US20070287682A1 - Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor - Google Patents

Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor Download PDF

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US20070287682A1
US20070287682A1 US11/788,728 US78872807A US2007287682A1 US 20070287682 A1 US20070287682 A1 US 20070287682A1 US 78872807 A US78872807 A US 78872807A US 2007287682 A1 US2007287682 A1 US 2007287682A1
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cells
kit
blood vessel
catheter
patient
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Elizabeth Nabel
Gary Nabel
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University of Michigan
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University of Michigan
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • AHUMAN NECESSITIES
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    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00Surgical instruments, devices or methods, e.g. tourniquets
    • A61B17/22Implements for squeezing-off ulcers or the like on the inside of inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; Calculus removers; Calculus smashing apparatus; Apparatus for removing obstructions in blood vessels, not otherwise provided for
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6957Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a device or a kit, e.g. stents or microdevices
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • AHUMAN NECESSITIES
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    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0019Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • AHUMAN NECESSITIES
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    • A61LMETHODS 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
    • A61L29/00Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • A61L29/14Materials characterised by their function or physical properties, e.g. lubricating compositions
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    • A61B17/22Implements for squeezing-off ulcers or the like on the inside of inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; Calculus removers; Calculus smashing apparatus; Apparatus for removing obstructions in blood vessels, not otherwise provided for
    • A61B2017/22038Implements for squeezing-off ulcers or the like on the inside of inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; Calculus removers; Calculus smashing apparatus; Apparatus for removing obstructions in blood vessels, not otherwise provided for with a guide wire
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    • A61B2017/22051Implements for squeezing-off ulcers or the like on the inside of inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; Calculus removers; Calculus smashing apparatus; Apparatus for removing obstructions in blood vessels, not otherwise provided for with an inflatable part, e.g. balloon, for positioning, blocking, or immobilisation
    • A61B2017/22054Implements for squeezing-off ulcers or the like on the inside of inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; Calculus removers; Calculus smashing apparatus; Apparatus for removing obstructions in blood vessels, not otherwise provided for with an inflatable part, e.g. balloon, for positioning, blocking, or immobilisation with two balloons
    • AHUMAN NECESSITIES
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    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
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    • A61B17/22Implements for squeezing-off ulcers or the like on the inside of inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; Calculus removers; Calculus smashing apparatus; Apparatus for removing obstructions in blood vessels, not otherwise provided for
    • A61B2017/22082Implements for squeezing-off ulcers or the like on the inside of inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; Calculus removers; Calculus smashing apparatus; Apparatus for removing obstructions in blood vessels, not otherwise provided for after introduction of a substance
    • AHUMAN NECESSITIES
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    • A61B17/22Implements for squeezing-off ulcers or the like on the inside of inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; Calculus removers; Calculus smashing apparatus; Apparatus for removing obstructions in blood vessels, not otherwise provided for
    • A61B2017/22082Implements for squeezing-off ulcers or the like on the inside of inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; Calculus removers; Calculus smashing apparatus; Apparatus for removing obstructions in blood vessels, not otherwise provided for after introduction of a substance
    • A61B2017/22084Implements for squeezing-off ulcers or the like on the inside of inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; Calculus removers; Calculus smashing apparatus; Apparatus for removing obstructions in blood vessels, not otherwise provided for after introduction of a substance stone- or thrombus-dissolving
    • AHUMAN NECESSITIES
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    • A61LMETHODS 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
    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • AHUMAN NECESSITIES
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    • A61LMETHODS 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
    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/258Genetic materials, DNA, RNA, genes, vectors, e.g. plasmids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/62Encapsulated active agents, e.g. emulsified droplets
    • A61L2300/626Liposomes, micelles, vesicles
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/64Animal cells

Definitions

  • the present invention relates to the treatment of diseases by the site-specific instillation or transformation of cells and kits therefor.
  • the pathogenesis of atherosclerosis is characterized by three fundamental biological processes. These are: 1) proliferation of intimal smooth muscle cells together with accumulated macrophages; 2) formation by the proliferated smooth muscle cells of large amounts of connective tissue matrix; and 3) accumulation of lipid, principally in the form of cholesterol esters and free cholesterol, within cells as well as in surrounding-connective tissue.
  • Endothelial cell injury is an initiating event and is manifested by interference with the permeability barrier of the endothelium, alterations in the non-thrombogenic properties of the endothelial surface, and promotion of procoagulant properties of the endothelium.
  • Monocytes migrate between endothelial cells, become active as scavenger cells, and differentiate into macrophages.
  • Macrophages then synthesize and secrete growth factors including platelet derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor alpha (TGF- ⁇ ). These growth factors are extremely potent in stimulating the migration and proliferation of fibroblasts and smooth muscle cells in the atherosclerotic plaque.
  • platelets may interact with the injured endothelial cell and the activated macrophage to potentiate the elaboration of growth factors and thrombus formation.
  • thrombus formation in acute myocardial ischemia and restenosis following coronary angioplasty involves common cellular events, including endothelial injury and release of potent growth factors by activated macrophages and platelets.
  • Coronary angioplasty produces fracturing of the atherosclerotic plaque and removal of the endothelium. This vascular trauma promotes platelet aggregation and thrombus formation at the PTCA site. Further release of mitogens from platelets and macrophages, smooth muscle cell proliferation and monocyte infiltration result in restenosis.
  • a solution to restenosis is to prevent platelet aggregation, thrombus formation, and smooth muscle cell proliferation.
  • Thrombus formation is also a critical cellular event in the transition from stable to unstable coronary syndromes.
  • the pathogenesis most likely involves acute endothelial cell injury and or plaque rupture, promoting dysjunction of endothelial cell attachment, and leading to the exposure of underlying macrophage foam cells. This permits the opportunity for circulating platelets to adhere, aggregate, and form thrombi.
  • thrombolytic agents such as tissue plasminogen activator (tPA) results in lysis of thrombus in approximately 70% of patients experiencing an acute myocardial infarction. Nonetheless, approximately 30% of patients fail to reperfuse, and of those patients who undergo initial reperfusion of the infarct related artery, approximately 25% experience recurrent thrombosis within 24 hours. Therefore, an effective therapy for rethrombosis remains a major therapeutic challenge facing the medical community today.
  • tissue plasminogen activator tPA
  • an effective therapy for rethrombosis is by far not the only major therapeutic challenge existing today.
  • Others include the treatment of other ischemic conditions, including unstable angina, myocardial infarction or chronic tissue ischemia; or even the treatment of systemic and inherited diseases or cancers. These might be treated by the effective administration of anticoagulants, vasodilatory, angiogenic, growth factors or growth inhibitors to a patient.
  • anticoagulants vasodilatory, angiogenic, growth factors or growth inhibitors
  • one object of the present invention is to provide a novel method for the site-specific administration of a therapeutic agent.
  • Site-specific instillation of normal cells can be used to replace damaged cells, while instillation of transformed cells can be used to cause the expression of either a defective endogenous gene or an exogenous gene or the suppression of an endogenous gene product.
  • Instillation of cells in the walls of the patient's blood vessels can be used to cause the steady perfusion of a therapeutic agent in the blood stream.
  • FIGS. 1 and 2 illustrate the use of a catheter in accordance with the invention to surgically or percutaneously implant cells in a blood vessel or to transform in vivo cells present on the wall of a patient's blood vessel.
  • the present invention is used to treat diseases, such as inherited diseases, systemic diseases, diseases of the cardiovascular system, diseases of particular organs, or tumors by instilling normal or transformed cells or by transforming cells.
  • diseases such as inherited diseases, systemic diseases, diseases of the cardiovascular system, diseases of particular organs, or tumors by instilling normal or transformed cells or by transforming cells.
  • the cells which may be instilled in the present method include endothelium, smooth muscle, fibroblasts, monocytes, macrophages, and parenchymal cells. These cells may produce proteins which may have a therapeutic or diagnostic effect and which may be naturally occurring or arise from recombinant genetic material.
  • FIG. 1 this figure illustrates the practice of the present invention with a catheter having a design as disclosed in U.S. Pat. No. 4,636,195, which is hereby incorporated by reference.
  • This catheter may be used to provide normal or genetically altered cells on the walls of a vessel or to introduce vectors for the local transformation of cells.
  • 5 is the wall of the blood vessel.
  • the figure shows the catheter body 4 held in place by the inflation of inflatable balloon means 1 and 2 .
  • the section of the catheter body 4 situated between balloon means 1 and 2 is equipped with instillation port means 3 .
  • the catheter may be further equipped with a guidewire means 6 .
  • FIG. 2 illustrates the use of a similar catheter, distinguished from the catheter illustrated in FIG. 1 by the fact that it is equipped with only a single inflatable balloon means 2 and a plurality of instillation port means 3 .
  • This catheter may contain up to twelve individual instillation port means 3 , with five being illustrated.
  • the catheter may be introduced into the major artery supplying the tissue.
  • Cells containing recombinant genes or vectors can be introduced through a central instillation port after temporary occlusion of the arterial circulation. In this way, cells or vector DNA may be delivered to a large amount of parenchymal tissue distributed through the capillary circulation.
  • Recombinant genes can also be introduced into the vasculature using the double balloon catheter technique in the arterial circulation proximal to the target organ. In this way, the recombinant genes may be secreted directly into the circulation which perfuse the involved tissue or may be synthesized directly within the organ.
  • the therapeutic agents are secreted by vascular cells supplying specific organs affected by the disease.
  • ischemic cardiomyopathy may be treated by introducing angiogenic factors into the coronary circulation. This approach may also be used for peripheral vascular or cerebrovascular diseases where angiogenic factors may improve circulation to the brain or other tissues. Diabetes mellitus may be treated by introduction of glucose-responsive insulin secreting cells in the portal circulation where the liver normally sees a higher insulin concentration than other tissues.
  • the present method may also be used for delivery of recombinant genes to parenchymal tissues, because high concentrations of viral vector and other vectors can be delivered to a specific circulation.
  • deficiencies of organ-specific proteins may also be treated.
  • ⁇ -antitrypsin inhibitor deficiency or hyperchloresterolemia may be treated by introduction of ⁇ -antitrypsin or the LDL receptor gene.
  • this approach may be used for the treatment of malignancy. Secretion of specific recombinant toxin genes into the circulation of inoperable tumors provides a therapeutic effect. Examples include acoustic neuromas or certain hemangiomas which are otherwise unresectable.
  • these therapeutic recombinant genes are introduced in cells supplying the circulation of the involved organ.
  • the arterial and capillary circulations are the preferred locations for introduction of these cells, venous systems are also suitable.
  • the present invention provides for the expression of proteins which ameliorate this condition in situ.
  • proteins which ameliorate this condition in situ.
  • vascular cells are found at these sites, they are used as carriers to convey the therapeutic agents.
  • the invention thus, in one of its aspects, relies on genetic alteration of endothelial and other vascular cells or somatic cell gene therapy, for transmitting therapeutic agents (i.e., proteins, growth factors) to the localized region of vessel injury.
  • therapeutic agents i.e., proteins, growth factors
  • the gene which is to be implanted into the cell must be identified and isolated.
  • the gene to be expressed must be cloned and available for genetic manipulation.
  • the gene must be introduced into the cell in a form that will be expressed or functional.
  • the genetically altered cells must be situated in the vascular region where it is needed.
  • the altered cells or appropriate vector may be surgically, percutaneously, or intravenously introduced and attached to a section of a patient's vessel wall.
  • some of the cells existing on the patient's vessel wall are transformed with the desired genetic material or by directly applying the vector.
  • vascular cells which are not genetically modified can be introduced by these methods to replace cells lost or damaged on the vessel surface.
  • Any blood vessel may be treated in accordance with this invention; that is, arteries, veins, and capillaries. These blood vessels may be in or near any organ in the human, or mammalian, body.
  • a cell line is established and stored in liquid nitrogen. Prior to cryopreservation, an aliquot is taken for infection or transfection with a vector, viral or otherwise, containing the desired genetic material.
  • Endothelial or other vascular cells may be derived enzymatically from a segment of a blood vessel, using techniques previously described in J. W. Ford, et al., In Vitro, 17, 40 (1981).
  • the vessel is excised, inverted over a stainless steel rod and incubated in 0.1% trypsin in Ca ++ - and Mg ++ -free Hank's balanced salt solution (BSS) with 0.125% EDTA at pH 8 for 10 min at 37° C.
  • BSS Hank's balanced salt solution
  • Cells (0.4 to 1.5 ⁇ 10 6 ) are collected by centrifugation and resuspended in medium 199 (GIBCO) containing 10% fetal bovine serum, endothelial cell growth supplement (ECGS, Collaborative Research, Waltham, Mass.) at 25 ⁇ g/ml, heparin at 15 U/ml, and gentamicin (50 ⁇ g/ml).
  • GEBCO medium 199
  • ECGS Endothelial cell growth supplement
  • heparin at 15 U/ml
  • gentamicin 50 ⁇ g/ml
  • Cells are added to a 75 cm 2 tissue culture flask precoated with gelatin (2 mg/ml in distilled water). Cells are fed every second day in the above medium until they reach confluence.
  • the ECGS and heparin may be omitted from the medium when culturing porcine endothelium. If vascular smooth muscle cells or fibroblasts are desired the heparin and ECGS can be omitted entirely from the culturing procedure.
  • Aliquots of cells are stored in liquid nitrogen by resuspending to approximately 10 6 cells in 0.5 ml of ice cold fetal calf serum on ice.
  • An equal volume of ice cold fetal calf serum containing 10% DMSO is added, and cells are transferred to a prechilled screw cap Corning freezing tube. These cells are transferred to a ⁇ 70° C. freezer for 3 hours before long term storage in liquid nitrogen.
  • the cells are then infected with a vector containing the desired genetic material.
  • the patient is prepared for catheterization either by surgery or percutaneously, observing strict adherence to sterile techniques.
  • a cutdown procedure is performed over the target blood vessel or a needle is inserted into the target blood vessel after appropriate anesthesia.
  • the vessel ( 5 ) is punctured and a catheter, such as described in U.S. Pat. No. 4,636,195, which is hereby incorporated by reference (available from USCI, Billerica, Mass.) is advanced by guidewire means ( 6 ) under fluoroscopic guidance, if necessary, into the vessel ( 5 ) ( FIG. 1 ).
  • This catheter means ( 4 ) is designed to introduce infected endothelial cells into a discrete region of the artery.
  • the catheter has a proximal and distal balloon means ( 2 ) and ( 1 ), respectively, (e.g., each balloon means may be about 3 mm in length and about 4 mm in width), with a length of catheter means between the balloons.
  • the length of catheter means between the balloons has a port means connected to an instillation port means ( 3 ).
  • a region of the blood vessel is identified by anatomical landmarks and the proximal balloon means ( 2 ) is inflated to denude the endothelium by mechanical trauma (e.g., by forceful passage of a partially inflated balloon catheter within the vessel) or by mechanical trauma in combination with small amounts of a proteolytic enzyme such as dispase, trypsin, collagenase, papain, pepsin, chymotrypsin or cathepsin, or by incubation with these proteolytic enzymes alone.
  • a proteolytic enzyme such as dispase, trypsin, collagenase, papain, pepsin, chymotrypsin or cathepsin, or by incubation with these proteolytic enzymes alone.
  • lipases may be used.
  • the region of the blood vessel may also be denuded by treatment with a mild detergent or the like, such as NP-40, Triton X100, deoxycholate, or SDS.
  • the denudation conditions are adjusted to achieve essentially complete loss of endothelium for cell transfers or approximately 20 to 90%, preferably 50 to 75%, loss of cells from the vessel wall for direct infection. In some instances cell removal may not be necessary.
  • the catheter is then advanced so that the instillation port means ( 3 ) is placed in the region of denuded endothelium. Infected, transfected or normal cells are then instilled into the discrete section of artery over thirty minutes. If the blood vessel is perfusing an organ which can tolerate some ischemia, e.g., skeletal muscle, distal perfusion is not a major problem, but can be restored by an external shunt if necessary, or by using a catheter which allows distal perfusion. After instillation of the infected endothelial cells, the balloon catheter is removed, and the arterial puncture site and local skin incision are repaired. If distal perfusion is necessary, an alternative catheter designed to allow distal perfusion may be used.
  • Surgical techniques are used as described above. Instead of using infected cells, a high titer desired genetic material transducing viral vector (105 to 106 particles/ml) or DNA complexed to a delivery vector is directly instilled into the vessel wall using the double balloon catheter technique.
  • This vector is instilled in medium containing serum and polybrene (10 ⁇ g/ml) to enhance the efficiency of infection. After incubation in the dead space created by the catheter for an adequate period of time (0.2 to 2 hours or greater), this medium is evacuated, gently washed with phosphate-buffered saline, and arterial circulation is restored. Similar protocols are used for post operative recovery.
  • the vessel surface can be prepared by mechanical denudation alone, in combination with small amounts of proteolytic enzymes such as dispase, trypsin, collagenase or cathepsin, or by incubation with these proteolytic enzymes alone.
  • proteolytic enzymes such as dispase, trypsin, collagenase or cathepsin, or by incubation with these proteolytic enzymes alone. The denudation conditions are adjusted to achieve the appropriate loss of cells from the vessel wall.
  • Viral vector or DNA-vector complex is instilled in Dulbecco's modified Eagle's medium using purified virus or complexes containing autologous serum, and adhesive molecules such as polybrene (10 ⁇ g/ml), poly-L-lysine, dextran sulfate, or any polycationic substance which is physiologically suitable, or a hybrid antibody directed against the envelope glycoprotein of the virus or the vector and the relevant target in the vessel wall or in the tissue perfused by the vessel to enhance the efficiency of infection by increasing adhesion of viral particles to the relevant target cells.
  • the hybrid antibody directed against the envelope glycoprotein of the virus or the vector and the relevant target cell can be made by one of two methods. Antibodies directed against different epitopes can be chemically crosslinked (G.
  • a different protocol for instillation can also be used.
  • This second approach involves the use of a single balloon means (2) catheter with multiple port means (3) which allow for high pressure delivery of the retrovirus into partially denuded arterial segments.
  • the vessel surface is prepared as described above and defective vector is introduced using similar adhesive molecules.
  • the use of a high pressure delivery system serves to optimize the interaction of vectors with cells in adjacent vascular tissue.
  • the present invention also provides for the use of growth factors delivered locally by catheter or systemically to enhance the efficiency of infection.
  • herpes virus, adenovirus, or other viral vectors are suitable vectors for the present technique.
  • Direct transformation of organ or tissue cells may be accomplished by one of two methods.
  • a high pressure transfection is used. The high pressure will cause the vector to migrate through the blood vessel walls into the surrounding tissue.
  • injection into a capillary bed optionally after injury to allow leaking, gives rise to direct infection of the surrounding tissues.
  • the time required for the instillation of the vectors or cells will depend on the particular aspect of the invention being employed. Thus, for instilling cells or vectors in a blood vessel a suitable time would be from 0.01 to 12 hrs, preferably 0.1 to 6 hrs, most preferably 0.2 to 2 hrs. Alternatively for high pressure instillation of vectors or cells, shorter times might be preferred.
  • genetic material generally refers to DNA which codes for a protein. This phrase also encompasses RNA when used with an RNA virus or other vector based on RNA.
  • Transformation is the process by which cells have incorporated an exogenous gene by direct infection, transfection or other means of uptake.
  • vector is well understood and is synonymous with the often-used phrase “cloning vehicle”.
  • a vector is non-chromosomal double-stranded DNA comprising an intact replicon such that the vector is replicated when placed within a unicellular organism, for example by a process of transformation.
  • Viral vectors include retroviruses, adenoviruses, herpesvirus, papovirus, or otherwise modified naturally occurring viruses.
  • Vector also means a formulation of DNA with a chemical or substance which allows uptake by cells.
  • the present invention provides for inhibiting the expression of a gene.
  • Four approaches may be utilized to accomplish this goal. These include the use of antisense agents, either synthetic oligonucleotides which are complementary to the mRNA (Maher III, L. J. and Dolnick, B. J. Arch. Biochem. Biophys., 253, 214-220 (1987) and (Zamecnik, P. C., et al., Proc. Natl. Acad. Sci., 83, 4143-4146 (1986)), or the use of plasmids expressing the reverse complement of this gene (Izant, J. H.
  • RNA sequences can specifically degrade RNA sequences (Uhlenbeck, O. C., Nature, 328, 596-600 (1987), Haseloff, J. and Gerlach, W. L., Nature, 334, 585-591 (1988)).
  • the third approach involves “intracellular immunization”, where analogues of intracellular proteins can interfere specifically with their function (Friedman, A. D., Triezenberg, S. J. and McKnight, S. L., Nature, 335, 452-454 (1988)), described in detail below.
  • the first approaches may be used to specifically eliminate transcripts in cells.
  • the loss of transcript may be confirmed by S1 nuclease analysis, and expression of binding protein determined using a functional assay.
  • Single-stranded oligonucleotide analogues may be used to interfere with the processing or translation of the transcription factor mRNA.
  • synthetic oligonucleotides or thiol-derivative analogues (20-50 nucleotides) complementary to the coding strand of the target gene may be prepared.
  • These antisense agents may be prepared against different regions of the mRNA. They are complementary to the 5′ untranslated region, the translational initiation site and subsequent 20-50 base pairs, the central coding region, or the 3′ untranslated region of the gene.
  • the antisense agents may be incubated with cells transfected prior to activation.
  • the efficacy of antisense competitors directed at different portions of the messenger RNA may be compared to determine whether specific regions may be more effective in preventing the expression of these
  • RNA can also function in an autocatalytic fashion to cause autolysis or to specifically degrade complementary RNA sequences (Uhlenbeck, O. C., Nature, 328, 596-600 (1987), Haseloff, J. and Gerlach, W. L., Nature, 334, 585-591 (1988), and Hutchins, C. J., et al, Nucleic Acids Res., 14, 3627-3640 (1986)).
  • the requirements for a successful RNA cleavage include a hammerhead structure with conserved RNA sequence at the region flanking this structure. Regions adjacent to this catalytic domain are made complementary to a specific RNA, thus targeting the ribozyme to specific cellular mRNAs.
  • the mRNA encoding this gene may be specifically degraded using ribozymes.
  • any GUG sequence within the RNA transcript can serve as a target for degradation by the ribozyme. These may be identified by DNA sequence analysis and GUG sites spanning the RNA transcript may be used for specific degradation. Sites in the 5′ untranslated region, in the coding region, and in the 3′ untranslated region may be targeted to determine whether one region is more efficient in degrading this transcript. Synthetic oligonucleotides encoding 20 base pairs of complementary sequence upstream of the GUG site, the hammerhead structure and ⁇ 20 base pairs of complementary sequence downstream of this site may be inserted at the relevant site in the cDNA.
  • the ribozyme may be targeted to the same cellular compartment as the endogenous message.
  • the ribozymes inserted downstream of specific enhancers, which give high level expression in specific cells may also be generated.
  • These plasmids may be introduced into relevant target cells using electroporation and cotransfection with a neomycin resistant plasmid, pSV2-Neo or another selectable marker.
  • the expression of these transcripts may be confirmed by Northern blot and S1 nuclease analysis. When confirmed, the expression of mRNA may be evaluated by S1 nuclease protection to determine whether expression of these transcripts reduces steady state levels of the target mRNA and the genes which it regulates. The level of protein may also be examined.
  • Genes may also be inhibited by preparing mutant transcripts lacking domains required for activation. Briefly, after the domain has been identified, a mutant form which is incapable of stimulating function is synthesized. This truncated gene product may be inserted downstream of the SV-40 enhancer in a plasmid containing the neomycin resistance gene (Mulligan, R. and Berg, P., Science, 209, 1422-1427 (1980) (in a separate transcription unit). This plasmid may be introduced into cells and selected using G418. The presence of the mutant form of this gene will be confirmed by S1 nuclease analysis and by immunoprecipitation. The function of the endogenous protein in these cells may be evaluated in two ways.
  • the expression of the normal gene may be examined.
  • the known function of these proteins may be evaluated.
  • this mutant intercellular interfering form is toxic to its host cell, it may be introduced on an inducible control element, such as metallothionein promoter. After the isolation of stable lines, cells may be incubated with Zn or Cd to express this gene. Its effect on host cells can then be evaluated.
  • recombinant vectors in which, for example, retroviruses and plasmids are made to contain exogenous RNA or DNA, respectively.
  • the recombinant vector can include heterologous RNA or DNA, by which is meant RNA or DNA that codes for a polypeptide ordinarily not produced by the organism susceptible to transformation by the recombinant vector.
  • heterologous RNA or DNA by which is meant RNA or DNA that codes for a polypeptide ordinarily not produced by the organism susceptible to transformation by the recombinant vector.
  • the production of recombinant RNA and DNA vectors is well understood and need not be described in detail. However, a brief description of this process is included here for reference.
  • a retrovirus or a plasmid vector can be cleaved to provide linear RNA or DNA having ligatable termini. These termini are bound to exogenous RNA or DNA having complementary like ligatable termini to provide a biologically functional recombinant RNA or DNA molecule having an intact replicon and a desired phenotypical property.
  • RNA and DNA recombination A variety of techniques are available for RNA and DNA recombination in which adjoining ends of separate RNA or DNA fragments are tailored to facilitate ligation.
  • the exogenous, i.e., donor, RNA or DNA used in the present invention is obtained from suitable cells.
  • the vector is constructed using known techniques to obtain a transformed cell capable of in vivo expression of the therapeutic agent protein.
  • the transformed cell is obtained by contacting a target cell with a RNA or DNA-containing formulation permitting transfer and uptake of the RNA or DNA into the target cell.
  • RNA or DNA-containing formulations include, for example, retroviruses, plasmids, liposomal formulations, or plasmids complexes with polycationic substances such as poly-L-lysine, DEAC-dextran and targeting ligands.
  • the present invention thus provides for the genetic alteration of cells as a method to transmit therapeutic or diagnostic agents to localized regions of the blood vessel for local or systemic purposes.
  • the range of recombinant proteins which may be expressed in these cells is broad and varied. It includes gene transfer using vectors expressing such proteins as tPA for the treatment of thrombosis and restenosis, angiogenesis or growth factors for the purpose of revascularization, and vasoactive factors to alleviate vasoconstriction or vasospasm.
  • This technique can also be extended to genetic treatment of inherited disorders, or acquired diseases, localized or systemic.
  • the present invention may also be used to introduce normal cells to specific sites of cell loss, for example, to replace endothelium damaged during angioplasty or catheterization.
  • ischemic diseases thrombotic diseases
  • genetic material coding for tPA or modifications thereof urokinase or streptokinase is used to transform the cells.
  • ischemic organ e.g., heart, kidney, bowel, liver, etc.
  • genetic material coding for recollateralization agents such as transforming growth factor ⁇ (TGF- ⁇ ), transforming growth factor ⁇ (TGF- ⁇ ), angiogenin, tumor necrosis factor ⁇ , tumor necrosis factor ⁇ , acidic fibroblast growth factor or basic fibroblast growth factor
  • vasomotor diseases genetic material coding for vasodilators or vasoconstrictors may be used. These include atrial natriuretic factor, platelet-derived growth factor or endothelin.
  • genetic material coding for insulin may be used.
  • the present invention can also be used in the treatment of malignancies by placing the transformed cells in proximity to the malignancy.
  • genetic material coding for diphtheria toxin, pertussis toxin, or cholera toxin may be used.
  • genetic material coding for soluble CD4 or derivatives thereof may be used.
  • genetic material coding for the needed substance for example, human growth hormone, is used. All of these genetic materials are readily available to one skilled in this art.
  • the present invention provides a kit for treating a disease in a patient which contains a catheter and a solution which contains either an enzyme or a mild detergent, in which the catheter is adapted for insertion into a blood vessel and contains a main catheter body having a balloon element adapted to be inserted into said vessel and expansible against the walls of the blood vessel so as to hold the main catheter body in place in the blood vessel, and means carried by the main catheter body for delivering a solution into the blood vessel, and the solution which contains the enzyme or mild detergent is a physiologically acceptable solution.
  • the solution may contain a proteolytic enzyme, such as dispase, trypsin, collagenase, papain, pepsin, or chymotrypsin. In addition to proteolytic enzymes, lipases may be used.
  • the solution may contain NP-40, Triton X100, deoxycholate, SDS or the like.
  • the kit may contain a physiological acceptable solution which contains an agent such as heparin, poly-L-lysine, polybrene, dextran sulfate, a polycationic material, or bivalent antibodies.
  • This solution may also contain vectors or cells (normal or transformed).
  • the kit may contain a catheter and both a solution which contains an enzyme or mild detergent and a solution which contains an agent such as heparin, poly-L-lysine, polybrene, dextran sulfate, a polycationic material or bivalent antibody and which may optionally contain vectors or cells.
  • the kit may contain a catheter with a single balloon and central distal perfusion port, together with acceptable solutions to allow introduction of cells in a specific organ or vectors into a capillary bed or cells in a specific organ or tissue perfused by this capillary bed.
  • the kit may contain a main catheter body which has two spaced balloon elements adapted to be inserted in a blood vessel with both being expansible against the walls of the blood vessel for providing a chamber in the blood vessel, and to hold the main catheter body in place.
  • the means for delivering a solution into the chamber is situated in between the balloon elements.
  • the kit may contain a catheter which possesses a plurality of port means for delivering the solution into the blood vessel.
  • the present invention represents a method for treating a disease in a patient by causing a cell attached onto the walls of a vessel or the cells of an organ perfused by this vessel in the patient to express an exogeneous therapeutic agent protein, wherein the protein treats the disease or may be useful for diagnostic purposes.
  • the present method may be used to treat diseases, such as an ischemic disease, a vasomotor disease, diabetes, a malignancy, AIDS or a genetic disease.
  • the present method may use exogeneous therapeutic agent proteins, such as tPA and modifications thereof, urokinase, streptokinase, acidic fibroblast growth factor, basic fibroblast growth factor, tumor necrosis factor ⁇ , tumor necrosis factor ⁇ , transforming growth factor ⁇ , transforming growth factor ⁇ , atrial natriuretic factor, platelet-derived growth factor, endothelian, insulin, diphtheria toxin, pertussis toxin, cholera toxin, soluble CD4 and derivatives thereof, and growth hormone to treat diseases.
  • exogeneous therapeutic agent proteins such as tPA and modifications thereof, urokinase, streptokinase, acidic fibroblast growth factor, basic fibroblast growth factor, tumor necrosis factor ⁇ , tumor necrosis factor ⁇ , transforming growth factor ⁇ , transforming growth factor ⁇ , atrial natriuretic factor, platelet-derived growth factor, endothelian, insulin, diphtheria to
  • the present method may also use exogenous proteins of diagnostic value.
  • a marker protein such as ⁇ -galatosodase, may be used to monitor cell migration.
  • the cells caused to express the exogenous therapeutic agent protein be endothelial cells.
  • endothelial cells may be stably implanted in situ on the arterial wall by catheterization and express a recombinant marker protein, ⁇ -galactosidase, in vivo.
  • an inbred pig strain the Yucatan minipig (Charles River Laboratories, Inc., Wilmington, Mass.), was chosen as an animal model ( 1 ).
  • a primary endothelial cell line was established from the internal jugular vein of an 8 month-old female minipig. The endothelial cell identity of this line was confirmed in that the cells exhibited growth characteristics and morphology typical of porcine endothelium in tissue culture. Endothelial cells also express receptors for the acetylated form of low density lipoprotein (AcLDL), in contrast to fibroblasts and other mesenchymal cells (2). When analyzed for AcLDL receptor expression, greater than 99% of the cultured cells contained this receptor, as judged by fluorescent AcLDL uptake.
  • AcLDL acetylated form of low density lipoprotein
  • BAG murine amphotropic s-galactosidase-transducing retroviral vector
  • the femoral and iliac arteries were exposed, and a catheter was introduced into the vessel ( FIG. 1 ).
  • Intimal tissues of the arterial wall were denuded mechanically by forceful passage of a partially inflated balloon catheter within the vessel.
  • the artery was rinsed with heparinized saline and incubated with the neutral protease, dispase (50 U/ml), which removed any remaining luminal endothelial cells.
  • Residual enzyme was rapidly inactivated by ⁇ 2 globulin in plasma upon deflating the catheter balloons and allowing blood to flow through the vessel segment.
  • the cultured endothelial cells which expressed ⁇ -galactosidase were introduced using a specially designed arterial catheter (USCI, Billerica, Mass.) that contained two balloons and a central instillation port ( FIG. 1 ).
  • Segments of the artery innoculated with ⁇ -galactosidase-expressing endothelium were removed 2 to 4 weeks later.
  • Gross examination of the arterial specimen after staining using the X-gal chromagen showed multiple areas of blue coloration, compared to an artery seeded with uninfected endothelium, indicative of ⁇ -galactosidase activity.
  • Light microscopy documented ⁇ -galactosidase staining primarily in endothelial cells of the intima in experimentally seeded vessels.
  • a major concern of gene transplantation in vivo relates to the production of replication-competent retrovirus from genetically engineered cells. In these tests, this potential problem has been minimized through the use of a replication defective retrovirus. No helper virus was detectable among these lines after 20 passages in vitro. Although defective viruses were used because of their high rate of infectivity and their stable integration into the host cell genome (4), this approach to gene transfer is adaptable to other viral vectors.
  • a second concern involves the longevity of expression of recombinant genes in vivo. Endothelial cell expression of ⁇ -galactosidase appeared constant in vessels examined up to six weeks after introduction into the blood vessel in the present study.
  • the present data show that genetically-altered endothelial cells can be introduced at the time of intervention to minimize local thrombosis.
  • This technique can also be used in other ischemic settings, including unstable angina or myocardial infarction.
  • antithrombotic effects can be achieved by introducing cells expressing genes for tissue plasminogen activator or urokinase.
  • This technology is also useful for the treatment of chronic tissue ischemia. For example, elaboration of angiogenic or growth factors (7) to stimulate the formation of collateral vessels to severely ischemic tissue, such as the myocardium.
  • somatic gene replacement for systemic inherited diseases is feasible using modifications of this endothelial cell gene transfer technique.
  • Endothelial cells were derived from external jugular veins using the neutral protease dispase (8). Excised vein segments were filled with dispase (50 U/ml in Hanks' balanced salt solution) and incubated at 30° C. for 20 minutes. Endothelium obtained by this means was maintained in medium 199 (GIBCO, Grand Island, N.Y.) supplemented with fetal calf serum (10%), 50 ⁇ g/ml endothelial cell growth supplement (ECGS) and heparin (100 ⁇ g/ml). These cells were infected with BAG retrovirus, and selected for resistance to G-418.
  • a double balloon catheter was used for instillation of endothelial cells.
  • the catheter has a proximal and distal balloon, each 6 mm in length and 5 mm in width, with a 20 mm length between the balloons.
  • the central section of the catheter has a 2 mm pore connected to an instillation port.
  • Proximal and distal balloon inflation isolates a central space, allowing for instillation of infected cells through the port into a discrete segment of the vessel.
  • the catheter was then positioned with the central space located in the region of denuded endothelium, and both balloons were inflated.
  • the denuded segment was irrigated with heparinized saline, and residual adherent cells were removed by instillation of dispase (20 U/ml) for 10 min.
  • the denuded vessel was further irrigated with a heparin solution and the BAG-infected endothelial cells were instilled for 30 min.
  • the balloon catheter was subsequently removed, and antegrade blood flow was restored.
  • the vessel segments were excised 2 to 4 weeks later.
  • a portion of the artery was placed in 0.5% glutaraldehyde for five minutes and stored in phosphate-buffered saline, and another portion was mounted in a paraffin block for sectioning.
  • the presence of retroviral expressed s-galactosidase was determined by a standard histochemical technique (19).
  • ⁇ -Galactosidase activity was documented by histochemical staining in (A) primary endothelial cells from the Yucatan minipig, (B) a subline derived by infection with the BAG retroviral vector, (C) a segment of normal control artery, (D) a segment of artery instilled with endothelium infected with the BAG retroviral vector, (E) microscopic cross-section of normal control artery, and (F) microscopic cross-section of artery instilled with endothelium infected with the BAG retroviral vector.
  • Endothelial cells in tissue culture were fixed in 0.5% glutaraldehyde prior to histochemical staining.
  • the enzymatic activity of the E. coli ⁇ -galactosidase protein was used to identify infected endothelial cells in vitro and in vivo.
  • the ⁇ -galactosidase transducing Mo-MuLV vector (2), (BAG) was kindly provided by Dr. Constance Cepko. This vector used the wild type Mo-MuLV LTR as a promoter for the ⁇ -galactosidase gene.
  • the simian virus 40 (SV-40) early promoter linked to the Tn5 neomycin resistance gene provides resistance to the drug G-418 and is inserted downstream of the s-galactosidase gene, providing a marker to select for retrovirus-containing, ⁇ -galactosidase expressing cells.
  • This defective retrovirus was prepared from fibroblast ⁇ am cells (3,10), and maintained in Dulbecco's modified Eagle's medium (DMEM) and 10% calf serum. Cells were passaged twice weekly following trypsinization.
  • the supernatant with titers of 10 4 -10 5 /ml G-418 resistant colonies, was added to endothelial cells at two-thirds confluence and incubated for 12 hours in DMEM with 10% calf serum at 37° C. in 5% CO 2 in the presence of 8 ⁇ g/ml of polybrene.
  • Viral supernatants were removed, and cells maintained in medium 199 with 10% fetal calf serum, ECGS (50 ⁇ g/ml), and endothelial cell conditioned medium (20%) for an additional 24 to 48 hours prior to selection in G-418 (0.7 ⁇ g/ml of a 50% racemic mixture).
  • G-418 resistant cells were isolated and analyzed for ⁇ -galactosidase expression using a standard histochemical stain ( 9 ). Cells stably expressing the ⁇ -galactosidase enzyme were maintained in continuous culture for use as needed. Frozen aliquots were stored in liquid nitrogen.

Abstract

A method for the direct treatment towards the specific sites of a disease is disclosed. This method is based on the delivery of proteins by catheterization to discrete blood vessel segments using genetically modified or normal cells or other vector systems. Endothelial cells expressing recombinant therapeutic agent or diagnostic proteins are situated on the walls of the blood vessel or in the tissue perfused by the vessel in a patient. This technique, provides for the transfer of cells or vectors and expression of recombinant genes in vivo and allows the introduction of proteins of therapeutic or diagnostic value for the treatment of diseases.

Description

    BACKGROUND OF THE INVENTION
  • 1. Field of the Invention
  • The present invention relates to the treatment of diseases by the site-specific instillation or transformation of cells and kits therefor.
  • 2. Discussion of the Background
  • The effective treatment of many systemic and inherited diseases remains a major challenge to modern medicine. The ability to deliver therapeutic agents to specific sites in vivo would be an asset in the treatment of, e.g., localized diseases. In addition the ability to cause a therapeutic agent to perfuse through the circulatory system would be effective for the treatment of, e.g., systemic diseases.
  • For example, it would be desirable to administer in a steady fashion an antitumor agent or toxin in close proximity to a tumor. Similarly, it would be desirable to cause a perfusion of, e.g., insulin in the blood of a person suffering from diabetes. However, for many therapeutic agents there is no satisfactory method of either site-specific or systemic administration.
  • In addition, for many diseases, it would be desirable to cause, either locally or systemically, the expression of a defective endogenous gene, the expression of a exogenous gene, or the suppression of an endogenous gene. Again, these remain unrealized goals.
  • In particular, the pathogenesis of atherosclerosis is characterized by three fundamental biological processes. These are: 1) proliferation of intimal smooth muscle cells together with accumulated macrophages; 2) formation by the proliferated smooth muscle cells of large amounts of connective tissue matrix; and 3) accumulation of lipid, principally in the form of cholesterol esters and free cholesterol, within cells as well as in surrounding-connective tissue.
  • Endothelial cell injury is an initiating event and is manifested by interference with the permeability barrier of the endothelium, alterations in the non-thrombogenic properties of the endothelial surface, and promotion of procoagulant properties of the endothelium. Monocytes migrate between endothelial cells, become active as scavenger cells, and differentiate into macrophages.
  • Macrophages then synthesize and secrete growth factors including platelet derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor alpha (TGF-α). These growth factors are extremely potent in stimulating the migration and proliferation of fibroblasts and smooth muscle cells in the atherosclerotic plaque. In addition, platelets may interact with the injured endothelial cell and the activated macrophage to potentiate the elaboration of growth factors and thrombus formation.
  • Two major problems in the clinical management of coronary artery disease include thrombus formation in acute myocardial ischemia and restenosis following coronary angioplasty (PTCA). Both involve common cellular events, including endothelial injury and release of potent growth factors by activated macrophages and platelets. Coronary angioplasty produces fracturing of the atherosclerotic plaque and removal of the endothelium. This vascular trauma promotes platelet aggregation and thrombus formation at the PTCA site. Further release of mitogens from platelets and macrophages, smooth muscle cell proliferation and monocyte infiltration result in restenosis.
  • Empiric therapy with antiplatelet drugs has not prevented this problem, which occurs in one-third of patients undergoing PTCA. A solution to restenosis is to prevent platelet aggregation, thrombus formation, and smooth muscle cell proliferation.
  • Thrombus formation is also a critical cellular event in the transition from stable to unstable coronary syndromes. The pathogenesis most likely involves acute endothelial cell injury and or plaque rupture, promoting dysjunction of endothelial cell attachment, and leading to the exposure of underlying macrophage foam cells. This permits the opportunity for circulating platelets to adhere, aggregate, and form thrombi.
  • The intravenous administration of thrombolytic agents, such as tissue plasminogen activator (tPA) results in lysis of thrombus in approximately 70% of patients experiencing an acute myocardial infarction. Nonetheless, approximately 30% of patients fail to reperfuse, and of those patients who undergo initial reperfusion of the infarct related artery, approximately 25% experience recurrent thrombosis within 24 hours. Therefore, an effective therapy for rethrombosis remains a major therapeutic challenge facing the medical community today.
  • As noted above, an effective therapy for rethrombosis is by far not the only major therapeutic challenge existing today. Others include the treatment of other ischemic conditions, including unstable angina, myocardial infarction or chronic tissue ischemia; or even the treatment of systemic and inherited diseases or cancers. These might be treated by the effective administration of anticoagulants, vasodilatory, angiogenic, growth factors or growth inhibitors to a patient. Thus, there remains a strongly felt need for an effective therapy in all of these clinical settings.
  • SUMMARY OF THE INVENTION
  • Accordingly, one object of the present invention is to provide a novel method for the site-specific administration of a therapeutic agent.
  • It is another object of the present invention to provide a method for the perfusion of a therapeutic agent in the blood stream of a patient.
  • It is another object of the present invention to provide a method for causing the expression of an exogenous gene in a patient.
  • It is another object of the present invention to provide a method for causing the expression of a defective endogenous gene in a patient.
  • It is another object of the present invention to provide a method for suppressing the expression of an endogenous gene in a patient.
  • It is another object of the present invention to provide a method for site-specifically replacing damaged cells in a patient.
  • It is another object of the present invention to provide a method for the treatment of a disease by causing either the site-specific administration of a therapeutic agent or the perfusion of a therapeutic agent in the bloodstream of a patient.
  • It is another object of the present invention to provide a method for the treatment of a disease by causing either the expression of an exogenous gene, the expression of a defective endogenous gene, or the suppression of the expression of an endogenous gene in a patient.
  • It is another object of the present invention to provide a method for the treatment of a disease by site-specifically replacing damaged cells in a patient.
  • It is another object of the present invention to provide a kit for site-specifically instilling normal or transformed cells in a patient.
  • It is another object of the present invention to provide a kit for site-specifically transforming cells in vivo.
  • These and other objects of this invention which will become apparent during the course of the following detailed description of the invention have been discovered by the inventors to be achieved by (a) a method which comprises either (i) site-specific instillation or either normal (untransformed) or transformed cells in a patient or (ii) site-specific transformation of cells in a patient and (b) a kit which contains a catheter for (i) site-specific instillation of either normal or transformed cells or (ii) site-specific transformation of cells.
  • Site-specific instillation of normal cells can be used to replace damaged cells, while instillation of transformed cells can be used to cause the expression of either a defective endogenous gene or an exogenous gene or the suppression of an endogenous gene product. Instillation of cells in the walls of the patient's blood vessels can be used to cause the steady perfusion of a therapeutic agent in the blood stream.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same become better understood by reference to the following detailed description when considered in connection with the accompanying figures, wherein:
  • FIGS. 1 and 2 illustrate the use of a catheter in accordance with the invention to surgically or percutaneously implant cells in a blood vessel or to transform in vivo cells present on the wall of a patient's blood vessel.
  • DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • Thus, in one embodiment, the present invention is used to treat diseases, such as inherited diseases, systemic diseases, diseases of the cardiovascular system, diseases of particular organs, or tumors by instilling normal or transformed cells or by transforming cells.
  • The cells which may be instilled in the present method include endothelium, smooth muscle, fibroblasts, monocytes, macrophages, and parenchymal cells. These cells may produce proteins which may have a therapeutic or diagnostic effect and which may be naturally occurring or arise from recombinant genetic material.
  • Referring now to the figures, wherein like reference numerals designate identical or corresponding parts throughout the several views, and more particularly to FIG. 1 thereof, this figure illustrates the practice of the present invention with a catheter having a design as disclosed in U.S. Pat. No. 4,636,195, which is hereby incorporated by reference. This catheter may be used to provide normal or genetically altered cells on the walls of a vessel or to introduce vectors for the local transformation of cells. In the figure, 5 is the wall of the blood vessel. The figure shows the catheter body 4 held in place by the inflation of inflatable balloon means 1 and 2. The section of the catheter body 4 situated between balloon means 1 and 2 is equipped with instillation port means 3. The catheter may be further equipped with a guidewire means 6. FIG. 2 illustrates the use of a similar catheter, distinguished from the catheter illustrated in FIG. 1 by the fact that it is equipped with only a single inflatable balloon means 2 and a plurality of instillation port means 3. This catheter may contain up to twelve individual instillation port means 3, with five being illustrated.
  • In the case of delivery to an organ, the catheter may be introduced into the major artery supplying the tissue. Cells containing recombinant genes or vectors can be introduced through a central instillation port after temporary occlusion of the arterial circulation. In this way, cells or vector DNA may be delivered to a large amount of parenchymal tissue distributed through the capillary circulation. Recombinant genes can also be introduced into the vasculature using the double balloon catheter technique in the arterial circulation proximal to the target organ. In this way, the recombinant genes may be secreted directly into the circulation which perfuse the involved tissue or may be synthesized directly within the organ.
  • In one embodiment, the therapeutic agents are secreted by vascular cells supplying specific organs affected by the disease. For example, ischemic cardiomyopathy may be treated by introducing angiogenic factors into the coronary circulation. This approach may also be used for peripheral vascular or cerebrovascular diseases where angiogenic factors may improve circulation to the brain or other tissues. Diabetes mellitus may be treated by introduction of glucose-responsive insulin secreting cells in the portal circulation where the liver normally sees a higher insulin concentration than other tissues.
  • In addition to providing local concentrations of therapeutic agents, the present method may also be used for delivery of recombinant genes to parenchymal tissues, because high concentrations of viral vector and other vectors can be delivered to a specific circulation. Using this approach, deficiencies of organ-specific proteins may also be treated. For example, in the liver, α-antitrypsin inhibitor deficiency or hyperchloresterolemia may be treated by introduction of α-antitrypsin or the LDL receptor gene. In addition, this approach may be used for the treatment of malignancy. Secretion of specific recombinant toxin genes into the circulation of inoperable tumors provides a therapeutic effect. Examples include acoustic neuromas or certain hemangiomas which are otherwise unresectable.
  • In clinical settings, these therapeutic recombinant genes are introduced in cells supplying the circulation of the involved organ. Although the arterial and capillary circulations are the preferred locations for introduction of these cells, venous systems are also suitable.
  • In its application to the treatment of local vascular damage the present invention provides for the expression of proteins which ameliorate this condition in situ. In one embodiment, because vascular cells are found at these sites, they are used as carriers to convey the therapeutic agents.
  • The invention thus, in one of its aspects, relies on genetic alteration of endothelial and other vascular cells or somatic cell gene therapy, for transmitting therapeutic agents (i.e., proteins, growth factors) to the localized region of vessel injury. To successfully use gene transplantation in the cells, four requirements must be fulfilled. First, the gene which is to be implanted into the cell must be identified and isolated. Second, the gene to be expressed must be cloned and available for genetic manipulation. Third, the gene must be introduced into the cell in a form that will be expressed or functional. Fourth, the genetically altered cells must be situated in the vascular region where it is needed.
  • In accordance with the present invention the altered cells or appropriate vector may be surgically, percutaneously, or intravenously introduced and attached to a section of a patient's vessel wall. Alternatively, some of the cells existing on the patient's vessel wall are transformed with the desired genetic material or by directly applying the vector. In some instances, vascular cells which are not genetically modified can be introduced by these methods to replace cells lost or damaged on the vessel surface.
  • Any blood vessel may be treated in accordance with this invention; that is, arteries, veins, and capillaries. These blood vessels may be in or near any organ in the human, or mammalian, body.
  • Introduction of Normal or Genetically Altered Cells into a Blood Vessel:
  • This embodiment of the invention may be illustrated as follows:
  • I. Establishment of Endothelial or Other Vascular Cells in Tissue Culture.
  • Initially, a cell line is established and stored in liquid nitrogen. Prior to cryopreservation, an aliquot is taken for infection or transfection with a vector, viral or otherwise, containing the desired genetic material.
  • Endothelial or other vascular cells may be derived enzymatically from a segment of a blood vessel, using techniques previously described in J. W. Ford, et al., In Vitro, 17, 40 (1981). The vessel is excised, inverted over a stainless steel rod and incubated in 0.1% trypsin in Ca++- and Mg++-free Hank's balanced salt solution (BSS) with 0.125% EDTA at pH 8 for 10 min at 37° C.
  • Cells (0.4 to 1.5×106) are collected by centrifugation and resuspended in medium 199 (GIBCO) containing 10% fetal bovine serum, endothelial cell growth supplement (ECGS, Collaborative Research, Waltham, Mass.) at 25 μg/ml, heparin at 15 U/ml, and gentamicin (50 μg/ml). Cells are added to a 75 cm2 tissue culture flask precoated with gelatin (2 mg/ml in distilled water). Cells are fed every second day in the above medium until they reach confluence.
  • After two weeks in culture, the ECGS and heparin may be omitted from the medium when culturing porcine endothelium. If vascular smooth muscle cells or fibroblasts are desired the heparin and ECGS can be omitted entirely from the culturing procedure. Aliquots of cells are stored in liquid nitrogen by resuspending to approximately 106 cells in 0.5 ml of ice cold fetal calf serum on ice. An equal volume of ice cold fetal calf serum containing 10% DMSO is added, and cells are transferred to a prechilled screw cap Corning freezing tube. These cells are transferred to a −70° C. freezer for 3 hours before long term storage in liquid nitrogen.
  • The cells are then infected with a vector containing the desired genetic material.
  • II. Introduction of cells expressing normal or exogenous proteins into the vasculature.
  • A. Introduction of Cells Expressing Relevant Proteins by Catheterization.
  • The patient is prepared for catheterization either by surgery or percutaneously, observing strict adherence to sterile techniques. A cutdown procedure is performed over the target blood vessel or a needle is inserted into the target blood vessel after appropriate anesthesia. The vessel (5) is punctured and a catheter, such as described in U.S. Pat. No. 4,636,195, which is hereby incorporated by reference (available from USCI, Billerica, Mass.) is advanced by guidewire means (6) under fluoroscopic guidance, if necessary, into the vessel (5) (FIG. 1). This catheter means (4) is designed to introduce infected endothelial cells into a discrete region of the artery. The catheter has a proximal and distal balloon means (2) and (1), respectively, (e.g., each balloon means may be about 3 mm in length and about 4 mm in width), with a length of catheter means between the balloons. The length of catheter means between the balloons has a port means connected to an instillation port means (3). When the proximal and distal balloons are inflated, a central space is created in the vessel, allowing for instillation of infected cells though the port.
  • A region of the blood vessel is identified by anatomical landmarks and the proximal balloon means (2) is inflated to denude the endothelium by mechanical trauma (e.g., by forceful passage of a partially inflated balloon catheter within the vessel) or by mechanical trauma in combination with small amounts of a proteolytic enzyme such as dispase, trypsin, collagenase, papain, pepsin, chymotrypsin or cathepsin, or by incubation with these proteolytic enzymes alone. In addition to proteolytic enzymes, lipases may be used. The region of the blood vessel may also be denuded by treatment with a mild detergent or the like, such as NP-40, Triton X100, deoxycholate, or SDS.
  • The denudation conditions are adjusted to achieve essentially complete loss of endothelium for cell transfers or approximately 20 to 90%, preferably 50 to 75%, loss of cells from the vessel wall for direct infection. In some instances cell removal may not be necessary. The catheter is then advanced so that the instillation port means (3) is placed in the region of denuded endothelium. Infected, transfected or normal cells are then instilled into the discrete section of artery over thirty minutes. If the blood vessel is perfusing an organ which can tolerate some ischemia, e.g., skeletal muscle, distal perfusion is not a major problem, but can be restored by an external shunt if necessary, or by using a catheter which allows distal perfusion. After instillation of the infected endothelial cells, the balloon catheter is removed, and the arterial puncture site and local skin incision are repaired. If distal perfusion is necessary, an alternative catheter designed to allow distal perfusion may be used.
  • B. Introduction of Recombinant Genes Directly into Cells on the Wall of a Blood Vessel or Perfused by a Specific Circulation In Vivo; Infection or Transfection of Cells on the Vessel Wall and Organs.
  • Surgical techniques are used as described above. Instead of using infected cells, a high titer desired genetic material transducing viral vector (105 to 106 particles/ml) or DNA complexed to a delivery vector is directly instilled into the vessel wall using the double balloon catheter technique. This vector is instilled in medium containing serum and polybrene (10 μg/ml) to enhance the efficiency of infection. After incubation in the dead space created by the catheter for an adequate period of time (0.2 to 2 hours or greater), this medium is evacuated, gently washed with phosphate-buffered saline, and arterial circulation is restored. Similar protocols are used for post operative recovery.
  • The vessel surface can be prepared by mechanical denudation alone, in combination with small amounts of proteolytic enzymes such as dispase, trypsin, collagenase or cathepsin, or by incubation with these proteolytic enzymes alone. The denudation conditions are adjusted to achieve the appropriate loss of cells from the vessel wall.
  • Viral vector or DNA-vector complex is instilled in Dulbecco's modified Eagle's medium using purified virus or complexes containing autologous serum, and adhesive molecules such as polybrene (10 μg/ml), poly-L-lysine, dextran sulfate, or any polycationic substance which is physiologically suitable, or a hybrid antibody directed against the envelope glycoprotein of the virus or the vector and the relevant target in the vessel wall or in the tissue perfused by the vessel to enhance the efficiency of infection by increasing adhesion of viral particles to the relevant target cells. The hybrid antibody directed against the envelope glycoprotein of the virus or the vector and the relevant target cell can be made by one of two methods. Antibodies directed against different epitopes can be chemically crosslinked (G. Jung, C. J. Honsik, R. A. Reisfeld, and H. J. Muller-Eberhard, Proc. Natl. Acad. Sci. USA, 83, 4479 (1986); U. D. Staerz, O. Kanagawa, and M. J. Bevan, Nature, 314, 628 (1985); and P. Perez, R. W. Hoffman, J. A. Titus, and D. M. Segal, J. Exp. Med., 163, 166 (1986)) or biologically coupled using hybrid hybridomas (U. D. Staerz and M. J. Bevan, Proc. Natl. Acad. Sci. USA, 83, 1453 (1986); and C. Milstein and A. C. Cuello, Nature, 305, 537 (1983)). After incubation in the central space of the catheter for 0.2 to 2 hours or more, the medium is evacuated, gently washed with phosphate buffered saline, and circulation restored.
  • Using a different catheter design (See FIG. 2), a different protocol for instillation can also be used. This second approach involves the use of a single balloon means (2) catheter with multiple port means (3) which allow for high pressure delivery of the retrovirus into partially denuded arterial segments. The vessel surface is prepared as described above and defective vector is introduced using similar adhesive molecules. In this instance, the use of a high pressure delivery system serves to optimize the interaction of vectors with cells in adjacent vascular tissue.
  • The present invention also provides for the use of growth factors delivered locally by catheter or systemically to enhance the efficiency of infection.
  • In addition to retroviral vectors, herpes virus, adenovirus, or other viral vectors are suitable vectors for the present technique.
  • It is also possible to transform cells within an organ or tissue. Direct transformation of organ or tissue cells may be accomplished by one of two methods. In a first method a high pressure transfection is used. The high pressure will cause the vector to migrate through the blood vessel walls into the surrounding tissue. In a second method, injection into a capillary bed, optionally after injury to allow leaking, gives rise to direct infection of the surrounding tissues.
  • The time required for the instillation of the vectors or cells will depend on the particular aspect of the invention being employed. Thus, for instilling cells or vectors in a blood vessel a suitable time would be from 0.01 to 12 hrs, preferably 0.1 to 6 hrs, most preferably 0.2 to 2 hrs. Alternatively for high pressure instillation of vectors or cells, shorter times might be preferred.
  • Obtaining the Cells Used in this Invention:
  • The term “genetic material” generally refers to DNA which codes for a protein. This phrase also encompasses RNA when used with an RNA virus or other vector based on RNA.
  • Transformation is the process by which cells have incorporated an exogenous gene by direct infection, transfection or other means of uptake.
  • The term “vector” is well understood and is synonymous with the often-used phrase “cloning vehicle”. A vector is non-chromosomal double-stranded DNA comprising an intact replicon such that the vector is replicated when placed within a unicellular organism, for example by a process of transformation. Viral vectors include retroviruses, adenoviruses, herpesvirus, papovirus, or otherwise modified naturally occurring viruses. Vector also means a formulation of DNA with a chemical or substance which allows uptake by cells.
  • In another embodiment the present invention provides for inhibiting the expression of a gene. Four approaches may be utilized to accomplish this goal. These include the use of antisense agents, either synthetic oligonucleotides which are complementary to the mRNA (Maher III, L. J. and Dolnick, B. J. Arch. Biochem. Biophys., 253, 214-220 (1987) and (Zamecnik, P. C., et al., Proc. Natl. Acad. Sci., 83, 4143-4146 (1986)), or the use of plasmids expressing the reverse complement of this gene (Izant, J. H. and Weintraub, H., Science, 229, 345-352, (1985); Cell, 36, 1077-1015 (1984)). In addition, catalytic RNAs, called ribozymes, can specifically degrade RNA sequences (Uhlenbeck, O. C., Nature, 328, 596-600 (1987), Haseloff, J. and Gerlach, W. L., Nature, 334, 585-591 (1988)). The third approach involves “intracellular immunization”, where analogues of intracellular proteins can interfere specifically with their function (Friedman, A. D., Triezenberg, S. J. and McKnight, S. L., Nature, 335, 452-454 (1988)), described in detail below.
  • The first approaches may be used to specifically eliminate transcripts in cells. The loss of transcript may be confirmed by S1 nuclease analysis, and expression of binding protein determined using a functional assay. Single-stranded oligonucleotide analogues may be used to interfere with the processing or translation of the transcription factor mRNA. Briefly, synthetic oligonucleotides or thiol-derivative analogues (20-50 nucleotides) complementary to the coding strand of the target gene may be prepared. These antisense agents may be prepared against different regions of the mRNA. They are complementary to the 5′ untranslated region, the translational initiation site and subsequent 20-50 base pairs, the central coding region, or the 3′ untranslated region of the gene. The antisense agents may be incubated with cells transfected prior to activation. The efficacy of antisense competitors directed at different portions of the messenger RNA may be compared to determine whether specific regions may be more effective in preventing the expression of these genes.
  • RNA can also function in an autocatalytic fashion to cause autolysis or to specifically degrade complementary RNA sequences (Uhlenbeck, O. C., Nature, 328, 596-600 (1987), Haseloff, J. and Gerlach, W. L., Nature, 334, 585-591 (1988), and Hutchins, C. J., et al, Nucleic Acids Res., 14, 3627-3640 (1986)). The requirements for a successful RNA cleavage include a hammerhead structure with conserved RNA sequence at the region flanking this structure. Regions adjacent to this catalytic domain are made complementary to a specific RNA, thus targeting the ribozyme to specific cellular mRNAs. To inhibit the production of a specific target gene, the mRNA encoding this gene may be specifically degraded using ribozymes. Briefly, any GUG sequence within the RNA transcript can serve as a target for degradation by the ribozyme. These may be identified by DNA sequence analysis and GUG sites spanning the RNA transcript may be used for specific degradation. Sites in the 5′ untranslated region, in the coding region, and in the 3′ untranslated region may be targeted to determine whether one region is more efficient in degrading this transcript. Synthetic oligonucleotides encoding 20 base pairs of complementary sequence upstream of the GUG site, the hammerhead structure and −20 base pairs of complementary sequence downstream of this site may be inserted at the relevant site in the cDNA. In this way, the ribozyme may be targeted to the same cellular compartment as the endogenous message. The ribozymes inserted downstream of specific enhancers, which give high level expression in specific cells may also be generated. These plasmids may be introduced into relevant target cells using electroporation and cotransfection with a neomycin resistant plasmid, pSV2-Neo or another selectable marker. The expression of these transcripts may be confirmed by Northern blot and S1 nuclease analysis. When confirmed, the expression of mRNA may be evaluated by S1 nuclease protection to determine whether expression of these transcripts reduces steady state levels of the target mRNA and the genes which it regulates. The level of protein may also be examined.
  • Genes may also be inhibited by preparing mutant transcripts lacking domains required for activation. Briefly, after the domain has been identified, a mutant form which is incapable of stimulating function is synthesized. This truncated gene product may be inserted downstream of the SV-40 enhancer in a plasmid containing the neomycin resistance gene (Mulligan, R. and Berg, P., Science, 209, 1422-1427 (1980) (in a separate transcription unit). This plasmid may be introduced into cells and selected using G418. The presence of the mutant form of this gene will be confirmed by S1 nuclease analysis and by immunoprecipitation. The function of the endogenous protein in these cells may be evaluated in two ways. First, the expression of the normal gene may be examined. Second, the known function of these proteins may be evaluated. In the event that this mutant intercellular interfering form is toxic to its host cell, it may be introduced on an inducible control element, such as metallothionein promoter. After the isolation of stable lines, cells may be incubated with Zn or Cd to express this gene. Its effect on host cells can then be evaluated.
  • Another approach to the inactivation of specific genes is to overexpress recombinant proteins which antagonize the expression or function of other activities. For example, if one wished to decrease expression of TPA (e.g., in a clinical setting of disseminate thrombolysis), one could overexpress plasminogen activator inhibitor.
  • Advances in biochemistry and molecular biology in recent years have led to the construction of “recombinant” vectors in which, for example, retroviruses and plasmids are made to contain exogenous RNA or DNA, respectively. In particular instances the recombinant vector can include heterologous RNA or DNA, by which is meant RNA or DNA that codes for a polypeptide ordinarily not produced by the organism susceptible to transformation by the recombinant vector. The production of recombinant RNA and DNA vectors is well understood and need not be described in detail. However, a brief description of this process is included here for reference.
  • For example, a retrovirus or a plasmid vector can be cleaved to provide linear RNA or DNA having ligatable termini. These termini are bound to exogenous RNA or DNA having complementary like ligatable termini to provide a biologically functional recombinant RNA or DNA molecule having an intact replicon and a desired phenotypical property.
  • A variety of techniques are available for RNA and DNA recombination in which adjoining ends of separate RNA or DNA fragments are tailored to facilitate ligation.
  • The exogenous, i.e., donor, RNA or DNA used in the present invention is obtained from suitable cells. The vector is constructed using known techniques to obtain a transformed cell capable of in vivo expression of the therapeutic agent protein. The transformed cell is obtained by contacting a target cell with a RNA or DNA-containing formulation permitting transfer and uptake of the RNA or DNA into the target cell. Such formulations include, for example, retroviruses, plasmids, liposomal formulations, or plasmids complexes with polycationic substances such as poly-L-lysine, DEAC-dextran and targeting ligands.
  • The present invention thus provides for the genetic alteration of cells as a method to transmit therapeutic or diagnostic agents to localized regions of the blood vessel for local or systemic purposes. The range of recombinant proteins which may be expressed in these cells is broad and varied. It includes gene transfer using vectors expressing such proteins as tPA for the treatment of thrombosis and restenosis, angiogenesis or growth factors for the purpose of revascularization, and vasoactive factors to alleviate vasoconstriction or vasospasm. This technique can also be extended to genetic treatment of inherited disorders, or acquired diseases, localized or systemic. The present invention may also be used to introduce normal cells to specific sites of cell loss, for example, to replace endothelium damaged during angioplasty or catheterization.
  • For example, in the treatment of ischemic diseases (thrombotic diseases), genetic material coding for tPA or modifications thereof, urokinase or streptokinase is used to transform the cells. In the treatment of ischemic organ (e.g., heart, kidney, bowel, liver, etc.) failure, genetic material coding for recollateralization agents, such as transforming growth factor α (TGF-α), transforming growth factor β (TGF-β), angiogenin, tumor necrosis factor α, tumor necrosis factor α, acidic fibroblast growth factor or basic fibroblast growth factor can be used. In the treatment of vasomotor diseases, genetic material coding for vasodilators or vasoconstrictors may be used. These include atrial natriuretic factor, platelet-derived growth factor or endothelin. In the treatment of diabetes, genetic material coding for insulin may be used.
  • The present invention can also be used in the treatment of malignancies by placing the transformed cells in proximity to the malignancy. In this application, genetic material coding for diphtheria toxin, pertussis toxin, or cholera toxin may be used.
  • In the use of the present invention in the treatment of AIDS, genetic material coding for soluble CD4 or derivatives thereof may be used. In the treatment of genetic diseases, for example, growth hormone deficiency, genetic material coding for the needed substance, for example, human growth hormone, is used. All of these genetic materials are readily available to one skilled in this art.
  • In another embodiment, the present invention provides a kit for treating a disease in a patient which contains a catheter and a solution which contains either an enzyme or a mild detergent, in which the catheter is adapted for insertion into a blood vessel and contains a main catheter body having a balloon element adapted to be inserted into said vessel and expansible against the walls of the blood vessel so as to hold the main catheter body in place in the blood vessel, and means carried by the main catheter body for delivering a solution into the blood vessel, and the solution which contains the enzyme or mild detergent is a physiologically acceptable solution. The solution may contain a proteolytic enzyme, such as dispase, trypsin, collagenase, papain, pepsin, or chymotrypsin. In addition to proteolytic enzymes, lipases may be used. As a mild detergent, the solution may contain NP-40, Triton X100, deoxycholate, SDS or the like.
  • Alternatively, the kit may contain a physiological acceptable solution which contains an agent such as heparin, poly-L-lysine, polybrene, dextran sulfate, a polycationic material, or bivalent antibodies. This solution may also contain vectors or cells (normal or transformed). In yet another embodiment the kit may contain a catheter and both a solution which contains an enzyme or mild detergent and a solution which contains an agent such as heparin, poly-L-lysine, polybrene, dextran sulfate, a polycationic material or bivalent antibody and which may optionally contain vectors or cells.
  • The kit may contain a catheter with a single balloon and central distal perfusion port, together with acceptable solutions to allow introduction of cells in a specific organ or vectors into a capillary bed or cells in a specific organ or tissue perfused by this capillary bed.
  • Alternatively, the kit may contain a main catheter body which has two spaced balloon elements adapted to be inserted in a blood vessel with both being expansible against the walls of the blood vessel for providing a chamber in the blood vessel, and to hold the main catheter body in place. In this case, the means for delivering a solution into the chamber is situated in between the balloon elements. The kit may contain a catheter which possesses a plurality of port means for delivering the solution into the blood vessel.
  • Thus, the present invention represents a method for treating a disease in a patient by causing a cell attached onto the walls of a vessel or the cells of an organ perfused by this vessel in the patient to express an exogeneous therapeutic agent protein, wherein the protein treats the disease or may be useful for diagnostic purposes. The present method may be used to treat diseases, such as an ischemic disease, a vasomotor disease, diabetes, a malignancy, AIDS or a genetic disease.
  • The present method may use exogeneous therapeutic agent proteins, such as tPA and modifications thereof, urokinase, streptokinase, acidic fibroblast growth factor, basic fibroblast growth factor, tumor necrosis factor α, tumor necrosis factor β, transforming growth factor α, transforming growth factor β, atrial natriuretic factor, platelet-derived growth factor, endothelian, insulin, diphtheria toxin, pertussis toxin, cholera toxin, soluble CD4 and derivatives thereof, and growth hormone to treat diseases.
  • The present method may also use exogenous proteins of diagnostic value. For example, a marker protein, such as β-galatosodase, may be used to monitor cell migration.
  • It is preferred, that the cells caused to express the exogenous therapeutic agent protein be endothelial cells.
  • Other features of the present invention will become apparent in the course of the following Descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.
  • The data reported below demonstrate the feasibility of endothelial cell transfer and gene transplantation; that endothelial cells may be stably implanted in situ on the arterial wall by catheterization and express a recombinant marker protein, β-galactosidase, in vivo.
  • Because atherogenesis in swine has similarities to humans, an inbred pig strain, the Yucatan minipig (Charles River Laboratories, Inc., Wilmington, Mass.), was chosen as an animal model (1). A primary endothelial cell line was established from the internal jugular vein of an 8 month-old female minipig. The endothelial cell identity of this line was confirmed in that the cells exhibited growth characteristics and morphology typical of porcine endothelium in tissue culture. Endothelial cells also express receptors for the acetylated form of low density lipoprotein (AcLDL), in contrast to fibroblasts and other mesenchymal cells (2). When analyzed for AcLDL receptor expression, greater than 99% of the cultured cells contained this receptor, as judged by fluorescent AcLDL uptake.
  • Two independent β-galactosidase-expressing endothelial lines were isolated following infection with a murine amphotropic s-galactosidase-transducing retroviral vector (BAG), which is replication-defective and contains both β-galactosidase and neomycin resistance genes (3). Cells containing this vector were selected for their ability to grow in the presence of G-418. Greater than 90% of selected cells synthesized β-galactosidase by histochemical staining. The endothelial nature of these genetically altered cells was also confirmed by analysis of fluorescent AcLDL uptake. Infection by BAG retrovirus was further verified by Southern blot analysis which revealed the presence of intact proviral DNA at approximately one copy per genome.
  • Endothelial cells derived from this inbred strain, being syngeneic, were applicable for study in more than one minipig, and were tested in nine different experimental subjects. Under general anesthesia, the femoral and iliac arteries were exposed, and a catheter was introduced into the vessel (FIG. 1). Intimal tissues of the arterial wall were denuded mechanically by forceful passage of a partially inflated balloon catheter within the vessel. The artery was rinsed with heparinized saline and incubated with the neutral protease, dispase (50 U/ml), which removed any remaining luminal endothelial cells. Residual enzyme was rapidly inactivated by α2 globulin in plasma upon deflating the catheter balloons and allowing blood to flow through the vessel segment. The cultured endothelial cells which expressed β-galactosidase were introduced using a specially designed arterial catheter (USCI, Billerica, Mass.) that contained two balloons and a central instillation port (FIG. 1).
  • When these balloons were inflated, a protected space was created within the artery into which cells were instilled through the central port 3 (FIG. 1). These endothelial cells, which expressed β-galactosidase, were allowed to incubate for 30 minutes to facilitate their attachment to the denuded vessel. The catheter was then removed, the arterial branch ligated, and the incision closed.
  • Segments of the artery innoculated with β-galactosidase-expressing endothelium were removed 2 to 4 weeks later. Gross examination of the arterial specimen after staining using the X-gal chromagen showed multiple areas of blue coloration, compared to an artery seeded with uninfected endothelium, indicative of β-galactosidase activity. Light microscopy documented β-galactosidase staining primarily in endothelial cells of the intima in experimentally seeded vessels.
  • In contrast, no evidence of similar staining was observed in control segments which had received endothelial cells containing no β-galactosidase. β-Galactosidase staining was occasionally-evident in deeper intimal tissues, suggesting entrapment or migration of seeded endothelium within the previously injured vessel wall. Local thrombosis was observed in the first two experimental subjects. This complication was minimized in subsequent studies by administering acetylsalicylic acid prior to the endothelial cell transfer procedure and use of heparin anticoagulation at the time of innoculation. In instances of thrombus formation, β-galactosidase staining was seen in endothelial cells extending from the vessel wall to the surface of the thrombus.
  • A major concern of gene transplantation in vivo relates to the production of replication-competent retrovirus from genetically engineered cells. In these tests, this potential problem has been minimized through the use of a replication defective retrovirus. No helper virus was detectable among these lines after 20 passages in vitro. Although defective viruses were used because of their high rate of infectivity and their stable integration into the host cell genome (4), this approach to gene transfer is adaptable to other viral vectors.
  • A second concern involves the longevity of expression of recombinant genes in vivo. Endothelial cell expression of β-galactosidase appeared constant in vessels examined up to six weeks after introduction into the blood vessel in the present study.
  • These tests have demonstrated that genetically-altered endothelial cells can be introduced into the vascular wall of the Yucatan minipig by arterial catheterization. Thus, the present method can be used for the localized biochemical treatment of vascular disease using genetically-altered endothelium as a vector.
  • A major complication of current interventions for vascular disease, such as balloon angioplasty or insertion of a graft into a diseased vessel, is disruption of the atherosclerotic plaque and thrombus formation at sites of local tissue trauma (5). In part, this is mediated by endothelial cell injury (6). The present data show that genetically-altered endothelial cells can be introduced at the time of intervention to minimize local thrombosis.
  • This technique can also be used in other ischemic settings, including unstable angina or myocardial infarction. For instance, antithrombotic effects can be achieved by introducing cells expressing genes for tissue plasminogen activator or urokinase. This technology is also useful for the treatment of chronic tissue ischemia. For example, elaboration of angiogenic or growth factors (7) to stimulate the formation of collateral vessels to severely ischemic tissue, such as the myocardium. Finally, somatic gene replacement for systemic inherited diseases is feasible using modifications of this endothelial cell gene transfer technique.
  • Experimental Section:
  • A. Analysis of AcLDL Receptor Expression in Normal and β-Galactosidase-Transduced Porcine Endothelial Cells.
  • Endothelial cell cultures derived from the Yucatan minipig, two sublines infected with BAG retrovirus or 3T3 fibroblast controls were analyzed for expression of AcLDL receptor using fluorescent labelled AcLDL.
  • Endothelial cells were derived from external jugular veins using the neutral protease dispase (8). Excised vein segments were filled with dispase (50 U/ml in Hanks' balanced salt solution) and incubated at 30° C. for 20 minutes. Endothelium obtained by this means was maintained in medium 199 (GIBCO, Grand Island, N.Y.) supplemented with fetal calf serum (10%), 50 μg/ml endothelial cell growth supplement (ECGS) and heparin (100 μg/ml). These cells were infected with BAG retrovirus, and selected for resistance to G-418. Cell cultures were incubated with (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbacyanine perchlorate) (Dil) AcLDL (Biomedical Technologies, Stoughton, Mass.) (10 μg/ml) for 4-6 hrs. at 37° C., followed by three rinses with phosphate-buffered saline containing 0.5% glutaraldehyde. Cells were visualized by phase contrast and fluorescent microscopy.
  • B. Method of Introduction of Endothelial Cells by Catheterization.
  • A double balloon catheter was used for instillation of endothelial cells. The catheter has a proximal and distal balloon, each 6 mm in length and 5 mm in width, with a 20 mm length between the balloons. The central section of the catheter has a 2 mm pore connected to an instillation port. Proximal and distal balloon inflation isolates a central space, allowing for instillation of infected cells through the port into a discrete segment of the vessel. For a schematic representation of cell introduction by catheter, see FIGS. 1 and 2.
  • Animal care was carried out in accordance with “Principles of Laboratory Animal Care” and “Guide for the Care and Use of Laboratory Animals” (NIH publication No. 80-23, Revised 1978). Female Yucatan minipigs (80-100 kg) were anesthetized with pentobarbital (20 mg/kg), intubated, and mechanically ventilated. These subjects underwent sterile surgical exposure of the iliac and femoral arteries. The distal femoral artery was punctured, and the double-balloon catheter was advanced by guidewire into the iliac artery. The external iliac artery was identified; the proximal balloon was partially inflated and passed proximally and distally so as to mechanically denude the endothelium. The catheter was then positioned with the central space located in the region of denuded endothelium, and both balloons were inflated. The denuded segment was irrigated with heparinized saline, and residual adherent cells were removed by instillation of dispase (20 U/ml) for 10 min. The denuded vessel was further irrigated with a heparin solution and the BAG-infected endothelial cells were instilled for 30 min. The balloon catheter was subsequently removed, and antegrade blood flow was restored. The vessel segments were excised 2 to 4 weeks later. A portion of the artery was placed in 0.5% glutaraldehyde for five minutes and stored in phosphate-buffered saline, and another portion was mounted in a paraffin block for sectioning. The presence of retroviral expressed s-galactosidase was determined by a standard histochemical technique (19).
  • C. Analysis of Endothelial Cells In Vitro and In Vivo.
  • β-Galactosidase activity was documented by histochemical staining in (A) primary endothelial cells from the Yucatan minipig, (B) a subline derived by infection with the BAG retroviral vector, (C) a segment of normal control artery, (D) a segment of artery instilled with endothelium infected with the BAG retroviral vector, (E) microscopic cross-section of normal control artery, and (F) microscopic cross-section of artery instilled with endothelium infected with the BAG retroviral vector.
  • Endothelial cells in tissue culture were fixed in 0.5% glutaraldehyde prior to histochemical staining. The enzymatic activity of the E. coli β-galactosidase protein was used to identify infected endothelial cells in vitro and in vivo. The β-galactosidase transducing Mo-MuLV vector (2), (BAG) was kindly provided by Dr. Constance Cepko. This vector used the wild type Mo-MuLV LTR as a promoter for the β-galactosidase gene. The simian virus 40 (SV-40) early promoter linked to the Tn5 neomycin resistance gene provides resistance to the drug G-418 and is inserted downstream of the s-galactosidase gene, providing a marker to select for retrovirus-containing, β-galactosidase expressing cells. This defective retrovirus was prepared from fibroblast φ am cells (3,10), and maintained in Dulbecco's modified Eagle's medium (DMEM) and 10% calf serum. Cells were passaged twice weekly following trypsinization. The supernatant, with titers of 104-105/ml G-418 resistant colonies, was added to endothelial cells at two-thirds confluence and incubated for 12 hours in DMEM with 10% calf serum at 37° C. in 5% CO2 in the presence of 8 μg/ml of polybrene. Viral supernatants were removed, and cells maintained in medium 199 with 10% fetal calf serum, ECGS (50 μg/ml), and endothelial cell conditioned medium (20%) for an additional 24 to 48 hours prior to selection in G-418 (0.7 μg/ml of a 50% racemic mixture). G-418 resistant cells were isolated and analyzed for β-galactosidase expression using a standard histochemical stain (9). Cells stably expressing the β-galactosidase enzyme were maintained in continuous culture for use as needed. Frozen aliquots were stored in liquid nitrogen.
  • PUBLICATIONS CITED
    • 1. J. S: Reitman, R. W. Mahley, D. L. Fry, Atherosclerosis 43, 119 (1982).
    • 2. R. E. Pitos, T. L. Innerarity, J. N. Weinstein, R. W. Mahley, Arteriosclerosis 1, 177 (1981); T. J. C. Van Berkel, J. F. Kruijt FEBS Lett. 132, 61 (1981); J. C. Voyta, P. A. Netland, D. P. Via, E. P. Zetter, J. Cell. Biol., 99, 81A (abstr.) (1984); J. M. Wilson, D. E. Johnston, D. M. Jefferson, R. C. Mulligan, Proc. Natl. Acad. Sci. U.S.A., 84, 4421 (1988).
    • 3. J. Price, D. Turner, C. Cepko, Proc. Natl. Acad. Sci. U.S.A., 84, 156 (1987).
    • 4. R. Mann, R. C. Mulligan, D. Baltimore, Cell 33, 153 (1983); C. L. Cepko, B. E. Roberts, R. C. Mulligan, Cell 37, 1053 (1984); M. A. Eglitis, W. F. Anderson, Biotechniques 6, 608 (1988).
    • 5. S. G. Ellis, G. S. Roubin, S. B. King, J. S. Douglas, W. S. Weintraub et al., Circulation 77, 372 (1988); L. Schwartz, M. G. Bourassa, J. Lesperance, H. E. Aldrige, F. Kazim, et al., N. Engl. J. Med. 318, 1714 (1988).
    • 6. P. C. Block, R. K. Myler, S. Stertzer, J. T. Fallon, N. Engl. J. Med. 305, 382 (1981); P. M. Steele, J. H. Chesebro, A. W. Stanson, Circ. Res. 57, 105 (1985); J. R. Wilentz, T. A. Sanborn, C. C. Handenschild, C. R. Valeri, T. J. Ryan, D. P. Faxon, Circulation 75, 636 (1987); W. McBride, R. A. Lange, L. D. Hillis, N. Engl. J. Med. 318, 1734 (1988).
    • 7. J. Folkmah, M. Klagsbrun, Science 235, 442 (1987); S. J. Leibovich, P. J. Polyerini, H. Michael Shepard, D. M. Wiseman, V. Shively, N. Nuseir, Nature 329, 630 (1987); J. Folkman, M. Klagsbrun, Nature 329, 671 (1987).
    • 8. T. Matsumura, T. Yamanka, S. Hashizume, Y. Irie, K. Nitta, Japan. J. Exp. Med. 45, 377 (1975); D. G. S. Thilo, S. Muller-Kusel, D. Heinrich, I. Kauffer, E. Weiss, Artery, 8, 25a (1980).
    • 9. A. M. Dannenberg, M. Suga, in Methods for Studying Mononuclear Phagocytes, D. O. Adams, P. J. Edelson, H. S. Koren, Eds. (Academic Press, New York, 1981), pp 375-395.
    • 10. R. D. Cone, R. C. Mulligan, Proc. Natl. Acad. Sci. U.S.A. 81, 6349 (1984).
  • Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims (29)

1-17. (canceled)
18. A kit for treating a disease in a patient, comprising:
a catheter comprising a body and balloon element adapted to be inserted into a blood vessel of said patient and being expansible against the walls of said vessel so as to hold said catheter body in place; and
a physiologically acceptable solution comprising cells,
wherein said catheter body includes means for delivering said composition into said blood vessel.
19. The kit of claim 18, wherein said catheter body comprises two spaced balloon elements, adapted to be inserted in a blood vessel and both being expansible against the wall of the blood vessel, for providing a chamber in said blood vessel and so as to hold said body in place, and where the means for delivering the composition into the blood vessel is situated in between the balloon elements.
20. The kit of claim 18, wherein the cells are normal (untransformed) cells.
21. The kit of claim 18, wherein the cells are transformed cells.
22. The kit of claim 18, wherein the means for delivering the composition into the blood vessel comprises a plurality of pores.
23. The kit of claim 18, wherein the number of pores is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.
24. The kit of claim 18, wherein the cells are endothelial cells, smooth muscle cells, fibroblasts, monocytes, macrophages, or parenchymal cells.
25. The kit of claim 18, wherein the cells produce a protein which has a therapeutic or diagnostic effect in the patient.
26. The kit of claim 18, wherein the cells produce an angiogenic factor.
27. The kit of claim 18, wherein the cells are glucose-responsive insulin-secreting cells.
28. The kit of claim 18, wherein the cells produce a toxin.
29. The kit of claim 18, wherein the composition is stored in liquid nitrogen.
30. The kit of claim 1, wherein the catheter is a catheter as described in U.S. Pat. No. 4,636,195.
31. The kit of claim 18, wherein the kit further comprises a guidewire.
32. The kit of claim 18, wherein the composition further comprises heparin, poly-L-lysine, polybrene, dextran sulfate, a polycationic material, or a bivalent antibody.
33. The kit of claim 21, wherein the cell comprises a nucleic acid sequence encoding tPA, urokinase, streptokinase, acidic fibroblast growth factor, basic fibroblast growth factor, tumor necrosis factor α, tumor necrosis factor β, transforming growth factor α, transforming growth factor β, atrial natriuretic factor, platelet-derived growth factor, endothelian, insulin, diphtheria toxin, pertussis toxin, cholera toxin, soluble CD4, a growth hormone, a marker protein, or derivatives thereof.
34. A kit for treating a disease in a patient in need thereof, comprising:
a) a medical device for insertion into a blood vessel; and
b) an antisense agent which is complementary to a DNA or mRNA encoded by a gene in said patient.
35. The kit of claim 34, wherein the antisense agent is complementary to the 5′ untranslated region of the mRNA.
36. The kit of claim 34, wherein the antisense agent is complementary to the coding region of the mRNA.
37. The kit of claim 34, wherein the antisense agent is complementary to the 3′ untranslated region of the mRNA.
38. The kit of claim 34, wherein the antisense agent is a synthetic oligonucleotide.
39. The kit of claim 34, wherein the antisense agent is antisense DNA.
40. The kit of claim 34, wherein the medical device is a catheter.
41. The kit of claim 34, wherein the medical device is a syringe and needle.
42. The kit of claim 34, wherein the gene encodes an angiogenic factor.
43. The kit of claim 34, wherein the antisense agent is in solution.
44. The kit of claim 34, wherein the antisense agent is a plasmid which expresses the revise complement of the gene.
45. The kit of claim 34, wherein the gene encodes a protein that induces angiogenesis.
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Families Citing this family (101)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7238673B2 (en) * 1989-03-31 2007-07-03 The Regents Of The University Of Michigan Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor
US5698531A (en) * 1989-03-31 1997-12-16 The Regents Of The University Of Michigan Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor
FR2688514A1 (en) * 1992-03-16 1993-09-17 Centre Nat Rech Scient Defective recombinant adenoviruses expressing cytokines and antitumour drugs containing them
ATE307212T1 (en) 1992-11-18 2005-11-15 Arch Dev Corp ADENOVIRUS-DIRECTED GENE TRANSFER TO THE HEART AND VASCULAR SMOOTH MUSCLE
AU7404994A (en) * 1993-07-30 1995-02-28 Regents Of The University Of California, The Endocardial infusion catheter
US20020193338A1 (en) * 1994-02-18 2002-12-19 Goldstein Steven A. In vivo gene transfer methods for wound healing
US5942496A (en) 1994-02-18 1999-08-24 The Regent Of The University Of Michigan Methods and compositions for multiple gene transfer into bone cells
US5962427A (en) 1994-02-18 1999-10-05 The Regent Of The University Of Michigan In vivo gene transfer methods for wound healing
US6551618B2 (en) 1994-03-15 2003-04-22 University Of Birmingham Compositions and methods for delivery of agents for neuronal regeneration and survival
US6037329A (en) 1994-03-15 2000-03-14 Selective Genetics, Inc. Compositions containing nucleic acids and ligands for therapeutic treatment
JP3961019B2 (en) 1995-02-28 2007-08-15 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Gene transfer mediated angiogenesis therapy
US20030148968A1 (en) * 1995-02-28 2003-08-07 Hammond H. Kirk Techniques and compositions for treating cardiovascular disease by in vivo gene delivery
US5922687A (en) * 1995-05-04 1999-07-13 Board Of Trustees Of The Leland Stanford Junior University Intracellular delivery of nucleic acids using pressure
US6322548B1 (en) 1995-05-10 2001-11-27 Eclipse Surgical Technologies Delivery catheter system for heart chamber
US20020068049A1 (en) * 1998-09-10 2002-06-06 Henderson Daniel R. Tissue specific adenoviral vectors
US7803782B2 (en) * 2003-05-28 2010-09-28 Roche Madison Inc. Intravenous delivery of polynucleotides to cells in mammalian limb
US6265387B1 (en) * 1995-10-11 2001-07-24 Mirus, Inc. Process of delivering naked DNA into a hepatocyte via bile duct
US6627616B2 (en) 1995-12-13 2003-09-30 Mirus Corporation Intravascular delivery of non-viral nucleic acid
US20010009904A1 (en) * 1997-12-30 2001-07-26 Jon A. Wolff Process of delivering a polynucleotide to a cell via the vascular system
US7507722B1 (en) 1999-11-05 2009-03-24 Roche Madison Inc. Intravascular delivery of nucleic acid
US6379966B2 (en) 1999-02-26 2002-04-30 Mirus Corporation Intravascular delivery of non-viral nucleic acid
US20020001574A1 (en) 1995-12-13 2002-01-03 Jon A. Woiff Process of delivering a polynucleotide to a muscle cell via the vascular system
JP3930052B2 (en) 1996-02-15 2007-06-13 バイオセンス・インコーポレイテッド Catheter-based surgery
US6143037A (en) * 1996-06-12 2000-11-07 The Regents Of The University Of Michigan Compositions and methods for coating medical devices
US6443974B1 (en) 1996-07-28 2002-09-03 Biosense, Inc. Electromagnetic cardiac biostimulation
US6387700B1 (en) * 1996-11-04 2002-05-14 The Reagents Of The University Of Michigan Cationic peptides, Cys-Trp-(LYS)n, for gene delivery
US6335010B1 (en) * 1996-11-08 2002-01-01 University Of California At San Diego Gene therapy in coronary angioplasty and bypass
US6547787B1 (en) 1997-03-13 2003-04-15 Biocardia, Inc. Drug delivery catheters that attach to tissue and methods for their use
US6416510B1 (en) 1997-03-13 2002-07-09 Biocardia, Inc. Drug delivery catheters that attach to tissue and methods for their use
US6818016B1 (en) * 1997-06-27 2004-11-16 The Regents Of The University Of Michigan Methods for coating stents with DNA and expression of recombinant genes from DNA coated stents in vivo
US6696423B1 (en) 1997-08-29 2004-02-24 Biogen, Inc. Methods and compositions for therapies using genes encoding secreted proteins such as interferon-beta
US7435723B2 (en) * 1997-11-21 2008-10-14 Mirus Bio Corporation Process for delivery of polynucleotides to the prostate
US6221425B1 (en) 1998-01-30 2001-04-24 Advanced Cardiovascular Systems, Inc. Lubricious hydrophilic coating for an intracorporeal medical device
US20030113303A1 (en) * 1998-02-05 2003-06-19 Yitzhack Schwartz Homing of embryonic stem cells to a target zone in tissue using active therapeutics or substances
US20030129750A1 (en) * 1998-02-05 2003-07-10 Yitzhack Schwartz Homing of donor cells to a target zone in tissue using active therapeutics or substances
ES2293473T3 (en) 1998-02-05 2008-03-16 Biosense Webster, Inc. INTRACARDIAC ADMINISTRATION OF FARMACO.
NZ505955A (en) 1998-02-06 2005-04-29 Collateral Therapeutics Inc Variants of the angiogenic factor vascular endothelial cell growth factor: VEGF-A, and pharmaceutical use
US6395253B2 (en) 1998-04-23 2002-05-28 The Regents Of The University Of Michigan Microspheres containing condensed polyanionic bioactive agents and methods for their production
US6102887A (en) 1998-08-11 2000-08-15 Biocardia, Inc. Catheter drug delivery system and method for use
WO2000033891A1 (en) * 1998-12-04 2000-06-15 Medivas, Llc Methods for detection of vulnerable plaques using a detectable lipid-avid agent
US6224566B1 (en) 1999-05-04 2001-05-01 Cardiodyne, Inc. Method and devices for creating a trap for confining therapeutic drugs and/or genes in the myocardium
US6565528B1 (en) 1999-05-07 2003-05-20 Scimed Life Systems, Inc. Apparatus and method for delivering therapeutic and diagnostic agents
EP1187652B1 (en) * 1999-06-02 2006-10-11 Boston Scientific Limited Devices for delivering a drug
US7147633B2 (en) 1999-06-02 2006-12-12 Boston Scientific Scimed, Inc. Method and apparatus for treatment of atrial fibrillation
US6770740B1 (en) 1999-07-13 2004-08-03 The Regents Of The University Of Michigan Crosslinked DNA condensate compositions and gene delivery methods
US20040110684A1 (en) * 1999-08-02 2004-06-10 Universite Catholique De Louvain Novel pharmaceutical compositions for modulating angiogenesis
US7214369B2 (en) * 2003-05-05 2007-05-08 Mirus Bio Corporation Devices and processes for distribution of genetic material to mammalian limb
US6676679B1 (en) 1999-11-05 2004-01-13 Boston Scientific Corporation Method and apparatus for recurrent demand injury in stimulating angiogenesis
EP1246649B1 (en) * 1999-11-05 2006-10-18 Mirus Bio Corporation Intravascular delivery of nucleic acid
US6748258B1 (en) * 1999-11-05 2004-06-08 Scimed Life Systems, Inc. Method and devices for heart treatment
US7642248B2 (en) * 1999-11-05 2010-01-05 Roche Madison Inc Devices and processes for distribution of genetic material to mammalian limb
EP1229845A2 (en) * 1999-11-05 2002-08-14 Microheart, Inc. Method and apparatus for demand injury in stimulating angiogenesis
US20040072785A1 (en) * 1999-11-23 2004-04-15 Wolff Jon A. Intravascular delivery of non-viral nucleic acid
US8460367B2 (en) 2000-03-15 2013-06-11 Orbusneich Medical, Inc. Progenitor endothelial cell capturing with a drug eluting implantable medical device
US8088060B2 (en) 2000-03-15 2012-01-03 Orbusneich Medical, Inc. Progenitor endothelial cell capturing with a drug eluting implantable medical device
US9522217B2 (en) * 2000-03-15 2016-12-20 Orbusneich Medical, Inc. Medical device with coating for capturing genetically-altered cells and methods for using same
WO2001070117A2 (en) 2000-03-23 2001-09-27 Microheart, Inc. Pressure sensor for therapeutic delivery device and method
US7214223B2 (en) * 2000-03-24 2007-05-08 Boston Scientific Scimed, Inc. Photoatherolytic catheter apparatus and method
AU2001238518B2 (en) * 2000-04-04 2006-09-07 Boston Scientific Limited Medical devices suitable for gene therapy regimens
US6478776B1 (en) 2000-04-05 2002-11-12 Biocardia, Inc. Implant delivery catheter system and methods for its use
US20030056244A1 (en) * 2000-05-02 2003-03-20 Ning Huang Feed additive compositions and methods
US7588554B2 (en) 2000-06-26 2009-09-15 Boston Scientific Scimed, Inc. Method and apparatus for treating ischemic tissue
US6685672B1 (en) 2000-07-13 2004-02-03 Edwards Lifesciences Corporation Multi-balloon drug delivery catheter for angiogenesis
US7220232B2 (en) * 2000-08-24 2007-05-22 Timi 3 Systems, Inc. Method for delivering ultrasonic energy
CA2421005A1 (en) * 2000-08-24 2002-02-28 Timi 3 Systems, Inc. Systems and method for applying ultrasonic energy
US20020091339A1 (en) * 2000-08-24 2002-07-11 Timi 3 Systems, Inc. Systems and methods for applying ultrasound energy to stimulating circulatory activity in a targeted body region of an individual
US6790187B2 (en) * 2000-08-24 2004-09-14 Timi 3 Systems, Inc. Systems and methods for applying ultrasonic energy
US20030069526A1 (en) * 2000-08-24 2003-04-10 Timi 3 Systems, Inc. Applicators that house and support ultrasound transducers for transcutaneous delivery of ultrasound energy
US7335169B2 (en) * 2000-08-24 2008-02-26 Timi 3 Systems, Inc. Systems and methods for delivering ultrasound energy at an output power level that remains essentially constant despite variations in transducer impedance
WO2002015804A1 (en) * 2000-08-24 2002-02-28 Timi 3 Systems, Inc. Systems and methods for applying ultrasound energy
US7241270B2 (en) * 2000-08-24 2007-07-10 Timi 3 Systems Inc. Systems and methods for monitoring and enabling use of a medical instrument
US20020049395A1 (en) * 2000-08-24 2002-04-25 Timi 3 Systems for applying ultrasound energy to the thoracic cavity
AU2002213192A1 (en) * 2000-10-13 2002-04-22 The Trustees Of Columbia University In The City Of New York A method for inhibiting new tissue growth in blood vessels in a patient subjected to blood vessel injury
US6812217B2 (en) * 2000-12-04 2004-11-02 Medtronic, Inc. Medical device and methods of use
US6692458B2 (en) 2000-12-19 2004-02-17 Edwards Lifesciences Corporation Intra-pericardial drug delivery device with multiple balloons and method for angiogenesis
CA2439185A1 (en) * 2001-02-23 2002-09-06 Novartis Ag Vector constructs
US6808518B2 (en) * 2001-09-28 2004-10-26 Ethicon, Inc. Methods and devices for treating diseased blood vessels
US20030086903A1 (en) * 2001-11-02 2003-05-08 Genvec, Inc. Therapeutic regimen for treating cancer
US7229423B2 (en) * 2003-02-05 2007-06-12 Timi 3 System, Inc Systems and methods for applying audible acoustic energy to increase tissue perfusion and/or vasodilation
AU2009210402A1 (en) * 2002-07-24 2009-09-10 Timi 3 Systems, Inc. Systems and methods for monitoring and enabling use of a medical instrument
WO2004011060A2 (en) 2002-07-26 2004-02-05 Mirus Corporation Delivery of molecules and complexes to mammalian cells in vivo
US20040044329A1 (en) * 2002-08-29 2004-03-04 Trudell Leonard A. Catheter for cardiac injection and method for delivery of therapeutic agents to specified tissues
US20040136960A1 (en) * 2003-01-10 2004-07-15 Wolff Jon A. Devices and processes for distribution of genetic material to mammalian limb
US20080208084A1 (en) * 2003-02-05 2008-08-28 Timi 3 Systems, Inc. Systems and methods for applying ultrasound energy to increase tissue perfusion and/or vasodilation without substantial deep heating of tissue
JP2005006779A (en) * 2003-06-17 2005-01-13 Terumo Corp Lumen of living body cleaning device
US10517883B2 (en) 2003-06-27 2019-12-31 Zuli Holdings Ltd. Method of treating acute myocardial infarction
US8870796B2 (en) 2003-09-04 2014-10-28 Ahof Biophysical Systems Inc. Vibration method for clearing acute arterial thrombotic occlusions in the emergency treatment of heart attack and stroke
US8721573B2 (en) 2003-09-04 2014-05-13 Simon Fraser University Automatically adjusting contact node for multiple rib space engagement
US8734368B2 (en) 2003-09-04 2014-05-27 Simon Fraser University Percussion assisted angiogenesis
CA2439667A1 (en) 2003-09-04 2005-03-04 Andrew Kenneth Hoffmann Low frequency vibration assisted blood perfusion system and apparatus
CA2545815A1 (en) * 2003-11-14 2005-06-09 Genvec, Inc. Adenoviral vectored tnf-a and chemoradiation to treat cancer
GB0406728D0 (en) * 2004-03-25 2004-04-28 Hydrodynamic Gene Delivery Ltd Gene therapy
US7534775B2 (en) * 2004-04-08 2009-05-19 Sangamo Biosciences, Inc. Methods and compositions for modulating cardiac contractility
EP1732614B1 (en) * 2004-04-08 2008-12-24 Sangamo Biosciences Inc. Compositions for treating neuropathic and neurodegenerative conditions
EP2946666B1 (en) * 2004-04-30 2017-11-15 OrbusNeich Medical, Inc. Medical device with coating for capturing genetically-altered cells and methods of using same
JP5058822B2 (en) 2005-01-25 2012-10-24 ファイブ プライム セラピューティクス, インコーポレイテッド Compositions and methods for treating cardiac conditions
US20080097377A1 (en) * 2006-06-13 2008-04-24 The Florida International University Board Of Trustees Temperature-controlled catheter system and method
WO2008103336A1 (en) * 2007-02-21 2008-08-28 Cook Incorporated Methods for intravascular engraftment in heart
KR20100129295A (en) 2008-02-19 2010-12-08 셀라돈 코포레이션 Compositions for enhanced uptake of viral vectors in the myocardium
US9605844B2 (en) * 2009-09-01 2017-03-28 Cree, Inc. Lighting device with heat dissipation elements
EP3400290B1 (en) 2016-01-08 2023-04-05 Replimune Limited Oncolytic virus strain

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4332893A (en) * 1980-06-13 1982-06-01 Rosenberg Ralph A Process for the production of an insulin-producing cell line of pancreatic beta cells
US4353888A (en) * 1980-12-23 1982-10-12 Sefton Michael V Encapsulation of live animal cells
US4636195A (en) * 1982-04-02 1987-01-13 Harvey Wolinsky Method and apparatus for removing arterial constriction
US4824436A (en) * 1985-04-09 1989-04-25 Harvey Wolinsky Method for the prevention of restenosis
US4874746A (en) * 1987-12-22 1989-10-17 Institute Of Molecular Biology, Inc. Wound headling composition of TGF-alpha and PDGF
US5087617A (en) * 1989-02-15 1992-02-11 Board Of Regents, The University Of Texas System Methods and compositions for treatment of cancer using oligonucleotides
US5580859A (en) * 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
US5661133A (en) * 1991-11-12 1997-08-26 The Regents Of The University Of Michigan Expression of a protein in myocardium by injection of a gene
US5662896A (en) * 1988-03-21 1997-09-02 Chiron Viagene, Inc. Compositions and methods for cancer immunotherapy
US5674722A (en) * 1987-12-11 1997-10-07 Somatix Therapy Corporation Genetic modification of endothelial cells
US5707969A (en) * 1989-03-31 1998-01-13 The Regents Of The University Of Michigan Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor
US6001350A (en) * 1987-12-11 1999-12-14 Somatix Therapy Corp Genetic modification of endothelial cells

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0273085A1 (en) * 1986-12-29 1988-07-06 IntraCel Corporation A method for internalizing nucleic acids into eukaryotic cells
US5997859A (en) * 1988-03-21 1999-12-07 Chiron Corporation Method for treating a metastatic carcinoma using a conditionally lethal gene
CA2489769A1 (en) * 1989-03-21 1990-10-04 Philip L. Felgner Expression of exogenous polynucleotide sequences in a vertebrate
JPH04504216A (en) 1989-03-31 1992-07-30 ザ・リージエンツ・オブ・ザ・ユニバーシテイ・オブ・ミシガン Treatment of diseases by site-specific infusion of cells or site-specific transformation of cells, and kits for the treatment
CA2044593C (en) 1989-11-03 2004-04-20 Kenneth L. Brigham Method of in vivo delivery of functioning foreign genes
US6203991B1 (en) * 1998-08-21 2001-03-20 The Regents Of The University Of Michigan Inhibition of smooth muscle cell migration by heme oxygenase I

Patent Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4332893A (en) * 1980-06-13 1982-06-01 Rosenberg Ralph A Process for the production of an insulin-producing cell line of pancreatic beta cells
US4353888A (en) * 1980-12-23 1982-10-12 Sefton Michael V Encapsulation of live animal cells
US4636195A (en) * 1982-04-02 1987-01-13 Harvey Wolinsky Method and apparatus for removing arterial constriction
US4824436A (en) * 1985-04-09 1989-04-25 Harvey Wolinsky Method for the prevention of restenosis
US6001350A (en) * 1987-12-11 1999-12-14 Somatix Therapy Corp Genetic modification of endothelial cells
US5674722A (en) * 1987-12-11 1997-10-07 Somatix Therapy Corporation Genetic modification of endothelial cells
US4874746A (en) * 1987-12-22 1989-10-17 Institute Of Molecular Biology, Inc. Wound headling composition of TGF-alpha and PDGF
US5662896A (en) * 1988-03-21 1997-09-02 Chiron Viagene, Inc. Compositions and methods for cancer immunotherapy
US5087617A (en) * 1989-02-15 1992-02-11 Board Of Regents, The University Of Texas System Methods and compositions for treatment of cancer using oligonucleotides
US5589466A (en) * 1989-03-21 1996-12-31 Vical Incorporated Induction of a protective immune response in a mammal by injecting a DNA sequence
US5580859A (en) * 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
US5707969A (en) * 1989-03-31 1998-01-13 The Regents Of The University Of Michigan Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor
US5661133A (en) * 1991-11-12 1997-08-26 The Regents Of The University Of Michigan Expression of a protein in myocardium by injection of a gene
US5661133B1 (en) * 1991-11-12 1999-06-01 Univ Michigan Collateral blood vessel formation in cardiac muscle by injecting a dna sequence encoding an angiogenic protein

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