WO2001023537A1 - Procede servant a promouvoir l'administration assistee par champ electrique de materiaux biologiques dans des cellules - Google Patents

Procede servant a promouvoir l'administration assistee par champ electrique de materiaux biologiques dans des cellules Download PDF

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Publication number
WO2001023537A1
WO2001023537A1 PCT/US2000/021982 US0021982W WO0123537A1 WO 2001023537 A1 WO2001023537 A1 WO 2001023537A1 US 0021982 W US0021982 W US 0021982W WO 0123537 A1 WO0123537 A1 WO 0123537A1
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biological material
electric field
cells
biological
binding reagent
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PCT/US2000/021982
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English (en)
Inventor
Alan D. King
Robert W. Malone
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Cyto Pulse Sciences, Inc.
University Of Maryland, Baltimore
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Priority to CA002390716A priority Critical patent/CA2390716A1/fr
Priority to EP00964909A priority patent/EP1159414A4/fr
Publication of WO2001023537A1 publication Critical patent/WO2001023537A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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

Definitions

  • the present invention relates generally to a method of using biopolymer binding reagents in conjunction with electroporation techniques to enable and enhance uptake of a biological material into cells.
  • the invention thereby provides improved delivery efficiency of biological material to cells, both in vitro and in vivo.
  • the improved method finds particular utility in the transfer of genetic material into cells and has broad applicability for a wide range of gene therapy and polynucleotide vaccine applications.
  • ex vivo and invasive in vivo systems have been relegated to the treatment of life threatening diseases such as cancer or rare metabolic diseases.
  • Ex vivo therapies are associated with risks and challenges in harvesting, modifying, maintaining, and administering cell products.
  • Invasive in vivo gene delivery systems require surgical procedures, a process which itself is associated with multiple types of risks .
  • All gene therapies confront at least two problems: (1) selecting genes or polynucleotides to use and (2) efficiently and specifically delivering genes or polynucleotides into cells or tissues. It is the latter issue that tends to be most problematic.
  • viral vector systems are generally highly efficient in terms of delivery and expression. However, they rely on specific virus-cell interactions, and are subject to a variety of host defense and virus-specific constraints which result in safety, manufacturing, and reproducibility problems .
  • the invention herein addresses the problems discussed above. Specifically, the invention involves the employment of biopolymer binding reagents, e. g. polynucleotide binding reagents, in combination with electroporation techniques to enhance the efficiency of biopolymer transfer, e. g. polynucleotide transfer, to the biological cells of interest. Electrical fields enhance polynucleotide uptake into cells via both increased cell permeability and electrophoretic effects. As such, electroporation can augment the activity of other delivery technologies, particularly non-viral gene transfer methods.
  • biopolymer binding reagents e. g. polynucleotide binding reagents
  • electroporation techniques to enhance the efficiency of biopolymer transfer, e. g. polynucleotide transfer
  • the administration of polynucleotide binding reagents prior to or in combination with the application of the electrical field can result in the enhancement of (1) pericellular concentration of the polynucleotide and (2) cell membrane fusion and/or destabilization.
  • the two together create a synergistic effect on the efficiency of polynucleotide transfer.
  • Advantages associated with the novel techniques of the invention include the enhanced delivery and transfection efficiency as well as a reduction in the required electrical field strength (relative to standard electroporation conditions) .
  • the efficacy is achieved with a reduced dose of polynucleotide binding reagent.
  • additional advantages may derive from these primary advantages, including reduced treatment- associated toxicity.
  • the combined electroporation/biopolymer-binding- reagent-based therapies of the present invention find utility for both ex vivo and in vivo gene therapy delivery systems.
  • the invention herein is not limited to the transport of polynucleotides. Rather, it is also applicable to the transport of other biopolymers across cell membranes, such biopolymers including but not limited to proteins and antigens.
  • the combined electroporation/biopolymer-binding-reagent-based therapies of the present invention are also useful in in vitro and in vivo transfer of biopolymers into biological cells.
  • the polynucleotide preparations of the present invention may be used for gene therapy in general and, more specifically, for delivering exogenous copies of a therapeutic gene or polynucleotide to a specific cellular tissue target in vivo.
  • Enhanced polynucleotide delivery of the present invention also finds utility not only in vaccine therapies, but also for the following genetic therapies: for inborn metabolic diseases, such as cystic fibrosis; the expression of immunomodulatory agents, such as cytokines or costimulatory molecules; and the delivery of such therapeutic polynucleotides into cells of the immune system including antigen presenting cells.
  • the techniques of the present invention find utility in pre-clinical applications directed to modifying immune responses to cancer and infectious diseases.
  • the invention involves the genetic modification of dendritic cells.
  • biological materials such as polynucleotides or other biopolymers
  • polynucleotide refers to a covalently-linked sequence of nucleotides in which the 3' and 5' ends on each nucleotide are joined by phosphodiester bonds .
  • the polynucleotides contemplated by the present invention include DNA and RNA, both sense and antisense strands, as well as catalytic RNAs, oligonucleotides and ribozymes.
  • the polynucleotides may be naturally occurring or recombinant, of biological origin, such as viral DNA or RNA, or of synthetic origin, such as cDNA.
  • polynucleotide construct refers to an expression cassette comprising a polynucleotide in combination with transcription and translation elements required for expression of the polynucleotide.
  • constructs contemplated by the present invention include but are not limited to plasmid, phagemid, and cosmid expression vectors.
  • biopolymer refers to any macromolecule which can be found within a living thing, such as proteins (essentially long chains of amino acids) , nucleic acids (such as DNA or RNA) , and polysaccharides (long chains of simple sugars) .
  • the biopolymers contemplated by the present invention include but are not limited to antigens, proteins, peptides, carbohydrates, and lipoproteins.
  • polynucleotide binding reagents and “biopolymer binding reagents” refer to agents that result in the enhanced pericellular concentration of the polynucleotide or biopolymer and the enhanced cell membrane fusion and/or destabilization, thereby providing a synergistic effect on the efficiency of polynucleotide or biopolymer transfer (transfection) when applied in conjunction with an electric field.
  • the polynucleotide and biopolymer binding reagents contemplated by the present invention include but are not limited to cationic lipids, polar molecules, charged organic polymers, viral structural proteins, and Polymixin B.
  • the polynucleotide binding reagent is in the form of a liposome or a colloid.
  • the cationic lipids may be provided alone or in combination with other organic molecules, polysaccharides or peptides. As cationic lipids have been relatively inefficient or ineffective when utilized in vivo, formulation or dose modifications such as are known in the art may be required to facilitate an in vivo application of the present invention.
  • charged organic polymers that are employed include, but are not limited to, polycationic amino polymers, poly L-lysines, polyamidoamine dendrimers, jpolyethyleneimine, polyethanolamine, and poly-L-ornithine and dendrimer, oligomers, and copolymers thereof.
  • electroporeation refers to the process wherein an electric field pulse is applied to a cellular membrane to induce an effective state of poration in said membrane .
  • the pores formed by electroporation are commonly referred to as “electropores” .
  • electropore ⁇ allows molecules (such as drugs, biopolymers, and genetic material) , ions, and water to pass from one side of a membrane to the other.
  • the electropores generally appear on that surface of the cell that is closest to the electrodes and are generally transient in nature. Provided proper parameters are utilized, the electropore ⁇ spontaneously reseal allowing the electroporated cell to fully recover normal function.
  • the electric field may range from 1 to 10,000 volts per centimeter, more preferably 1 to 1000 volts per cm.
  • the duration of the electric field may be from 1 microsecond to 20 milliseconds.
  • the electric field may be delivered as a single pulse or as multiple pulses.
  • the electric field may take the form of a square wave pulse, a sine wave pulse, a DC shifted sine wave or other pulse shape.
  • polynucleotides are polynucleotide sequences that encode a protein or fragment thereof or antisense RNA or catalytic RNA, which is endogenous or foreign to the particular host species.
  • the term “recombinant polynucleotide” refers to a polynucleotide of genomic, cDNA, semisynthetic or synthetic origin which is distinct in form, linkage or association from the form, linkage, or association in which the polynucleotide exists in nature.
  • recombinant DNA refers to a DNA molecule produced by operatively linking two DNA segments.
  • a recombinant DNA molecule is a hybrid molecule comprising at least two nucleotide sequences not normally found together in nature.
  • Recombinant DNA molecules not having a common biological origin i.e., evolutionarily different
  • the term “purified polynucleotide” refers to a polynucleotide which is essentially free, i.e., containing less than about 50%, preferably less than about 70%, even more preferably less than about 90% of polypeptides with which the polynucleotide is naturally associated.
  • the polynucleotide preparation delivers a sequence specific polynucleotide of interest into a target cell or tissue.
  • the polynucleotide may encode for a gene, vaccine antigen, an immunoregulatory agent, or a therapeutic agent.
  • the polynucleotide of interest may be either a foreign gene or an endogenous gene.
  • the polynucleotide of interest need not encode a protein. Rather, the polynucleotide of interest may be a therapeutic polynucleotide (e.g., an antisense RNA or catalytic RNA) or one which will affect the biology of a cell, tissue or host.
  • Such polynucleotides find particular utility in the field of gene discovery (e.g., identification and functional characterization of new genes) and rational drug design (e.g., identification and validation genetic targets for pharmaceutical manipulation) .
  • foreign gene means a polynucleotide encoding a protein or fragment thereof or antisense RNA or catalytic RNA, which is foreign to the recipient animal cell or tissue, such as a vaccine antigen, immunoregulatory agent, or therapeutic agent.
  • an “endogenous gene” means a polynucleotide encoding a protein or part thereof or antisense RNA or catalytic RNA which is naturally present in the recipient animal cell or tissue.
  • the polynucleotide of interest encodes a vaccine antigen.
  • the term "vaccine antigen” refers to an agent capable of stimulating the immune system of a living organism, inducing the production of an increased level of antibodies, the production of a cellular immune response, or the activation other immune responsive cells involved in the immune response pathway against said antigen.
  • the vaccine antigen expression may be performed to elicit an immune response and/or to induce tolerance to the encoded antigen.
  • expression of antigens in cells which lack co-stimulatory molecule expression can enable the development of tolerance to the antigen.
  • the vaccine antigen may be a protein or antigenic fragment thereof from viral pathogens, bacterial pathogens, and parasitic pathogens.
  • the vaccine antigen may be a synthetic polynucleotide, constructed using recombinant DNA methods which encode antigens or parts thereof from viral, bacterial, or parasitic pathogens. These pathogens can be infectious in humans, domestic animals or wild animal hosts.
  • the antigen can be any molecule that is expressed by any viral, bacterial, or parasitic pathogen prior to or during entry into, colonization of, or replication in their animal hos .
  • the viral pathogens from which the viral antigens are derived, include, but are not limited to: Orthomyxoviruses, such as influenza virus; Retroviruses, such as RSV and S1V; Herpesviruses , such as EBV, CMV, or herpes simplex virus; Lentiviruses, such as human immunodeficiency virus; Rhabdoviruses, such as rabies ; Picornoviruses, such as poliovirus; Poxviruses, such as vaccinia; Rotavirus; and Parvoviruses .
  • Orthomyxoviruses such as influenza virus
  • Retroviruses such as RSV and S1V
  • Herpesviruses such as EBV, CMV, or herpes simplex virus
  • Lentiviruses such as human immunodeficiency virus
  • Rhabdoviruses such as rabies
  • Picornoviruses such as poliovirus
  • Poxviruses such as vaccinia
  • protective antigens of viral pathogens include: the human immunodeficiency virus antigens Nef, p24, gpl20, gp41, Taq, Rev, and Pol et al, Nature, 313:277-280 (1985)) and T cell and B cell epitopes of gpl20(Palker et al, J. Immunol., 142:3612-3619 (1989)); the hepatitis B surface antigen (Wu et al, Proc. Nati. Acad. Sci., USA, 86:4726-4730 (1989)); rotavirus antigens, such as VP4 (Mackow et al, Proc. Nati. Acad.
  • influenza virus antigens such as hemagglutinin or nucleoprotein (Robinson et al . , Supra; Webster et al, Supra); and herpes simplex virus thymidine kinase (Whitley et al, New Generation Vaccines, pages 825-854) .
  • the bacterial pathogens, from which the bacterial antigens are derived include but are not limited to, Mycobacterium spp., Helicobacter pylori, Salmonella spp., Shigella spp., E. coil, Rickettsia spp., Listeria spp., Legionella pneumoniae, Pseudomonas spp., Vibrio spp., and Borellia burgdorferi .
  • protective antigens of bacterial pathogens include the Shigella sonnei form 1 antigen (Formal et ai, Infect. Immun., 34:746-750 (1981)); the 0-antigen of V. cholerae Inaba strain 569B (Forrest et al, J. Infect. Dis., 159:145-146 (1989); protective antigens of enterotoxigenic E. coli, such as the CFA/I fimbrial antigen (Yamamoto et al, Infect.
  • the parasitic pathogens from which the parasitic antigens are derived, include but are not limited to, Plasmodium spp., Trypanosome spp., Giardia spp., Boophilus spp., Babesia spp., Enta oeba spp., Eimeria spp., Leish ania spp., Schistosome spp., Brugin spp., Fascida spp., Dirofilaria spp., Wuchereria spp., and Onchocerea spp ..
  • protective antigens of parasitic pathogens include the circumsporozoite antigens of Plasmodium spp.
  • the polynucleotide of interest can encode a therapeutic agent to animal cells or animal tissue.
  • the polynucleotide can encode tumor-specific, transplant, or autoimnune antigens or parts thereof.
  • the polynucleotide can encode synthetic genes, which encode tumor-specific, transplant, or autoimmune antigens or parts thereof.
  • Additional immunostimulatory polypeptide encoding polynucleotides such as those encoding adjuvants or chemokines, may be administered to stimulate the immune response associated with the expression of the antigen encoding polynucleotide.
  • tumor specific antigens include but are not limited to prostate specific antigen (Gattuso et al, Human Pachol . , 26:123-126 (1995)), TAG-72 and CEA
  • mice have been shown in mice that immunization with non-malignant cells expressing a tumor antigen provides a vaccine effect, and also helps the animal mount an immune response to clear malignant tumor cells displaying the same antigen ( oeppen et al, Anal. N.Y. Acad. Sci., 690:244- 255 (1993)).
  • transplant antigens examples include the CD3 receptor on T cells (Alegre et al, Digest. Dis. Sci., 40:58-64 (1995)) . Treatment with an antibody to CD3 receptor has been shown to rapidly clear circulating T cells and reverse most rejection episodes (Alegre et al, supra) .
  • autoimmune antigens include LAS Beta- chain (Topham et al, Proc. Nati. Acad. Sci., USA, 91:8005-8009 (1994)). Vaccination of mice with an 18 amino acid peptide from LAS Beta-chain has been demonstrated to provide protection and treatment to mice with experimental autoimmune encephalomyelitis (Topham et al, supra) .
  • the polynucleotide of interest can encode immunoregulatory molecules.
  • immunoregulatory molecules include, but are not limited to, growth factors, such as M-CSF, GM-CSF; and cytokines, such as IL-2, IL-4, IL-5 7 IL-6, IL-10, IL-12 or IFN-gamma. Delivery of cytokines expression cassettes to tumor tissue has been shown to stimulate potent systemic immunity and enhanced tumor antigen presentation without producing a systemic cytokine toxicity (Golumbek et al , Cane. Res., 53:5841-5844 (1993); Golumbek et al, Immmun. Res., 12:183-192 (1993); Pardoll, Curr. Opin. Oncol., 4:1 124-1 129 (1992); and Pardoll, Curr. Opin. Immunol. . 4:619-623 (1992)).
  • growth factors such as M-CSF, GM-CSF
  • cytokines such
  • the polynucleotide of interest may encode an antisense RNA or catalytic RNA.
  • the RNA can be targeted against any molecule present within the recipient cell or likely to be present within the recipient cell. These include but are not limited to RNA species encoding cell regulatory molecules, such as interlukin-6 (Mahieu et al, Blood,
  • oncogenes such as ras (Kashani-Sabet et al, Antisen. Res. Devel . , 2:3-15 (1992)), causative agents of cancer such as human papillomavirus (Steele et al, Cane. Res., 52:4706-4711 (1992)), enzymes, viral RNA'S and pathogen derived RNA's such as HIV-1 (Meyer et al, Gene, 129:263-268 (1993); Chatterjee et al, Sci., 258:1485-1488 (1992); and Yamada et al , Virol., 205:121-126 (1994)).
  • RNAs can also be targeted at non-transcribed DNA sequences, such as promoter or enhancer regions, or to any other molecule present in the recipient cells, such as but not limited to, enzymes involved in DNA synthesis or tRNA molecules [Scanlon et al, Proc. Natl. Acad. Sci. USA, 88:1059 1- 10595 (1991); and Baier et al, Mol. Immmunol . , 31:923-932 (1994) ⁇ .
  • the target cell and/or tissue is not critical to the invention.
  • the cells may be present in the intact animal, a primary cell culture, explant culture or a transformed cell line.
  • Certain cells or tissues contemplated by the invention include but are not limited to embryos and embryonic tissues, fetal tissues, oocytes, embryonic or pleuripotent tissue or cells.
  • the particular cells and tissue source are not critical to the present invention.
  • the host organism employed in the present invention is not critical thereto and includes all organisms within the kingdom animalia, such as those of the families mammalia, pisces, avian, and reptilia.
  • Preferred animals are mammals, such as humans, bovines, ovines, porcines, felines, buffalos, canines, goats, equines, donkeys, deer, and primates. The most preferred animal is a human.
  • tissue are preferred targets for expression of the polynucleocide of interest.
  • tissues of interest include but are not limited to epithelial tissues, mucosal tissues, respiratory tissues skeletal muscle, cardiac muscle, vascular endothelium, pancreatic and liver tissues, tumors, thyroid, chymus, synovium, and brain.
  • mucosal tissue or mucosal associated tissues contemplated include oral tissues, ocular tissues, gastro-intestinal tissues, gut-associated lymphoid tissues, bronchial-associated lymphoid tissues, nasal-associated lymphoid tissues, genital-associated lymphoid tissues, Waddeyer's ring, Peyer's patches, and tonsils.
  • Respiratory tract tissues are subject to a variety of pathologies, provide a first line of defense against many environmental toxins and pathogens, are selectively permeable to a range of molecules including biopolymers, and are largely associated with a rich, vascular bed.
  • technologies for genetic modification of respiratory tissues may be used to develop treatments for inborn errors of metabolism (such as Cystic Fibrosis) , to treat systemic disease via absorption to or from the circulatory compartment, or to modify either pathologic or beneficial immune responses (asthma, mucosal immunity) .
  • the polynucleotide may be constructed as an expression cassette or expression vector.
  • an expression cassette is composed of a promoter region, a transcriptional initiation site, a ribosome binding site (RBS) , an open reading frame (orf) encoding a protein (or fragment thereof) , with or without sites for RNA splicing (only in eukaryotes) , a translational stop codon, a transcriptional terminator and post-transcriptional poly-adenosine processing sites (only in eukaryotes) (Wormington, Curr. Opin. Cell. Biol . , 5:950-954 (1993); Reznikoff et al, Eds., Maximizing Gene Expression, Butterworths, Stoneham, Mass. (1986)).
  • the particular expression cassette employed in the present invention is not critical thereto, and can be selected from the many commercially available cassettes. Examples include but are not limited to pCEP4 and pRc/RSV (Invitrogen Corporation, San Diego, CA) ; pXTl, pSG5, pPbac and pMbac (Stratagene, La Jolla, CA) ; pPUR, pEGFP-1, pND and pMAM (ClonTech, Palo Alto, CA) ; and pSV-Beta-gal (Promega Corporation, Madison, WI) .
  • the cassette may be synthesized either de novo or by adaptation of a publicly or commercially available expression system.
  • reporter genes include but are not limited to beta-galactosidase, green fluorescent protein, and luciferase.
  • An enhanced green fluorescent protein gene is commercially available and can be amplified from a commercial vector (pEGFP-l, Clonetech, Palo Alto, CA) incorporating Sal I and BamH I sites into the primers.
  • the first 28 amino acids of the protein are from Drosophila Alcohol Dehydrogenase followed by the fused E. coli Beta-galactosidase sequences. The insect sequences are reported to give higher expression in mammalian cells presumably by providing eukaryotic translation initiation signals.
  • the individual elements within the expression cassette can be derived from multiple sources and may be selected to confer specificity in sites of action or longevity of the cassettes in the host cell or target tissue. Such manipulation of the expression cassette can be done by any standard molecular biology approach.
  • These expression cassettes may be in the form of plasmids, cosmids, phagemids and the like. They generally contain various promoters well-known to be useful for driving expression of genes in animal cells, such as the viral derived SV40, CMV and, RSV promoters or eukaryotic derived beta-casein, uteroglobin, beta-actin or tyrosinase promoters.
  • the particular promoter is not critical to the present invention, except in the case where the object is to obtain expression in only targeted cell types or tissues.
  • the promoter is selected to be one which is only active in the targeted cell type or tissue.
  • tissue specific promoters include, but are not limited to the tyrosinase promoter which is active in lung and spleen cells, but not testes, brain, heart, liver, or kidney [Vile et ad, Cane. Res., 54:6228-6234 (1994)]; the involucerin promoter which is only active in differentiating keratinocytes of the squamous epithelia [Carroll et ad, J. Cell Sci., 103:925-930 (1992)]; and the uteroglobin promoter which is active in lung and endometrium [Heiftenbein et al, Annad. N.Y. Acad. Sci., 622:69-79 (1991).].
  • tissue/cell specific enhancer sequences can be used to control expression.
  • tissue specific expression is to use a hormone responsive element (HRE) to specify which cell lineages a promoter will be active in, for example, the MMTV promoter requires the binding of a hormone receptor, such as progesterone receptor, to an upstream HRE before it is activated [Beato, FASEB J., 5:2044-2051(1991); and Truss et al, J. Steroid Biochem. Mol. Biol . , 41:241-248 (1992) ] .
  • HRE hormone responsive element
  • Additional genetic elements may be included in the expression cassette in order to modify its behavior inside the host cell [Hodgson, Bio/Technology, 13:222-225 (1995)] .
  • Such elements include viral genome components such as the DNA genome of a recombinant adenovirus or the self replicating "replicon" RNA of an alphavirus such as semliki forest or Sindbus virus.
  • Additional elements include but are not limited to mammalian artificial chromosome elements or elements from the autonomous replicating circular minichromosomes, such as found in DiFi colorectal cancer cells to allow stable non-integrated retention of the expression cassette [Huxley et al, Bio/Technology 12:586-590 (1994); and Untawale et al, Cane.
  • These additional genetic elements may also include substantial regions of viral genomes, so that integration and/or autonomous replication of the polynucleotide of interest will be enabled by the viral sequence elements.
  • inclusion of the AAV ITR sequences together with the rep protein ORF in the expression cassette can provide integration.
  • the amount and concentration of the polynucleotide to be administered vary depending on the species of the subject, as well as the desired response and the disease or condition that is being treated. Generally, it is expected that up to 100-200 microgm of DNA can be administered in a single dosage, although as little as about 0.3 microgm of DNA administered through skin or mucosa can induce long lasting immune responses. For purposes of the invention, however, it is sufficient that the polynucleotides be supplied at a dosage sufficient to cause expression of the polynucleotide of interest carried by the polynucleotide.
  • dose-response experiments can be used to determine efficacy, toxicity, and effective dose of a particular polynucleotide preparation and polynucleotide binding reagent . Such experiments are routine in the art and well within the skill of one with ordinary skill in the art .
  • the electroporation system comprises the PA-4000 electroporation system from Cyto Pulse Sciences, Inc., Columbia, Maryland, U. S. A..
  • the Cyto Pulse PA-4000 Pulser (or the PulseAgile (TM) ) provides control of all protocol parameters with the ability to set pulse width, amplitude, time between pulses and the electric field direction. This tool allows the design and implementation of optimal electroporation protocols.
  • the instructions, tutorials and protocols for the PA-4000 Pulser (as well as other electroporation equipment) are available from Cyto Pulse Sciences Inc..
  • the electroporation process can be broken down into the following parts: (1) pore formation;; (2) mass transport of material into the cell; and (3) cell viability factors.
  • Electroosmosis is a force which occurs as a result of charge differences between the cell membrane within the pore and water molecules adjacent to the charged membrane. While the membrane is negatively charged, the layer of water immediately adjacent to the cell membrane is positively charged. This results in movement of water within the pore toward the negative electrode. Movement of water into the cell on one end and out or the cell on the other end pulls dissolved molecules in the direction of water transport.
  • Electrophoresis drives the movement of negatively charged molecules such as DNA move toward the positive electrode. (Opposite to the direction of electroosmosis) . This force is linearly proportional to the voltage and time of voltage application. This means that the best transport by electrophoresis occurs in high voltage fields that are applied continuously. There are important factors such as heat production that limit the voltage and the duration of voltage application that can be applied to cell suspensions. Generally, the most practical and effective mass movement derived from electrophoresis is obtained when lower voltages are applied in multiple, medium length pulses.
  • Heat production within the solution is exponentially proportional to electrical current within the solution.
  • the cooling is not rapid enough to compensate for the rapid rise in temperature related to excessive electrical current.
  • One method to compensate for heat production due to electrical current is to reduce the applied voltage and deliver wider pulses.
  • the gain in heat reduction is exponentially related to voltage reduction while the loss of movement related to electrophoresis force is only linear. For example, a reduction of the voltage by half coupled with a simultaneous doubling of pulse width results in the same mass movement, while halving the heat produced.
  • multiple, wide, low voltage pulses are used to induce mass transport by electrophoresis .
  • Pulse voltages much beyond breakdown threshold result in formation of pores too large to spontaneously repair.
  • cells lyse or die from loss of cytoplasmic content.
  • the problem of excess cell death due to excess voltage is readily solved by reducing the voltage.
  • the diameter of individual cells does vary and there is a distribution of cell sizes. Because of this, some cell death is inevitable.
  • the larger cells will be _killed as optimal voltage for average cells is applied.
  • maximum poration has been observed using pulse parameters where about half of the cells are killed; this is because traditional protocols use initial pore forming conditions for pore maintenance or mass transport effects. Other cell specific factors add to variability in electroporation efficiency.
  • Cell cytoskeletal structure is an example. Increased density of cell cytoskeletin at the site of pore formation can make the cell more resistant to detrimental effects of excessive pore expansion. Roughness of the cell due to cell processes or villi are another example.
  • the temperatures of the cell membrane influences pore maintenance.
  • Cell membrane pores remain open for seconds to minutes at room temperature. Higher temperatures accelerate pore closure.
  • cell membranes are viscous and inflexible and pore closure is slower. Pore induction or formation is similarly affected by temperature variations. It is more difficult to induce pores in cold cell membranes.
  • cells can be electroporated at 27-37 degrees C and brought rapidly to 4 degrees C. These methods of prolonging pore life are rarely practical or for that matter very useful . Electroporation efficiency is much higher if the molecules to be introduced into cells (DNA, proteins, small molecules) are in the cell solution before application of pulses rather than after. Even though electropores are theoretically open for seconds to minutes, close association to cells at the time of electroporation is essential; this may be due to mass transport effects that occur during electroporation.
  • a method for transfecting biological cells with biological material includes a first step of preparing a mixture which includes the biological cells, the biological material, and a quantity of a biological material binding reagent which is present in a synergistic reagent amount which is less than a conventional reagent amount if an accompanying electric field having synergistic electric field values were not imposed, and a second step of imposing an electric field on the mixture wherein the
  • the electric field has parameters which have synergistic electric field values which are less than conventional electric field values if the accompanying synergistic reagent amount were not present in the mixture.
  • the biological cells are transfected with the biological material at a greater than conventional transfection amount, and the biological cells undergo a less than conventional, low frequency of cell death.
  • Tables I and II sets forth examples which demonstrates synergism with respect to transfection efficiency by synergistically combining the use of binding reagents with pulsed electric fields to treat biological cells with biological materials.
  • Table II sets forth examples which demonstrates synergism with respect to cell death by synergistically combining the use of binding reagents with pulsed electric fields to treat biological cells with biological materials.
  • Tables I and II also set forth examples which demonstrates conventional transfection efficiency and conventional cell death using conventional amounts of binding reagents and conventional imposition of electric fields for electroporation.
  • the first vertical column shows that binding reagents are used, without imposing an electric field for electroporation.
  • binding reagents are used in a conventional optimum amount, without imposing an electric field for electroporation, a conventional high amount of biological cells are transfected with the biological materials.
  • the first vertical column shows that when binding reagents are used, without imposing an electric field for electroporation.
  • the top horizontal row shows when an electric field is imposed for electroporation, without employing any binding reagents .
  • the top horizontal row shows that an electric field is imposed for electroporation, without employing any binding reagents.
  • an electric field is imposed for electroporation, without employing any binding reagents.
  • Tables I and II Further novel, surprising, unexpected, and inventive synergistic findings of the present invention are presented in Tables I and II. More specifically, as shown Table I in the entry in the second vertical column and the third horizontal row, indicated by the bracketed [EH] , when a conventionally optimum amount of binding reagents is employed in combination with a conventionally low amount of electric field imposition, a synergistically extra high level of cell transfection efficiency occurs. Similarly, as shown in Table II in the entry in the second vertical column and the third horizontal row, indicated by the bracketed [VL] , when a conventionally optimum amount of binding reagents is employed in combination with a conventionally low amount of electric field imposition, a synergistically very low level of cell death occurs.
  • Tables I and II Still further novel, surprising, unexpected, and inventive synergistic findings of the present invention are presented in Tables I and II. More specifically, as shown Table I in the entry in the third vertical column and the second horizontal row, indicated by the bracketed [EH] , when a conventionally low amount of binding reagents is employed in combination with a conventionally optimum amount of electric field imposition, a synergistically extra high level of cell transfection efficiency occurs. Similarly, as shown in Table II in the entry in the third vertical column and the second horizontal row, indicated by the bracketed [VL] , when a conventionally low amount of binding reagents is employed in combination with a conventionally optimum amount of electric field imposition, a synergistically very low level of cell death occurs .

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Abstract

L'invention concerne des procédés pharmaceutiques définis simples, non toxiques, destinés à modifier génétiquement des cellules et des tissus permettant le développement d'une variété de médecines moléculaires. Cette invention concerne un procédé servant à améliorer l efficacité du transfert de matériel biologique dans des cellules et à abaisser la fréquence de mort cellulaire, in vitro, in vivo, et ex vivo. Précisément, cette invention concerne l'utilisation synergique de réactifs de liaison de bioploymères, des réactifs de liaison de polynucléotides, par exemple, combinée avec des techniques d'électroporation, servant à augmenter l'efficacité du transfert de polynucléotides vers les cellules étudiées et à abaisser la fréquence de mort cellulaire. Les thérapies génétiques à base d'électroporation sont particulièrement utiles dans le transfert de matériel génétique dans des cellules et présente un champ d'application large pour une large gamme d'applications de thérapie génique et de vaccination polynucléotidique. Les procédés améliorés de la présente invention sont utiles avec les systèmes d'administration ex vivo et in vivo. De plus, cette invention ne se limite pas au transport de polynucléotides, mais plutôt, elle est applicable au transport d'autres biopolymères, tels que des protéines et des antigènes à travers les membranes cellulaires.
PCT/US2000/021982 1999-09-24 2000-09-25 Procede servant a promouvoir l'administration assistee par champ electrique de materiaux biologiques dans des cellules WO2001023537A1 (fr)

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EP00964909A EP1159414A4 (fr) 1999-09-24 2000-09-25 Procede servant a promouvoir l'administration assistee par champ electrique de materiaux biologiques dans des cellules

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Cited By (6)

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Publication number Priority date Publication date Assignee Title
AU2002360648B2 (en) * 2001-12-14 2009-01-08 Genetronics, Inc. Methods for particle-assisted polynucleotide immunization using a pulsed electric field
WO2009105833A1 (fr) 2008-02-28 2009-09-03 Murdoch University Nouvelles séquence de brachyspira, compositions immunogènes, procédé d'élaboration et utilisation correspondante
WO2009117773A1 (fr) 2008-03-27 2009-10-01 Murdoch University Nouvelles séquences de brachyspira, compositions immunogènes, leurs procédés de fabrication et d'utilisation
EP2224008A2 (fr) 2005-05-12 2010-09-01 Murdoch University Gènes et protéines de brachyspira hyodysenteriae et leur utilisation en diagnostic et thérapie
EP2626370A1 (fr) 2007-10-17 2013-08-14 The University Court of the University of Edinburgh Compositions immunogènes contenant des flagelles H7 d'Escherichia coli et leurs procédés d'utilisation
EP3018139A2 (fr) 2007-08-03 2016-05-11 Boehringer Ingelheim Vetmedica GmbH Gènes et protéines de brachyspira hyodysenteriae et leurs utilisations

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2002360648B2 (en) * 2001-12-14 2009-01-08 Genetronics, Inc. Methods for particle-assisted polynucleotide immunization using a pulsed electric field
EP2224008A2 (fr) 2005-05-12 2010-09-01 Murdoch University Gènes et protéines de brachyspira hyodysenteriae et leur utilisation en diagnostic et thérapie
EP3018139A2 (fr) 2007-08-03 2016-05-11 Boehringer Ingelheim Vetmedica GmbH Gènes et protéines de brachyspira hyodysenteriae et leurs utilisations
EP2626370A1 (fr) 2007-10-17 2013-08-14 The University Court of the University of Edinburgh Compositions immunogènes contenant des flagelles H7 d'Escherichia coli et leurs procédés d'utilisation
WO2009105833A1 (fr) 2008-02-28 2009-09-03 Murdoch University Nouvelles séquence de brachyspira, compositions immunogènes, procédé d'élaboration et utilisation correspondante
WO2009117773A1 (fr) 2008-03-27 2009-10-01 Murdoch University Nouvelles séquences de brachyspira, compositions immunogènes, leurs procédés de fabrication et d'utilisation
EP2530087A2 (fr) 2008-03-27 2012-12-05 Prionics AG séquences de Brachyspira, compositions immunogènes, procédés pour leur préparation et utilisation

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