WO1989010690A1 - Methodes d'electrofusion de particules biologiques sur des tissus - Google Patents

Methodes d'electrofusion de particules biologiques sur des tissus Download PDF

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Publication number
WO1989010690A1
WO1989010690A1 PCT/US1989/001774 US8901774W WO8910690A1 WO 1989010690 A1 WO1989010690 A1 WO 1989010690A1 US 8901774 W US8901774 W US 8901774W WO 8910690 A1 WO8910690 A1 WO 8910690A1
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cells
tissue
animal
biological particles
plant
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PCT/US1989/001774
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English (en)
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Robert J. Grasso
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University Of South Florida
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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
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas

Definitions

  • This invention relates to methods for electrofusing biological particles to cells of animal or plant tissues in vitro, in situ, or Ln vivo. Selected biological particles are placed in contact with cells of a selected tissue and fused to cells in the tissue by means of an electric field.
  • the methods of this invention may be used to produce animals and plants characterized by features 'and properties that differ from those typically displayed by the native species.
  • One embodiment of the methods of this invention has been used to produce an animal mode for ocular gonorrhea by electrofusing human cells with functional receptors for the human bacterial pathogen Neisseria gonorrhoeae to epithelial cells in the histologically intact superficial corneal tissue of living rabbits.
  • the methods of this invention may be used to produce other animal or plant models for the study of receptor-mediated processes, including infectivity studies required for the development of vaccines effective to protect against or lessen the severity of infections caused by microbial pathogens. This is achieved by electrofusing cells with membrane receptors for a pathogen to cells of tissues of plants or animals that are normally not susceptible to infection by that pathogen. Other applications are disclosed. Background Art
  • electrofusion techniques have also been employed to induce fusion of cells and other biological particles in vitro.
  • These electrofusion methods generally include the following steps. Cells to be fused are placed in suspension in a non-electrolyte medium. The cell suspension is placed in a specially-constructed fusion chamber that includes electrodes connected to a source of electric power. The cell suspension is subjected to a non-uniform alternating current field created by applying alternating current to the electrodes in the fusion chamber. The non-uniform electric field induces the cells in suspension to act as dipoles.
  • the "dipole-cells" align themselves in the inhomogeneous electric field and move toward regions of higher field strength. As the cells align and migrate toward regions of higher field strength, they are attracted to each other and form strings of contiguous cells that are usually described as "pearl chains.” After the cells have aligned and formed pearl chains, one or more short pulses of direct current are applied to the electrodes to cause electrical breakdown of the cell membranes of contiguous cells. After direct current fusion pulses have been applied, the non-uniform alternating current field is restored to keep the cells aligned and in contact while the cell membranes of contiguous cells coalesce and fuse to form single hybrid cells.
  • Joule heating may be reduced by using nonionic media with low conductivity, many cells are unable to tolerate such media for any useful length of time.
  • the alternating current field also induces the cells in suspension to rotate or spin. Cell rotation interferes with membrane contact in the "pearl chains” and may adversely affect the fusion process.
  • Another limitation of the method arises because the dielectrophoretic force required to align cells and form "pearl chains" depends on the size of the particle. Smaller particles require stronger fields to align the particles and cause formation of pearl chains.
  • Such high alternating current voltages are required for particles with a radius smaller than 0.5 li ⁇ that the process becomes impractical, particularly for the fusion of small liposomes.
  • Electrofusion of 3T3 cells (Teissie, J., et al., "Electric Pulse-Induced Fusion of 3T3 Cells in Monolayer Culture,” Science, 216, pp. 537-38 (1982)) and electrofusion of CHO cells (Blangero, C. and Teissie, J. , “Homokaryon Production by Electrofusion: A Convenient Way to Produce a Large Number of Viable Mammalian Fused Cells," Biochem. Biophys. Res. Comm. , 114(2), pp. 663-69 (1983); Orgambide, G. et al., “Electrofusion of Chinese Hamster Ovary Cells After Ethanol Incubation," Biochim. Biophys.
  • the cell membrane contact required for electrofusion has also been obtained with two different centrifugation techniques.
  • fusion was achieved by applying fusion pulses to a cell pellet obtained by centrifugation.
  • Zimmermann, U. "Electric Field-Mediated Fusion and Related Electrical Phenomena,” Biochim. Biophys. Acta, 694, pp. 227-77 (1982).
  • low density cell suspensions were subjected to fusion pulses before centrifugation. Fusion of the cells occurred in the pellet formed by centrifuging cells that had already been rendered "fusogenic" by exposure to the fusion pulses.
  • a filter or membrane technique for obtaining the cell juxtaposition and membrane contact required for electrofusion has also been outlined in the literature.
  • the cells to be fused are deposited in the pores of filters or artificial membranes by suction or by binding the cells to the surface of artificial membranes by electrostatic attraction.
  • the filters or membranes are placed between parallel electrodes and the distance between the filters or membranes is reduced until contact is made between the cells on the opposing filters or membranes.
  • Zimmermann, U. "Electrical Breakdown, Electropermeabilization and Electro ⁇ fusion,” Rev. Physiol. Biochem. Pharmacol., 150, 175-256 (1986).
  • This approach is limited to cell-to-cell electrofusion i vitro.
  • electrofusion methods described in the literature including the dielectrophoresis approach, are directed to the fusion of discrete biological particles to other discrete biological particles _in vitro. All of these methods are characterized by significant limitations and disadvantages. A need therefore exists for methods that avoid these limitations and disadvantages and permit iri vitro, in situ or in vivo electrofusion of biological particles to cells of animal or plant tissues.
  • Gonorrhea is a host-specific affliction caused by the human bacterial pathogen Neisseria gonorrhoeae ( "N. 12
  • gonorrhoeae Pathogenesis of the disease is initiated when N. gonorrhoeae binds to specific gonococcal attachment receptors found exclusively on the surface of membranes of human cells. Many attempts have been made to infect animals with this human pathogen since Neisser first described the organism in 1879. None of those attempts, however, have produced a practical, biologically-relevant animal model that produces reproducible gonococcal lesions.
  • Kita recently reported that mice treated with estradiol are susceptible to disseminated gonococcal infection produced by intraperitoneal injection of N. gonorrhoeae 57-120, apparently because the steroid hormone impairs PMN bacteriocidal activity. Kita, E., et al.. Infect. Immun., 49, pp. 238-243 (1985). Kita has also reported the vaginal infection of ddY mice with N. gonorrhoeae PH2. Kita, E., et al., J. Infect. Pis. , 143, pp. 67-70 (1981); Kita, E. and Kashiba, S., Br. J. Vener. Pis., 60, pp. 219-25 (1984).
  • the present invention relates to novel methods for electrofusing biological particles to cells of animal or plant tissues _in vitro, in situ or iri vivo. Selected biological particles are placed in contact with cells of a selected tissue. The biological particles are fused to cells of the tissue by means of an electric field.
  • animals and plants characterized by features other than those typical of the native species may be produced.
  • the methods of this invention may be used to produce animal or plant models for the study of receptor-mediated 15
  • One embodiment has been used to produce an animal model for the study of ocular gonorrhea by electrofusing human cells, with receptors for the human bacterial pathogen Neisseria gonorrhoeae, to epithelial cells in the histologically intact superficial corneal tissue of living rabbits.
  • Gonococcal adherence assays, specificity studies based on scanning electron microscopy techniques and production of purulent ocular gonorrhea m vivo have demonstrated that functional human gonococcal attachment receptors were electrofused to cells of the corneal epithelium of the rabbits.
  • the methods of this invention may be used for other applications where it is advantageous to fuse selected cells, liposomes or other biological particles to cells of plant or animal tissues in vitro, in situ or _in vivo.
  • the methods of this invention are safe, simple, practical and efficient, and avoid limitations and disadvantages that characterize coventional _ir ⁇ vitro cell-to-cell electrofusion techniques.
  • This invention relates to methods for electrofusing biological particles to cells of animal or plant tissues.
  • the methods of the invention may be used for in vitro electrofusion of biological particles to cells of tissues that have been excised from an animal or plant, in situ electrofusion of biological particles to cells of tissues in dead animals or plants, or _in vivo electrofusion of 16
  • the methods of this invention generally comprise the following steps: (1) biological particles and animal or plant tissue are selected and prepared for electrofusion; (2) the biological particles are placed in contact with cells of the selected tissue; and (3) the biological particles are fused to cells of the tissue by subjecting the biological particles and cells of the tissue to an electric field created by application of one or more pulses of electric current.
  • Biological particles that may be selected for electrofusion to cells in animal or plant tissue in accordance with the methods of this invention include animal cells, plant cells, microorganisms such as bacteria and yeast cells, liposomes, cell vesicles and cell organelles such as lysosomes, phagosomes, nuclei, mitochondria, Golgi bodies, chloroplasts and other vacuoles.
  • Some of these biological particles e.g., plant cells, bacteria and yeast cells, have cell walls that are known to interfere with electrofusion. Prior to electrofusion according to the methods of this invention, the membranes of such particles are exposed by stripping or removing the cell walls by conventional enzymatic techniques or other known methods. Zimmermann, U.
  • the methods of this invention may advantageously be used to fuse biological particles to any animal or plant tissue that may be exposed or excised so that biological particles may be placed in intimate contact with the membranes of the cells in the tissue.
  • Biological particles may be fused to histologically intact tissues or to tissues that have been treated, manipulated or modified prior to electrofusion. Cell walls of cells in plant tissues are stripped or removed using conventional techniques prior to electrofusion in accordance with this invention.
  • the selected tissue is, of course, excised from a donor plant or animal using known procedures. If the tissue is not used immediately after collection, appropriate storage media are employed to preserve and maintain the integrity of the excised tissue.
  • Tissues used in accordance with _in vitro, in situ or m vivo applications of the methods of this invention are preferably rinsed free of debris with a physiologically compatible buffered isotonic solution such as phosphate-buffered saline (“PBS”) solution, before selected 18
  • PBS phosphate-buffered saline
  • biological particles are placed in contact with and electrofused to cells of the selected tissue.
  • the pH of the solution is adjusted in accordance with known techniques so that it is compatible with the tissue selected for electrofusion.
  • the contact required for electrofusion is preferably obtained by applying a mechanical force sufficient to cause intimate contact between selected biological particles and cells of a selected tissue.
  • the contact required for electrofusion is achieved by depositing the particles by centrifugation onto a support such as a filter (e.g., Millipore HAWP filters (Millipore; New Bedford, MA)) or an electrostatically charged disc (e.g., Zetaprobe blotting membrane disc (Bio-Rad; Richmond, CA) ) in an evenly-distributed layer.
  • a filter e.g., Millipore HAWP filters (Millipore; New Bedford, MA)
  • an electrostatically charged disc e.g., Zetaprobe blotting membrane disc (Bio-Rad; Richmond, CA)
  • the particle-laden support is placed cell-side down on the surface of a selected tissue.
  • a "fusion electrode” connected to a direct current pulse generator is positioned on the upper surface of the support.
  • Another electrode (“ground electrode”) connected to the direct current pulse generator is grounded to the tissue or plant or animal containing the tissue.
  • the fusion electrode is pressed down on the support with a force sufficient to create contact between the particles on the support and cells in the tissue. Particles are fused to cells of the tissue by applying direct current pulses to the fusion electrode. After removing the .fusion electrode, ground electrode and support, the tissue is washed with a buffered isotonic 20
  • Optimal conditions for electrofusion using mechanical force to create contact between the particles and cells of the selected tissue in any particular case may depend on the nature of the selected particles and tissue, the preparation and deposition of the particles on the support, the configuration of the fusion electrode and perhaps other factors. Such optimization may be achieved using conventional methods and means that are known to those of skill in the art.
  • human HL60 or U937 lymphoma cells deposited by centrifugation in an even layer on filters or electrostatically-charged discs may be electrofused to the histologically intact corneal epithelium of a rabbit in vitro, in situ or _in vivo, by applying with an eye-shaped electrode a mechanical force on the surface of the filter or disc sufficient to create a pressure of about 600 to 800 g/cm . Fusion is preferably accomplished under constant voltage conditions by applying to the electrode 3 square-wave 20 microsecond pulses of direct current with an amplitude of 20 volts at a pulse rate of 1 pulse per second.
  • the current which may be calculated using Ohm's law, depends on the electrical resistance of the tissue, which is about 3000 ohms for in vivo applications.
  • biological particles may be placed in contact with cells of a selected tissue and electrofused in 21
  • electrofusion may be carried out with direct current fields created by applying one or more pulses of direct current to the fusion electrode using a range of voltages and currents sufficient to cause fusion, but not so high that the biological particles or tissues are damaged.
  • the number, duration and pulse rate of the direct current pulses may also be varied.
  • a fusion electrode adapted to conform to the surface of the selected tissue.
  • a fusion electrode with a surface that matches the curvature of a rabbit eye may be used when fusing human cells to the corneal epithelia of anesthetized rabbits. 22
  • electrofusion may be carried out by exposing selected biological particles and cells of the selected tissue to an electric field sufficient to render the particles and cells of the tissue "fusogenic" before placing the particles in contact with cells of the tissue.
  • the contact required for electrofusion in accordance with the methods of this invention may be obtained by means other than the application of a mechanical force.
  • Contact between selected particles and cells of a selected tissue may be obtained, for example, by binding to the particles antibodies exhibiting specificity to cells of the selected tissue. Particles coated with such antibodies are strongly bound to cells of the selected tissue when the antigen-combining sites of the antibody bind with cells in the tissue.
  • Human HL60 cells for example, possess antibody receptor sites (Fc receptors) that bind IgG in a manner that leaves the antigen-combining sites of the immunoglobulins pointed away from the surface of the cell. The immunoglobulins on the surface of the HL60 cells remain free to bind with specific antigens.
  • selected tissue may be achieved by coating the HL60 cells with immunoglobulins that exhibit specificity to cells of the selected tissue.
  • immunoglobulins that exhibit specificity to cells of the selected tissue.
  • Particles coated with antibodies may be introduced to a selected tissue in various ways.
  • Cells coated with IgG for example, may be injected into an animal by intravenous injection. After the immunoglobulins on the coated cells bind to cells in the target tissue, the coated cells may be fused to cells of the tissue by applying a pulsed electric field.
  • Cells coated with antibodies may also be placed in contact with a selected tissue by depositing the coated cells on filters or membranes ' by centrifugation or suction, placing the filter or membrane cell-side down on the tissue and applying mechanical force on the upper surface of the filter or membrane. Very specific fusion products may be efficiently obtained in this way.
  • Photoreactive bifunctional cross-linking agents such as N-Hydroxysuccini- idyl 4-azidobenzoate, for example, may be incubated in the dark with selected cells or liposomes to covalently link one end of the linear cross-linking agent to proteins on the cells or liposomes.
  • cross-linking agent are then introduced to cells of the selected tissue and exposed to high intensity light so that the photoreactive end of the cross-linking agent binds to proteins on cells of the selected tissue by photolysis, firmly binding the cells or liposomes to cells in the selected tissue.
  • fusion is accomplished by applying an electric field.
  • the biological particle-tissue contact required for electrofusion in accordance with this invention may also be obtained by centrifugation.
  • Biological particles in suspension may be deposited on a selected tissue and placed in contact with cells of the tissue by centrifugation. Fusion may be accomplished by exposing the particles and cells of the tissue to an electric field to render them fusogenic before the particles are deposited on the tissue by centrifugation, or by subjecting the particles and cells of the tissue to an electric field after the particles are placed in contact with cells in the tissue by centrifugation. 25
  • Magnetically-coated particles are placed in contact with cells of a selected tissue by exposing the coated particles to a magnetic field with a gradient oriented in a direction that causes the particles to move toward the surface of the tissue.
  • the force of the field is sufficient to create close contact between the particles and cells in the tissue.
  • the biological particle-tissue contact required for electrofusion in accordance with the methods of this invention may also be obtained by using . a combination of means.
  • Particle-tissue contact may be achieved, for example, by combining bifunctional cross-linking agents, centrifugation and application of a mechanical force to achieve intimate contact between selected biological particles and cells of a selected tissue.
  • Biological particles selected for electrofusion in accordance with the methods of this invention may be modified or treated according to known procedures before being placed in contact with cells of a selected tissue.
  • Cells selected for electrofusion for example, may be infected with microorganisms that bind to the surface of the cell membrane or with organisms that are taken up by the cell and survive as intracellular parasites. Certain viruses, bacteria. 26
  • protozoa and mycotic agents may all be used as pathogens.
  • Cells may be treated with drugs or biological response modifiers such as lymphokines or monokines, or transfected with isolated genes or other PNA before being electrofused to cells of a selected tissue.
  • specific receptor molecules may be covalently bound to the surface of selected cells prior to electrofusion.
  • Bifunctional double cross-linking agents such as N-hydroxysuccinimidyl 4-azidobenzoate, for example, may be used to covalently bind microbial or toxin attachment components to cells before the cells are placed in contact with cells of a selected tissue for electrofusion.
  • Selected cells may also be fused with the same or other kinds of cells before being electrofused to cells in a selected tissue.
  • liposomes may be employed as carriers for drugs, covalently-linked receptor molecules, immunoregulatory agents, cloned genes and other agents.
  • Liposomes carrying anti-tumor drugs for example, may be directly electrofused to neo-plastic tissue. Liposomes, which are generally non- immunogenic, may be used to avoid rejection problems that arise as a result of the functional immune response of an animal host.
  • drugs, gene ' s and other molecules may be bound to or incorporated in a host animal's own leukocytes or erythrocytes before the leukocytes or erythrocytes are electrofused to cells of a selected tissue 27
  • the autograft created by electrofusion will not be rejected by the immune system of the host animal.
  • One embodiment of the methods of this invention has been used to produce a novel animal model for the study of ocular gonorrhea.
  • Functional gonococcal attachment receptors from the surface membranes of human U937 and HL60 cells were transferred to epithelial cells in the histologically intact superficial corneal tissue of living rabbits by electrofusion.
  • Acute purulent gonococcal keratoconjuncti- vitis was observed in the eyes of the rabbits after infecting the modified rabbit corneal tissue with the human bacterial pathogen Neisseria gonorrhoeae.
  • the example below is set forth in order that this invention may be more fully understood. This example is for the purpose of illustration only and it is not to be construed as limiting the scope of this invention in any way.
  • Nonadherent human HL60 promyelocytic leukemia cells (ATCC CCL 240; American Type Culture Collection, Rockville, MD) and nonadherent murine WEHI-3 myelocytic leukemia cells (ATCC TIB 68; American Type Culture Collection, Rockville, MD) were prepared for electrofusion by growing the cells in suspension cultures at 37°C in Costar plastic tissue culture flasks containing DMEM (GIBCO; Grand Island, NY) supplemented with 28
  • Adherent rabbit skin cells obtained from Dr. G.J. Lanca, Pepartment of Medical Microbiology and Immunology at the University of South Florida College of Medicine, Tampa, Florida, were prepared for electrofusion by growing cells in monolayer cultures at 37°C in plastic tissue culture flasks containing Eagle's MEM (GIBCO; Grand Island, NY) supplemented with 5% newborn bovine calf serum (Flow Laboratories; McLean, VA) .
  • the adherent rabbit skin cells were detached from the plastic flasks with 0.25% trypsin (GIBCO; Grand Island, NY). Prior to electrofusion, suspensions of HL60, WEHI-3 and rabbit skin cells were washed twice by centrifugation (200xg, 4°C, 10 min) in phosphate-buffered saline solution (pH 7.4; 150 mM NaCl; 3.4 mM KC1; 10.1 mM a 2 HP0 4 ; and l.g mM KH 2 P0 4 ) . The final washed sedimented pellets of HL60, WEHI-3 and rabbit skin cells were resuspended in the same phosphate-buffered saline solution at cell densities of about 10 8 to 109 per ml.
  • ketamine-HCl/kg body weight After the rabbits were tranquilized, anesthesia was induced by administering medical/dental grade N-,0 (Puritan Bennett; Gonzales, FL) through a nose cone until no ocular muscle responses occurred when the eyes were flushed with PBS. Effective anesthesia was maintained throughout the electrofusion procedure by repeated interrupted inhalation of N 2 o for 5 seconds administered intermittently at 10 second intervals. The anesthetized rabbits were allowed to breathe room air. normally during the intervening 5 second periods. The rabbits recovered from anesthesia within approximately 1 minute after their last exposure to N 2 o.
  • HL60 cells, murine WEHI-3 cells and rabbit skin cells were electrofused to the intact superficial corneal epithelial tissue of the anesthetized rabbits as summarized below in Table 1. Electrofusion was carried out by separately collecting washed HL60 cells, murine WEHI-3 cells and rabbit skin cells by centrifugation (200xg, 4°C, 10 in) on HAWP 8mm diameter Millipore filters (Millipore; New Bedford, MA) positioned at the bottom of centrifuge tubes. After discarding the supernatants, the filters were removed with forceps and placed cell-side down on the PBS-washed corneal surfaces of the anesthetized rabbits.
  • HAWP 8mm diameter Millipore filters Millipore; New Bedford, MA
  • a concave titanium electrode housed in an insulating handle and machined to reflect the radius of curvature of the rabbit corneal surfaces, was positioned on the upper side of the filters. Another electrode was attached to the buccal 30
  • the eye-shaped titanium fusion electrode positioned on the upper side of the filter was pressed manually with a force sufficient to create a pressure of about 600 to 800 g/cm ⁇ on the upper surface of the filter. Fusion was accomplished under constant voltage conditions by applying to the fusion electrode 3 square-wave 20 microsecond pulses of direct current with an amplitude of 20 volts at a pulse rate of 1 pulse per second. After fusion pulses had been applied, the fusion electrode, ground electrode and filters were removed from the rabbits. The corneal tissue of the rabbits was then thoroughly washed with PBS to remove any unfused cells.
  • N. gonorrhoeae Pgh 3-2 J.C. McMichael, ImmunoMed Corpora ⁇ tion; Tampa, FL
  • Pasteur pipette After 10 minutes the eyes were thoroughly washed free of any unbound bacteria with PBS dispensed from a squeeze bottle.
  • Infection was carried out with N_ ⁇ gonorrhoeae Pgh supplemented with 1% Isovitalex (DIFCO; Petroit, MI) in an atmosphere of 95% air plus 5% C0 2 .
  • Gonococcal cell suspensions (about 10 colony forming units per ml) were prepared by flooding plates containing fresh overnight stable type 2 colonies with 31
  • Pluses and minuses indicate whether treatments were (+) or were not (-) performed on the respective left and right corneas of the 4 rabbits.
  • Pluses and minuses indicate postinfection observations made in accordance with the following grading scheme for assessing the severity of ocular gonorrhea: no symptoms (-); trace (+/-); slight (+); mild (++); moderate (+++); and severe (++++). 32
  • Table 1 depicts the design of this example.
  • Rabbit #1 served as a control animal. Fusion pulses were applied to the left cornea of rabbit #1 in the absence of HL60, WEHI-3 or rabbit skin cells. Human HL60 cells were placed on the right cornea of rabbit #1, but no fusion pulses were applied. Human HL60 cells, mouse WEHI-3 cells and rabbit skin cells were respectively electrofused to both eyes of rabbits #2, #3, and #4. Unfused cells were removed by washing the eyes with sterile PBS. Both eyes of rabbit #1, but only the right eyes of rabbits #2, #3, and #4 were infected with N_ ⁇ gonorrhoeae Pgh 3-2. Gonococci that did not attach to the cornea were removed by rinsing the eyes with sterile PBS. Both eyes of all four of the rabbits appeared normal immediately after they had been infected with bacteria and rinsed free of unattached gonococci.
  • the foregoing example demonstrates that the methods of this invention may be used to produce a clinically-relevant animal model for ocular gonorrhea. It should be understood, however, that the methods of this invention may be used for many other applications.
  • the methods may be used, for example, to create other animal models for the study of the pathogenesis of infectious diseases important in human and veterinary medicine where infection is initiated by the binding of the etiological agent directly to host or tissue-specific receptors.
  • infectious diseases include, but are not limited to, acquired immune deficiency syndrome (AIDS) caused by HIV infections, swine rhinitis and sheep pneumonia caused by mycoplasmae infections, and other diseases caused by rhino viruses or corona viruses.
  • Animal models for such diseases may be created by transferring appropriate attachment receptors to cells of selected tissues of common laboratory animals by electrofusion _in vivo.
  • Liposomes carrying potent antitumor agents may be directly fused to cells in neoplastic tissue in accordance with this invention. Site-specific delivery of the antitumor agents achieves a high concentration of the drug where it is needed most and minimizes or avoids undesirable side effects that are normally encountered when the drug is administered systemically.
  • the methods of this invention may also be used in gene therapy to correct inherited genetic disorders. Genetic disorders caused by defective genes that result in metabolic deficiencies, for example, may be treated by electrofusing cells or liposomes containing functional genes that code for a particular enzyme directly to cells in the afflicted animal.
  • the methods of this invention may be used for any application where it is advantageous to fuse selected biological particles to cells of selected tissues of plants or animals.

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Abstract

Méthodes d'électrofusion de particules biologiques sur des cellules de tissus animaux ou végétaux in vitro, in situ ou in vivo. On met des particules biologiques sélectionnées en contact avec des cellules d'un tissu sélectionné et on les unit par fusion aux cellules dudit tissu au moyen d'un champ électrique. Lesdites méthodes peuvent servir à produire des animaux et des plantes présentant des caractéristiques et des propriétés différentes de celles qui sont inhérentes à leur espèce. Dans un des modes de réalisation desdites méthodes, on a produit un modèle animal pour la blennorrhée oculaire en unissant par fusion des cellules humaines comportant des récepteurs fonctionnels pour la bactérie pathogène humaine Neisseria Gonorrhoeae sur des cellules épithéliales de tissu cornéen superficiel histologiquement intact de lapins vivants. Lesdites méthodes peuvent servir à produire d'autres modèles animaux ou végétaux pour l'étude de processus faisant intervenir des récepteurs, y compris les études d'infectivité nécessaires pour mettre au point des vaccins efficaces de protection contre les infections causées par des microbes pathogènes ou pour limiter les effets de ces microbes. Pour cela, on unit par fusion des cellules comportant des récepteurs membraneux d'agents pathogènes à des cellules de tissus d'animaux ou de plantes qui ne sont pas normalement sujets aux infections causées par cet agent pathogène. L'invention porte aussi sur d'autres applications.
PCT/US1989/001774 1988-05-02 1989-04-27 Methodes d'electrofusion de particules biologiques sur des tissus WO1989010690A1 (fr)

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US5439440A (en) * 1993-04-01 1995-08-08 Genetronics, Inc. Electroporation system with voltage control feedback for clinical applications
US5501662A (en) * 1992-05-22 1996-03-26 Genetronics, Inc. Implantable electroporation method and apparatus for drug and gene delivery
US5702359A (en) * 1995-06-06 1997-12-30 Genetronics, Inc. Needle electrodes for mediated delivery of drugs and genes
US5993434A (en) * 1993-04-01 1999-11-30 Genetronics, Inc. Method of treatment using electroporation mediated delivery of drugs and genes
US6132419A (en) * 1992-05-22 2000-10-17 Genetronics, Inc. Electroporetic gene and drug therapy
WO2003070933A2 (fr) * 2002-02-22 2003-08-28 I.D.M. Immuno-Designed Molecules Procede d'electrofusion de cellules
US6697669B2 (en) * 1998-07-13 2004-02-24 Genetronics, Inc. Skin and muscle-targeted gene therapy by pulsed electrical field
US7570992B2 (en) 1998-07-13 2009-08-04 Genetronics, Inc. Electrical field therapy with reduced histopathological change in muscle
US7922709B2 (en) 1998-07-13 2011-04-12 Genetronics, Inc. Enhanced delivery of naked DNA to skin by non-invasive in vivo electroporation

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US5501662A (en) * 1992-05-22 1996-03-26 Genetronics, Inc. Implantable electroporation method and apparatus for drug and gene delivery
US6132419A (en) * 1992-05-22 2000-10-17 Genetronics, Inc. Electroporetic gene and drug therapy
US5439440A (en) * 1993-04-01 1995-08-08 Genetronics, Inc. Electroporation system with voltage control feedback for clinical applications
US5993434A (en) * 1993-04-01 1999-11-30 Genetronics, Inc. Method of treatment using electroporation mediated delivery of drugs and genes
US6763264B2 (en) * 1993-04-01 2004-07-13 Genetronics, Inc. Method of treatment using electroporation mediated delivery of drugs and genes
US5702359A (en) * 1995-06-06 1997-12-30 Genetronics, Inc. Needle electrodes for mediated delivery of drugs and genes
US6697669B2 (en) * 1998-07-13 2004-02-24 Genetronics, Inc. Skin and muscle-targeted gene therapy by pulsed electrical field
US7570992B2 (en) 1998-07-13 2009-08-04 Genetronics, Inc. Electrical field therapy with reduced histopathological change in muscle
US7922709B2 (en) 1998-07-13 2011-04-12 Genetronics, Inc. Enhanced delivery of naked DNA to skin by non-invasive in vivo electroporation
WO2003070933A2 (fr) * 2002-02-22 2003-08-28 I.D.M. Immuno-Designed Molecules Procede d'electrofusion de cellules
WO2003070933A3 (fr) * 2002-02-22 2004-01-22 Idm Immuno Designed Molecules Procede d'electrofusion de cellules

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