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WO1990011734A1 - Traitement de maladies par instillation specifique au site de cellules ou transformation specifique au site de cellules, et kits utilises dans ce traitement - Google Patents

Traitement de maladies par instillation specifique au site de cellules ou transformation specifique au site de cellules, et kits utilises dans ce traitement Download PDF

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Publication number
WO1990011734A1
WO1990011734A1 PCT/US1990/001662 US9001662W WO9011734A1 WO 1990011734 A1 WO1990011734 A1 WO 1990011734A1 US 9001662 W US9001662 W US 9001662W WO 9011734 A1 WO9011734 A1 WO 9011734A1
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WIPO (PCT)
Prior art keywords
cells
disease
blood vessel
vessel
solution
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PCT/US1990/001662
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English (en)
Inventor
Elizabeth G. Nabel
Gary J. Nabel
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The Regents Of The University Of Michigan
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Publication of WO1990011734A1 publication Critical patent/WO1990011734A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods
    • A61B17/22—Implements for squeezing-off ulcers or the like on inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; for invasive removal or destruction of calculus using mechanical vibrations; for removing obstructions in blood vessels, not otherwise provided for
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods
    • A61B17/22—Implements for squeezing-off ulcers or the like on inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; for invasive removal or destruction of calculus using mechanical vibrations; for removing obstructions in blood vessels, not otherwise provided for
    • A61B2017/22038—Implements for squeezing-off ulcers or the like on inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; for invasive removal or destruction of calculus using mechanical vibrations; for removing obstructions in blood vessels, not otherwise provided for with a guide wire
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods
    • A61B17/22—Implements for squeezing-off ulcers or the like on inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; for invasive removal or destruction of calculus using mechanical vibrations; for removing obstructions in blood vessels, not otherwise provided for
    • A61B2017/22051—Implements for squeezing-off ulcers or the like on inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; for invasive removal or destruction of calculus using mechanical vibrations; for removing obstructions in blood vessels, not otherwise provided for with an inflatable part, e.g. balloon, for positioning, blocking, or immobilisation
    • A61B2017/22054—Implements for squeezing-off ulcers or the like on inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; for invasive removal or destruction of calculus using mechanical vibrations; 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods
    • A61B17/22—Implements for squeezing-off ulcers or the like on inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; for invasive removal or destruction of calculus using mechanical vibrations; for removing obstructions in blood vessels, not otherwise provided for
    • A61B2017/22082—Implements for squeezing-off ulcers or the like on inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; for invasive removal or destruction of calculus using mechanical vibrations; for removing obstructions in blood vessels, not otherwise provided for after introduction of a substance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods
    • A61B17/22—Implements for squeezing-off ulcers or the like on inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; for invasive removal or destruction of calculus using mechanical vibrations; for removing obstructions in blood vessels, not otherwise provided for
    • A61B2017/22082—Implements for squeezing-off ulcers or the like on inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; for invasive removal or destruction of calculus using mechanical vibrations; for removing obstructions in blood vessels, not otherwise provided for after introduction of a substance
    • A61B2017/22084—Implements for squeezing-off ulcers or the like on inner organs of the body; Implements for scraping-out cavities of body organs, e.g. bones; for invasive removal or destruction of calculus using mechanical vibrations; for removing obstructions in blood vessels, not otherwise provided for after introduction of a substance stone- or thrombus-dissolving

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- ⁇ ) .
  • PDGF platelet derived growth factor
  • FGF fibroblast growth factor
  • EGF epidermal growth factor
  • TGF- ⁇ transforming growth factor alpha
  • thrombus formation in acute myocardial ischemia and restenosis following coronary angioplasty 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.
  • 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.
  • FIGURES 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, dr 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, dr 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.
  • FIGURE 1 this figure illustrates the practice of the present invention with a catheter having a design as disclosed in U.S. Patent 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 l and 2 is equipped with instillation port means 3..
  • the catheter may be further equipped with a guidewire means 6.
  • FIGURE 2 illustrates the use of a similar catheter, distinguished from the catheter illustrated in Figure 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. . 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 x 10 ) are collected by centrifugation and resuspended in medium 199 (GIBCO) containing 10% fetal bovine serum, endothelial cell growth supplement (ECGS, Collaborative Research, Waltham, MA) at 25 ⁇ g/ml, heparin at 15 U/ml, and gentamicin (50 ⁇ g/ml) .
  • 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 fibroblaits 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 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. Patent 4,636,195, which is hereby incorporated by reference (available from USCI, Billerica, MA) is advanced by guidewire means (6) under fluoroscopic guidance, if necessary, into the vessel (5) ( Figure 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 (10 to 10 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. Jung, C.J. Honsik, R.A. Reisfeld, and H.J. Muller-Eberhard, Proc. Natl. Acad. Sci. USA. 83, 4479 (1986); U.D. Staerz, 0. 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.
  • 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.
  • growth factors delivered locally by catheter or systemically to enhance the efficiency of infection.
  • retroviral vectors 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. and Weintraub, H. , Science, 229, 345-352, (1985); Cell.
  • 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 Si 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 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.
  • 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 ele ⁇ troporation 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 SI nuclease analysis. When confirmed, the expression of mRNA may be evaluated by SI 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 neo ycin 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 SI 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.
  • 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.
  • 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-3) , angiogenin, tumor necrosis factor ⁇ , tumor necrosis factor ⁇ , acidic fibroblast growth factor or basic fibroblast growth factor can be used.
  • 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.
  • 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, diphth
  • the present method may also use exogenous proteins of diagnostic value.
  • a marker protein such as j8-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, MA) 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 low density lipoprotein
  • BAG murine amphotropic j ⁇ -galactosidase-transducing retroviral vector
  • the femoral and iliac arteries were exposed, and a catheter was introduced into the vessel (Figure 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 j8-galactosidase were introduced using a specially designed arterial catheter (USCI, Billerica, MA) that contained two balloons and a central instillation port (Figure 1) .
  • 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 0-galactosidase appeared constant in vessels examined up to six weeks after introduction into the blood vessel in the present study.
  • a major complication of current interventions for vascular disease 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.
  • antithrombotic effects can be achieved by introducing cells expressing genes for tissue plasminogen 5 activator or urokinase. This technology is also useful for the treatment of chronic tissue ischemia.
  • Endothelial cells were derived from external jugular veins 20 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%), 25 50 ⁇ mg/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 3-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 0-galactosidase protein was used to identify infected endothelial cells in vitro and in vivo.
  • the 3-galactosidase transducing Mo-MuLV vector (2) , (BAG) was kindly provided by Dr. Constance Cepko. This vector used the wild type MoMuLV LTR as a promoter for the 3-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 3-galactosidase gene, providing a marker to select for retrovirus-containing, /3-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 trypsmization.
  • G-418 resistant cells were isolated and analyzed for ?-galactosidase expression using a standard histochemical stain (9) .
  • Cells stably expressing the 0-galactosidase enzyme were maintained in continuous culture for use as needed. Frozen aliquots were stored in liquid nitrogen.

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Abstract

Méthode de traitement direct de maladie axé sur les sites spécifiques. La méthode est fondée sur l'apport de protéines par cathétérisation de segments individuels de vaisseaux sanguins en utilisant des cellules normales ou génétiquement modifiées ou d'autres systèmes vecteurs. Des cellules endothéliales exprimant un agent thérapeutique de recombinaison ou des protéines diagnostiques sont situées sur des parois (5) du vaisseau sanguin ou dans les tissus subissant une perfusion par l'intermédiaire du vaisseau dans le patient. Cette technique permet le transfert de cellules ou vecteurs et l'expression de gènes de recombinaison in vivo et permet l'introduction de protéines à action thérapeutique ou diagnostique pour le traitement de maladies.
PCT/US1990/001662 1989-03-31 1990-04-02 Traitement de maladies par instillation specifique au site de cellules ou transformation specifique au site de cellules, et kits utilises dans ce traitement WO1990011734A1 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0591385A1 (fr) * 1991-06-28 1994-04-13 The Regents Of The University Of Michigan Traitement de pathologies par instillation sitospecifique de cellules ou par transformation sitospecifique de cellules et trousses prevues a cet effet
EP0591408A1 (fr) * 1991-06-28 1994-04-13 The Regents Of The University Of Michigan Traitement de pathologies par instillation sitospecifique de cellules ou par transformation sitospecifique de cellules et trousses prevues a cet effet
EP0671954A1 (fr) * 1992-02-05 1995-09-20 Thomas Jefferson University Therapie genique par interferons pour le traitement des pathologies vasculaires
WO1995025547A1 (fr) * 1994-03-24 1995-09-28 University Of Washington Dispositif et methodes d'implantation de cellules ayant subi une transduction
US5641497A (en) * 1992-05-22 1997-06-24 Children's Hospital Of Philadelphia Gastrointestinal defensins, cDNA sequences and method for the production and use thereof
WO1999033500A2 (fr) 1997-12-31 1999-07-08 Pharmasonics, Inc. Procedes, systemes et kits pour l'apport intravasculaire d'acides nucleiques
US5941868A (en) * 1995-12-22 1999-08-24 Localmed, Inc. Localized intravascular delivery of growth factors for promotion of angiogenesis
US6297219B1 (en) 1989-03-31 2001-10-02 The Regents Of The University Of Michigan Site-specific instillation of cells or site-specific transformation of cells and kits therefor
US6696423B1 (en) 1997-08-29 2004-02-24 Biogen, Inc. Methods and compositions for therapies using genes encoding secreted proteins such as interferon-beta
US6794369B2 (en) 1997-12-31 2004-09-21 Pharmasonics Methods, systems, and kits for intravascular nucleic acid delivery
WO2005028608A3 (fr) * 2003-08-29 2006-01-05 Kist Europe Forschungsges Mbh Processeur de cellules servant a traiter des maladies
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

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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

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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

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(NABEL), "Recombinant Gene Expression In Vivo within Endothelial Cells of the Arterial Wall", SCIENCE, issued June 1989, Volume 244, pages 1342-1344, see the entire document. *
(SHELDEN), "Implantation of Genetically Engineerd Fibroblasts into Mice: Implications for Gene Therapy", SCIENCE, issued May 1987, Volume 236, pages 714-718, see the entire document. *
(ZWIEBEL); "High-Level Recombinant Gene Expression in Rabbit Endothelial Cells Transduced by Retroviral Vectors", SCIENCE, issued January 1989, Volume 243, pages 220-222, see the entire document. *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6297219B1 (en) 1989-03-31 2001-10-02 The Regents Of The University Of Michigan Site-specific instillation of cells or site-specific transformation of cells and kits therefor
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
US7226589B2 (en) 1989-03-31 2007-06-05 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
EP1013287A2 (fr) * 1991-06-28 2000-06-28 The Regents Of The University Of Michigan Traitement de pathologies par instillation sitospécifique de cellules ou par transformation sitospécifique de cellules et trousses prévues à cet effet
EP1927368A3 (fr) * 1991-06-28 2008-06-11 The Regents Of The University Of Michigan Traitement de maladies par la perfusion ou la transformation spécifique au site de cellules et kits correspondants
EP0591385A4 (en) * 1991-06-28 1996-11-27 Univ Michigan Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor
EP0591408A4 (en) * 1991-06-28 1996-11-27 Univ Michigan Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor
EP0591408A1 (fr) * 1991-06-28 1994-04-13 The Regents Of The University Of Michigan Traitement de pathologies par instillation sitospecifique de cellules ou par transformation sitospecifique de cellules et trousses prevues a cet effet
EP1213032A1 (fr) * 1991-06-28 2002-06-12 The Regents Of The University Of Michigan Traitement de pathologies par instillation sitospécifique de cellules ou par transformation sitospécifique de cellules et trousses prévues à cet effet
EP1013287A3 (fr) * 1991-06-28 2000-07-05 The Regents Of The University Of Michigan Traitement de pathologies par instillation sitospécifique de cellules ou par transformation sitospécifique de cellules et trousses prévues à cet effet
EP0591385A1 (fr) * 1991-06-28 1994-04-13 The Regents Of The University Of Michigan Traitement de pathologies par instillation sitospecifique de cellules ou par transformation sitospecifique de cellules et trousses prevues a cet effet
EP0671954A1 (fr) * 1992-02-05 1995-09-20 Thomas Jefferson University Therapie genique par interferons pour le traitement des pathologies vasculaires
EP0671954A4 (fr) * 1992-02-05 1995-10-11
US5641497A (en) * 1992-05-22 1997-06-24 Children's Hospital Of Philadelphia Gastrointestinal defensins, cDNA sequences and method for the production and use thereof
WO1995025547A1 (fr) * 1994-03-24 1995-09-28 University Of Washington Dispositif et methodes d'implantation de cellules ayant subi une transduction
US5941868A (en) * 1995-12-22 1999-08-24 Localmed, Inc. Localized intravascular delivery of growth factors for promotion of angiogenesis
US6696423B1 (en) 1997-08-29 2004-02-24 Biogen, Inc. Methods and compositions for therapies using genes encoding secreted proteins such as interferon-beta
US7256181B2 (en) 1997-08-29 2007-08-14 Biogen Idec Ma Inc. Methods and compositions for therapies using genes encoding secreted proteins such as interferon-beta
WO1999033500A2 (fr) 1997-12-31 1999-07-08 Pharmasonics, Inc. Procedes, systemes et kits pour l'apport intravasculaire d'acides nucleiques
US6794369B2 (en) 1997-12-31 2004-09-21 Pharmasonics Methods, systems, and kits for intravascular nucleic acid delivery
US6372498B2 (en) 1997-12-31 2002-04-16 Pharmasonics, Inc. Methods, systems, and kits for intravascular nucleic acid delivery
WO2005028608A3 (fr) * 2003-08-29 2006-01-05 Kist Europe Forschungsges Mbh Processeur de cellules servant a traiter des maladies

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