JPH06509329A - Treatment of diseases by site-specific infusion of cells or site-specific transformation of cells and kits therefor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] site-specific infusion of cells or Treatment of diseases by site-specific transformation of cells and kits therefor Techniques The present invention is directed to site-specific infusion of cells or site-specific transformation of cells. Related to disease treatment and kits therefor.

Back 1U orthopedic surgery Effective treatments for many systemic and congenital diseases are essential to modern medicine. This remains a major challenge. Ability to deliver therapeutic drugs to specific parts of the body are of great importance, for example, for the treatment of local diseases. Also, cure If therapeutic drugs can be perfused through the circulatory system, they can be used to treat systemic diseases, etc. effective.

For example, ensuring that antitumor drugs or toxins can be delivered in close proximity to the tumor site. That is desirable. Similarly, insulin is present in the blood of diabetics, for example. It is also desirable to be able to perfuse the However, many treatments Regarding therapeutic agents, whether administered locally or systemically, the results are satisfactory. There is no way to do that.

In addition, in many diseases locally or systemically defective endogenous genes , expression of an exogenous gene, or suppression of an endogenous gene. Although it would be desirable to be able to do so, these are also issues that have not yet been achieved.

In particular, the pathogenesis of atherosclerosis involves the following three basic biological processes. It is characterized by These are = 1) proliferation of smooth muscle cells in the vascular intima and macrophages; 2) production of a large amount of connective tissue matrix by proliferation of smooth muscle cells; 3) Accumulation of lipids in cells and the connective tissue surrounding them (basically in the form of sterol esters and free cholesterol).

The first event that occurs is endothelial cell damage, which manifests as interference with the endothelial permeable membrane. This changes the anticoagulant properties of the endothelial surface and enhances the procoagulant properties of the endothelium. Monocytes inside Migrate between skin cells, become active as scavenger cells, and separate into macrophages. become

Macrophages then receive platelet-derived growth factor (PDGF), a cell growth factor. (FGF), epidermal growth factor (EGF), and transforming growth factor alpha (TGF- α) is synthesized and secreted. These growth factors inhibit atherosclerotic plaques. potently stimulates the migration and proliferation of fibroblasts and smooth muscle cells in the body. moreover, Platelets are formed through interaction with damaged endothelial cells and activated macrophages. leading to the production of long factors and the formation of blood clots.

Two major problems in the clinical management of coronary artery disease are acute myocardial ischemia. Thrombus formation and restenosis after coronary angioplasty (PTCA). Both of these growth factors due to endothelial damage, activated macrophages and platelets Common cellular events such as population release are involved. When coronary angioplasty is performed, atherosclerosis This will destroy the atherosclerotic plaque and remove the endothelium. This vascular trauma , promotes platelet aggregation and thrombus formation at the PTCA site. platelets and macrophages Further release of mitogens from the medium stimulates smooth muscle cell proliferation and monocyte infiltration. moisture leads to restenosis.

This problem cannot be prevented by empirical medical practice using antiplatelet agents, and patients with PTCA It is happening in 173 of . The solution to restenosis is to prevent platelet aggregation and blood clot formation. and inhibition of smooth muscle cell proliferation.

Thrombus generation is also a critical cell in the transition from stable to unstable coronary syndromes. It is a phenomenon. The pathogenesis often involves acute endothelial cell damage or plaque formation. destruction, promoting dysfunction of endothelial cell attachments and underlying macrophage foci. This will expose the ohmic cells. This allows the adhesion of circulating platelets. This creates opportunities for , aggregation, and thrombus formation.

by intravenous injection of thrombolytic agents such as tissue plasminogen activator (tPA). The results showed that it dissolves blood clots in approximately 70% of patients with acute myocardial infarction. It is. Despite this, approximately 30% of patients are unable to achieve reperfusion and suffer from infarction. Approximately 25% of patients who are reperfused with a strained artery will see the blood clot reoccur within 24 hours. have experienced an outbreak. Therefore, effective treatment for rethrombo This remains an important issue in the future.

As mentioned above, effective treatments for re-thrombosis are currently the only serious It's not a necessary issue. unstable angina, myocardial infarction and chronic tissue ischemia treatment of other ischemic conditions, such as systemic and congenital diseases, Treatment of cancer and other diseases is also an important issue. These diseases are treated with anticoagulants, vasodilators, blood Treatment can be achieved by appropriate administration of tube-forming agents, growth factors, and growth inhibitors to the patient. can be treated. Thus, effective treatments are available in all of these clinical areas. Therapy is desperately needed.

Kabuto-cho @Aji Accordingly, one object of the present invention is to develop a novel method for site-specific administration of therapeutic agents. The purpose is to provide a method.

Another object of the invention is to provide a method for perfusing therapeutic agents into the bloodstream of a patient. It is something that

Another object of the present invention is to provide a method for expressing an exogenous gene in a patient. It is something to do.

Another object of the present invention is to provide a method for expressing a defective endogenous gene in a patient. This is what we provide.

Another object of the present invention is to provide a method for suppressing the expression of endogenous genes in a patient's body. This is what we provide.

Another object of the present invention is to site-specifically replace damaged cells within a patient's body. This method provides a method for

Another object of the invention is the site-specific administration or delivery of therapeutic agents into the patient's bloodstream. It provides a method of treating diseases either by perfusion.

Another object of the present invention is to improve the expression of exogenous genes, defective endogenous genes in the patient's body. cause either expression of an endogenous gene or suppression of expression of an endogenous gene The present invention provides a method for treating diseases.

Another object of the present invention is to site-specifically replace damaged cells within a patient's body. It provides a method for treating diseases by

Another object of the present invention is to site-specifically introduce normal or transformed cells into a patient's body. The present invention provides a kit for differential infusion.

Another object of the present invention is to provide a kit for site-specific transformation of cells in vivo. This is what we provide.

These and other objects of the invention, which will become apparent in the following detailed description, are This was made clear by the enlighteners accomplishing the following: a) (i) Site identification of normal (non-transformed) or transformed cells to the patient (11) site-specific transformation of patient cells; and b) a kit comprising a catheter, the kit comprising: (1) normal cells or (11) site-specific infusion of transformed cells; For specific transformation.

Site-specific instillation of healthy cells can be used to replace damaged cells. , whereas instillation of transformed cells can detect defective endogenous genes or exogenous genes. Can be used to express either a gene or suppress an endogenous gene product . Instillation of cells into the patient's blood vessel walls is used to ensure perfusion of therapeutic agents into the bloodstream. be able to.

The invention and its many attendant advantages will be explained in more detail below with reference to the accompanying drawings. As you understand the explanation, you can easily reach a more complete understanding and evaluation. .

Figures 1 and 2 show how the catheter of the present invention can be used to surgically or percutaneously remove cells from blood vessels. for transplantation into the human body or for in vivo transformation of cells present in the patient's blood vessels. Here we show you how to use it.

1■ = - of 1 Here, in one embodiment, a congenital disease, a systemic disease, a cardiovascular disease, The present invention can be used to treat diseases of specific organs or diseases such as tumors. have used methods of instillation of transformed cells, or cell transformation.

Cells injected in the main stream include endothelial cells, smooth muscle cells, miscellaneous blast cells, and monoblasts. These include spheres, macrophages, and parenchymal cells. These cells have no therapeutic effect It is a protein that has diagnostic properties and can be found in naturally occurring or recombinant genes. It produces proteins derived from genetic substances.

Next, referring to the diagrams, here the same or corresponding parts are shown in all diagrams. The same numbers are given in both cases. In Figure 1, disclosed in U.S. Patent 4,836,195 2 shows an embodiment of the invention using a catheter of the design shown in FIG. (patent incorporated herein by reference). This catheter is normal or genetic. Deliver the transformed cells to the walls of blood vessels or use the base for local transformation of cells. can be used to introduce vectors. In the figure, each is the wall of the blood vessel.

In the figure, the catheter is inserted by inflating the inflatable balloon means 1 and 2. The handle portion 4 is shown being held in place. Balloon means ↓ and Beauty? A drip inlet means is provided in the portion between -. This catheter has Additionally, a guide wire means may be provided. Figure 2 shows the use of a similar catheter. is shown, the catheter includes an inflatable balloon means 2 and a plurality of This catheter differs from the catheter of FIG. 1 in that it is provided with a drip inlet means. This catheter The cell can be equipped with up to 12 independent drip inlet means a; 5flIAW is shown.

In the case of organ supply, this catheter is guided into the main artery supplying the tissue. entered. Cells containing recombinant genes or vectors can unify the arterial circulation. After temporary occlusion, it is introduced through the central drip inlet. In this way, Cell or vector DNA is imported into a large amount of parenchymal tissue through capillary circulation.

Recombinant genes also allow the double balloon catheter technique to be used in target organs. It can be used in the immediate arterial circulation and introduced into the vasculature. Like this If so, the recombinant genes can be secreted directly into the circulatory system that perfuses the involved tissues. It can be synthesized directly in the organ.

In one embodiment, the therapeutic agent is secreted by vascular cells that are affected by a disease. supplied to specific organs. For example, ischemic cardiomyopathy has angiogenic effects on the coronary circulatory system. It can be treated by introducing factors. This approach also Peripheral blood vessels and cerebrum where angiogenic factors can improve circulation in the brain and other tissues Can be used for vascular diseases. Diabetes mellitus is a glucose-responsive insulin insulin-secreting cells into the portal circulation, where the liver normally has higher insulin concentrations than other tissues. can be treated by introducing

In addition to increasing the local concentration of therapeutic agents, it is also possible to assemble mainstream combinant genes. It can also be used to feed textiles. This is because there are high concentrations of urinary chloride in the main stream. Can viral and other vectors be delivered to specific circulatory systems? It is et al. Organ-specific protein deficiencies can also be treated using this approach. can do. For example, in the liver, α-antitribusin inhibitor deficiency or Cholesterolemia can be treated by introducing α-antitribucin or LDL receptor genes. It can be treated with. Additionally, this approach can be used to treat malignant diseases. can also be used. Recombinant treatment for the circulatory system of inoperable tumors Secretion of the syngene has therapeutic effects. Examples include unresectable auditory tumors. Rui has some type of hemangioma.

In clinical situations, these therapeutic recombinant genes are The cells are then introduced into cells that are supplied cyclically. The best place to introduce these cells is the artery. The venous and capillary systems are preferred, but the venous system may also be used.

In the case of treatment of localized vascular damage, the present invention provides a method for locally ameliorating this condition. Express protein. In one embodiment, vascular cells are found at the site. Therefore, they were used as carriers for therapeutic drugs.

The invention thus provides, in one aspect, endothelial and other vascular cells. Therapeutic agents (i.e., proteins, growth factors) to the damaged area. Successful gene transplant into cells In order to do so, four conditions must be met. First, transplant it into cells. The gene of interest must be identified and isolated. Second, try to express The genes used must be cloned and made available for genetic manipulation. stomach. Third, genes are introduced into cells in such a way that they can be expressed or perform their functions. must be introduced. Fourth, genetically modified cells need It must be located correctly in the blood vessel site where it is intended.

According to the invention, transformed cells or suitable vectors can be surgically, percutaneously, or , or introduced intravenously and attached to a portion of the patient's blood vessel wall. Or the patient some of the cells present in the walls of human blood vessels, using the desired genetic material or Direct vector application and transformation. In some cases, lost from the blood vessel surface These methods can be used to replace damaged or damaged cells. It is also possible to introduce vascular cells that have not been converted to .

The present invention allows treatment of any blood vessel, i.e. arteries, veins, capillary system. can be done. These blood vessels can be found anywhere in the human or mammalian body. It can be in or near an organ.

Blood vessel tonight JJ missing l go J e 1 and 1 town Nu exchange - J libi thin a pu sun no, hereinafter, the present invention An embodiment of this will be described.

■Establishment of endothelial or other vascular cells in tissue culture Initially, one cell line is established and stored in liquid nitrogen. Take out a certain amount before storing at low temperature be infected or infected with a vector, virus, or other material containing the desired genetic material. Import◎ Endothelial or other vascular cells as previously published J, W, Ford et al.

Using the method of in Vitrq, 17.40 (1981), enzymatically Take out from part of. Excise the vessel and invert onto a stainless steel rod. Set aside and add a balanced salt solution (BSS ), in a medium containing 0.125% EDTA, p. Incubate at H8, 37°C for 10 minutes.

Cell populations (0.4 to 1.5 x lO') were collected by centrifugation and 10% fetal Bovine serum, 25 μg/ml endothelial cell growth supplement (ECGS).

Co11aborative Research, Waltham, Massachusetts ), 15 U/ml heparin, and 50 μg/ml gentamicin. Resuspend in culture medium 199 (GIBCO). cells with gelatin (distilled) 2 mg/ml in water) to a pre-coated 75 cm” tissue culture flask.

The cell population is fed the above medium every two days until mating is reached.

After 2 cycles of culture, when culturing pig endothelium, culture ECG5 and heparin. Remove from liquid. If you want to culture vascular smooth muscle cells or linear blast cells, ECG5 and heparin are completely excluded from the culture process. aliquot of cells Approximately 10 cells were resuspended in 0.5 ml of water-cold fetal calf serum on ice. Store in liquid nitrogen by holding. Equal volume of water containing 10% DMSO A screw-cap Corning refrigerated tube containing chilled fetal calf serum and chilled cells was added. Arrange in a cryotube. These cells were incubated for 3 hours before long-term storage in liquid nitrogen. Transfer to a -70°C freezer for a while.

The cells are then infected with a vector containing the desired genetic material.

Il, introduction of cells expressing normal or exogenous proteins into the vasculature.

A, Catheterization of cells expressing relevant proteins.

The patient is catheterized either surgically or percutaneously, applying strict aseptic technique. Prepare to be able to do so. Perform a phlebotomy of the target vessel or apply appropriate anesthesia. After anesthesia, insert the needle into the target blood vessel. Puncture the blood vessel (5) and A catheter such as No. 4,636,195 (this is a USCI catheter).

Available from B111erica, Massachusetts. The disclosure of this U.S. patent (incorporated herein), under a fluoroscopic kite if necessary, by means of a guide wire. (6) into the blood vessel (5) (Figure 1). This catheter means (4) is designed to introduce infected endothelial cells into separate areas of the artery. .

The catheter has distal and proximal balloon means (2) and (1); (each balloon means is, for example, about 3 mm long and about 4 mm wide); Between the balloon means is a length of catheter means. cat between balloons The ether means has one mouth leading to the drip inlet (3). distal and When the proximal balloon means is inflated, a central space is formed within the blood vessel. The infected cells can then be instilled through the mouth.

Anatomical landmarks identify the location of the vessel, and proximal The balloon means (2) is inflated and the endothelium is mechanically traumatized (e.g. into a blood vessel). mechanical trauma (forcible passage of an inflated balloon catheter) or mechanical trauma. A small amount of Disper7, trypsin, collagenase, papain, pepsin, Kimoto proteolytic enzymes such as lipsin or cathepsins, or a combination of these Stratification is achieved by incubation using only protease. In addition to proteolytic enzymes, lipolytic can also be used. The site of the blood vessel is also treated with NP-40, TritonXloo , deoxycholate, or even by treatment with mild detergents such as SDS. Can be layered.

The conditions for stratification are basically to completely remove the endothelium for cell transfer, or to remove the endothelium due to direct infection. In some cases, about 20 to 90%, preferably 50 to 75%, of the cells are removed from the blood vessel wall. Adjust so that In some cases, it may not be necessary to remove the cells.

The catheter is then advanced so that the IV inlet (3) is at the layered endothelium. Melt. A specific section of the artery is then infected, transfected, or normal. Cells are instilled for 30 minutes. If the blood vessels of an organ can tolerate some degree of ischemia, When perfused, for example, skeletal muscle, distal perfusion is not a particular problem; If necessary, use an external shunt or a catheter that allows for distal perfusion. can be recovered. After instilling the infected endothelial cells, the balloon catheter is Remove the tether and repair the artery puncture site and skin incision. If the distal If perfusion is required, use a separate catheter designed for distal perfusion. use

B. Direct introduction of recombinant genes into cells of blood vessel walls or specific in vivo Perfusion by the circulatory system: infection or transfection of organ walls or cells of organs.

A surgical technique similar to that described above is used. Transduction instead of using infected cells virus vectors (106 to 106 cells/m1) or carrier vectors. High titer desired genetic material, such as synthesized DNA, is transferred into a double balloon catheter. Inject directly into the organ wall using the tell technique. This vector reduces the effectiveness of infection. in culture medium containing serum and polybrene (10 μg/ml) to increase Injected. in the dead space created by the catheter for a sufficient period of time (0, After incubation (2 to 2 hours or more), the culture is degassed and phosphate buffered. Gently wash with saline and restore arterial circulation. The same goes for post-surgery recovery. The same procedure applies.

The surface of the organ can be layered mechanically or with a small amount of dispase or trypsin. , in combination with proteolytic enzymes such as collagenase or cathepsins, or It can also be prepared by culturing using only these proteolytic enzymes. stratification The conditions are adjusted to achieve adequate cell shedding from the organ wall.

Viral vectors or DNA-vector complexes are purified viruses or The complex containing sex serum was injected dropwise into Dulbecco's modified Eagle's medium. Ru. Alternatively, it can enhance the adhesion of virus particles to target cells to increase the effectiveness of infection. Polybrene (10 μg/ml), poly L-lysine, dextran sticky substances such as ruphate, or physiologically relevant polycationic substances or hybrid antibodies or antibodies directed against the coat glycoprotein of the virus or vector. Hybrid resistance to relevant targets in organ walls or in tissues perfused by organs. A complex containing a body is used. viral or vector coat glycoproteins and related Hybrid antibodies directed against target cells are made in one of two ways. different Antibodies against epitopes can be chemically cross-linked (G, J u n g, c.

J, Hon5ik, R, A, Relsfeld, and H, J, Muller- Eberhard, Proc, Natl, Acad, Sci, USA, 83°4 479 (1986); U, D, 5taerz, O, Kanagawa, and M. J. Bevan, Nature, 314, 628 (1985); and P. .

Perez, R.W., Hoffman, J.A., Titus, and Tori, M.

Segal, J. 13cp, Med, 163, 166 (1986)), or biologically coupled using a hybrid hybridoma (U, D.

5taerz and M, J. Bevan, Proc. Natl, Acad.

Sci, psΔ, 83.1453 (1986); and C, Milstein and A. C. Cuello, Nature, 305, 537 (1983)) Incubate in the central space of the e-catheter for 0.2 to 2 hours or more. Afterwards, the culture medium is degassed, gently washed with phosphate-buffered saline, and recirculated. restore it.

Different infusion protocols can be performed using different catheter designs (see Figure 2). You can also use the This second approach uses a single norrune With the catheter of method (2), retrovirus was detected in the partially stratified arterial segment. A device with a plurality of mouth means (3) that can convey the gas under high pressure is used. organ The surface of is prepared as above and similar adhesive molecules are used to bind the defect vector Introduce. In this case, the use of a high-pressure delivery system is important because the vector and adjacent vascular tissue It helps to optimize the interaction with the cells inside.

The present invention also provides for local delivery of growth factors by catheter to increase the effectiveness of infection. Methods for use locally or systemically are also presented. Retrovirus vector Crab, herpesvirus, adenovirus, or other viral vectors - is also a vector suitable for mainstream methods.

It is also possible to carry out transformation of cells within organs or tissues.

Direct transformation of organ or tissue cells can be performed in one of two ways. Ru. The first method uses high pressure transduction. Vector by high pressure migrates through the blood vessel wall and into the surrounding tissues. In the second method, the capillary bed By injection (this may be after damage to infiltrate) into the Directly infect surrounding tissues.

The time required for instillation of vectors and cells depends on which method of the present invention is used. It depends on the situation. What is the appropriate time to inject cells or vectors into blood vessels? 0.01 to 12 hours, preferably 0.01 to 12 hours. 1 to 6 hours, most preferably 0.2 to 2 hours. Alternatively, when performing high-pressure infusion of vectors or cells. A shorter time is preferable.

Honmitsu Tomo Eshi 10L The term "genetic material" generally refers to DNA for encoding proteins. refers to Regarding RNA, there are also RNA viruses or RNA-based viruses. This term applies when used in conjunction with other vectors.

Transformation involves introducing exogenous genes by direct infection, transduction, or other uptake methods. This is the process of integrating the offspring into the cell.

The term “vector” is a combination of the widely known and commonly used word “crony”. A vector is a non-chromosomal, double-helical DNA It consists of a replicon that, when placed in a unicellular organism, can be transformed, for example. is replicated by the process. Viral vectors include retroviruses , adenoviruses, herpesviruses, pavoviruses, or modified natural viruses. There are some types. Vectors also contain DNA and chemicals that can be taken up by cells. It also means a prescription crystal of a product or substance.

In another embodiment, the invention provides a method of suppressing gene expression. this Four approaches can be used to achieve the objective. These include Synthetic oligonucleotides complementary to mRNA, including the use of antisense agents (Maher III, L, J, and Dolnick, B, J, Arch.

Tj-i o-■” Siri 1 [9th generation 1.253. 214-220 (1987 ) and Zamecnik, P. C., et al., Proc. Natl., Acad, S. ci,.

83.4143-4146. (1986)), or generate the reverse complement of this gene. Expressing plasmids (Izant, J. H., and Weintraub, H.).

5-science, 229, 345-352. (1985); Ce1l, 36° 1077-1015 (1984)). In addition, ribo Catalytic RNA5 called zyme is particularly capable of degrading RNA sequences. (Uhl, enbeck, O, C,, Nature, 328, 596-60 0° (1987), Hasel, off, J., and Gerlack, W.L. .

jja t, -u,, r-, e, 334, 585-591. (1988)), No. The third approach is “intracellular immunization,” in which analogs of intracellular proteins are specifically inhibit their functions (Friedman, A.D., Triezenberg g, S. J., and McNight, S. L., Natgre, 335, 4. 52-454 (1988)), details of which are shown below.

The first approach can be used specifically to eliminate intracellular transcription. Removal of transcription Expression of binding proteins can be confirmed by S1 nuclease analysis and functional analysis. will be confirmed. A single agent for inhibiting transcription factor mRNA processing and translation. Single oligonucleotide analogs can be used. In summary, objective genetics derived from synthetic oligonucleotides or thiols complementary to the child coding strand Analogs (20-50 nucleotides) are prepared. These antisense agents Prepared for each different region of mRNA. They are the 5” untranslated region of the gene, The translation initiation site and <20-50 base pairs following it, the central coding region, and is complementary to the 3' untranslated region. Antisense agents are conjugated prior to activation. It is cultured together with the introduced cells. Which regions block the expression of these genes? In order to see if it is more effective in Compare the effectiveness of competing antisense drugs.

RNA can also act autocatalytically to cause autolysis, especially when complementary R It has the function of decomposing the NA sequence (Uhl, enbeck, O, C ,.

Nature, 328, 596-600 (1987), Haseloff, J. .

and Gerlack, ,W.L., ,Nature, 334,585-+59. 1° (J, 988), and Hutchins, C.J., et al., Retired Emperor Nuc. 1/jq i-5Res Engineering, 14.3627-3640 (1986)). R.N. The conditions for successful A cleavage are the hammerhead structure and the flank structure. There is a conserved RNA sequence attached to the binding site. The region adjacent to this catalytic region is ribozymes are complementary to specific RNAs, and thus ribozymes target specific cellular mRNAs. In order to prevent the production of a specific target gene characterized by The mRNA to be coded may be degraded using ribozyme. In summary, R.N. Any GUG sequence within the A transcript can be targeted for degradation using ribozymes. It can be. These were confirmed by DNA sequence analysis, and G UG sites are used for its specific degradation. Sites in the 5' untranslated region , sites in the coding region, and sites in the 3′ untranslated region that degrade this transcription. The goal is to determine whether it is more effective. On the GUG site A synthetic oligonucleotide encoding a 20 base pair complementary sequence of The Marhead structure and a complementary sequence of 120 base pairs downstream of this site indicate that cD It will be inserted into NA related sites. In this way, ribozymes target the same cellular compartment. Target as an endogenous message. inserted downstream of a special enhancer Bozymes are produced that are highly expressed in specific cells. these pluses Mid with neomycin resistance plasmid psV2-Neo or selective marker Both by electroporation and co-transduction, the relevant cells of interest will be introduced in Expression of these transcripts was determined by Northern blot and S1 nuclear Confirmed by enzyme analysis. Once confirmed, mRNA expression is controlled by 81 nuclease By protection, the expression of these transcripts is controlled by the target mRNA and its regulation. Assess whether the steady-state levels of genes that protein levels too I'll check again.

Genes can also be inhibited by providing mutant transcripts that lack regions necessary for activation. be harmed. In summary, once the region has been defined, mutations that fail to stimulate function Compose the mold. This truncated gene is transferred to a plasmid containing the neomycin resistance gene. Insert downstream of the 5V-40 enhancer (Mu 11 i gan, R, and Berg, P., 5science, 209. L422-1427 (19 80) (in a separate transcription unit). This plasmid is introduced into cells, and 041 Sort using 8. The presence of mutant forms of this gene was confirmed by S1 nuclease analysis and confirmed by immunoprecipitation. The function of endogenous proteins in these cells is It is evaluated by two methods. The first confirms normal gene expression. Second evaluate the known functions of these proteins. If this intercellular inhibitory mutation If the strain is virulent to its host, a metallothionein promoter, etc. Introduced into the bladder conductivity control element. After isolating a stable strain, Cells are cultured with Zn or Cd to express this gene. After that, the host details Assess the effect on cells.

Another approach to inactivating specific genes is to The method is to overexpress a combination protein that competes with the expression. and For example, if you want to reduce TPA expression (e.g., in the treatment of disseminated thrombolysis) method) involves excessive production of plasminogen activator inhibitors. All you have to do is make it appear.

Recent advances in biochemistry and molecular biology have led to the development of “recombinant” vectors. For example, retroviruses and plasmids therein may contain exogenous RNA or DNA. It is now possible to create things that each contain A. For special items, recombination is required. Non-homologous RNA or DNA (this is also a combination vector) It encodes a polypeptide that is not normally produced in organisms that can be transformed with It has also become possible to include RNA or DNA (in the sense of RNA or DNA). Recon The production of binant RNA or DNA vectors is well known and detailed. There is no need to explain it. However, I will briefly describe this process for your reference. I'll keep it.

For example, retroviral or plasmid vectors can be ligated by cleavage. It can be linear RNA or DNA with terminal ends. These ends are binding to exogenous RNA or DNA having complementary like ligatable termini; A complete replicon and a replica that has the desired expression characteristics and is capable of performing biological functions. Becomes a combination RNA or DNA molecule.

For RNA and DNA recombination, adjacent ends of separate RNA or DNA There are various techniques, such as adjusting fragments to make them easier to connect.

The exogenous, or donor, RNA or DNA used in the present invention may be any suitable It can be obtained from a group of cells. Using known methods, vectors are created and therapeutic drug tags are added. Transformed cells capable of in vivo expression of proteins are obtained. Transformed cells are target cells of a formulation that allows for the incorporation of RNA or DNA into target cells for transformation. It can be obtained by contacting RNA or DNA. such a prescription For example, retroviruses, plasmids, liposomal compositions, or Polycationic substances such as poly-luridine, DEAC-dextran and and target ligands.

The present invention thus provides the ability to administer therapeutic or diagnostic agents for local or systemic purposes. A method that utilizes genetic modification of cells as a method of delivery to the local area of blood vessels for the purpose of It provides: Recombinant proteins expressed in these cells The scope of treatment is wide and varied, including treatment of thrombosis and restenosis, and angiogenesis. tPA for therapy, or growth factors for revascularization purposes, and vasoconstriction or express proteins such as vasoactive factors, to reverse vasospasm It also includes gene conversion using vectors that This technique can also be used to treat genetic diseases, or genetic therapy for acquired, local or systemic diseases. It is called. The invention also allows the introduction of normal cells into specific sites where cells have been lost. for example, replacing damaged endothelium during angioplasty surgery or catheterization It can also be used for

For example, in the treatment of ischemic diseases (thrombosis), tPA or its modified products, Genetic material encoding urokinase or streptokinase, etc., is present in cells. used to transform. Ischemic organs (e.g. heart, kidneys, colon, In the treatment of diseases such as liver disease, transforming growth factor a (TGF-α), Transforming growth factor s (TGF-β), angiogenin, tumor necrosis factor α, tumor Necrosis factor beta, acidic fibroblast growth factor or basic fibroblast growth factor child-like collateral regeneration agent (recollateralization agent) Genetic material encoding ts) can be used. Diseases of the vasomotor system Treatment uses genetic material encoding vasodilators or vasoconstrictors can do. These include atrial natriuretic factor, which is induced from platelets; Contains induced growth factors or endothelin. In the treatment of diabetes, Genetic material encoding insulin can be used.

The present invention also provides methods for treating malignant diseases by placing transformed cells in close proximity to the malignant disease. It can be used to treat diseases. For this application, diphtheria toxin, Genetic material encoding pertussis toxin or cholera toxin is used.

When the present invention is used for the treatment of AIDS, soluble CD4 or its derivatives are Genetic material that can be used can be used. In the case of treatment of genetic diseases (and For example, growth hormone deficiency) requires genetic material that encodes the required substance, ( For example, human growth hormone) can be used. All this genetic material These materials are readily available to those skilled in the art.

In another alternative embodiment, the invention provides a kit for treating a disease in a patient. However, this kit does not require a catheter and either an enzyme or mild detergent. The catheter is a catheter that can be inserted into a blood vessel and has a The catheter is equipped with a balloon element that can be inserted into the blood vessel. Inflate against the wall of the blood vessel so that the tail can be properly held within the blood vessel. The main catheter section is designed to allow a means for delivering a solution into the blood vessel containing enzymes or mild detergents; The solder solution is a physiologically acceptable solution. This solution contains dispase, Proteolytic enzymes such as psin, collagenase, papain, pepsin, and chymotrypsin Contains elements. As a mild cleaning agent, this solution is NP-40, Tritonxtoo , deoxycholate, or SDS.

Alternatively, this kit includes heparin, poly such as L-lysine, polybrene, dextran sulfate, and polycationic substances. It may also contain drugs such as drugs or bivalent antibodies. This solution also or cells (normal or transformed). yet another In an embodiment, the kit includes a catheter and an enzyme or mild irrigant. solution, heparin, poly-L-lysine, polybrene, dextran sulfate , drugs such as polycationic substances, or solutions containing divalent antibodies (this solution may contain both vectors and cells).

This kit includes one balloon and a catheter with a central distal perfusion port, and introduction of cells into specific organs, or introduction of vectors into capillary beds, or cells to specific organs or tissues perfused by this capillary bed. may include a solution acceptable for carrying out the introduction of.

Alternatively, the kit can be spaced apart and inserted into a blood vessel. It has two balloon elements and can form one chamber inside the blood vessel. Both catheters should be Elements can now be inflated against the walls of blood vessels. It may include two main catheter sections. In this case, supply the solution to Chang/( A means for doing so is provided between the two balloons. This kit delivers a solution into the blood vessels. It may also be a catheter with multiple exit means for delivery.

Thus, the present invention provides a method for treating disease in a patient. exogenous therapeutic tan/1 to cells that are perfused from this organ of the patient. This protein is used to treat diseases and diagnose diseases. be useful for a purpose. The method of the present invention is applicable to ischemic diseases, vasomotor diseases, diabetes, b4 can be used to treat malignant diseases, AIDS, or genetic diseases.

In the present invention, the exogenous therapeutic protein is tPA or one other modification, urothelium. Kinase, streptokinase, acidic fibroblast growth factor, basic fib Roblast growth factor, tumor necrosis factor alpha, tumor necrosis factor beta, transforming growth factor alpha, Transforming growth factor β, atrial natriuretic factor, and platelet-derived growth long factor, endothelin, insulin, diphtheria toxin, pertussis toxin, Relatoxin, soluble CD4 or its derivatives, and growth hormone, etc. Can be used for treatment.

Exogenous proteins with diagnostic properties can also be used in secondary methods. Even if Ii, β- Marker proteins such as calactosidase can be used to monitor cell migration. Can be used.

The cells that express exogenous therapeutic proteins are preferably endothelial cells. Other characteristics of will become apparent through the description of the reference embodiments below. , these embodiments are for illustration of the invention and are not intended to limit the invention. do not have.

The data reported below demonstrate the potential of endothelial cell transplantation and gene transfer. Endothelial cells are stably transplanted into the artery wall by catheterization for 1 to 11 days; Expressing recombinant marker protein and β-galactosidase in vivo There is.

The progressive layer formation in pigs is similar to that in humans, so it is used as an animal model. Pig series, Yucatan Mini Big (Charles River Labora) Tories, Inc., Wilmington, Massachusetts) was selected ( 1). The first endothelial cell line was established in the internal jugular vein of an 8-year-old female minibig.

Identification of this type of endothelial cell indicates that this cell population was found in pig endothelium in tissue culture. It was confirmed by exhibiting unique growth and morphological characteristics.

These endothelial cells also contain fibroblasts and other mesenchymal cell groups ( 2), in contrast to the receptor for acetylated low-density lipoprotein (AcLDL). - expressed. Analysis of ACLDL receptor expression revealed that fluorescent ACLD More than 99% of cultured cells contain this receptor, as judged by L uptake. there was.

Two independent β-galactosidase-expressing endothelial cell lines were generated in murine amphotrophic cells. infected with β-calactosidase-transducing retroviral vector (BAG). It was then isolated. This vector is replication defective and contains β-galactosidase and neomyelinase. It contains both of the syn-resistant gene (3). Formation in the presence of G-418 .

Cell types containing this vector were chosen for their long-term ability. Histochemical staining More than 90% of the selected cell groups synthesized β-caractosidase. Also, this The endothelial properties of these genetically modified cell populations were also analyzed for fluorescent ACLDL uptake. It was confirmed. Infection with BAG retrovirus is also associated with Southern plots. It was demonstrated by analysis that the complete proviral DNA consists of approximately one genome. It has been revealed that there is one copy of each.

Endothelial cell populations taken from this inbred inbred species can be used for research on two or more mini-big animals. The test consisted of nine different experimental tasks. normal anesthesia procedure At the bottom, the femoral and ileal arteries were exposed and a catheter was introduced into the organ (Figure 1).

The intimal tissue of the artery wall is forcibly inserted into the organ using a slightly inflated Norune catheter. Through it, it was mechanically exposed. The artery is irrigated with heparinized saline and neutral probiotics. Cultured with Tease and Disparza (50 U/ml). This results in Any remaining luminal endothelial cells were also removed. Remove the remaining enzyme with a catheter balloon. After allowing blood to flow through the deflated organ compartment, α2 globulin is activated in the plasma. It was rapidly inactivated. A group of cultured endothelial cells expressing β-galactosidase was A specially designed arterial cartilage (Figure 1) with a central IV inlet The cells were introduced using a Tether (USCI, Bjllerica, 14A).

When these balloons are inflated, they create a protected space within the artery that Cells are injected into the tube through an IV inlet (Figure 1). β-galactosidase These endothelial cell populations expressing 3 Incubate for 0 minutes. The catheter is then removed, the arterial tributaries are ligated, and The incision was closed.

Arterial sections inoculated with β-galactosidase-expressing endothelium were removed after 2-4 weeks. there was. Gross examination of the arterial specimen after staining with X-gal chromagen reveals multiple Blue pigmented areas are seen in comparison to arteries implanted with uninfected endothelium. , indicating β-galactosidase activity. Optical microscopy mainly uses experimental β-galactosidase staining has been recorded in the endothelial cells of the lining of the attached organ.

In contrast, controls that received endothelial cells without β-galactosidase No similar coloration was observed in the plots. β-galactosidase coloring is It is often found in deep intimal tissue, where the implanted endothelium has been previously damaged. It suggests that they will be captured by the official wall or that they will be immigrated. Local thrombosis observed in the first two experimental tasks. This complication was explained in subsequent studies in endothelial cell transfer. Acetylsalicylic acid was administered prior to the explant procedure and heparin anticoagulation was administered at the time of inoculation. This could be suppressed by using drugs. In cases where blood clots form, β-galactosidase staining is observed in endothelial cells ranging from the organ wall to the surface of the thrombus. It was.

The primary concern regarding in-vivo gene transplantation is that from genetically generated cells, The production of replication-competent retroviruses. In these tests, replication By using a defective retrovirus, this problem could be minimized.

In these cell lines, even after 20 passages in vitro, the helper virus remains Not detected at all. High probability of infection and homology to the host cell genome A defective virus was used because it is stable, but it is difficult to use in gene transplantation. This approach can also be used with other viral vectors.

The second interest concerns the expression lifespan of Recon and Nando genes in living organisms. It is. In the current study, endothelial cell β-galactosidase expression within the organ It is stable for up to 6 weeks after introduction into the blood vessel.

These tests demonstrate that genetically modified endothelial cells are It has been shown that it can be introduced into the vascular wall of Yucatan Minibig. Therefore This technology uses genetically modified endothelium as a vector to treat vascular diseases. Can be used for topical biochemical treatment.

Vascular diseases such as balloon angioplasty and insertion of grafts into diseased organs A major complication of current interventional methods in the treatment of atherosclerotic plaques is tissue destruction and thrombus formation at the site of local tissue trauma (5). Partially endothelial cell damage mediates this complication (6). This data indicates that no intervention exists. genetically modified endothelial cells to reduce local thrombosis during Indicates that it can be used.

This technique can also be used to treat other ischemic conditions, including unstable angina and myocardial infarction. Available for use. For example, Kumifi plasminogen activator and Urokinar An antithrombotic effect can be obtained by introducing cells that express the gene for antithrombotic cells.

This technique is also useful in treating chronic tissue ischemia. For example, severe Angiogenesis or to stimulate the generation of collateral blood vessels in ischemic tissue, e.g. myocardium. It is this that causes the formation of growth factor (7). Finally, for systemic congenital diseases, Genetic replacement of somatic cells in the human eye is also possible by applying this endothelial cell gene transplantation method. It is.

Experiment part: A, normal pig endothelial cells and pig endothelial cells transfected with β-galactosidase. Analysis of AcLDL receptor expression in.

An endothelial cell line collected from Yucatan Minibig, containing BAG retrovirus or For the two sublineage species infected with the 3T3 fibroblast control, AcLDL receptor expression was investigated using photolabeled AcLDL.

Endothelial cells were isolated from the external jugular vein using the neutral protease Dysp1-ase (8). was collected. The excised vein part was treated with a disperser (50LJ/ml, Hanks). (in balanced salt solution) and incubated at 30°C for 20 minutes. Of what I got this way The skin was treated with fetal calf serum (10%) and endothelial cell growth supplement (EC) at 50 μg/ml. GS), and culture fi199 (G IBC○.

QrBrld Is 1 and New York). these Cells were infected with BAG retrovirus and those resistant to G-418 were selected. is. This cell type was Tramethylindocarbacyanine verchlorate) (Dil) AcLDL ( Biomedical Technologies, Stoughton, Massachusetts Tushu) (10 μg/ml) for 4-6 hours at 37°C, and 0.5% glucose. Washed three times with phosphate buffered saline containing taraldehyde. Phase contrast and fluorescence Cells were visualized under a microscope.

B, Endothelial cell introduction method using a catheter.

A double balloon catheter was used for intravenous infusion of endothelial cells. this catheter has proximal and distal balloons, each measuring 6 mm in length and 5 mm in width. The distance between them is 20 mm. In the center of the catheter there is a 2 There is a mm hole. The proximal and distal balloons create a central space and Infected cells can be injected into a specific compartment of the blood vessel through an inlet. It's becoming a sea urchin. See Figures 1 and 2 for an illustration of cell introduction via catheter. I want to be.

Animal management is carried out by 'Principles of LaboratoryAnim' al　Care” and “Guj, de for the Care and U se of Laboratory Animals” (NIHPubl 1 cation No., revised in 80-23.1.978). female yukata Bend Parbital (20mg/ g) The animal was anesthetized, intubated, and mechanically ventilated. The ileum and large The femoral artery was exposed using aseptic surgery. Puncture the distal femoral artery and use a kite wire to A double balloon catheter was then placed into the ileal artery. Identify the external ileal artery; proximal Inflate the balloon slightly to mechanically denud the endothelium distally and proximally. I let it pass. The central space of the catheter is then aligned with the denuded endothelium. I adjusted the balloons and inflated both balloons. Heparinize the denuded compartment The remaining attached cells were washed with diluted physiological saline and treated with dispase (20 U/m 1) was removed by drip infusion for 10 minutes. The denuded organs (blood vessels) are coated with heparin. After washing with solution, B'AG infected endothelial cells were instilled for 30 minutes. After that, the bar The catheter was removed and blood flow returned to an antegrade state. the organ part for 2-4 weeks It was removed after a while. Place a section of the artery in a 0.5% glutaraldehyde solution for 5 minutes. Store in phosphate-buffered saline and place in paraffin for cutting and subdividing pond pieces. covered with a block. Presence of retrovirus expressing β-galactosidase is standard This was confirmed by histochemical techniques (19).

C, Analysis of endothelial cells in vitro and in vivo.

β-Rautosidase activity was recorded by histochemical staining method for: Ta. (A) Primary endothelial cells removed from Yucatan Minibig, (B) BAG Subline infected with retroviral vector, (C) normal artery control (D) Endothelium infected with BAG retrovirus vector was instilled. (E) Microscopic section of normal control artery, and (F) BAG Microscopic sectioning of an artery instilled with endothelium infected with a retroviral vector.

Endothelial cells in tissue culture were treated with 0.5% glutaraldehyde before histochemical staining. Fixed inside. infected in vitro and in vivo. endothelial cells To confirm, the enzymatic activity of E. coli β-galactosidase protein was used. β-galactosidase transduction Mo-MuLV vector (2), (B AG) was kindly provided by Dr. Con5tance Cepko.

This vector converts the wild type M o M u L V L T R into a β-ga It is used as a promoter for the lactosidase gene. Tn5 neomycin Simian virus 40 (SV-40) early promoter linked to resistance gene It confers resistance to the drug G-418, which is linked to the β-galactosidase gene. and select cells expressing β-caractosidase containing retrovirus. Use it as a marker to do so. This defective retrovirus is a fibroblast ψa m cells (3,10) and cultured in Dulbecco's modified Eagle's medium (DMEM) or and 10% calf serum. Cells were passaged for 2 passages - weeks after trypsinization. I paid for it. The supernatant (titer to'-io''/ml, G-418 resistant) was added to the cells. B co = -) was added in 2 parts and 3 matings for 12 min in DMEM supplemented with 10% calf serum. time, 37°C, 5% CO*.

Culture was performed in the presence of 8 μg/ml polybrene. Remove the viral supernatant 10% fetal calf serum, ECG3 (60 μg/ml) was added to the cells. In culture solution 199, Zarani G-418 (50% racemic mixture 0.7 μg/ml) Store in endothelial cell conditioned medium (20%) for 24 to 48 hours prior to sorting. did. G-418-resistant cells were isolated and expressed using standard histochemical staining (9). - Analysis of galactosidase expression was performed. Stable β-galactosidase enzyme The expressing cells were continuously cultured for future needs. Frozen aliquots are made using liquid nitrogen. Saved in the water.

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Needless to say, the present invention is susceptible to many modifications and variations in light of the above teachings. Ru. Therefore, within the scope of the appended claims, the invention resides in the invention as particularly described herein. Please understand that other methods may also be used.

Continuation of front page (51) Int, C1,' Identification symbol Internal office reference number A61K 48100 8314-4CA61M 25100 // A61K 37102 ADU 8314-4C37/24 ADP 8 314-4C 37154ABN 8314-4C C12N 5108 I

Claims (1)

[Claims] 1. A kit necessary to treat a patient's disease, including one catheter and an enzyme or (i) the catheter handle is inserted into a blood vessel; The main catheter part is inserted into the blood vessel. In addition, the main catheter section can be held at a desired position within the blood vessel. a balloon element such that it can be inflated against the wall of the blood vessel; the main catheter section is provided with means for supplying a solution into the catheter; and (ii) the solution is a physiologically acceptable solution. 2. The solution contains enzymes such as disbase, trypsin, collagenase, bavine, at least selected from the group consisting of pepsin, chymotrypsin, and lipase; The kit according to claim 1, which comprises one or more products. 3. The solution contains NP-40, TritonX100, deoxycholate, and It includes at least one product class selected from the group consisting of SDS, The kit according to claim 1. 4. The main catheter section has one channel within the blood vessel for insertion into the blood vessel. blood to allow for the formation of the catheter and to hold the main catheter in the desired position. Two spaced apart balloons that can be inflated against the wall of the tube element means and means for supplying the solution to the chamber by the balloon; The kit according to claim 1, wherein the kit is arranged between elements. 5. The means for supplying the solution into the blood vessel is comprised of a plurality of outlet means. The kit according to claim 1. 6. A kit necessary to treat a patient's disease, including one catheter means and physiology composed of a legally acceptable solution, (i) the catheter means is insertable into a blood vessel and the main catheter The main catheter can be inserted into the blood vessel and the main catheter can be positioned within the blood vessel as desired. A band that can be inflated against the wall of the vessel to hold it in place. The main catheter includes a rune element and a means for supplying a solution into the vessel. (ii) the physiologically acceptable solution comprises: , hevarin, poly-L-lysine, polybrene, dextran sulfate, poly At least one type selected from the group consisting of a cationic substance and a divalent antibody Kit containing the above substances. 7. Claim 6, wherein the physiologically acceptable solution further comprises DNA. Kit as described. 8. Claim 6, wherein the physiologically acceptable solution further comprises a growth factor. Kit as described in section. 9. A method necessary to treat a patient's disease, which Cells attached to organs or tissues are treated with exogenous therapeutic agents that treat the disease. A method characterized by expressing protein. 10. If the disease is ischemic disease, vasomotor disease, diabetes, malignant disease, AIDS, or 10. The method of claim 9, wherein is a genetic disease. 11. 10. The method according to claim 9, wherein the disease is a systemic disease. 12. The exogenous therapeutic agent protein is tPA or its modification, urokinase, Leptokinase, acidic fibroblast growth factor, basic fibroblast growth factor, tumor necrosis factor alpha, tumor necrosis factor beta, transforming growth factor alpha, transforming growth factor child beta, atrial natriuretic factor, platelet-derived growth factor, endo Serine, insulin, diphtheria toxin, pertussis toxin, cholera toxin, Also selected from the group consisting of soluble CD4 and derivatives thereof, and growth hormone. 10. The method according to claim 9. 13. Cells include endothelial cells, vascular smooth muscle cells, fibroblasts, connective tissue cells, and macrophage cells. Claim 9 is selected from the group consisting of phages, monocytes, and parenchymal cells. The method described in section. 14. A method for treating a disease characterized by site-specific intravenous injection of cells. 15. 15. The method of claim 14, wherein the cell is a transformed cell. 16. 15. The method according to claim 14, wherein the cells are normal cells. 17. Treatment of diseases characterized by site-specific cell transformation in vivo Law.