990).
  • the use of a specific composition consisting of polyacrylic acid has been disclosed in International Patent Publication No. WO 94/24983. Naked DNA has been administered as disclosed in International Patent Publication No. WO 90/11092.
  • Gene therapy has quickly become a major area of research in drug development.
  • a key technological barrier to commercialization of gene therapy is the need for practical and effective gene delivery methods.
  • the primary problem of gene injection by conventional needle-syringe methods is that genetic material must be injected in large quantities into the target site because of the inefficiency of attempting to diffuse genetic material into the cells' nuclei and the need to overwhelm enzyme systems that immediately move to destroy the injected nucleic acid molecules.
  • Therapeutic injection technology using a needle-syringe has progressed relatively slowly.
  • Gene transfer strategies targeting tumor endothelium to provide sustained, high, and local concentrations of anti-angiogenesis mediators, immunocytokines, or cytotoxic proteins thus mimmizing systemic toxicity have potential therapeutic value.
  • Current gene delivery systems transfect cells in vivo in a manner largely determined by blood flow and site of introduction (Mulligan, R.C. (1993), Science 260, 926-932; Feigner, P.L. and Rhodes, G. (1991), Nature 349, 351-352). Because of this, the ability of these systems to deliver therapeutic genes to target cells in vivo is limited.
  • cationic liposomes have been widely used for gene transfer into endothelial cells in vivo (Brigham, K.B., et al. (1989), Am. J. Med. Sci. 298, 278 - 281; Hofland, H.E.J., et al. (1997), Pharm. Res. 14, 742 - 749; Liu, F., et al. (1997), Gene Therapy 4, 517 - 523; Mahato, R.I., et al. (1998), Hum. Gene Ther. 9, 2083 - 2099; Rolland, A.P. (1998), Critical Reviews in Therapeutic Drug Carrier Systems 15, 143 - 198).
  • Ultrasound-mediated delivery has potential as a powerful new method for enhancing and targeting administration of therapeutic compounds into and across cells and tissues. Ultrasound-enhanced delivery to cells has been demonstrated in vitro by uptake of extracellular fluid, drugs, and DNA into cells (Liu, J., et al. (1998), Pharm. Res. 15, 918-924; Mitragotri, et al.
  • This invention features compositions and methods for enhancing the administration to and uptake of nucleic acids in an organism.
  • the data presented herein demonstrates that ultrasound delivery of formulated nucleic acid molecules is a more favorable method for nucleic acid delivery to tumors when compared with non-ultrasound delivery methods.
  • sonoporation is used to describe use of ultrasound treatment to facilitate the transfection of cells with formulations including nucleic acids.
  • sonoporation is used to mean ultrasound treatment without any limitation to specific mechanisms of cellular changes that may be induced by the treatment.
  • the invention provides a method to deliver nucleic acid molecules formulated with an agent that facilitates transfection (preferably a cationic lipid or PINCTM agent as described below) to an organism by using an apparatus configured and arranged to administer molecules by applying ultrasound waves to the cells of an organism.
  • an agent that facilitates transfection preferably a cationic lipid or PINCTM agent as described below
  • the present invention allows for superior delivery of nucleic acid molecules into cells (preferably tumor cells) in vivo by the combination of an ultrasound device and formulated nucleic acid molecules.
  • the present invention also allows for treatment of diseases and vaccination, especially with respect to various cancers.
  • An ultrasound method is described that provides for specific targeting of gene transfer into primary tumors, preferably after systemic administration of cationic lipid/plasmid complexes.
  • Intravenous administration of N-[(l-(2,3-dioleyloxy) propyl)] -N-N-N- trimethylammonium chloride (DOTMA)-based transfection complexes into tail vein of subcutaneous squamous cell tumor bearing mice led to plasmid uptake and reporter gene expression in tumor lesions. Expression was also observed in non-tumor tissues including lung and liver.
  • Application of ultrasound to tumor lesions after i.v. administration of lipid plasmid complexes enhanced reporter gene expression in tumor by 6 - 270 fold. The enhancement in gene expression was treatment site specific since no increase was observed in non-tumor tissue.
  • the present invention features an ultrasound method that provides for specific enhancement in gene transfer to tumor lesions transfected by systemic, subcutaneous or intratumoral administration of transfection facilitating agent/DNA complexes.
  • the enhancement in gene transfer was restricted to the local tumor treatment site since no effects were observed in tissues distal to the site of ultrasound administration. Therefore, ultrasound treatment may prove to be a useful method to enhance systemic gene delivery into tumor without affecting non-target tissue.
  • the invention provides a method for delivering a nucleic acid molecule to a tumor, preferably a primary tumor, comprising the steps of systemically administering a transfection facilitating agent/plasmid complex preferably a liposome/plasmid complex, to the tumor and sonoporating the tumor.
  • Sonoporation typically follows administration of the gene, but sonoporation prior to (preferably immediately prior to) administration also results in enhancement of gene delivery and expression.
  • delivery or “delivering” is meant transportation of nucleic acid molecules to desired cells or any cells.
  • the nucleic acid molecules may be delivered to multiple cell lines, including the desired target. Delivery results in the nucleic acid molecules coming in contact with the cell surface, cell membrane, cell endosome, within the cell membrane, nucleus or within the nucleus, or any other desired area of the cell from which transfection can occur within a variety of cell lines which can include but are not limited to; tumor cells, epithelial cells, Langerhan cells, Langhans cells, littoral cells, keratinocytes, dendritic cells, macrophage cells, Kupffer cells, muscle cells and lymphocytes.
  • the formulation is delivered to the cells by sonoporation and the nucleic acid molecule component is not significantly sheared upon delivery, nor is cell viability directly effected by the sonoporation process.
  • nucleic acid refers to both RNA and DNA including: cDNA, genomic DNA, plasmid DNA or condensed nucleic acid, nucleic acid formulated with cationic lipids, nucleic acid formulated with peptides, antisense molecules, cationic substances, RNA or mRNA.
  • the nucleic acid administered is plasmid DNA that includes a "vector".
  • the nucleic acid can be, but is not limited to, a plasmid DNA vector with a eukaryotic promoter which expresses a protein with potential therapeutic action, such as, for example; hGH, VEGF, EPO, IGF-1, LPO, Factor IX, IFN- alpha, IFN-beta, IL-2, IL-12, or the like.
  • a eukaryotic promoter which expresses a protein with potential therapeutic action, such as, for example; hGH, VEGF, EPO, IGF-1, LPO, Factor IX, IFN- alpha, IFN-beta, IL-2, IL-12, or the like.
  • plasmid refers to a construct made up of genetic material (i.e., nucleic acids). It includes genetic elements arranged such that an inserted coding sequence can be transcribed in eukaryotic cells. Also, while the plasmid may include a sequence from a viral nucleic acid, such viral sequence preferably does not cause the incorporation of the plasmid into a viral particle, and the plasmid is therefore a non- viral vector. Preferably a plasmid is a closed circular DNA molecule.
  • vector refers to a construction comprised of genetic material designed to direct transformation of a targeted cell.
  • a vector contains multiple genetic material, preferably contiguous fragments of DNA or RNA, positionally and sequentially oriented with other necessary elements such that the nucleic acid can be transcribed and when necessary translated in the transfected cells.
  • the term "transfection facilitating agent" as used herein refers to an agent that forms a complex with the nucleic acid. This molecular complex is associated with nucleic acid molecule in either a covalent or a non-covalent manner.
  • the transfection facilitating agent should be capable of transporting nucleic acid molecules in a stable state and of releasing the bound nucleic acid molecules into the cellular interior.
  • DNA extraction methods, methods of immunofluorescence, or well-known reporter gene methods such as for example CAT, or
  • LacZ containing plasmids could be used in order to determine the transfection efficiency.
  • the transfection facilitating agent should also be capable of being associated with nucleic acid molecules and may be lyophilized or freeze dried and rehydrated prior to delivery and sonoporation.
  • the transfection facilitating agent may prevent lysosomal degradation of the nucleic acid molecules by endosomal lysis.
  • the transfection facilitating agent may allow for efficient transport ofthe nucleic acid molecule through the cytoplasm of the cell to the nuclear membrane and into the nucleus and provide protection.
  • transfection facilitating agents are non-condensing polymers, oils and surfactants.
  • Non- condensing polymers have been found to be particularly suitable for injection into the site of desired expression such as in intra-tumoral administration. These may be suitable for use as compounds which prolong the localized bioavailability of a nucleic acid: polyvinylpyrrolidones; polyvinylalcohols; propylene glycols; polyethylene glycols; polyvinylacetates; poloxamers (Pluronics)(block copolymers of propylene oxide and ethylene oxide, relative amounts ofthe two subunits may vary in different poloxamers); poloxamines (Tetronics); ethylene vinyl acetates; celluloses, including salts of carboxymethylcelluloses, methylcelluloses, hydroxypropyl-celluloses, hydroxypropylmethylcelluloses; salts of hyaluronates; salts of alginates; heteropolysaccharides (pectins); phosphatidylcholines (lecithins); miglyols; polylactic acid
  • cationic condensing agents such as cationic lipids, peptides, or lipopetides, or for example, dextrans, chitosans, dendrimers, polyethyleneimine (PEI), or polylysine, may associate with the nucleic acid molecule and may facilitate transfection in conjunction with sonoporation.
  • the PINC enhances the delivery ofthe nucleic acid molecule to mammalian cells in vivo, and preferably the nucleic acid molecule includes a coding sequence for a gene product to be expressed in the cell.
  • PINC has been found useful for direct injection into muscle, tumors or organs.
  • the relevant gene product is a polypeptide or protein.
  • the PINC may be used under conditions so that the PINC does not form a gel, or so that no gel form is present at the time of administration at about 30-40°C. Thus, in these compositions, the PINC is present at a concentration of 30%> (w/v) or less.
  • the PINC concentration is still less, for example, 20% or less, 10% or less, 5% or less, or 1% or less.
  • these compositions differ in compound concentration and functional effect from uses of these or similar compounds in which the compounds are used at higher concentrations, for example in the ethylene glycol mediated transfection of plant protoplasts, or the formation of gels for drug or nucleic acid delivery.
  • the PINCs are not in gel form in the conditions in which they are used as PINCs, though certain ofthe compounds may form gels under some conditions.
  • non-condensing means that an associated nucleic acid is not condensed or collapsed by the interaction with the PINC at the concentrations used in the compositions.
  • the PINCs differ in type and/or concentration from such condensing polymers. Examples of commonly used condensing polymers include polylysine, and cascade polymers (spherical polycations).
  • the term “protects” or “protective” or “protected” as used herein refers to an effect of the interaction between such a compound and a nucleic acid such that the rate of degradation ofthe nucleic acid is decreased in a particular environment, thereby prolonging the localized bioavailability ofthe nucleic acid molecule. Such degradation may be due to a variety of different factors, which specifically include the enzymatic action of a nuclease.
  • the protective action may be provided in different ways, for example, by exclusion of the nuclease molecules or by exclusion of water.
  • PINC interleukin-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinasethacrylate, phosphatethyl-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinase-like kinas
  • These interactions can facilitate transfection by, for example, helping associate the nucleic acid molecule-PENC complex closely with the cell wall as a result of biochemical interactions between the PLNC and the cell wall and thereby mediate transfection. These interactions may also provide protection from nucleases by closely associating with the nucleic acid molecule.
  • the term “enhances the delivery” means that at least in conditions such that the amounts of PINC and nucleic acid is optimized, a greater biological effect is obtained than with the delivery of nucleic acid in saline.
  • the level of expression obtained with the PINC:nucleic acid composition is greater than the expression obtained with the same quantity of nucleic acid in saline for delivery by a method appropriate for the particular PINC/coding sequence combination.
  • the PINC is polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), a PVP-PVA co-polymer, N-methyl-2- pyrrolidone (NM2P), ethylene glycol, or propylene glycol.
  • PVP polyvinyl pyrrolidone
  • PVA polyvinyl alcohol
  • NM2P N-methyl-2- pyrrolidone
  • the nucleic acid is preferably not a viral vector, i.e., the nucleic acid is a non- viral vector.
  • the PINC is bound with a targeting ligand.
  • targeting ligands can be of a variety of different types, including but not limited to galactosyl residues, fucosal residues, mannosyl residues, carntitine derivatives, monoclonal antibodies, polyclonal antibodies, peptide ligands, and DNA-binding proteins.
  • the targeting ligands may bind with receptors on cells such as antigen-presenting cells, hepatocytes, myocytes, epithelial cells, endothelial cells, and cancer cells.
  • the term "bound with” means that the parts have an interaction with each other such that the physical association is thermodynamically favored, representing at least a local minimum in the free energy function for that association. Such interaction may involve covalent binding, or non- covalent interactions such as ionic, hydrogen bonding, van der Waals interactions, hydrophobic interactions, and combinations of such interactions.
  • the targeting ligand may be of various types, in one embodiment the ligand is an antibody. Both monoclonal antibodies and polyclonal antibodies may be utilized.
  • the nucleic acid may also be present in various forms.
  • the nucleic acid is not associated with a compounds(s) that alter the physical form, however, in other embodiments the nucleic acid is condensed (such as with a condensing polymer), formulated with cationic lipids, formulated with peptides, or formulated with cationic polymers.
  • the protective, interactive non-condensing compound is polyvinyl pyrrolidone, and/or the plasmid is in a solution having between 0.5% and 50% PVP, more preferably about 5% PVP.
  • the DNA preferably is at least about 80% supercoiled, more preferably at least about 90% supercoiled, and most preferably at least about 95% supercoiled.
  • the compounds which protect the nucleic acid and/or prolong the localized bioavailabihty of a nucleic acid may achieve one or more ofthe following effects, due to their physical, chemical or rheo logical properties: (1) protect nucleic acid, for example plasmid DNA, from nucleases due to steric, viscosity, or other effects such as shearing; (2) increase the area of contact between nucleic acid, such as plasmid DNA, through extracellular matrices and over cellular membranes, into which the nucleic acid is to be taken up; (3) concentrate nucleic acid, such as plasmid DNA, at cell surfaces due to water exclusion; (4) indirectly facilitate uptake of nucleic acid, such as plasmid DNA, by disrupting cellular membranes due to osmotic, hydrophobic or lytic effects; (5) indirectly facilitate uptake of nucleic acids by allowing diffusion of protected nucleic acid chains through tissue at the administration site; and (6) indirectly facilitate uptake of nucleic acid molecules through pore
  • nucleic acid administered to an organism in a composition comprising a transfection facilitating agent will be available for uptake by cells for a longer period of time than if administered in a composition without such a compound, for example when administered in a saline solution.
  • nucleic acid to cells could occur, for example, due to increased duration of contact between the composition containing the nucleic acid and a cell or due to protection of the nucleic acid from attack by nucleases.
  • the compounds that prolong the localized bioavailability of a nucleic acid are suitable for internal administration.
  • suitable for internal administration is meant that the compounds are suitable to be administered within the tissue of an organism, for example within a muscle or within a joint space, epidermally, intradermally or subcutaneously.
  • Properties making a compound suitable for internal administration can include, for example, the absence of a high level of toxicity to the organism as a whole.
  • the plasmid may also be complexed with a liposome formed from the one or more cationic lipids.
  • the cationic lipid is DOTMA and the neutral co-lipid is cholesterol (chol).
  • DOTMA is l,2-di-O-octadecenyl-3-trimethylammonium propane, which is described and discussed in Eppstein et al., U.S. Patent 4,897,355, issued January 30, 1990, which is incorporated herein by reference.
  • other lipids and lipid combinations may be used in other embodiments. A variety of such lipids are described in Gao & Huang, 1995, Gene Therapy 2:710-722, which is hereby incorporated by reference.
  • the charge ratio ofthe cationic lipid and the DNA is also a significant factor, in preferred embodiments the DNA and the cationic lipid are present is such amounts that the negative to positive charge ratio is about 1:3. While preferable, it is not necessary that the ratio be 1 :3. Thus, preferably the charge ratio for the compositions is between about 1 : 1 and 1:10, more preferably between about 1:2 and 1:5.
  • cationic lipid refers to a lipid which has a net positive charge at physiological pH, and preferably carries no negative charges at such pH.
  • An example of such a lipid is DOTMA.
  • neutral co-lipid refers to a lipid which has is usually uncharged at physiological pH.
  • An example of such a lipid is cholesterol.
  • Cationic lipid formulations have been found useful for systemic delivery.
  • negative to positive charge ratio for the DNA and cationic lipid refers to the ratio between the net negative charges on the DNA compared to the net positive charges on the cationic lipid.
  • the DNA preferably is at least about 80% supercoiled, more preferably at least about 90% supercoiled, and most preferably at least about 95% supercoiled.
  • the composition preferably includes an isotonic carbohydrate solution, such as an isotonic carbohydrate solution that consists essentially of about 10% lactose.
  • the composition the cationic lipid and the neutral co-lipid are prepared as a liposome having an extrusion size of about 800 nanometers.
  • the liposomes are prepared to have an average diameter of between about 20 and 800 nm, more preferably between about 50 and 400 nm, still more preferably between about 75 and 200 nm, and most preferably about 100 nm.
  • Microfluidization is the preferred method of preparation ofthe liposomes.
  • sonoporation device relates to an apparatus that is capable of causing or causes uptake of nucleic acid molecules into the cells of an organism by ultrasound means.
  • the cell membrane may thus destabilize and result in the formation of passageways or pores in the cell membrane.
  • the type of sonoporation device is not considered a limiting aspect of the present invention.
  • the primary importance of a sonoporation device is, in fact, the capability ofthe device to deliver formulated nucleic acid molecules into the cells of an organism.
  • apparatus as used herein relates to the set of components that upon combination allow the delivery of formulations of nucleic acid molecules and transfection facilitating agents into the cells of an organism by sonoporation delivery methods.
  • skin refers to the outer covering of a mammal consisting of epidermal and dermal tissue and appendages such as sweat ducts and hair follicles. Skin can comprise the hair of a mammal in cases where the mammal has an epidermis that is covered by hair. In mammals which have enough hair to be considered fur or a pelt it is preferable to shave the hair, leaving primarily skin.
  • the term "organism” as used herein refers to common usage by one of ordinary skill in the art.
  • the organism can include; micro-organisms, such as yeast or bacteria, plants, birds, reptiles, fish or mammals.
  • the organism can be a companion animal or a domestic animal.
  • the organism is a mammal and is therefore any warm-blooded organism. More preferably the mammal is a human.
  • the method results in an immune response, preferably a humoral immune response targeted for the protein product encoded by the nucleic acid molecule, such as an antibody response.
  • the immune response preferably is a cytotoxic T-lymphocyte response.
  • the term "immune response” as used herein refers to the mammalian natural defense mechanism that can occur when foreign material is internalized.
  • the immune response can be a global immune response involving the immune system components in their entirety.
  • the immune response results from the protein product encoded by the formulated nucleic acid molecule.
  • the immune response can be, but is not limited to: antibody production, T-cell proliferation /differentiation, activation of cytotoxic T-lymphocytes, and/or activation of natural killer cells.
  • the immune response is a humoral immune response.
  • the immune response preferably, is a cytotoxic T-lymphocyte response.
  • the term "humoral immune response” refers to the production of antibodies in response to internalized foreign material.
  • the foreign material is the protein product encoded by a formulated nucleic acid molecule internalized by injection with a needle free device.
  • the method results in enhanced transfection of cells as a result of a better method for gene delivery, when compared to sonoporation of non-formulated (naked) nucleic acid.
  • the enhanced transfection can be measured by transfection reporter methods commonly known in the art such as, for example, assays for CAT gene product activity, or LacZ gene product activity, and the like.
  • the "commercial package” or the “container” can include instructions furnished to allow one of ordinary skill in the art to make formulated nucleic acid molecules.
  • the instructions may furnish steps to make the compounds used for formulating nucleic acid molecules. Additionally, the instructions may include methods for testing the formulated nucleic acid molecules that entail establishing if the formulated nucleic acid molecules are damaged upon injection after electroporation.
  • the kit may also include notification of an FDA approved use and instructions.
  • the term "transfection” as used herein refers to the process of introducing DNA (e.g., formulated DNA expression vector) into a cell, thereby, allowing cellular transformation. Following entry into the cell, the transfected DNA may: (1) recombine with that ofthe host; (2) replicate independently as a plasmid or temperate phage; or (3) be maintained as an episome without replication prior to elimination.
  • transformation relates to transient or permanent changes in the characteristics (expressed phenotype) of a cell induced by the uptake of a vector by that cell.
  • Genetic material is introduced into a cell in a form where it expresses a specific gene product or alters the expression or effect of endogenous gene products.
  • Transformation of the cell may be associated with production of a variety of gene products including protein and RNA. These products may function as intracellular or extracellular structural elements, ligands, hormones, neurotransmitters, growth regulating factors, enzymes, chemotaxins, serum proteins, receptors, carriers for small molecular weight compounds, drugs, immunomodulators, oncogenes, cytokines, tumor suppressors, toxins, tumor antigens, antigens, antisense inhibitors, triple strand forming inhibitors, ribozymes, or as a ligand recognizing specific structural determinants on cellular structures for the purpose of modifying their activity. This list is only an example and is not meant to be limiting.
  • Administration refers to the route of introducing the formulated nucleic acid molecules ofthe invention into the body of cells or organisms. Administration includes the use of sonoporation methods to targeted areas ofthe mammalian body such as tumors, the muscle cells and the lymphatic cells in regions such as the lymph nodes.
  • the nucleic acid molecules ofthe invention can be formulated with at least one transfection facilitating agent type of molecule.
  • the molecular complexes can be formulated with cationic lipids or PINCs such as polyvinyl-pyrrolidone as described herein. Formulation techniques are provided herein by example.
  • Figure 1 shows the effect of ultrasound treatment on cationic liposome mediated systemic gene transfer in mouse s.c. SCCVTI tumor and lung.
  • CAT expression levels in tumor (A) and lung (B) were measured 18-20 hr after DNA administration.
  • Figure 2 shows the effect of ultrasound treatment on tumor/lung expression ratio after systemic administration of cationic lipid/plasmid complexes at different DNA doses.
  • Figure 3 shows the influence of sonoporation time on CAT expression in s.c. SCCVII tumors after tail vein administration of DOTMA: CHOL/plasmid complexes.
  • Figure 4 shows the effect of time interval between DNA administration and sonoporation on CAT expression in s.c. SCCVII tumors after tail vein administration of DOTMA:CHOL/plasmid complexes.
  • Figure 5 show the effect of sonoporation prior to DNA administration.
  • Figure 6 shows the relative sonoporation enhancement of plasmid DNA uptake by s.c. SCCVII tumors versus lung after tail vein administration of plasmid/ DOTMA: CHOL complexes.
  • Figure 7 shows the effect of ultrasound treatment on IL-12 expression in s.c. tumors by tail vein injection of IL-12 plasmid complexed with DOTMA:CHOL liposomes.
  • Figure 8 shows the effect of ultrasound treatment on inhibition of tumor growth following systemic administration of IL-12 transfection complexes
  • the delivery, preferably to tumors, of formulations of nucleic acid molecules and transfection facilitating agents by the use of sonoporation device represents a novel approach to gene delivery.
  • the present invention offers a nucleic acid delivery apparatus that provides, for example, an increased number of transfected cells, and also an increased immune response when compared to previous methods as a direct result of providing a more efficient method for transforming cell lines and, thereby increase the production of therapeutic proteins or proteins that potentially trigger an immune response.
  • the invention provides the advantage of allowing the uptake of formulated nucleic acid molecules by specifically targeted cells in vivo.
  • the present invention provides an enhanced delivery of nucleic acid molecules and also provides a more efficient gene delivery system which can be used to generate an immune response, modulate aspects ofthe cell cycle or cell physiology, or provide a method to achieve other gene delivery related therapeutic methods such as anti-tumor therapy.
  • Sonoporation of formulated nucleic acid molecules to an organism depends on several factors which are discussed below, including transfection efficiency and the composition of the formulated nucleic acid molecule.
  • Cationic lipid-based formulations have been widely described to achieve gene transfer into normal endothelial tissue by systemic administration (Brigham, K.B., et al. (1989), Am. J. Med. Sci. 298, 278 - 281 ; Hofland, H.E.J., et al. (1997), Pharm. Res. 14, 742 - 749; Liu, F., et al. (1997), Gene Therapy 4, 517 - 523; Mahato, R.I., et al. (1998), Hum. Gene Ther. 9, 2083 - 2099; Rolland, A.P.
  • the present application describes an ultrasound treatment method that provides for specific increase in gene transfer into mouse primary tumors transfected by systemic administration of cationic lipid/plasmid complexes.
  • the sonoporation treatment did not affect gene expression in non-tumor tissues and did not produce adverse effects on the animals as assessed by gross tissue examination and general physical health ofthe animals. Ultrasound has been a well established ⁇ iagnostic and therapeutic tool in medicine for last several decades.
  • the magnitude of ultrasound enhancement of tumor gene transfer was affected by the sonoporation exposure time, which indicates that selection of appropriate duration of ultrasound exposure is important to achieve gene transfer at desirable level.
  • the observation that enhancement of gene transfer depends only weakly on sonoporation energy is useful information, since it permits greater flexibility in designing ultrasound protocols.
  • the effect of ultrasound was inversely related to the time interval between DNA a(-ministration and sonoporation application suggesting sonoporation effect is acute and may not involve new protein synthesis.
  • Co - localization of DNA plasmid with CD31 endothelial cell marker indicates tumor endothelial cells as the primary sites of DNA uptake in sonoporated tumors.
  • Increase in the perinuclear distribution of DNA by sonoporation further substantiates that the increase DNA uptake is involved in the effect of sonoporation on gene expression.
  • the ultrasound treatment appeared to be well tolerated by the animals during and after the treatment as determined by animal survival, mobility and general well being.
  • ultrasound enhanced transdermal drug delivery without causing damage to skin or underlying tissue or altering the permeability properties ofthe epidermis (Gallo, S.A., et al. (1997), Biophys. J. 72, 2805-2811).
  • the sonoporation treatment does not affect DNA integrity since the size and sequence of plasmid DNA isolated from yeast cells transformed with sonication appeared identical to that obtained from cells transformed in the absence of sonoporation (Liu, J., et al. (1998), Pharm. Res. 15, 918-924).
  • ultrasound is a promising new and safe method for tumor targeting of systemically administered genes.
  • a specific enhancement in gene transfer to tumor by sonoporation thus minimizing delivery to normal tissue is critical for the development of safe and effective systemic gene therapy methods for treatment of cancer.
  • Formulations of nucleic acid molecules can be prepared as disclosed in Example 1. Substitute polymers are selected as determined by application. Generally, a weight/volume ratio is used as exemplified in both ofthe provided examples.
  • nucleic acids in many formulations is limited due to degradation ofthe nucleic acids by cellular components of organisms, such as for instance nucleases.
  • protection of the nucleic acids when delivered in vivo can greatly enhance the resulting expression, and thereby enhance a desired pharmacological or therapeutic effect.
  • certain types of compounds that interact with a nucleic acid e.g., DNA
  • do not condense the nucleic acid provide in vivo protection to the nucleic acid, and correspondingly enhance the expression of an encoded gene product.
  • PINC systems are non-condensing systems that allow the plasmid to maintain flexibility and diffuse freely throughout the muscle while being protected from nuclease degradation. While the PINC systems are primarily discussed below, it will be understood that cationic lipid based systems and systems utilizing both PINCS and cationic lipids are also within the scope ofthe present invention.
  • a common structural component of the PINC systems is that they are amphiphilic molecules, having both a hydrophilic and a hydrophobic portion.
  • the hydrophilic portion of the PINC is meant to interact with plasmids by hydrogen bonding (via hydrogen bond acceptor or donor groups), Van der Waals interactions, or/and by ionic interactions.
  • PVP and N-mefhyl-2-pyrrolidone (NM2P) are hydrogen bond acceptors
  • PVA and Propylene Glycol (PG) are hydrogen bond donors.
  • the high coefficient of variation for reporter gene expression with plasmid formulated in saline has been described previously (Davis, H.L., et al., 1993, Hum. Gene Ther. 4:151-9).
  • PVP polyvinyl pyrrolidone
  • PVP is able to protect all forms of the plasmid from rapid nuclease degradation.
  • the surface modification of plasmids by PVP may also facilitate the uptake of plasmids by muscle cells.
  • the structure-activity relationship described above can be used to design novel copolymers that will also have enhanced interaction with plasmids. It is expected that there is "an interactive window of opportunity" whereby enhanced binding affinity of the PINC systems will result in a further enhancement of gene expression after their intramuscular injection due to more extensive protection of plasmids from nuclease degradation. It is expected that there will be an optimal interaction beyond which either condensation of plasmids will occur or "triplex" type formation, either of which can result in decreased bioavailability in muscle and consequently reduced gene expression.
  • the PINC compounds are generally amphiphilic compounds having both a hydrophobic portion and a hydrophilic portion.
  • the hydrophilic portion is provided by a polar group. It is recognized in the art that such polar groups can be provided by groups such as, but not limited to, pyrrolidone, alcohol, acetate, amine or heterocyclic groups such as those shown on pp. 2-73 and 2-74 of CRC Handbook of Chemistry and Physics (72nd Edition), David R.
  • Lide editor, including pyrroles, pyrazoles, imidazoles, triazoles, dithiols, oxazoles, (iso)thiazoles, oxadiazoles, oxatriazoles, diaoxazoles, oxathioles, pyrones, dioxins, pyridines, pyridazines, pyrimidines, pyrazines, piperazines, (iso)oxazines, indoles, indazoles, carpazoles, and purines and derivatives of these groups, hereby incorporated by reference.
  • hydrophobic groups which, in the case of a polymer, are typically contained in the backbone of the molecule, but which may also be part of a non- polymeric molecule.
  • hydrophobic backbone groups include, but are not limited to, vinyls, ethyls, acrylates, acrylamides, esters, celluloses, amides, hydrides, ethers, carbonates, phosphazenes, sulfones, propylenes, and derivatives of these groups.
  • the polarity characteristics of various groups are quite well known to those skilled in the art as illustrated, for example, by discussions of polarity in any introductory organic chemistry textbook.
  • a targeting ligand in addition to the nucleic acid/PINC complexes described above for delivery and expression of nucleic acid sequences, in particular embodiments it is also useful to provide a targeting ligand in order to preferentially obtain expression in particular tissues, cells, or cellular regions or compartments.
  • a targeted PINC complex includes a PINC system (monomeric or polymeric
  • the PINC system is covalently or non-covalently attached to (bound to) a targeting ligand (TL) which binds to receptors having an affinity for the ligand.
  • TL targeting ligand
  • Such receptors may be on the surface or within compartments of a cell.
  • targeting provides enhanced uptake or intracellular trafficking of the nucleic acid.
  • the targeting ligand may include, but is not limited to, galactosyl residues, fucosal residues, mannosyl residues, carnitine derivatives, monoclonal antibodies, polyclonal antibodies, peptide ligands, and DNA-binding proteins.
  • Examples of cells which may usefully be targeted include, but are not limited to, antigen-presenting cells, hepatocytes, myocytes, epithelial cells, endothelial cells, and cancer cells.
  • a targeting method for cytotoxic agents is described in Subramanian et al., International Application No. PCT/US96/08852, International Publication No. WO 96/39124, hereby incorporated by reference.
  • This application describes the use of polymer affinity systems for targeting cytotoxic materials using a two-step targeting method involving zip polymers, i.e., pairs of interacting polymers. An antibody attached to one ofthe interacting polymers binds to a cellular target. That polymer then acts as a target for a second polymer attached to a cytotoxic agent.
  • other two-step (or multi- step) systems for delivery of toxic agents are also described.
  • nucleic acid coding sequences can be delivered and expressed using a two-step targeting approach involving a non-natural target for a PINC system or PLNC- targeting ligand complex.
  • a PINC -plasmid complex can target a binding pair member which is itself attached to a ligand which binds to a cellular target (e.g., a MAB).
  • the PINC can be complexed to a targeting ligand, such as an antibody. That antibody can be targeted to a non-natural target which binds to, for example, a second antibody.
  • Administration refers to the route of introduction of a plasmid or carrier of DNA into the body. Administration can be directly to a target tissue or by targeted delivery to the target tissue after systemic administration. In particular, the present invention can be used for treating conditions by administration of the formulation to the body in order to establish controlled expression of any specific nucleic acid sequence within tissues at certain levels that are useful for gene therapy.
  • vector vector
  • formulations for delivery are described above and involve sonoporation ofthe target cells.
  • any selected vector construct will depend on the particular use for the expression vectors. In general, a specific formulation for each vector construct used will focus on vector delivery with regard to the particular targeted tissue, the sonoporation delivery parameters, followed by demonstration of efficacy. Delivery studies will include uptake assays to evaluate cellular uptake of the vectors and expression of the DNA of choice. Such assays will also determine the localization of the target DNA after uptake, and establishing the requirements for maintenance of steady-state concentrations of expressed protein. Efficacy and cytotoxicity can then be tested. Toxicity will not only include cell viability but also cell function.
  • Muscle cells have the unique ability to take up DNA from the extracellular space after simple injection of DNA particles as a solution, suspension, or colloid into the muscle. Expression of DNA by this method can be sustained for several months.
  • Delivery of formulated DNA vectors involves incorporating DNA into macromolecular complexes that undergo endocytosis by the target cell. Such complexes may include lipids, proteins, carbohydrates, synthetic organic compounds, or inorganic compounds. Preferably, the complex includes DNA, a cationic lipid, and a neutral lipid in particular proportions.
  • the characteristics of the complex formed with the vector determines the bioavailability of the vector within the body. Other elements ofthe formulation function as ligand which interact with specific receptors on the surface or interior of the cell.
  • DNA transporters refers to molecules which bind to DNA vectors and are capable of being taken up by epidermal cells. DNA transporters contain a molecular complex capable of noncovalently binding to DNA and efficiently transporting the DNA through the cell membrane. It is preferable that the transporter also transport the DNA through the nuclear membrane. See, e.g. , the following applications all of which (including drawings) are hereby incorporated by reference herein: (1) Woo et al., U.S. Serial No.
  • a DNA transporter system can consist of particles containing several elements that are independently and non-covalently bound to DNA. Each element consists of a ligand which recognizes specific receptors or other functional groups such as a protein complexed with a cationic group that binds to DNA. Examples of cations that may be used are spermine, spermine derivatives, histone, cationic peptides and/or polylysine.
  • One element is capable of binding both to the DNA vector and to a cell surface receptor on the target cell.
  • examples of such elements are organic compounds that interact with the asialoglycoprotein receptor, the folate receptor, the mannose-6-phosphate receptor, or the carnitine receptor.
  • a second element is capable of binding both to the DNA vector and to a receptor on the nuclear membrane.
  • the nuclear ligand is capable of recognizing and transporting a transporter system through a nuclear membrane.
  • An example of such ligand is the nuclear targeting sequence from SV40 large T antigen or histone.
  • a third element is capable of binding to both the DNA vector and to elements that induce episomal lysis. Examples include inactivated virus particles such as adenovirus, peptides related to influenza virus hemagglutinin, or the GALA peptide described in the Szoka patent cited above. Administration may also involve lipids as described in preferred embodiments above.
  • the lipids may form liposomes which are hollow spherical vesicles composed of lipids arranged in unilamellar, bilamellar, or multilamellar fashion and an internal aqueous space for entrapping water soluble compounds, such as DNA, ranging in size from 0.05 to several microns in diameter.
  • Lipids may be useful without forming liposomes. Specific examples include the use of cationic lipids and complexes containing DOPE that interact with DNA and with the membrane ofthe target cell to facilitate entry of DNA into the cell.
  • the chosen method of delivery should result in expression of the gene product encoded within the nucleic acid cassette at levels that exert an appropriate biological effect.
  • the rate of expression will depend upon the disease, the pharmacokinetics of the vector and gene product, and the route of administration, but should be in the range 0.001-100 mg/kg of body weight/day, and preferably 0.01-10 mg/kg of body weight/day. This level is readily determinable by standard methods. It could be more or less depending on the optimal dosing.
  • the duration of treatment will extend through the course ofthe disease symptoms, possibly continuously.
  • the number of doses will depend upon the disease, delivery vehicle, and efficacy data from clinical trials.
  • An immune response can be measured by, but is not limited to, the amount of antibodies produced for a protein encoded and expressed by the injected nucleic acid molecule.
  • the present invention can be used to deliver nucleic acid vaccines in a more efficient manner than is conventionally done at the present time.
  • Nucleic acid vaccines or the use of plasmid encoding antigens or therapeutic molecules such as Human Growth Hormone, has become an area of intensive research and development in the last half decade.
  • Comprehensive reviews on nucleic acid based vaccines have been published (M.A. Liu, et al.(Eds.), 1995,
  • nucleic acid based treatment for reducing tumor-cell immunogenicity, growth, and proliferation is indicative of gene therapy for diseases such as tumorigenic brain cancer (Fakhrai et al., Proc. Natl. Acad. Sci., 93:2909-2914, 1996).
  • An important goal of gene therapy is to affect the uptake of nucleic acid by cells, thereby causing an immune response to the protein encoded by the injected nucleic acid.
  • Uptake of nucleic acid by cells is dependent on a number of factors, one of which is the length of time during which a nucleic acid is in proximity to a cellular surface.
  • the present invention provides formulations that increase the length of time during which a nucleic acid is in proximity to a cellular surface, and penetrate the cell thereby delivering nucleic acid molecules into the cell.
  • Nucleic acid based vaccines are an attractive alternative vaccination strategy to subunit vaccines, purified viral protein vaccines, or viral vector vaccines.
  • Each of the traditional approaches has limitations that are overcome if the antigen(s) is expressed directly in cells of the body.
  • these traditional vaccines are only protective in a strain-specific fashion. Thus, it is very difficult, and even impossible using traditional vaccine approaches to obtain long lasting immunity to viruses that have several sera types or viruses that are prone to mutation.
  • Nucleic acid based vaccines offer the potential to produce long lasting immunity against viral epitopes that are highly conserved, such as with the nucleoprotein of viruses. Injecting plasmids encoding specific proteins by the present invention results in increased immune responses, as measured by antibody production.
  • the present invention includes new methods of providing nucleic acid vaccines by delivering a formulated nucleic acid molecule with a sonoporation device as described herein.
  • nucleic acid vaccines are enhanced by one of at least three methods: (1) the use of delivery systems to increase the stability and distribution of plasmid within the muscle, (2) by the expression (or delivery) of molecules to stimulate antigen presentation/transfer, or (3) by the use of adjuvants that may modulate the immune response.
  • V. Polymeric and non-polymeric formulations for plasmid delivery The present invention provides polymeric and non-polymeric formulations that address problems associated with injection of nucleic acids suspended in saline. Unformulated (naked nucleic acid molecules) plasmids suspended in saline have poor bioavailability in muscle due to rapid degradation of plasmid by extracellular nucleases.
  • One possible approach to overcome the poor bioavailability is to protect plasmid from rapid nuclease degradation by for example condensing the plasmid with commonly used cationic complexing agents.
  • the use of rigid condensed particles containing plasmid for efficient transfection of a larger number of muscle cells has not been successful to date.
  • Cationic lipid and polylysine plasmid complexes do not cross the external lamina to gain access to the caveolae and T tubules ([Wolff, J.A., et al., 1992, J. Cell. Sci. 103:1249-1259).
  • the strategy identified for increasing the bioavailability of plasmid in muscle was to: protect plasmid from rapid extracellular nuclease degradation, disperse and retain intact plasmid in the muscle and/or tumor, and facilitate the uptake of plasmid by muscle and/ or tumor cells.
  • a specific method of accomplishing this, which preferably is used in conjunction with sonoporation is the use of protective, interactive, non-condensing systems (PINC).
  • the present invention described herein can be utilized for the delivery and expression of many different coding sequences.
  • the demonstrated effectiveness for the PINC systems (PCT Application No. PCT/US95/17038, WO9621470) for delivery to muscle indicate that such formulations are effective for delivery and sonoporation of a large variety of coding sequences to muscle and/or tumor.
  • tumor cells are also targeted for sonoporation.
  • the present invention provides methods for treating cancerous conditions associated with the formation of tumors or aggregated cell colonies such as those found in conditions such as skin cancer and the like. Specific suggestions for delivery and sonoporation of coding sequences to muscle cells include those summarized in Table 2 below. Table 2: Applications for Plasmid-Based Gene Therapy by Intramuscular Injection
  • the condition or disease preferably is a cancer, such as epithelial glandular cancer, including adenoma and adenocarcinoma; squamous and transitional cancer, including polyp, papilloma, squamous cell and transitional cell carcinoma; connective tissue cancer, including tissue type positive, sarcoma and other (oma's); hematopoietic and lymphoreticular cancer, including lymphoma, leukemia and Hodgkin's disease; neural tissue cancer, including neuroma, sarcoma, neurofibroma and blastoma; mixed tissues of origin cancer, including teratoma and teratocarcinoma.
  • a cancer such as epithelial glandular cancer, including adenoma and adenocarcinoma
  • squamous and transitional cancer including polyp, papilloma, squamous cell and transitional cell carcinoma
  • connective tissue cancer including tissue type positive, sarcoma and other (
  • cancerous conditions that are applicable to treatment include cancer of any ofthe following: adrenal gland, anus, bile duct, bladder, brain tumors: adult, breast, cancer of an unknown primary site, carcinoids of the gastrointestinal tract, cervix, childhood cancers, colon and rectum, esophagus, gall bladder, head and neck, islet cell and other pancreatic carcinomas, Kaposi's sarcoma, kidney, leukemia, liver, lung: non-small cell, lung: small cell, lymphoma: ATDS-associated, lymphoma: Hodgkin's disease, Lymphomas: non-Hodgkin's disease, melanoma, mesothelioma, metastatic cancer, multiple myeloma, ovary, ovarian germ cell tumors, pancreas, parathyroid, penis, pituitary, prostate, sarcomas of bone and soft tissue, skin, small intestine, stomach, testis, thymus, thyroid, troph
  • the expression plasmid pCMV-CAT contained a CMV enhancer and promoter, an
  • SV40 intron a chloramphenicol acetyltransferase (CAT) gene and a SV40 late poly A signal.
  • CAT chloramphenicol acetyltransferase
  • the expression plasmid pCMV-IL-12 contained two transcription units in tandem, one for the p35 subunit and one for the p40 subunit, each transcription unit included a CMV enhancer and promoter, a synthetic intron, an IL-12 subunit coding sequence and a human growth hormone poly A signal.
  • the pDNA was isolated and purified from Escherichia coli by chromatography. The purity of pDNA preparations was determined by 1% agarose gel electrophoresis followed by SYBRTM Green (Molecular Probes, Eugene, OR) staining, and DNA concentration was measured by UV absorption at 260 nm.
  • the percentage of supercoiled pDNA and OD 260/280 ratios of these pDNA preparations was in the range of 70-95% and 1.8-1.9, respectively.
  • Endotoxin levels of pDNA preparations were determined using the chromogenic limulus amebocyte lysate assay (Chromogenic End-Point, LAL BioWhittaker, Walkersville, MD) and were ⁇ 50 endotoxin units (EU)/mg.
  • Liposomes containing N-[l-(2-3-dioleyloxy)propyl)]-N-N-N-trimethylammonium chloride (DOTMA) and cholesterol (CHOL) were prepared at 4:1 DOTMA:CHOL molar ratio. Briefly, lipids were mixed in chloroform and evaporated to a thin film in a 50 ml round bottom flask using a rotary evaporator. The film was hydrated in sterile water, probe-sonicated, centrifuged at 32000 x g for 30 minutes, and sterile filtered to obtain small unilamelar vesicles (SUV).
  • SUV small unilamelar vesicles
  • DOTMA:CHOL/plasmid complexes were formed in 10% lactose by controlled mixing of liposomes with DNA plasmid at 3:1 (+/-) charge ratio unless stated otherwise.
  • the plasmid concentration in the formulation was 300 microgram/ml.
  • serial dilutions with 10% lactose were made to obtain DNA concentrations of 33 microgram/ml, 50 microgram /ml, and 150 microgram /ml, respectively.
  • Subcutaneous solid tumors were created in 6 - 8 week old female C3H mice (20 - 22 g) (Charles River Laboratories Raleigh, NC) by s.c. injection of 4 x 10 5 squamous carcinoma cells. The average tumor size before DNA injection was typically in the range of ⁇ 30 - 40 mm 3 (6-7 days after implantation).
  • pCMV-CAT was complexed with DOTMA: CHOL (4:1, mol/mol) with cationic lipid to plasmid ratio of 3:1 (+/-) and administered intravenously into the tail vein of mice.
  • tumors were covered with Spectrogel 50 conducting gel (Spectrogel XX) and sonoporated for 1, 2, 5, or 15 minutes at
  • Tumors and lungs were homogenized in 0.35 ml and 1 ml TENT (Tris 10 mM, EDTA 1 mM, NaCl 0.1 M, Triton X-100 0.5 %)buffer, respectively.
  • the tissue homogenate was centrifuged at 10,000 x g for 15 min and supernatant was assayed for the gene product, for example CAT or IL-12, using a specific ELISA (Boehringer Mannheim, Indianapolis, IN).
  • Psoralen-fluorescent-labeled pCMV-CAT was complexed with DOTMA:CHOL (4:1, mol mol) at 3:1 (+/-) lipid/plasmid charge ratio and injected into s.c. tumor bearing mice via tail vein at a dose of 90 ⁇ g DNA/mouse.
  • the tumors were sonoporated at 1.5 W/cm 2 for various periods immediately before and after DNA injection.
  • mice were anesthetized by intraperitoneal administration of a mixture of ketamine (42.8 mg/ml), xylazine (8.6 mg/ml) and acepromazine (1.4 mg/ml) at a dose of 0.5-0.7 ml kg, and whole-body perfusion was performed with 1 % BSA PBS solution to clear entrapped blood. Tumors were removed and embedded in O.C.T embedding medium. Tissue cryosections (5 micrometers) were examined for plasmid fluorescence with an Olympus
  • tumor paraffin cryosections (JUNG CM3000) were first incubated with a rat anti-mouse CD31 antibody (Pharmingen, San Diego, CA), and then with a rabbit anti-rat IgG labeled with Texas Red fluorophore (Vector Laboratories, Inc., Burlingame, CA). The tumor sections were fixed in formaldehyde and counterstained with VectashieldTM mounting medium containing DAPI to highlight nuclei. Controls included unstained sections and incubation with secondary antibody alone. Tissue sections were viewed using an Olympus BX-60 fluorescence microscope and photographed using a Montague spot camera.
  • Tissues were digested by incubation with digestion buffer (100 mM NaCl, 10 mM Tris-HCl, [pH 8.0], 25 mM EDTA [pH 8.0], 0.5% SDS, and protemase K [0.1 milligram/ml]) at 50° C.
  • the samples were extracted with an equal volume of Tris-buffered phenol (pH 8.0), followed by extraction with chloroform:isoamyl alcohol (24:1, v/v) and ethanol precipitation.
  • the DNA precipitates were dissolved in TE buffer (10 mM Tris [pH 7.5], 1 mM EDTA), and DNA concentration was measured by UV absorption at 260 nm.
  • the amount of plasmid DNA associated with the tissue was quantified by a polymerase chain reaction (PCR) assay using Taqman PCR (Perkin-Elmer, Foster City, CA).
  • PCR polymerase chain reaction
  • the primers used in the reaction were a forward primer, 5'-TGA CCT CCA TAG AAG ACA CCG GGA C-3 1 (Genosys Biotechnologies, The Woodlands, TX), which primes in the CMV 5' untranslated region (UTR), and a reverse primer, 5' AGG CCG TAA TAT CCA GCT GAACG-3', which primes in the CAT coding region.
  • the probe sequence was 5'-CCA GCC TCC GGA CTC TAG AGG A-3'.
  • the initial copy numbers of unknown samples were determined by using an Applied Biosystem 7700 sequence detector to compare them with a standard curve generated from purified pCMV-CAT of known initial copy numbers.
  • the sonoporation enhancement of gene expression was tumor specific since no increase was observed in lung (Fig. IB).
  • the sonoporation treatment increased the tumor/non-tumor expression ratio (Fig. 2).
  • the tumor/lung expression ratios in non-sonoporated animals were 0.001, 0.0001, and 0.0009 at 15, 45, and 90 microgram DNA, respectively.
  • the tumor/lung ratios increased to 0.3, 0.01, and 0.004 at 15, 45, and 90 microgram dose, respectively.
  • the magnitude of sonoporation enhancement of tumor gene transfer was dependent on the duration of sonoporation (Fig. 3).
  • Tumors were collected 15 min after plasmid injection and tissue cryosections (5 micrometers) were prepared and examined under a fluorescence microscope for distribution of fluorescent plasmid (green). Nuclei are stained in blue with DAPI. The sections were counterstained with a rhodamine labeled anti-CD31 endothelial cell marker. The plasmid fluorescence appeared to be higher in sonoporated tumors compared to non-sonoporated tumors. Sonoporation appeared to increase the perinuclear distribution ofthe DNA when compared with the control. Co-localization of fluorescent plasmid and antibody to endothelial cell surface marker demonstrates the endothelial localization of sonoporated DNA. The sonoporation treatment did not produce any adverse effect at the sonoporation site or on general well-being ofthe animals.
  • FIG. 6 shows the effect of ultrasound treatment on IL-12 expression in s.c. tumors.
  • IL-12 levels in ultrasound treated tumors at 0.5 W/cm 2 and 1.5 W/cm 2 were 0.44 ⁇ 0.18 ng/g and 0.52 ⁇ 0.31 ng/g tumor, respectively.
  • IL- 12 levels in non-sonoporated tumors were 0.064 - 0.066 ng/g.
  • systemic tail vein injection of DOTMA: CHOL/pCMV-IL- 12 complexes followed by local sonoporation resulted in preferential IL-12 expression in tumors.
  • FIG. 7 shows the effect of ultrasound treatment on inhibition of tumor growth following systemic administration of IL-12 transfection complexes.
  • the combination of systemic administration of IL-12 transfection complexes and ultrasound treatment produced a significant inhibition of tumor growth.
  • the combination of IL-12 transfection complex systemic administration and ultrasound treatment yielded a 65% reduction in tumor growth rate compared to the lactose control group. Tumor inhibition from combination treatment was significantly higher than that obtained with IL-12 gene treatment alone.
  • the effect of ultrasound treatment on tumor growth was specific to IL- 12 gene since no effect of ultrasound treatment was observed in lactose or CAT gene injected group.

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Abstract

L'invention concerne des produits et des procédés permettant d'administrer une molécule d'acide nucléique(ou davantage) à une tumeur et de traiter cette dernière par ultrason. Les produits et les procédés permettent l'administration et l'expression préférentielles de molécules d'acides nucléiques dans des cellules situées dans une tumeur soumise à un traitement par ultrason, par rapport à l'expression dans des tissus non traités par ultrason.
PCT/US2000/020631 1999-07-28 2000-07-28 Traitement par ultrason des tumeurs WO2001008709A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2003066104A2 (fr) * 2002-02-08 2003-08-14 Institut National De La Sante Et De La Recherche Medicale (Inserm) Composition pharmaceutique ameliorant le transfert de gene in vivo
FR2835749A1 (fr) * 2002-02-08 2003-08-15 Inst Nat Sante Rech Med Composition pharmaceutique ameliorant le transfert de gene in vivo
WO2003066104A3 (fr) * 2002-02-08 2004-03-25 Inst Nat Sante Rech Med Composition pharmaceutique ameliorant le transfert de gene in vivo
US7709452B2 (en) 2002-02-08 2010-05-04 Institut National De Le Sante Et De La Recherche Medicale Pharmaceutical composition which improves in vivo gene transfer
US8367631B2 (en) 2002-02-08 2013-02-05 Institut National De La Sante Et De La Recherche Medicale Pharmaceutical composition which improves in vivo gene transfer
WO2007028981A1 (fr) * 2005-09-08 2007-03-15 University Of Dundee Appareil et procédé de sonoporation
EP3346005A1 (fr) * 2008-01-31 2018-07-11 CureVac AG Acides nucléiques de formule (i) (nugixmgnnv)a et dérivés associés en tant qu'agent/adjuvant de stimulation immunitaire

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