WO1999044487A2 - Mise au point genetique de greffes de pontage - Google Patents

Mise au point genetique de greffes de pontage Download PDF

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Publication number
WO1999044487A2
WO1999044487A2 PCT/US1999/004500 US9904500W WO9944487A2 WO 1999044487 A2 WO1999044487 A2 WO 1999044487A2 US 9904500 W US9904500 W US 9904500W WO 9944487 A2 WO9944487 A2 WO 9944487A2
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WIPO (PCT)
Prior art keywords
antagonist
cells
graft
prosthetic
bypass graft
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Legal status (The legal status 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 status listed.)
Ceased
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PCT/US1999/004500
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English (en)
Inventor
Darwin Eton
Hong Yu
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University of Southern California USC
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University of Southern California USC
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Priority to AU29769/99A priority Critical patent/AU2976999A/en
Publication of WO1999044487A2 publication Critical patent/WO1999044487A2/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • 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
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
    • A61F2/06—Blood vessels
    • A61F2/062—Apparatus for the production of blood vessels made from natural tissue or with layers of living 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/126—Immunoprotecting barriers, e.g. jackets, diffusion chambers
    • 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
    • C12N2510/02—Cells for production

Definitions

  • This invention relates to prosthetic bypass grafts, particularly to grafts with improved patency containing genetically engineered cells expressing selected genetic materials of interest and to methods for the use of such prosthetic grafts.
  • stenosis or occlusion of arteries that supply vital organs.
  • the pathologic mechanism most commonly implicated in such disease states is atherosclerosis. Examples include angina pectoris and myocardial infarction due to coronary artery disease; transient ischemic attacks and strokes due to cerebral vascular disease; renal vascular hypertension, and ultimately, renal failure due to renal artery stenosis; and claudication of the lower extremities, which is caused by vascular disease of peripheral arteries and, in its most severe form, can result in amputation.
  • An accepted and widely used therapeutic approach to advanced atherosclerotic disease is to bypass the site of major stenosis or occlusion with a prosthetic vessel made of synthetic material, such as Dacron ® or Gore-Tex ®. More than 350,000 vascular grafts are implanted each year.
  • a prosthetic vessel is thrombogenic (i.e., it has the propensity to develop clots), which leads to a very high rate of graft failure.
  • Tissue plasminogen activator a thrombolytic proteinase
  • Tissue plasminogen activator a thrombolytic proteinase
  • ECs and SMCs can be overexpressed by ECs and SMCs following intracellular transfer of the tPA gene (Ekhterae. D. and J.C. Stanley (1995) J. Vase. Surg. 21 :953-82; Shayani, V. et al. (1994) J. Surg. Res. 57:495-504).
  • This enzyme converts inactive plasminogen into active plasmin, which then degrades fibrin complexes, a major component of thrombus.
  • the present invention relates to novel prosthetic bypass grafts with improved patency rates.
  • the present invention provides a graft comprised of two concentric cylindrical prosthetic tubes confining a space capable of containing genetically engineered cells expressing one or more selected genetic materials of interest or other therapeutic agents.
  • the outer prosthetic tube is a standard graft, whereas the inner prosthetic tube is a semi-permeable membrane.
  • Preferred prosthetic materials for use within the invention include a broad range of biologically compatible polymers known to have medically useful structure and surface characteristics.
  • Useful polymers within this context include, e.g., polyethyleneterephthalate, polytetrafluoroethylene, expanded polytetrafluoroethylene, polymers of lactide-glycolide, polyglactin, polydioxanone, polyurethanes, polypropylenes and polyesters.
  • the porosity and the thickness of the semi- permeable membrane are such that (i) the genetically engineered cells are protected from the shear stress of flowing blood as well as from exposure to inflammatory cells and platelets, and (ii) free diffusion and exchange of the thrombolytic enzyme, essential nutrients from the circulation, and waste product of metabolism is accomplished.
  • Cells of this invention include, but are not limited to, endothelial cells, smooth muscle cells, fibroblasts, and stem cells. These cells have stably incorporated in them genetic material of interest (e.g., that which encodes a protein of therapeutic value) and provide constitutive synthesis and delivery thereof useful in the prevention or treatment of diseases.
  • cells are genetically engineered to overproduce a thrombolytic enzyme or mutant forms thereof which are resistant to degradation. The genetically altered cells secrete the thrombolytic enzyme which diffuses onto the luminal surface of the graft to keep the graft free of thrombus.
  • Another aspect of the invention relates to grafts capable of releasing a second therapeutic drug.
  • This drug may be housed within the graft with its release controlled by the semipermeable membrane.
  • the genetically engineered cells in the thromboresistant graft produce a second therapeutically active protein. A sustained release of the protein into the circulation is thereby produced.
  • methods of treatment involve implanting a prosthetic graft of the invention. Such methods are useful for treating stenotic or occluded arteries.
  • Figures la and lb are perspective views illustrating two variations of the bypass graft.
  • Figure 2 illustrates the antigen concentration (A) and enzyme activity (B) in the supernatants of cultured EC and tPA transduced EC (EC/tPA).
  • Figure 3 shows cell growth in a semipermeable membrane graft. Endothelial cells were stained with X-gal one day (A) and one month (B) after placement in "tea bag" of semipermeable membrane. Dark staining represents cells expressing ⁇ -Gal enzyme.
  • cells refers to cells of the present invention including, but not limited to, endothelial cells, smooth muscle cells, fibroblasts, and stem cells, that express incorporated genetic material encoding a polypeptide or a protein of interest or a gene encoding a selectable marker. Cells which express incorporated genetic material are referred to herein as transduced cells.
  • genetic material refers to genetic material of interest that has been incorporated into cells and expressed in the resulting genetically engineered cells.
  • Genetic material includes any nucleic acid molecule.
  • nucleic acid molecule encompasses single-stranded and double- stranded DNA molecules, including genomic DNA, cDNA, DNA produced by amplification reaction (such as polymerase chain reaction ["PCR”]), and DNA produced by olgonucleotide synthesis, as well as RNA molecules, such as mRNA.
  • Genomic DNA can include non-transcribed and transcribed regions (such as 5' and 3' non-coding regions, introns, and coding regions of the genetic material of interest).
  • cDNA and mRNA molecules contain sequences corresponding to transcribed regions.
  • genetically engineered relates to ways of introducing genetic material of interest into cells.
  • standard graft refers to tubular grafts of biocompatible materials suitable for use as prosthetic bypass grafts.
  • tissue plasminogen activator or "tPA” refers to a protein with the general property of activating the plasminogen protein.
  • tP A refers to the human tissue plasminogen activator.
  • tPA variant encompasses proteins and protein fragments substantially homologous to human tissue plasminogen activator that retain the property of activating tissue plasminogen.
  • substantially homologous refers to proteins possessing at least 70% amino acid identity when best aligned with tPA.
  • Best alignment is an alignment of two or more sequences, with appropriate gaps introduced, that maximizes identity of identity of corresponding amino acid residues.
  • agent refers to any genetically encoded secreted product of a cell that has biological activity.
  • Reagents include but are not limited to both linear and cyclic peptides, lipids, carbohydrates, nucleic acids, polyketides and antibiotics. Prosthetic Bypass Grafts
  • the present invention relates to prosthetic grafts that are able to deliver therapeutic enzymes directly into the circulation.
  • Well known obstacles encountered in the design of such grafts include (i) platelet adherence, (ii) fibrin crosslinking, (iii) host cell immune responses, (iv) flow induced shear injury of the cells seeded onto the graft surface, and (v) chemically induced detachment of engineered cells (for example by tPA).
  • a semipermeable membrane is incorporated into the graft design as a physical barrier to protect the genetically engineered cells from the shear stress of blood flow, as well as inhibit immune responses.
  • the use of genetically engineered cells overexpressing a thrombolytic enzyme and the ability to control the amount to be delivered into the circulation will inhibit fibrin crosslinking.
  • platelet adherence can be inhibited by both systemic and/or local therapy (examples include cyclooxygenase inhibitors and hirudin).
  • the novel graft consists of two concentric cylindrical prosthetic tubes enclosing a space that contains genetically engineered cells which express selected genetic material of interest.
  • the outer prosthetic tube is a standard graft, whereas the inner prosthetic tube is a semi-permeable membrane (Figs, la, b).
  • Preferred prosthetic materials for use within the invention include a broad range of biologically compatible polymers known to have medically useful structure .and surface characteristics.
  • Useful polymers within this context include, but are not limited to, polyethyleneterephthalate (e.g., Dacron ®), polytetrafluoroethylene, expanded polytetrafluoroethylene (PTFE, e.g., Gore-Tex ®, W. L.
  • polyolefins polyisobutylene and ethylene-alphaolefin copolymers
  • acrylic polymers and copolymers vinyl halide polymers and copolymers, such as polyvinyl chloride; polyvinyl ethers, such as polyvinyl methyl ether; polyvinylidene halides, such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile, polyvinyl ketones; polyvinyl aromatics, such as polystyrene; polyvinyl esters, such as polyvinyl acetate; copolymers of vinyl monomers with each other and olefins, such as ethylene- methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins, .and ethylene-vinyl acetate copolymers; polyamides, such as Nylon 66 and polycaprolactam; alkylene ethylene ethylene-vinyl
  • the graft can be constructed in a variety of sizes to accommodate a particular vascular application.
  • the reservoir of such an improved graft containing the genetically engineered cells will prevent or treat complications associated with conventional grafts including, but not limited to, protection of the cells from: the shear stress of blood flow; exposure to inflammatory cells; platelet deposition; coagulation; thrombosis; neointimal hyperplasia; and fibrosis.
  • the short half lives of drugs such as hormone polypeptides will not be a limitation in such a continuous delivery system.
  • the porosity (internodal distance) of the semipermeable membrane can be varied to affect the rate of drug release.
  • the porosity and thickness of the semipermeable membrane will permit free diffusion and exchange of the thrombolytic enzyme, essential nutrients from the circulation, and waste products of metabolism, but will prevent cell and platelet trans-membrane passage.
  • the tested semipermeable membrane will be manufactured as a cylinder and placed concentrically into a compatible standard graft.
  • the genetically engineered cells can be introduced into the graft at a time determined by one with skill in the art.
  • the genetically altered cells preferably will be injected between the two materials at the time of implantation.
  • the graft comprises an injection port means integral with the external standard graft to facilitate this injection and a vent means to prevent undue pressure during the injection process.
  • the cells can be introduced as a suspension or in any other appropriate form known to one with skill in the art. For example, cells could be microencapsulated in polymeric materials.
  • the cells can be introduced to the internal surface of the tubular external graft before putting the internal semipermeable membrane in place.
  • This prosthesis can be utilized to provide local drug delivery by utilizing arterial blood flow for prevention or treatment of any disease or condition distal to the site of arterial implantation of the device.
  • the device described herein may contain a substance in its reservoir that inhibits platelet deposition and thrombus formation or promotes thrombolysis and thrombus dissolution.
  • Examples of such substances include, but are not limited to, plasmin, urokinase (UK), single chain prourokinase (scuPA), streptokinase, prostaglandins, cyclooxygenase inhibitors, phosphodiesterase inhibitors, thromboxane synthetase inhibitors; antagonists of glycoprotein receptors including GP lb, GP Ilb/IIIa, antagonists of collagen receptors, and antagonists of platelet thrombin receptors.
  • plasmin urokinase
  • scuPA single chain prourokinase
  • streptokinase prostaglandins
  • cyclooxygenase inhibitors prostaglandins
  • cyclooxygenase inhibitors cyclooxygenase inhibitors
  • phosphodiesterase inhibitors phosphodiesterase inhibitors
  • thromboxane synthetase inhibitors antagonists of glycoprotein receptors including GP l
  • the substance in the reservoir can be any substance, including any drug, and the device can be used for local delivery of such substances to prevent or treat a variety of disease syndromes or to promote or enhance desired activity within the body.
  • the substance can be an anticoagulant, including but not limited to, heparin, hirudin, hirulog, hirugen, activated and non-activated protein C; synthetic or naturally occurring antagonists of: thrombin, Factor Xa, or other activated or non-activated coagulation proteases, coagulation factors, e.g., FBI, FIX, FNIII, FV, FVII, or tissue factor. Since the semi-permeable membrane of the present invention serves as a barrier to cellular immunity, it is possible to use allogenic cells in the graft.
  • the substance in the reservoir may affect platelet metabolic function.
  • examples of such substances include, but are not limited to, prostaglandins, cyclooxygenase inhibitors, phosphodiesterase or thromboxane synthetase inhibitors, inhibitors of calcium transport, or elevators of cyclic adenosine monophosphate (cyclic AMP) such as forskolin, or cyclic AMP agonists.
  • cyclic AMP cyclic adenosine monophosphate
  • the devices of the present invention include, but are not limited to, local drug delivery to treat cancer or to maintain perfusion of tissue or organ transplants while the body establishes revascularization of the subject tissue, or as a dialysis access graft.
  • the reservoir can be positioned proximal to the dialysis access site on the graft and utilized to deliver drugs which prevent hyperplasia, thrombosis and occlusion at the distal end of the graft.
  • drugs which prevent hyperplasia, thrombosis and occlusion at the distal end of the graft.
  • diseases such as diabetes release of appropriate amounts of insulin can be envisioned.
  • the disclosed device Utilizing the disclosed device, one skilled in the ait can determine suitable dosage requirements and treatment regimens for any substance placed in the reservoir of the device. Dosages and regimens will vary, of course, depending upon the tissue targeted for therapy and upon the particular drug utilized and can be determined by optimization procedures known in the art.
  • the device described herein is able to achieve very high drug concentrations locally while minimizing total drug requirements and circulating drug levels, therefore allowing for the efficient use of agents which are available in limited amounts or which could produce side effects.
  • cells are engineered to produce one or more desired proteins or reagents.
  • the semipermeable membrane protects the cells from cellular immunity, a number of cell types can be incorporated.
  • the preferred embodiments of the invention use mammalian cells including smooth muscle cells, epithelial cells and fibroblasts. Allogenic cells may also be used including, but not limited to, bacterial cells, yeast or other fungal cells, or the cells of higher organisms.
  • a gene encoding a protein product of interest can be introduced into said cells by the methods of genetic engineering. Methods chosen are within the knowledge of one skilled in the art, and will vary according to the cell type used. Genetic material of interest incorporated into cells according to the method described can be any selected DNA of interest (e.g., all or a portion of a gene encoding a product of interest) or any selected RNA of interest.
  • it can be DNA or RNA which is present and expressed in normal cells; DNA or RNA which does not normally occur in cells; DNA or RNA which normally occurs in cells but is not expressed in them at levels which are biologically significant (levels sufficient to produce the normal physiological effect of the protein or polypeptide it encodes); DNA or RNA which occurs in cells and has been modified in such a manner that it can be expressed in such cells; and any DNA or RNA which can be modified to be expressed in cells, alone or in any combination thereof.
  • This genetic material of interest is referred to herein as incorporated genetic material. Genetic material of interest incorporated into cells will enable the transduced cells to overproduce the desired therapeutic gene and protein.
  • Retroviruses refer to RNA viruses; that is, the viral genome is RNA.
  • This genomic RNA is, however, reverse transcribed into a DNA copy which is integrated stably and efficiently into the chromosomal DNA of transduced cells. This stably integrated DNA copy is referred to as a provirus and is inherited by daughter cells as any other gene.
  • Genetic material of interest can also be introduced into cells in such a manner that it is not incorporated stably into the recipient cells, but is expressed episomally (remains distinct or separate from the recipient cell genome), for example by using liposomes.
  • chemical or physical means can be used to introduce genetic material of interest into cells.
  • a chemical means is the commonly used calcium phosphate transfection procedure
  • an example of a physical means is electroporation whereby cells are exposed to an electric current which enables the entry into the cell of genetic material of interest.
  • the protein product of interest may be in itself a therapeutic protein.
  • the genetic material is chosen such that it encodes one of the desired protein products discussed above or a variant thereof.
  • the genetic material of interest is chosen such that the protein product is expressed in therapeutically useful amount by the genetically engineered cells.
  • the protein product chosen may not be in itself therapeutic, but may catalyze or help to catalyze the formation of a therapeutic reagent that is secreted by the genetically engineered cells.
  • VSV-G vesicular stomatitis virus G glycoprotein
  • 293T cells American Type Culture Collection, CRL-1573
  • plasmids vector plasmid, gag-pol genes, and env gene of amphotropic or NSV-G gene.
  • the vector plasmid is LtS ⁇ for vector delivering tPA gene (Dichek, D.A. et al.
  • Transductions of EC with retroviral vectors were accomplished by placing 5 ml of the LtS ⁇ viral supernatants with polybrene (8 ⁇ g/ml) onto the cultured EC in a 100 mm plate ( ⁇ lxl 0 6 cells). One day after the transduction, the cells were cultured in the medium containing 0.6 mg/ml neomycin analog G418 (Gibco/BRL) for 10- 14 days to select the transduced cells. The pool of the selected cells were named as
  • tPA concentration EC/tPA and EC were plated on 24-well plates with 2x10 cells/well and were cultured until 90% confluent. The culture medium was then replaced with 1 ml of serum-free William medium. The cells were continually cultured at 37 °C for 24, 48, 72, and 96 hrs and the supernatants at each time point were collected and stored at -70 °C for different assays described below.
  • the fibrinolytic enzyme activity of the cell culture supernatant was determined by chromogenic assay using Chromolize tPA assay kit from Biopool (Ventura, CA).
  • the concentration of tPA in the supernatant was determined by measuring the tPA antigen concentration with TintElize tPA ELISA assay kit from Biopool according to the manufacturer's directions.
  • Endothelial and Smooth Muscle Cell Harvest and Transduction Endothelial cells can be obtained by standard procedures from umbilical vein, saphenous vein or other sources (e.g., Balconi et al. (1986) Med. Biol. 64: 231-245; Ryan et al. (1985) Tissue Cell 17:171-176; Budd et al. (1989) Br. J. Surg. 76: 1259-1261). Cells can be harvested by mechanical or, preferably, enzymatic methods. In specific, excreta human vein samples can be obtained from patients undergoing bypass procedures.
  • the vein samples are flushed with RPM1 1640 media containing 20 mM HEPES, 2 mM L-glutamine, 50 units/ml penicillin, 50 ⁇ g/ml gentamicin and 2.5 ⁇ g/ml amphotericin B, clamped at one end, filled with 0.1% collagenase I in Dulbecco's phosphate buffered saline (DPBS); and incubated 15 min at 37 °C to dislodge the endothelial cells.
  • the dissociated ECs are pelleted by centrifugation at room temperature, 1200 rpm (200xg) for 5 min. Cells are resuspended and plated on fibronectin (0.1% in PBS) pre-coated dishes in Ml 99 medium (Gibco/BRL,
  • the vein is incised longitudinally and the intimal layer is scraped off with a scalpel.
  • the cellular composition of the underlying media is primarily vascular smooth muscle.
  • the opened vein is placed into tissue culture flasks. These tissues are agitated in DPBS containing collagenase I (1.8 mg/ml) and elastase (0.2 mg/ml) for 1 hour at 37 °C. Single-cell suspension supernatants are removed and pelleted by centrifugation at 1200 rpm for 5 min.
  • These SMCs are resuspended and cultured in Ml 99 medium containing 20% FBS, L-glutamine (2 mM), and the antibiotics mentioned above.
  • ECs are characterized by cellular morphological observation through microscopy as well as fluorescent staining for von Willebrand factor and low-density lipoprotein uptake.
  • SMCs are identified by positive reaction in a smooth muscle ⁇ -actin assay. Both ECs and SMCs are passaged at a 1 :3 ratio and be used for experiments between passages 2 and 15.
  • the cultured cells are transduced with retroviral vectors by mixing the cultured cells with the viral supernatants.
  • Transduced cells are selected with neomycin analog G418 to produce a population of cells secreting the targeted gene product.
  • the enzyme activity and antigen concentration of tPA in medium conditioned with transduced cells is measured using Chromolize tPA and TintElize tPA assay kits from Biopool (Ventura, CA).
  • Northern blot analysis of transduced ECs and SMCs is performed to identify the persistence and stability of gene expression in the transduced cells.
  • the total cellular RNA is extracted from the ECs and SMCs (Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162:156-9). Electrophoresis of 7-10 ⁇ g of each sample through 1.2% formaldehyde agarose gels is performed.
  • RNA is then blotted onto nitrocellulose membranes (Schleicher and Schuell, Inc., Keene, NH), vacuum dried, and hybridized with a radiolabeled fragment of plasminogen activator cDNA.
  • Membranes are then washed in 0.2 N saline citrate and 0.1% sodium dodecyl sulfate at 55 °C and subsequently exposed to phosphor screens for quantification on a Phosphorimager
  • the semi-permeable membranes are tested for their permeability by sealing and culturing transduced cells in 1 -2 cm "tea bags" made from the membrane.
  • the activity of the specific genetic material e.g., polypeptide, protein, and the like is measured on both sides of the membrane.
  • grafts are implanted into a porcine animal model using standard surgery and/or stent graft technology. Patency rates, tissue response (e.g. myointimal hyperplasia), and serological evidence of thrombolysis are compared to control animals undergoing similar procedures using untransduced cells.
  • cells are harvested from the animal, then amplified and transduced with tPA vectors. These transduced cells are injected into a space created between a 6 mm internal diameter cylindrical semi-permeable membrane mounted in a 7 mm outer diameter tubule PTFE graft. The length of the graft is 3-4 cm. A transverse aortotomy is performed above the iliac bifurcation.
  • the graft is entirely premounted on a short modified balloon expandable stent (Johnson and Johnson, Warren, NJ). The assembly is deployed over a balloon catheter into the infrarenal aorta just below the kidneys.
  • Example 1 Enzyme Activity of TPA Transduced ECs.
  • the membrane design parameters include inertness, strength, durability, and pore size that is smaller than a platelet but large enough for protein and lipid diffusion. It was shown that cells packaged within these membranes can survive for two weeks, and secreted tPA was detected on both sides of the membrane. No evidence of these cells being able to traverse the membranes has been obtained in vitro. Cells packaged within a "tea bag" made of these membranes can survive and grow for one month

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Abstract

L'invention concerne une greffe prothétique, composée de deux tubes prothétiques coaxiaux, lesquels enferment un espace contenant des cellules mises au point par génie génétique et destinées à surproduire une enzyme thrombolytique. Ces cellules peuvent comprendre des cellules endothéliales, des cellules de muscles lisses, des fibroblastes, et des cellules souches. Le tube extérieur est une greffe classique, et le tube intérieur est une membrane semi-perméable qui sert à protéger les cellules, mises au point par génie génétique, de la contrainte de cisaillement du débit sanguin ainsi que de l'exposition à des cellules et plaquettes inflammatoires, tout en permettant un échange et une diffusion libres de l'enzyme thrombolytique, de substrats métaboliques provenant de la circulation, et de déchets du métabolisme.
PCT/US1999/004500 1998-03-02 1999-03-01 Mise au point genetique de greffes de pontage Ceased WO1999044487A2 (fr)

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Application Number Priority Date Filing Date Title
AU29769/99A AU2976999A (en) 1998-03-02 1999-03-01 Genetic engineering of prosthetic bypass grafts

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US7644098P 1998-03-02 1998-03-02
US60/076,440 1998-03-02

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102004048458A1 (de) * 2004-10-05 2006-04-06 Admedes Schuessler Gmbh Stentsystem zum Implantieren in einen lebenden Körper
WO2006085044A1 (fr) * 2005-02-14 2006-08-17 Vascutek Limited Vaisseau sanguin artificiel
WO2009134991A2 (fr) 2008-04-30 2009-11-05 Ethicon, Inc. Vaisseau sanguin obtenu par ingénierie tissulaire

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102004048458A1 (de) * 2004-10-05 2006-04-06 Admedes Schuessler Gmbh Stentsystem zum Implantieren in einen lebenden Körper
DE102004048458B4 (de) * 2004-10-05 2020-08-13 Admedes Schuessler Gmbh Aneurysmen-Stent zum Implantieren in einen lebenden Körper und Verwendung desselben als ballonexpandierter Aneurysmen-Stent
WO2006085044A1 (fr) * 2005-02-14 2006-08-17 Vascutek Limited Vaisseau sanguin artificiel
US7780622B2 (en) 2005-02-14 2010-08-24 Vascutek Limited Artificial blood vessel
WO2009134991A2 (fr) 2008-04-30 2009-11-05 Ethicon, Inc. Vaisseau sanguin obtenu par ingénierie tissulaire
WO2009134991A3 (fr) * 2008-04-30 2010-07-29 Ethicon, Inc. Vaisseau sanguin obtenu par ingénierie tissulaire
JP2011519616A (ja) * 2008-04-30 2011-07-14 エシコン・インコーポレイテッド 組織工学による血管
US9290742B2 (en) 2008-04-30 2016-03-22 Cordis Corporation Tissue engineered blood vessel

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