WO1989007944A1 - Procede et dispositif de neovascularisation a localisation controlee - Google Patents

Procede et dispositif de neovascularisation a localisation controlee Download PDF

Info

Publication number
WO1989007944A1
WO1989007944A1 PCT/US1989/000742 US8900742W WO8907944A1 WO 1989007944 A1 WO1989007944 A1 WO 1989007944A1 US 8900742 W US8900742 W US 8900742W WO 8907944 A1 WO8907944 A1 WO 8907944A1
Authority
WO
WIPO (PCT)
Prior art keywords
neovascularization
support
hbgf
collagen
biological response
Prior art date
Application number
PCT/US1989/000742
Other languages
English (en)
Inventor
John A. Thompson
W. French Anderson
Thomas Maciag
Original Assignee
American National Red Cross
The United States Of America, As Represented By Th
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by American National Red Cross, The United States Of America, As Represented By Th filed Critical American National Red Cross
Publication of WO1989007944A1 publication Critical patent/WO1989007944A1/fr
Priority to KR1019890701950A priority Critical patent/KR900700122A/ko
Priority to DK202790A priority patent/DK202790A/da

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
    • C01G23/00—Compounds of titanium
    • C01G23/04—Oxides; Hydroxides
    • C01G23/047—Titanium dioxide
    • C01G23/053—Producing by wet processes, e.g. hydrolysing titanium salts
    • 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
    • A61K38/18—Growth factors; Growth regulators
    • 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
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82B—NANOSTRUCTURES FORMED BY MANIPULATION OF INDIVIDUAL ATOMS, MOLECULES, OR LIMITED COLLECTIONS OF ATOMS OR MOLECULES AS DISCRETE UNITS; MANUFACTURE OR TREATMENT THEREOF
    • B82B3/00—Manufacture or treatment of nanostructures by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
    • C01G23/00—Compounds of titanium
    • C01G23/04—Oxides; Hydroxides
    • C01G23/047—Titanium dioxide
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A61L2300/254—Enzymes, proenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A61L2300/414—Growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/43—Hormones, e.g. dexamethasone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/64—Animal cells
    • 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
    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • the invention relates to a device and method for directing the formation of new blood vessels and artificial organs. Specifically, the invention relates to a device and method for directing neovascularization with a biological response modifier adsorbed onto a support.
  • Angiogenesis is the formation of blood vessels in situ and involves the orderly migration, proliferation, and differentiation of vascular cells and occurs during development. Angiogenesis is an infrequent event in the -2-
  • HBGF heparin-binding growth factor
  • the gene family for producing the heparin-binding growth factor family of polypeptides includes HBGF-1 (acidic fibroblast growth factor), HBGF-2 (basic fibroblast growth factor), and three additional HBGF-like structures, hst/KS, int-2, and FGF-5, each of which is encoded by an oncogene.
  • the prototype HBGF polypeptides are potent inducers of endothelial cell migration and/or proliferation in vitro and are known to modulate the expression of endothelial cell derived proteases. Further, HBGF-1 and HBGF-2 are tightly adsorbed to the extracellular matrix presumably by their avid affinity for the glycosaminoglycan heparin.
  • HBGF-I Class I heparin-binding growth factor
  • HBGF-II Class II heparin-binding growth factor
  • HBGF-I and HBGF-II share a structural similarity of 55 percent and both are synthesized as polypeptides lacking an apparent signal peptide sequence. Human cells which express the HBGF-I mRNA transcript do not secrete the polypeptide in vitro. Further, HBGF-II has been shown to be associated with the extracellular matrix and heparin protects HBGF-I from proteolytic modification by plasmin.
  • PCT International Publication Number WO 87/01728 discloses recombinant fibroblast growth factors. These growth factors are examples of biological response modifiers. This disclosure identifies the importance of the growth factors for constructing vascular systems in healing tissues. The invention of this disclosure is directed to recombinant DNA sequences for encoding bovine and human acidic and basic FGF and vectors bearing these DNA sequences. This publication does not disclose a device or method for site directed neovascularization.
  • This article discloses a sponge implant model for wound healing in animals.
  • the sponge consists of an inert polyvinyl alcohol that is implanted under the skin of the animal. Growth factor is then injected directly into the sponge. The wound undergoes rapid healing and an increase in blood vessels occurs at the wound site.
  • the blood vessels resulting from this invention do not form complete, permanent vascular structures that are directed by a support to which the growth factor is adsorbed.
  • This article does not disclose a device or method for site directed neovascularization.
  • U.S. Patent Number 4,699,141 to Lamberton, et al. discloses a container and method for neovascularization.
  • This invention has a sponge body that is wetted throughout with a solution of fibrinogen and heparin.
  • the sponge body is placed adjacent to or around a noncapillary blood vessel. Capillaries then grow into the sponge.
  • the sponge can then be used as a receptacle for desired cells such as pancreas cells.
  • This patent does not disclose a device or method wherein the growth of blood vessels is directed in a specific direction or between specific sites.
  • Neither the heparin nor collagen in this invention modify a biological response. Both the heparin and collagen are substrates upon which a biological response modifier acts.
  • the capillary growth developed by this invention is a result of the inflammatory response of the vessel to a foreign body or the sponge.
  • the blood vessels of this invention are not directed in their growth and do not form permanent structures or long term structures. These blood vessels are not permanent because the fibrinogen support is absorbed by the organism before maturation of the blood vessels can occur.
  • the blood vessels developed by the Lamberton, et al. invention are, essentially, a bundle of cells or capillaries within a sponge.
  • This invention is identified as being a receptacle for "desired cells.”
  • Such a receptacle is desirable for developing an "artificial organ”.
  • the development of the receptacle requires an undesirably long period of time of about 6 weeks.
  • the invention is an in vivo site directed neovascularization device.
  • the device includes a support.
  • the support can be an absorbable support, a non-absorbable support, or both.
  • the device also includes a biological response modifier for inducing neovascularization.
  • the biological response modifier is adsorbed to support.
  • the invention also includes a method for directing in vivo neovascularization.
  • the method requires adsorbing a biological response modifier for inducing neovascularization onto a support.
  • the step of contacting a therapeutically effective amount of said adsorbed biological response modifier to at least one selected tissue then occurs.
  • the method then involves directing or culturing neovascular cell growth at the contacted, selected tissue for a sufficient time to obtain a vascular structure.
  • the method of this invention is useful for providing artificial organs.
  • Objects of the present invention are to provide: (1) a new device for inducing site-directed neovascularization; (2) a method for in vivo formation of new blood vessel or a vascular bed; (3) mammalian cells collected about the implanted device of the present invention for multiplication, cloning, manipulation and implantation thereof; (4) a vascular bed for transplantation; and (5) other objects made evident from the following detailed description of the invention.
  • Figure 1 illustrates ECGF binding to collagen supports.
  • Figure 2 illustrates the effect of implanting ECGF immobilized on collagen sponges and the results thereof (arrows to sponges) are shown.
  • Figure 3 illustrates the H & E histological stain of sponges (IP in rat) are shown.
  • Figure 4 illustrates the site-directed gelfoam implant (Sg) with GF (growth factor) between liver (left, L) and spleen (right, Sp).
  • Figure 5 illustrates genetically engineered rat hepatocytes recovered from collagen sponges adsorbed with ECGF at 4 to 6 weeks of post-implantation.
  • Figure 6 illustrates a cross-section of a blood vessel developed according to this invention.
  • Figure 7 illustrates an angiogeneic response induced by HBGF-1 in situ four weeks after surgery.
  • Figure 8 illustrates the posterior portion of a fiber implant containing vascular strings that are generally connected to the mesentary tissue around the bowel loop.
  • Figure 9 illustrates multiple vascular connections between the fiber implant and mesenterial vessels and vascular turbosity within the implant.
  • Figure 10 illustrates an x-ray view of the multiple vascular connections of Figure 9.
  • Figure 11 illustrates a histological examination of a longitudinal section that reveals the presence of multiple vascular lumina surrounded by thick, collagenous and muscular walls of the neovessel structure.
  • Figure 12 illustrates the vascular bundle of Figure 6 at higher magnification which reveals the rich collagen component of the vascular structure and abundance of endothelial cell-lined capillary structures.
  • Figure 13 illustrates serum bilirubin levels of a Gunn rat implanted with hepatocytes seeded onto collagen (Type IV) and HBGF-1 coated PTFE fibers.
  • Figure 14A illustrates a Gortex shunt tube, containing a collagen I (Gelfoam) sponge, impregnated with HBGF-1, implanted onto the aorta of a rat for one month, then excised and cross-sectioned. -9-
  • Figures 14B, 14C and 14D illustrates a Gortex shunt tube containing a bundle of Gortex angel-hair fibers coated with Type I collagen and impregnated with HBGF-1.
  • the invention includes both a composition or "device” and a method for using that device.
  • the device is used in vivo to stimulate and direct neovascularization.
  • the neovascularization is accompanied by the growth of other cellular tissue including nerves.
  • the device requires a support.
  • the support must be capable of adsorbing a biological response modifier or adhering to a composition that can adsorb a biological response modifier.
  • the biological response modifier is a compound that stimulates and induces neovascularization.
  • the invention further includes a method for inducing neovascularization that can include the development of artificial organs and/or genetically engineered tissues.
  • a biological response modifier can be at least one compound or agent that stimulates or facilitates vascular cell growth from a tissue or organ.
  • a biological response modifier is a biochemical agent, such as a growth factor, hormone, or their chimeric derivative, that directly or indirectly induces a transcriptional or translational cellular event.
  • a biological response modifier directly or indirectly exerts an effect through a high affinity receptor. This effect produces vascular cell growth.
  • Compounds that exert a direct stimulation of a receptor include hormones.
  • a receptor include hormone prototypes or precursors and hydrolases.
  • Hydrolases such as a plasminogen activator, collagenase, or heparinase, initiate a biological response by enzymatically activating or releasing latent, stored, or zymogen precursors of direct biological response modifiers.
  • Biological response modifiers desirable angiogenic growth f ctors include a member of the group consisting of HBGF-I, HBGF-II, platelet-derived growth factor (PDGF), macrophage-derived growth factor (MDGF), epidermal growth factor (EGF), tumor angiogenesis factor (TAF), endothelial cell growth factor (ECGF), fibroblast growth factor (FGF), hypothalamus-derived growth factor (HDGF), retina-derived growth factor (RDGF), and mixtures thereof.
  • the preferred embodiment of the invention uses HBGF-I.
  • Desirable hydrolases include a member selected from the group consisting of heparinase, collagenase, plasmin, a plasminogen activator, thro bin, heparatinase, and mixtures thereof.
  • Hormones such as the growth factors are particularly desirable for use in this invention as biological response modifiers. Hormones specifically elicit cell growth and differentiation. The use of hormones as biological response modifiers cause neovascularization to rapidly occur and to form a complete vascular structure. The resulting blood vessel stimulated by hormones is more than just a mass of cells in that it has a tubular cavity and connective tissue between its cells. The resulting blood vessel produced -11-
  • hormones from the use of hormones is complete within itself and can be excised and transplanted into another portion of the body.
  • the other biological response modifiers produce similar results, but do not provide as rapid a growth as hormones and, in particular, the HBGF-I and HBGF-II hormones.
  • the invention includes a biocompatible support to which the biological response modifier is adsorbed.
  • the support can be either or both an absorbable or non-absorbable biocompatible matrix.
  • the support must be i plantable into an organism and is, desirably, rigid and strong enough to be transplantable after neovascularization has occurred.
  • the biocompatible support must have the rigidity and strength to support neovascularization.
  • absorbable supports include a member selected from the group consisting of collagen Type I, known commercially by the trade name "Gelf ⁇ am", laminins, fibronectins, gelatins, glycosaminoglycans, glycolipids, proteolipids, mucopolysaccharides, glycoproteins, polypeptides, and mixtures thereof.
  • non-absorbable matrices include members of the group consisting of nylon, rayon, dacron, polypropylene, polyethylene, expanded PTFE, cross-linked collagen Type IV, and mixtures thereof. It is desirable that a selected support contain extracellular matrix protein to provide or to facilitate the adsorption of the biological response modifier to the biocompatible support. -12-
  • An extracellular matrix protein can be the material from which the biocompatible support is formed or a component added to the biocompatible support to fully provide or, alternatively, facilitate the adsorption of the biological response modifier to the biocompatible support.
  • An extracellular matrix protein component can include a pure or mixed composition of proteins or polypeptides. The proteins and polypeptides can be either natural or synthetic.
  • the extracellular matrix protein component is desirably derived from extracellular structural molecules. These extracellular structural molecules include a member selected from the group consisting of collagens, laminins, fibronectins, gelatins, glycosaminoglycans, glycoproteins, proteoglycans, and mixtures thereof.
  • Expanded polytetrafluoroethylene has been found to be most suitable non-absorbable support for this invention. This support provides the following benefits.
  • PTFE has a general lack of an inflammatory response which is the basis for the current acceptance of PTFE in the surgical community.
  • PTFE can be coated conveniently with various components of the extra
  • HBGF-1 and HBGF-2 can be immobilized to collagen-coated PTFE by previously established methods.
  • PTFE polymers are routinely engineered to various specifications to meet a multitude of required configurations.
  • the configuration of the non-absorbable PFTE is a more critical aspect of the long-term implant model.
  • All multicellular organisms utilize a three-dimensional architecture of branching fiber networks to solve the problem of increasing surface area in a given volume. Seeding of such a network with HBGF polypeptides before implantation allows for high localized concentrations of the mitogen.
  • Non-woven multifilament angel-hair fibers of expanded PTFE are commercially available from W.L. Gore and Associates, Inc., Flagstaff, Arizona. These fibers allow sufficient organized surface area for infiltrating cells to be exposed to the environment of the host. This permits the free exchange of nutrients and toxic waste to occur while neovascularization processes occur.
  • cell shape as determined by cytoskeletal components and attachment to a specific matrix generally is regarded to play a significant role in both cell proliferation and differentiation.
  • a support can be provided for use in this invention in any desired shape and size.
  • a support as small as one lmm ⁇ is suitable to provide a base for neovascularization. Desirable shapes for a support can -14-
  • Supports are desirably capable of being secured within an organism. Suitable means for securing a support can include a staple, biocompatible glue, or other surgical procedures such as suturing or tying the support to a tissue.
  • a desirable support is obtained by filling a tube or sleeve of expanded PTFE with expanded PTFE fibers or "angel hair".
  • Supports formed from tubes or sleeves provide a pouch for an artificial organ.
  • the tubular form of the support and the bundle of fibers within the tube are particularly desirable for directing neovascularization.
  • Such embodiments can be receptacles for implanted cells when the invention is used to provide an artificial organ.
  • the most effective concentrations for a biological response modifier can be any concentration that elicits a growth response from the target cells, but is not toxic to those cells.
  • Effective or therapeutic concentrations of angiogenetic growth factors are between about 1 to about 10 nanograms per cubic millimeter of a support.
  • a support for this calculation includes both the absorbable support and the non-absorbable support.
  • a support is provided in an amount suitable to establish the length and width of the desired blood vessel. For example, if a blood vessel is desired between two tissues and there exists a distance between those two tissues, then a corresponding length of support is implanted into the organism to provide the approximate length and width of this desired blood vessel. The amount of the biological response modifier is then adapted to the amount of support required to form this basic structure.
  • the invention can be practiced without a non-absorbable support.
  • a complex with gelatin, HBGF-1, or HBGF-2 is capable of inducing neovascularization in vivo at polypeptide concentrations consistent with the demonstration of this biological activity in vivo.
  • This neovascular response is capable of sustaining induced site-specific neovessel formation for up to four weeks in the neck and peritoneal cavity of the rat.
  • the device of this invention without a support has limited utility for the induction of long-term neovessels. This is because the three-dimensional architecture of the collagen sponges is ultimately disrupted by a reabsorption process that occurs within three to four weeks after implantation.
  • Nonabsorbable solid polymeric supports of well-defined specifications and containing bonded components of extracellular matrices induced the expression of long-term stable neovessels in vivo An example of such an embodiment is a nonabsorbable support bonded with both collagens Type I and Type IV and having both HBGF-1 and HBGF-2 attached to the collagens. -16-
  • a neovascularization device can also be seeded with desired cells prior to or subsequent to implantation in a host.
  • desired cells are mammalian cells and express a protein capable of performing a particular function.
  • the cells can be genetically engineered cells capable of expressing a heterologous protein.
  • the cells can be naturally occurring cells capable of providing a desired function or functions such as hepatocytes.
  • Desirable embodiments of the invention have cells seeded in or on the neovascularization device which are genetically engineered to express at least one heterologous protein.
  • a protein is preferably a therapeutic agent.
  • the expressed protein may or may not be secreted from the genetically engineered cells.
  • the genetically engineered cells used with this invention are transformed with at least one gene that encodes for the desired heterologous protein.
  • the cells are transformed with a suitable vector or expression vehicle which includes the desired gene.
  • the vector can also include a promoter for expression in the host cells.
  • the promotor for expression can be SV 40, LTR, metallothionein, PGK, CMV, ADA, TK, or others.
  • the vector can also include a suitable signal sequence or sequences for secreting the therapeutic agent from the cells. The selection of a suitable promotor is deemed to be within the skill of the art. -17-
  • the vector or expression vehicle is preferably a viral vector and in particular a retroviral vector.
  • suitable viral vectors which can be modified to include a gene for a therapeutic agent, include Harvey Sarcoma virus, ROUS Sarcoma virus, MPSV, Moloney murine leukemia virus, DNA viruses such as adenovirus and others.
  • the expression vehicle can be a plasmid. Transformation can be accomplished by liposome fusion, calcium phosphate or dextran sulfate transfection, electroporation, lipofection, tungsten particles, or other procedures. The selection of a suitable vehicle for transformation is deemed to be within the scope of those skilled in the art.
  • a retroviral vector When a retroviral vector is employed as the expression vehicle for transforming cells, steps should be taken to eliminate and/or minimize the chances for replication of the virus.
  • Various procedures are known in the art for providing helper cells which produce viral vector particles that are essentially free of replicating virus. Examples of such procedures are found in Markowitz, et al., "A Safe Packaging Line for Gene Transfer; Separating Viral Genes on Two Different Plasmids", Journal of Virology 62(4) (April 1988):1120-1124; Watanabe, et al., "Construction of a Helper Cell Line for Avian Reticuloendotheliosis Virus Cloning Vectors", Molecular and Cellular Biology 3(12) (Dec.
  • This procedure and other procedures can be employed for genetically engineering cells by use of a retroviral vector.
  • other material can be included in the vector. This material can include a selection gene such as a neomycin resistance gene, a sequence for enhancing expression, or other materials.
  • Genetically engineered mammalian cells can be implanted in a mammal by use of a neovascularization device. These genetically engineered cells are desirably implanted into a mammal of the same species.
  • the genetically engineered mammalian cells are cells originally derived from a patient, genetically engineered to include a gene for at least one therapeutic agent, and implanted into the patient from which they were derived by use of a neovascularization device in accordance with the invention. These autologous genetically engineered cells then provide "gene therapy" by in vivo production of the therapeutic agent for treatment of the patient. -19-
  • the genetically engineered cells can be engineered such that the therapeutic agent is secreted from the cells in order to exert its effect upon cells and tissues either in the immediate vicinity or in more distal locations.
  • the therapeutic agent if it is not secreted from the engineered cells, exerts its effect within or on the engineered cells and can cause the metabolism of substances that diffuse into or onto the cells.
  • therapeutic agents include adenosine deaminase (ADA) that functions within the cell to inactivate adenosine, a toxic metabolite that accumulates in severe combined immunodeficiency syndrome, or phenylalanine hydroxylase that functions within a cell to inactivate phenylalanine, a toxic metabolite in phenylketonuria.
  • the genetically engineered cells used with this invention are transformed with a gene for at least one heterologous protein.
  • This protein is preferably a therapeutic agent.
  • therapeutic agent is used in its broadest sense and means any agent or material which has a desired or beneficial effect on the host.
  • the therapeutic agent can be more than one type of protein. Desirable proteins include CD-4, Factor VIII, Factor IX, von Willebrand Factor, TPA, urokinase, hirudin, the interferons, tumor necrosis factor, the interleukins, hemotopoietic growth factors including G-CSF, GM-CSF, IL3, erythropoietin, antibodies. 944
  • glucocerebrosidase ADA
  • phenylalanine hydroxylase human growth hormone
  • insulin insulin and others.
  • the selection of a suitable gene is deemed to be within the scope of those skilled in the art.
  • Mixtures of cell types can also be used with this invention such s genetically engineered smooth muscle cells, fibroblasts, glial cells, keratinocy es, or others.
  • the effect in genetically engineered cells when used in gene therapy can be controlled by the selection of high producing clonal populations and/or the use of vectors with enhanced expression. This can provide, in vivo, therapeutically effective amounts of a desired therapeutic agent for treating a patient.
  • factors such as the half life of the therapeutic agent, volume of the vascular system, production rate of the therapeutic agent by cells, and the desired dosage level are considered.
  • the selection of such vectors and cells is dependent on the therapeutic agent and is within the scope of those skilled in the art.
  • the neovascularization device of the invention can also be employed to obtain cells from a host by implanting the device in a host and after a period of time removing the implanted neovascularization device from the host for recovery of cells which have been collected on the device.
  • Such cells can be differentiated and used for a variety of purposes. For -21-
  • this procedure can provide a source of autologous cells for genetic engineering and subsequent return to the host as genetically engineered cells for expression of a protein.
  • Cells collected in this manner can be genetically engineered and then returned to the host to provide an artificial organ.
  • the process for directing neovascularization first involves preparing the device of this invention as described above.
  • the device is prepared by adsorbing a biological response modifier, that is suitable for inducing neovascularization, onto a support.
  • the biological response modifier must be present on the support in such a concentration as to be therapeutically effective for eliciting cell growth.
  • the adsorbed biological response modifier is then contacted to at least one selected tissue.
  • the device is connected to at least two separate sites between which a blood vessel is desired. These two sites can be the same or separate tissues or organs.
  • the method then involves culturing neovascular cell growth at or from the contacted tissue. Culturing of the contacted cells must occur for a sufficient time to allow or enable neovascularization and the vascular structure to form.
  • Figure 1 demonstrates that ECGF binds to collagen supports. This is shown by an elution profile of HBGF-1 (ECGF) from collagen type IV-Sepharose and gelatin-Sepharose columns. Collagen Type IV-Sepharose and The gelatin-Sepharose (1 ml) were packed in a column 7944
  • Elution of column-associated ( i25 l)-HBGF-I was achieved with 1.5M NaCl in absorption buffer or 50 units of heparin (Upjohn, Kalamazoo, MI) in absorbtion buffer.
  • the NaCl-eluted column was regenerated with an absorption buffer wash and the heparin-eluted column was regenerated by consecutive washes with 1.5M NaCl in absorption buffer followed by another wash with absorbtion buffer.
  • the matrix affinity procedures were performed at room temperature (about 22°C to 25°C).
  • FIG. 2 demonstrates that ECGF binds to collagen supports.
  • the adsorbed factor was implanted in various anatomical sites to demonstrate the practicality of using growth factor-adsorbed implants to stimulate neovessel formation and the growth of vascular beds in areas of interest.
  • the effect of implanting ECGF immobilized on collagen sponges and the results thereof (arrows to sponges) are shown: -23-
  • FIG. 3 demonstrates that the device of this invention induces significant angiogenesis in situ. These implants were removed at various times for examination by common methods of histology in order to determine the microscopic nature of these dynamics.
  • Sg represents "sponge (C-l)"
  • Sp represents “spleen”
  • L represents “liver”
  • BV represents "blood vessel (aorta)”.
  • H & E histological stain of sponges (IP in rat) are shown:
  • A. sponge two weeks, IP, without ECGF
  • B. sponge one week, IP, plus ECGF
  • C. sponge two weeks, IP, plus ECGF
  • FIG 4 demonstrates that ECGF induces significant and stable angiogenic response in situ by the recruitment of appropriate cell types as shown in Figures 2 and 3. Implants were established to create site-directed bridges between a large variety of organs, vessels, tissues and the like. Illustrated .are the site-directed Gelfoam implant (Sg) with growth factor (GF) between liver (left, L) and spleen (right, Sp).
  • Sg site-directed Gelfoam implant
  • GF growth factor
  • Figure 5 demonstrates that the device of this invention serves to create neovessels independent of the implantation site in situ.
  • the device has an ability to serve as a recruitment vehicle for mammalian cells in general and as a vehicle to maintain the viability and physiological environment for and of the implanted and transplanted cells.
  • Genetically engineered rat hepatocytes recovered from collagen sponges adsorbed with ECGF after 4 to 6 weeks post-implantation are shown. Hepatocytes were removed to determine their viability.
  • Figure 5A shows the results with no growth factor. Note that in Figure 5A few cells appear to be unhealthy and there is no proliferation or growth of survivor cells.
  • Figure 5B shows the results with growth factor. Note that in Figure 5B healthy viable cells are accompanied by significant proliferation.
  • the device and method of this invention can provide angiogenesis and neovascularization from one or more sites on a single tissue or different tissues.
  • the development of a blood vessel from a single site of one tissue, such as an artery, provides a vessel that can be transplanted or that can be used as an artificial organ.
  • the development of a blood vessel between two or more sites located on the same or different tissues provides improved circulation between the sites.
  • FIG. 6 illustrates a cross section of a blood vascular structure developed by the device and method of this invention.
  • the blood vessel 1 contains endothelial cells 2, mesothelial cells 3, pericytes 4, smooth muscle cells 5, fibroblasts 6, and neuronal-like cells 7.
  • the cross section of the blood vessel 1 demonstrates the formation of capillary-like structures 8, arteries 9, and vein-like structures 10. This development of a complete vascular structure provides a rigid vessel that remains permanently in the organism and that can be transplanted within this organism.
  • a method of this invention can be used to provide an artificial organ by first directing the growth and development of a blood vessel from a tissue.
  • the developed blood vessel is then injected or seeded with cells from a selected tissue or organ.
  • the injected cells can be genetically altered before being seeded into the blood vessel.
  • the seeded cells can provide a desired metabolic effect.
  • These metabolic effects can include hepatic functions such as bilirubin metabolism and pancreatic functions such as insulin production.
  • Other metabolic functions can be provided by cells containing one or more hormone producing genes.
  • Artificial organs developed according to this invention can provide desired functions without being subject to a response from the organism's immune system.
  • Example 1 demonstrates various embodiments of the device or composition of the invention and the method by which the device is produced. This example uses HBGF-I with a radioactive iodine marker. In therapeutic use, the radioactive marker would not be present.
  • Example 1 is as follows.
  • the (l 2 5i)-HBGF-l adsorbed to immobilized gelatin and collagen Type IV can also be eluted with heparin as shown in Figures 1A and E. Approximately 20% of the growth factor, which remains bound after heparin elution, can be eluted with 1.5M NaCl.
  • Pretreatment of the gelatin and collagen Type IV matrix with 50 units of heparin significantly reduces the ability of either matrix to absorb (1 2 ⁇ I)-HBGF-1 as shown in Figures IB and F. Regeneration of either matrix with a 1.5M NaCl wash permits ( i25 I)-HBGF-l adsorption.
  • Example 2 demonstrates the method for implantation of the device of this invention and for eliciting neovascularization.
  • the use of immobilized gelatin with HBGF-I represents the preferred embodiment of the invented method.
  • Example 2 is as follows.
  • Example 2 demonstrates that HBGF-I binds to both immobilized gelatin and to collagen Type IV. It is shown that HBGF-I, adsorbed to gelatin sponges, promotes 07944
  • angiogenesis in the rat at concentrations of the growth factor which are consistent with the growth factor's activity as an endothelial cell mitogen in vitro. This concentration is about 10 ⁇ 3 times lower than the concentration used in vitro in the art.
  • HBGF-1 binds to immobilized gelatin and collagen Type IV, therefore, the possibility was evaluated that commercial gelatin sponges sold by the tradename "Gelfoam” adsorbed with HBGF-1 could be utilized as a method for inducing angiogensis in situ. Survival surgery was performed on the rat in order to implant gelatin sponges which were treated with HBGF-1. HBGF-1-adsorbed Gelfoam was independently placed in the neck and peritoneal cavities in the rat. A significant angiogenic response was observed in situ one week after surgery with lng HBGF-1 per mm2 ( Figure 2). Blood vessels, which migrated away from the tissue site of implantation, were observed macroscopically to be exclusively within the gelatin sponge.
  • angiogenesis and neovascularization has been achieved between various tissues and organs as demonstrated by Figures 2 through 5.
  • Neovascularization has been similarly accomplished between the following loci (data not shown): liver to spleen; liver to kidney; spleen to kidney; liver to aorta; liver to vena cava; liver to omentum (omentum, containing pancreatic tissue); aorta/to vena cava; spleen to aorta; spleen to vena cava; spleen to omentum kidney to aorta; kidney to vena cava; kidney to omentum; omentum to aorta; and omentum to vena cava.
  • Example 3 demonstrates the device of the invention having a non-absorbable support.
  • the experiments performed to derive this example were conducted with either Type I or Type IV collagen and involved implantation onto the liver or the spleen of a rat. 7944
  • Comparative Example A demonstrates that the use of the same materials and procedures of Example 3 without HBGF-1 did not induce neovascularization.
  • HBGF-1 adsorbed, collagen-coated (Type I or IV) expanded PTFE fibers were surgically implanted in the peritoneal cavity (onto the liver or the spleen) of the rat.
  • a significant angiogenic response was specifically induced by HBGF-1 in situ and the results four weeks after surgery are shown in Figure 7.
  • Blood vessels which have migrated from the tissue site of implantation, could be observed macroscopically within and around the implanted fibers.
  • the anterior portion of the fiber implant which was attached to the liver, exhibited substantial neovessel growth from the liver into the interior of the implant (Figure 7).
  • HBGF-1 HBGF-1 at concentrations between 1 to 100 ng/mm ⁇ of fiber surface area.
  • concentration of HBGF-1 required to induce an angiogenic response in the fiber implant model is consistent with the results obtained with the Gelfoam implant model and the mitogenic activity of the polypeptide in vitro.
  • Example 4 demonstrates that the blood vessel produced in Example 3 displayed a large organized solid matrix including a network of neovessel formations.
  • HBGF-1 is capable of signaling a variety of the squamous mesothelial cells of the serosa and the proximal cells of the tunica adventita to initiate angiogenesis.
  • mesoderm- and neuroectoderm-derived cells are consistent with the ability of HBGF-1 to act as a mitogen in vitro for epithelial cells, fibroblasts, smooth muscle cells, mesothelial cells, endothelial -35-
  • neuronal-like structures are also consistent with the nerve growth factor (NGF)-like biological activity of HBGF-1 to induce neurite extension and survival of PC12 cells in vitro.
  • NGF nerve growth factor
  • Example 5 demonstrates that the presence of a large organized solid matrix, containing a network of mature muscular neovessel formations of Example 4 and which are contiguous with the host's vascular tree in situ, permits successful selective cell transplantation.
  • Comparative Example B demonstrates that the use of the same materials and procedures of Example 5 without HBGF-1 did not sustain selective cell transplantation.
  • hepatocytes were harvested by collagenase perfusion of syngeneic Wistar (RHA) rats.
  • the Wistar rat is genetically identical to the Gunn rat except that it contains a normal bilirubin conjugation locus.
  • Example 5 HBGF-1 adsorbed collagen (Type IV) coated PTFE fibers were implanted next to the liver and after ten to fourteen days the peritoneal cavity was 7944
  • HBGF-1 fiber implant model functions in vivo as a receptacle for the successful site-specific introduction of cells capable of expressing a differentiated physiologic function.
  • Example 5 The long-term HBGF-1 fiber implant model of Example 5 induces a prominent angiotropic and neurotropic response when appropriately implanted in the rat.
  • Example 5 demonstrates the ability of HBGF-1 to induce, sustain, and maintain the anatomical coordination of highly sophisticated and widely diversified mammalian cell types in vivo.
  • the interrelationships between extracellular matrix components and differentiation-specific gene regulation can provide information critical for genetic engineering therapies.
  • This invention may also prove useful as a site-specific transgenic alternative with the ability to understand the temporal and coordinated expression of growth and differentiation signals during neuronal and angiogenic development in the adult.
  • Example 6 demonstrates the neovascular device of this invention wherein genetically engineered cells are seeded into the device.
  • Example 6 is as follows.
  • This rat growth hormone cDNA was electrophoretically isolated out of an agarose gel and purified via binding/elution to glass beads sold by the tradename, Geneclean Bio, 101, La Jolla, California. This growth hormone cDNA was then blunted using the large fragment of DNA polymerase Klenow known by the name, from New England Biolabs and nucleotide triphosphates as recommended by the manufacturer. This fragment was then purified with Geneclean product.
  • the B2 vector was constructed in order to replace the Neo R gene in N2 according to M.A. Eglitis, et al., Science 230 (1985):1395; D. Armentano, et al., J. Virol 61 (1987):1647 with a multiple cloning site.
  • N2 was first digested with Eco RI, thereby releasing both the 5' and 3' LTRs with the adjoining MoMLV flanking sequences.
  • the 3' LTR fragment was ligated into the EcoRI site of the plasmid GEM4 from Promega Biotech.
  • the 5' LTR fragment with its flanking gag sequence was then digested with Cla I, Hind III linkers were added, and the fragment was inserted into the Hind III site of pGEM4.
  • the pB2 vector was digested with the Hindi restriction endonuclease from New England Liolabs, and phosphatased using calf alkaline phosphatase from Boehringer Mannheim Biochemicals. The pB2 plasmid was then purified with the Geneclean product. The pB2 vector and the rat growth hormone cDNA were then ligated using T4 ligase from New England Biolabs, pG2 was then digested -39-
  • a 340 base pair SV40 promoted neomycin resistance gene fragment was isolated from the pSV2CAT plasmid (ATCC accession number 37155) by digesting with PvuII and Hindlll from New England Biolabs. This fragment was isolated by agarose gel electrophoresis and purified with the Geneclean product. The SV40-neomycin resistance fragment was then ligated using T4 ligase from New England Biolabs with pG2 and transformed into DH5 competent bacteria per the manufacturer's instructions (BRL). Colonies were screened and the resulting plasmid construct was called pG2N.
  • the SAX vector was obtained as described in Proc. Natl. Acad. Sci. USA 83 (1988):6563.
  • the recombinant vectors, N2, SAX, G2N, used in this example were each separately transfected into the currently available retroviral vector packaging cell lines, including the amphotropic packaging lines, PA317 Mol. Cell. Biol. 6(1986):2895, and the ecotropic line, Psi2, Cell 33(1983):153. These lines were developed in order to allow the production of helper virus-free retroviral vector particles.
  • the CD4 containing plasmid, p4B which was a gift of Richard Axel of College of Physicians and Surgeons Columbia University, New York, New York, was digested with the restriction endonucleases Eco RI and Bam HI from New England Biolabs, Beverly, Massachusetts, to release the CD4 gene which was isolated by agarose gel electrophoresis followed by purification via binding/elution to glass beads using the Geneclean product. Bio 101, La Jolla, California, in the manner recommended by the manufacturer. The CD4 fragment was ligated, using T4 DNA ligase as recommended by the supplier, into Eco RI plus Bam HI cut Bluescript cloning vector from Stratagene Co., La Jolla, California.
  • the ligation was then transformed into competent DH5 alpha bacteria from Bethesda Research Labs, Gaithersburg, Maryland, and white colonies were isolated and screened for proper insert size to yield the plasmid pCDW.
  • the plasmid SV2neo obtained from American Type Culture Collection, Rockville, Maryland, was digested with Hind III plus Hpa I.
  • This ligation was transformed into DH5 bacteria from Bethesda Research Labs and colonies screened for the presence of restriction enzyme sites unique to the polylinker to yield the vector pSVPL.
  • the pSCPL expression vector was further modified by the insertion of an Xho I linker using conditions and reagents suggested and supplied by New England Biolabs, into the Pvu II site on the 5' side of the SV40 early region promoter to produce pSVPLX.
  • the pCDW and pSVPLX plasmids were digested with enzymes Hind III plus Xba I from New England Biolabs and their DNAs isolated using the Geneclean product following agarose gel electrophoresis. Ligation of the CD4 fragment into the pSVPLX vector was performed and colonies were screened to yield pSVCDW in which the SV40 virus early region promoter is used to drive the expression of the complete CD4 gene product. The next step was to produce a form of the CD4 gene such that it would be exported from the cell as an extracellular product.
  • CD4 The production of a soluble form of CD4 was accomplished by the use of a specially designed oligonucleotide adaptor to produce a mutant form of the CD4 gene.
  • This adaptor has the unique property that when inserted into the Nhe I site of the CD4 gene it produces the precise premature termination of the CD4 protein amino acid sequence while regenerating the Nhe I site and creating a new Hpa I site.
  • This oligonucleotide adaptor synthesized by Midland Certified Reagent Co., was produced by annealing two phosphorylated oligonucleotides: (1) 5'CTAGCITGAGTGAGIT 3' and (2) AACTCACTCAAG. This product was then ligated into the site of pSVCDW.
  • the ligation reaction was then cleaved with Hpa I and then Xho I linkers were added.
  • the linker reaction was terminated by heating at 65°C for 15 minutes and then subjected to digestion with Xho I restriction endonuclease from New England Biolabs.
  • This reaction was then subjected to agarose gel electrophoresis and the fragment containing the SV4O-CD4 adaptor isolated using the Geneclean product.
  • the retroviral vector N2 was prepared to accept the SV40-CD4-adaptor fragment by digestion with Xho I and treatment with calf intestinal phosphatase from Boehringer Mannheim, Indianapolis, Indiana. 944
  • the ligation of a CD4 expression cassette was performed with an insert to vector ratio of 5:1 and then transformed in DH5 competent bacteria from Bethesda Research Labs. Constructs were analyzed by restriction enclonuclease digestion to screen for orientation and then grow up in large scale.
  • SSC The construct where the SV40 virus promoter is in the same orientation as the viral LTR promoters is known as SSC while the construction in the reverse orientation is called SCSX.
  • the SSC vector is packaged into PA 317 cell line as described by Miller, et al., supra, to provide PA 317 cells capable of producing soluble CD4 protein.
  • the SSC vector packaged PA 317 cells were used to transduce rabbit endothelial cells as described above.
  • the transduced endothelial cells expressed soluble CD4.
  • Collagen sponges containing adsorbed HBGF-1 of the type previously described were surgically implanted in the abdominal cavity of a rat near the liver. Sponges were surgically removed seven to ten days post-implantation and digested 30 to 60 minutes at 27°C with a solution of collagenase in phosphate buffered saline in a concentration of lmg/ml using a tissue culture incubator at 5 percent in CO2. Released cells were collected by centrifugation for 10 minutes at 1000 RPM at 20°C. The cells were washed once with phosphate buffered saline (PBS) and pelleted by centrifugation. Cells were resuspended with two volumes of 30 ml of media containing: M199 media (Gibco); ECGF (crude brain extract) 7.2mg; Heparin (Upjohn) 750 units; -43-
  • conditioned cellular media collected as supernatant from confluent dishes after 48 hours of either bovine aortic or human umbilical vein endothelial cells.
  • the other media contained: 10 percent fetal calf serum (Hyclone); 3000 units Penicillan G (Biofluids); and 3000 units streptomycin sulfate (Biofluids) and the cells were plated for 16 hours on 100 mm tissue culture disk coated with fibronectin (human) using lug/cm2. Plated cells were washed with PBS three times and fed 15ml of previously mentioned media. Media was changed every 2 days for the duration of the procedures.
  • Selected rat endothelial cells were transduced with N-7, SAX, G2N and SSC vectors by the following procedures:
  • the endothelial cells are seeded directly onto a HBGF-1 adsorbed, collagen coated PTFE fiber sponge, and the sponge is implanted back into the same animal used as the source of endothelial cells.
  • the site of implantation can be subcutaneous, intraperitoneal, or at or near the site of the organ that normally produces the new product encoded by the gene transduced into the endothelial cells.
  • the sponge implant generates its own vascularization within 5 to 10 days, as described in earlier examples.
  • the engineered endothelial cells are maintained on the implant such that the new gene product is delivered directly into the circulation after secretion from the cell. The production of the gene product is monitored either by direct measurement of its serum levels, by the biochemical or physiological effect of the agent, or both.
  • An HBGF-1 absorbed, collagen coated PTFE fiber sponge is preimplanted at the desired site, as described above, and at the time determined to be optional for that implant site for establishment of neovascularization.
  • the transformed cells are injected directly into the -45-
  • the advantage of this method is that the engineered cells are more rapidly and effectivel . established in the implant or migrate back into the parent organ (e.g., liver).
  • the product begins to enter the circulation much sooner than with method A above.
  • Production of the new gene product is measured as described in method A.
  • This procedure can be applied to a number of different cell types capable of being sampled, genetically engineered in vivo, and reinserted via the fiber sponge implant.
  • Such cells include fibroblasts, hepatocytes, smooth muscle cells, bone marrow cells and others.
  • the products delivered to the circulation can be any peptide or protein whose gene can be inserted into a cell and whose product is desired to be delivered.
  • Gortex shunt tubes were surgically implanted into the peritoneum of rats, in such a way as to form a loop, with each end contacting the aorta.
  • the tubes contained either a Gelfoam (Collagen I) sponge impregnated with HBGF-1 (1 ng/ml) or a bundle of "angel hair” Gortex fibers, coated with Collagen I and impregnated with HBGF-1 (1 ng/ml).
  • the tubes were left in the animals for one month, then surgically extracted, grossly examined for blood vessel formation, and the sponge prepared for histological examination. As shown in Figure 14A, the 7944
  • This experiment provides an example of directing neovascularization to a particular site, with a two component device.
  • the first component a tube or pouch
  • the first component can provide a receptacle in which implanted cells, genetically engineered or normal, can be seeded. It is possible that such a site may be immunologically privileged, and allow cells from another individual, or even another species, to survive and produce a desired product.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Transplantation (AREA)
  • Dermatology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Inorganic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Vascular Medicine (AREA)
  • Molecular Biology (AREA)
  • Environmental & Geological Engineering (AREA)
  • Immunology (AREA)
  • Nanotechnology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Geology (AREA)
  • General Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Heart & Thoracic Surgery (AREA)

Abstract

Un dispositif de néovascularisation contrôlée comprend un support biocompatible et un modificateur des réponses biologiques qui induit la néovascularisation et qui est adsorbé dans le support biocompatible. Un procédé de commande de la néovascularisation in vivo implique l'adsorption dans un support biocompatible d'un modificateur des réponses biologiques qui induit la néovascularisation. Puis on met en contact une quantité thérapeutiquement efficace du modificateur adsorbé des réponses biologiques avec au moins un tissu sélectionné. On commande la croissance des cellules vasculaires dans le tissu sélectionné en contact pendant une durée suffisante pour obtenir une structure vasculaire. Ce procédé est utile pour développer dans un organisme des organes artificiels et d'autres tissus, y compris des nerfs, et pour réimplanter, après les avoir génétiquement modifiées afin d'obtenir un produit voulu, des cellules prélevées dans un organisme.
PCT/US1989/000742 1988-02-24 1989-02-24 Procede et dispositif de neovascularisation a localisation controlee WO1989007944A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
KR1019890701950A KR900700122A (ko) 1988-02-24 1989-10-24 부위특정 신혈관신생을 위한 장치 및 방법
DK202790A DK202790A (da) 1988-02-24 1990-08-23 Indretning til maalrettet neovaskularisering og fremgangsmaade til samme

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US15973088A 1988-02-24 1988-02-24
US159,730 1988-02-24
US30159189A 1989-01-26 1989-01-26
US301,591 1989-01-26

Publications (1)

Publication Number Publication Date
WO1989007944A1 true WO1989007944A1 (fr) 1989-09-08

Family

ID=26856224

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1989/000742 WO1989007944A1 (fr) 1988-02-24 1989-02-24 Procede et dispositif de neovascularisation a localisation controlee

Country Status (6)

Country Link
EP (1) EP0424386A1 (fr)
JP (1) JPH03503167A (fr)
KR (1) KR900700122A (fr)
AU (1) AU4072189A (fr)
DK (1) DK202790A (fr)
WO (1) WO1989007944A1 (fr)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993008850A1 (fr) * 1991-10-30 1993-05-13 Massachusetts Institute Of Technology Implants polymeres prevascularises pour la transplantation d'organes
US5314471A (en) * 1991-07-24 1994-05-24 Baxter International Inc. Tissue inplant systems and methods for sustaining viable high cell densities within a host
US5344454A (en) * 1991-07-24 1994-09-06 Baxter International Inc. Closed porous chambers for implanting tissue in a host
WO1995014785A1 (fr) * 1993-11-23 1995-06-01 Rhone-Poulenc Rorer S.A. Composition pour la production de produits therapeutiques in vivo
US5545223A (en) * 1990-10-31 1996-08-13 Baxter International, Inc. Ported tissue implant systems and methods of using same
US5782912A (en) * 1990-10-31 1998-07-21 Baxter International, Inc. Close vascularization implant material
US5913998A (en) * 1995-06-07 1999-06-22 Gore Hybrid Technologies, Inc. Method of making an implantable containment apparatus for a therapeutical device
EP1142581A2 (fr) * 1990-11-27 2001-10-10 American National Red Cross Compositions contenant un agent de scellement tissulaire et des facteurs de croissance, qui accélérent la cicatrisation
US6309635B1 (en) 1986-11-20 2001-10-30 Children's Medical Center Corp. Seeding parenchymal cells into compression resistant porous scaffold after vascularizing in vivo
US6328765B1 (en) 1998-12-03 2001-12-11 Gore Enterprise Holdings, Inc. Methods and articles for regenerating living tissue
WO2004012759A2 (fr) * 2002-07-26 2004-02-12 Epoplus Gmbh & Co. Kg Utilisation de l'erythropoietine
US6773458B1 (en) 1991-07-24 2004-08-10 Baxter International Inc. Angiogenic tissue implant systems and methods
WO2005070450A2 (fr) * 2004-01-23 2005-08-04 Epoplus Gmbh & Co. Kg Utilisation d'erythropoietine faiblement dosee pour la stimulation de cellules precurseurs, ainsi que pour la regeneration d'organes et le ralentissement de la progression des dommages aux organes terminaux
WO2007057178A2 (fr) * 2005-11-17 2007-05-24 Gelita Ag Substrat favorisant l'angiogenese
USRE42479E1 (en) 1995-05-19 2011-06-21 Children's Medical Center Corporation Engineering of strong, pliable tissues

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987001728A1 (fr) * 1985-09-12 1987-03-26 Biotechnology Research Partners, Ltd. Facteurs de croissance de fibroblaste recombinant
US4699141A (en) * 1986-01-16 1987-10-13 Rhode Island Hospital Neovascularization

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987001728A1 (fr) * 1985-09-12 1987-03-26 Biotechnology Research Partners, Ltd. Facteurs de croissance de fibroblaste recombinant
US4699141A (en) * 1986-01-16 1987-10-13 Rhode Island Hospital Neovascularization

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
Biochemical and Biophysical Research Communications Volume 147, issued 15 September 1987 HAYEK et al, "An in Vivo Model for Study of the Angiogenic Effects of Basic Fibroblast Growth Factor", pages 876-880, entire document. *
Biochemical and Biophysical Research Communications, Volume 142, issued 30 January 1987, BAIRD et al, "Fibroblast Growth Factors are Present in the Extracellular Matrix Produced by Endothebal Cells in Vitro. Implications for a Role of Heporinase-Like Enzymes in the Neovascular Response, pages 428-435, especially page 433. *
British Journal of Experimental Pathology Volume 68, issued 1987, ANDRADE et al, "Quantitative in Vivo Studies on Angiogenesis in a Rat Sponge Model", pages 755-766, entire document. *
Cancer Research, Volume 43, issued June 1983, VLODAVSKY et al, "Lymphona Cell-Mediated Degradation of Sulfated Proteoglycans in the Subendothelial Extracellular Matrix: Relationship to Tumor Cell Metastiasis", pages 2704-2711, especially page 2710. *
CHEMICAL ABSTRACTS, Volume 109, issued 10 October 1988, RIFKIN, "Endothelial Cell Proteases and Cellular Invasion", Abstract No. 123103 *
Journal of Cell Biology, Volume 107 (6 part 3), issued 09 December 1988, MACIAG et al, "Heparin-Binding Growth Factor-I (HBGF-I) Bound to Gelatin Induces Site-Specfic Neovessel Formation in Vivo", Abstract No. 2697, page 479a. *
Proceedings of the National Academy of Sciences, USA, Volume 82, issued November 1985, BUCKLEY et al, "Sustained Release of Epidermal Growth Factor Accelerates Wound Repair," pages 7340-7344, entire document. *
See also references of EP0424386A4 *
Surgical Science Series, Volume 2, issued 1984, JACKSON et al, "Effect of Angiogenic Factors on the Vascularization of IVALON Sponge Implants", pages 190-201, see entire document. *

Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6309635B1 (en) 1986-11-20 2001-10-30 Children's Medical Center Corp. Seeding parenchymal cells into compression resistant porous scaffold after vascularizing in vivo
US5545223A (en) * 1990-10-31 1996-08-13 Baxter International, Inc. Ported tissue implant systems and methods of using same
US5593440A (en) * 1990-10-31 1997-01-14 Baxter International Inc. Tissue implant systems and methods for sustaining viable high cell densities within a host
US5733336A (en) * 1990-10-31 1998-03-31 Baxter International, Inc. Ported tissue implant systems and methods of using same
US5782912A (en) * 1990-10-31 1998-07-21 Baxter International, Inc. Close vascularization implant material
US5882354A (en) * 1990-10-31 1999-03-16 Baxter International Inc. Close vascularization implant material
EP1142581A2 (fr) * 1990-11-27 2001-10-10 American National Red Cross Compositions contenant un agent de scellement tissulaire et des facteurs de croissance, qui accélérent la cicatrisation
EP1142581A3 (fr) * 1990-11-27 2002-09-11 American National Red Cross Compositions contenant un agent de scellement tissulaire et des facteurs de croissance, qui accélérent la cicatrisation
US5314471A (en) * 1991-07-24 1994-05-24 Baxter International Inc. Tissue inplant systems and methods for sustaining viable high cell densities within a host
US5344454A (en) * 1991-07-24 1994-09-06 Baxter International Inc. Closed porous chambers for implanting tissue in a host
US6773458B1 (en) 1991-07-24 2004-08-10 Baxter International Inc. Angiogenic tissue implant systems and methods
WO1993008850A1 (fr) * 1991-10-30 1993-05-13 Massachusetts Institute Of Technology Implants polymeres prevascularises pour la transplantation d'organes
US6464998B1 (en) * 1993-11-23 2002-10-15 Aventis Pharma S.A. Composition for the in vivo production of therapeutic products
AU704919B2 (en) * 1993-11-23 1999-05-06 Institut National De La Sante Et De La Recherche Medicale Composition for the production of therapeutic products in vivo
FR2712812A1 (fr) * 1993-11-23 1995-06-02 Centre Nat Rech Scient Composition pour la production de produits thérapeutiques in vivo.
KR100375856B1 (ko) * 1993-11-23 2004-06-30 아방티 파르마 소시에테 아노님 치료물질의생체내생산을위한조성물
WO1995014785A1 (fr) * 1993-11-23 1995-06-01 Rhone-Poulenc Rorer S.A. Composition pour la production de produits therapeutiques in vivo
USRE42575E1 (en) 1995-05-19 2011-07-26 Children's Medical Center Corporation Engineering of strong, pliable tissues
USRE42479E1 (en) 1995-05-19 2011-06-21 Children's Medical Center Corporation Engineering of strong, pliable tissues
US5913998A (en) * 1995-06-07 1999-06-22 Gore Hybrid Technologies, Inc. Method of making an implantable containment apparatus for a therapeutical device
US6471687B2 (en) 1995-06-07 2002-10-29 Gore Enterprise Holdings, Inc. Implantable containment apparatus for a therapeutical device
US6328765B1 (en) 1998-12-03 2001-12-11 Gore Enterprise Holdings, Inc. Methods and articles for regenerating living tissue
EP2191838A1 (fr) * 2002-07-26 2010-06-02 EPOPLUS GmbH & Co. KG Erythropoetine pour la stimulation des cellules precurseurs endotheliales
EP1779862A1 (fr) * 2002-07-26 2007-05-02 EPOPLUS GmbH & Co. KG Erythropoietine pour la stimulation des cellules progenitrices endotheliales
EA009463B1 (ru) * 2002-07-26 2007-12-28 Эпоплус Гмбх Унд Ко. Кг Применение эритропоэтина
US7745387B2 (en) 2002-07-26 2010-06-29 Epoplus Gmbh & Co. Kg Use of erythropoietin
EA013966B1 (ru) * 2002-07-26 2010-08-30 Эпоплус Гмбх Унд Ко. Кг Применение эритропоэтина для лечения сахарного диабета
WO2004012759A3 (fr) * 2002-07-26 2004-06-03 Ferdinand Hermann Bahlmann Utilisation de l'erythropoietine
WO2004012759A2 (fr) * 2002-07-26 2004-02-12 Epoplus Gmbh & Co. Kg Utilisation de l'erythropoietine
WO2005070450A3 (fr) * 2004-01-23 2005-12-08 Epoplus Gmbh & Co Kg Utilisation d'erythropoietine faiblement dosee pour la stimulation de cellules precurseurs, ainsi que pour la regeneration d'organes et le ralentissement de la progression des dommages aux organes terminaux
WO2005070450A2 (fr) * 2004-01-23 2005-08-04 Epoplus Gmbh & Co. Kg Utilisation d'erythropoietine faiblement dosee pour la stimulation de cellules precurseurs, ainsi que pour la regeneration d'organes et le ralentissement de la progression des dommages aux organes terminaux
EA015350B1 (ru) * 2004-01-23 2011-06-30 Эпоплус Гмбх Унд Ко. Кг Применение эритропоэтина в низкой дозировке для стимуляции эндотелиальных клеток-предшественников, а также для регенерации органов и замедления прогрессирования повреждений конечных органов
EP2156843A3 (fr) * 2004-01-23 2011-12-28 EPOPLUS GmbH & Co. KG Utilisation d'erythropoietine faiblement dosée pour traiter l'insuffisance renale chronique ou aigue et pour traiter des plaies
WO2007057178A2 (fr) * 2005-11-17 2007-05-24 Gelita Ag Substrat favorisant l'angiogenese
WO2007057178A3 (fr) * 2005-11-17 2007-08-02 Gelita Ag Substrat favorisant l'angiogenese

Also Published As

Publication number Publication date
KR900700122A (ko) 1990-08-11
DK202790D0 (da) 1990-08-23
DK202790A (da) 1990-08-23
JPH03503167A (ja) 1991-07-18
EP0424386A4 (fr) 1991-02-05
AU4072189A (en) 1989-09-22
EP0424386A1 (fr) 1991-05-02

Similar Documents

Publication Publication Date Title
US6733747B2 (en) Prosthetic grafts
US6787357B2 (en) Plasma-derived fibrin-based matrices and tissue
WO1990006997A1 (fr) Cellules endotheliales modifiees par genie genetique et utilisation de ces cellules
Xu et al. Bridging Schwann cell transplants promote axonal regeneration from both the rostral and caudal stumps of transected adult rat spinal cord
WO1989007944A1 (fr) Procede et dispositif de neovascularisation a localisation controlee
JPH08508880A (ja) 生体内に治療用化合物を発現・分泌させるための生体適合移植片
JPH08509356A (ja) 細胞の移植システム及び方法
JP2001299367A (ja) 内皮細胞の遺伝的変性
EP1143983B1 (fr) Administration de cellules par pulverisation
WO1989005345A1 (fr) Modification genetique de cellules endotheliales
AU3552099A (en) Creation of three-dimensional tissues
JP2865354B2 (ja) 基質細胞を用いた遺伝子治療
CN105517587A (zh) 伤口愈合与组织工程
JP2012513782A (ja) 個々の移植物としての操作された組織および足場の迅速な調製および使用
JPH09509571A (ja) クローン原性線維芽細胞を調製する方法、線維芽細胞を遺伝子トランスフェクションする方法及びかくして得られた遺伝子トランスフェクション線維芽細胞
US20040156833A1 (en) Devices and methods for implanting transduced cells
DE69637185T2 (de) Isolierte Stromazellen und Methoden zu deren Verwendung
Langer I articles
WO1991001760A1 (fr) Cellules tumoreuses humaines implantees chez des animaux non humains
CA2436076A1 (fr) Methodes et compositions permettant de stimuler l'angiogenese
JP4431320B2 (ja) 自己組織化促進機能を持つ移植医療用材料
US20090214613A1 (en) Endothelized Artificial Matrix Comprising a Fibrin Gel, Which Is a Superproducer of Proangiogenic Factors
AU704371B2 (en) Retroviral vectors and recombinant cells containing same for the expression and secretion in vivo of a therapeutic compound
Suh et al. STUDY ON REPLACEMENT OF TRACHEAL DEFECT WITH AUTOGENOUS MUCOSA-LINED TRACHEAL PROSTHESIS MADE FROM POLYPROPHYLENE MESH
Jockenhoevel et al. CARDIOVASCULAR TISSUE ENGINEERING WITH FIBRIN GEL IN MICROSTRUCTURED CULTURE PLATES

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BR DK FI JP KR NO SU

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LU NL SE

WWE Wipo information: entry into national phase

Ref document number: 1989903426

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1989903426

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1989903426

Country of ref document: EP