CA2112376A1 - Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor - Google Patents
Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits thereforInfo
- Publication number
- CA2112376A1 CA2112376A1 CA002112376A CA2112376A CA2112376A1 CA 2112376 A1 CA2112376 A1 CA 2112376A1 CA 002112376 A CA002112376 A CA 002112376A CA 2112376 A CA2112376 A CA 2112376A CA 2112376 A1 CA2112376 A1 CA 2112376A1
- Authority
- CA
- Canada
- Prior art keywords
- cells
- dna
- patient
- disease
- vessel
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Abstract
A method for the direct treatment towards the specific sites of a disease is disclosed. This method is based on the delivery of proteins by catheterization to discrete blood vessel segments using genetically modified or normal cells or other vector systems.
Endothelial cells expressing recombinant therapeutic agent or diagnostic proteins are situated on the walls of the blood vessel or in the tissue perfused by the vessel in a patient. This technique provides for the transfer of cells or vectors and expression of recombinant genes in vivo and allows the introduction of proteins of therapeutic or diagnostic value for the treatment of diseases.
Endothelial cells expressing recombinant therapeutic agent or diagnostic proteins are situated on the walls of the blood vessel or in the tissue perfused by the vessel in a patient. This technique provides for the transfer of cells or vectors and expression of recombinant genes in vivo and allows the introduction of proteins of therapeutic or diagnostic value for the treatment of diseases.
Description
WO93/00~51 ' 1 2 1 1 2 3 7 6 PCT/US92/05242 Description Treatment of Diseases bv Site-S~ecific Instillation of Cells or Site-S~ecific Transformation of_Cells and Kits Therefor This is a continuation-in-part of U.S. Patent Application Serial No. 07/724,509, filed on June 28, l99l, which is a continuation-in-part of U.S. Patent Application Serial No. 07/331,336, filed on March 31, 1989, now abandoned.
Technical Field The present invention relates-to the treatment of diseases by the site-specific instillation or transformation of cells and kits therefor. The present invention also relates to a method for modulating the immune ~ystem of an animal by the in vivo introduction of recombinant genes.
Backaround Art .. . . .
The effective treatment of many acquired and inherited diseases remains a major challenge to modern medicine. The ability to deliver therapeutic agents to specific sites n vivo would an asset in the treatment of, e.g., localized diseases.~ In addition the ability to cause a therapeutic agent to perfuse through the circulatory system would be effective~for the treatment of, e.g., inherited diseases and acquired diseases or cancers.
.
~- For,example, it would be-desirable to administer in a steadyvfashion an antitumor agent or toxin in close -proximity to a tumor. Similarly, it would be desirable to cause a-perfusion of, e.g., insulin in the blood of a , 30 person suffering from diabetes. However, for many WO93/~51 ~ 1 ' PCT/US92/05242 .. . .
therapeutic agents there is no satisfactory method of either site-specific or systemic administration.
In addition, for many diseases, it would be desirable to cause, either locally or systemically, the expression of a defective endogenous gene, the expression of a exogenous gene, or the suppression of an endogenous gene. Again, these remain unrealized goals.
In particular, the pathogenesis of atheroscle'rosis 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 man~fested by interference with the permeability barrier of the endothelium, alterations in the nonthromb'ogenic properties of the endothelial surface, and promotion of procoagulant properties of the endothelium. Monocytes migrate between endothelial cells, become active as ;~cavenger cells, and differentiate into macrophages.
;i Macrophages then-~ynthe~ize and-secrete growth factors including platelet derived growth factor (PDGF),' fibroblast growth factor (FGF), epidermal growth factor tEGF), and transforming growth factor~alpha (TGF-~). These growth factors sre extremely potent in stimulating the migration and proliferation of fibroblasts and smooth muscle cells in the atherosclerotic plaque. In addition, platelets may interact with the injured endothelial cell and the WO93/~51 2 1 1 2 3 7 6 s92~0s~2 -3- - ~
activated macrophage to potentiate the elaboration of growth factors and thrombus formation.
Two major problems in the clinical management of coronary artery disease include thrombus formation in acute myocardial ischemia and restenosis following coronary angioplasty (PTCA). Both involve common cellular events, including endothelial injury and release of potent growth factors by activated macrophages and platelets. Coronary angioplasty produces fracturing of the atherosclerotic plaque and removal of the endothelium. This vascular trauma promotes platelet aggregation and thrombus formation at the PTCA site. Further release of mitogens from platelets and macrophages, smooth muscle cell proliferation and monocyte infiltration result in restenosis.
Empiric therapy with antiplatelet drugs has not prevented this problem, which occurs in one-third of patients undergoing PTCA. A solution to restenosis is to prevent platelet aggregation, thrombus formation, and gmooth 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 ~ - dy~unction of endothelial cell~~ttachment,~Jand leading to -the expo~ure-of underlying macrophage foam cells.-~This permits the opportunity for circulating platelets to adhere, aggregate, and form thrombi.
-- The intravenous administration of thrombolytic agents, - such as tissue plasminogen activator (tPA) results in lysis of thrombus in approximately 70% of patients experiencing I an acute myocardial infarction. Nonetheless, approximately W093/~51 ~ PCT/US92/05~2 . ., . ", .
" ~ . ~ i ,, . I
30% of patients fail to reperfuse, and of those patients who undergo initial reperfusion of the infarct related artery, approximately 25% experience recurrent thrombosis within 24 hours. Therefore, an effective therapy for rethrombosis remains a major therapeutic challenge facing the medical community today.
As noted above, an effective therapy for rethrombosis is by far not the only major therapeutic challenge existing today. Others include the treatment of other ischemic conditions, including unstable angina, myocardial infarction or chronic tissue ischemia, or even the treatment of acquired and inherited diseases or cancers.
These might be treated by the effective administration of anticoagulants, vasodilatory, angiogenic, growth factors or growth inhibitors to a patient. Thus, there remains a strongly felt need for an effective therapy in all of these clinica} settings.
In addition, it is desirable to be able to modulate the i Dune system of an animal. In particular, much effort has been directed toward the development of vaccines to provide immunological protection from infection. However, the development of safe vaccines which can be readily administered to large numbers of patients is problematic, -and for many diseases,-such as, e.g., AIDS, no safe`and 25~ effective~vaccine-is as-yet ava~lable. Further, it is also ~sometimes~desirable toispecifically suppress an animals immune response to prevent rejection-of-a transplant.
Efforts to suppress transplant rejection have resulted in the development of drugs which result in a general suppression of the iDune response, rather than specific supression to transplantation antigens, and such drugs are not always effective. Thus, there remains a need for a method to modulate the i Dune system of an animal.
WOg3/H 051 2 1 1 2 3 7 6 PCriUsg~/05242 Disclosure of the Invention Accordingly, one object of the present invention is to provide a novel method for the site-specific administration of a therapeutic agent.
S It is another object of the present invention to provide a method for the perfusion of a therapeutic agent in the blood stream of a patient.
It is another object of the present invention to provide a method for causing the expression of an exogenous gene in a patient.
It is another object of the present invention to provide a method for causing the expression of a defective endogenous gene in a patient.
It is another object of the present invention to provide a method for suppressing the expression of an endogenous gene in a patient. ~ ` `
It is another object of the present invention to provide a method for site-specifically replacing damaged cells in a patient.
, . , ... ~ ... ... ; .~ , ,, . i ;
It is another object of^the present invention~toii provide a method for the treatment of a disease by causing either the site-specific administration of a therapeutic a~ent or the perfusion of a therapeutic agent in the bloodstream of a patient. -It is another ob~ect of the present invention to ; provide a method for the treatmént of a disease by causing either the expression of an exogenous gene, the expression 21123;~ 6 PCT/US92~0 ~ 2 ~, . .. . .
of a defective endogenous gene, or the suppression of the expression of an endogenous gene in a patient.
It is another object of the present invention to provide a method for the treatment of a disease by site-specifically replacing damaged cells in a patient.
It is another object of the present invention to provide a kit for site-specifically instilling normal or transformed cells in a patient.
It is another object of the present invention to provide a kit for site-specifically transforming cells in vivo.
It is another object of the present invention to provide a method for modulating the immune system of an animal.
, .
It is another object of the present invention to provide a method for modulating the immune system of an animal to sensitize the animal to a foreign molecule.
It is another object of the present invention to provide a method to stimulate the immune system of an anir~l-to reject proteins in order to protect against infection~byj~a microorganism or~ivirus.~ r~
~ -~ It is another object of the present invention to provide a method for modulating the immune system of an animal to tolerize the animal to a foreign molecule.
, It-is~another object of the present invention to provide a method for modulating the immune system of an animal to reduce the tendenoy to reject a transplant.
W093/~05l 2 1 1 2 3 7 6 PCT/US92/0~2 It is another object of the present invention to provide a novel kit for transforming cells by systemic administration in vivo.
These and other objects of this invention which will become apparent during the course of the following detailed description of the invention have been discovered by the inventors to be achieved by (a) a method which comprises either (i) site-specific instillation of either normal (untransformed) or transformed cells in a patient or (ii) site-specific transformation of cells in a patient and (b) a kit which contains a catheter for (i) site-specific instillation of either normal or transformed cells or (ii) site-specific transformation of cells.
Site-specific instillation of normal cells can be used to replace damaged cells, while instillation of transformed cells can be used to cause the expression of either a defective endogenous gene or an exogenous gene or the suppression of an endogenous gene product. Instillation of cells in the walls of the patient's blood vessels can be used to cause the steady perfusion of a therapeutic agent in the blood stream.
The inventors have-also discovered that by transforming cells of an animal, in vivo, it is possible to ~s~ modulate the animal's immune-system. In particular, by transforming cells of an animal, with a recombinant gene, by ~ite-specific or systemic administration it is possible to modulate the animal's immune system to sensitize the animal to the molecule for which the recombinant gene encodes. Alternatively, by transforming cells of an anima?
with a recombinant gene,~ specifically at a site which determines the specificity of the immune system, such as, e.g., the thymus, it is possible to modulate the immune W093/~51.. .~ PCT/USg2/05~2 2112~7 6 -8-system of an animal to suppress the immune response to the molecule encoded by the recombinant gene.
Brief Description of the Drawinas A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same become better understood by reference to the following detailed description when considered in connection with the accompanying figures, wherein:-FIGURES 1 and 2 il-ustrate the use of a catheter in accordance with the invention to surgically or percutaneously implant cells in a blood vessel or to transform ~n vivo cells present on the wall of a patient's blood vessel;
FIGURE 3 illustrates the relationship between the % of target cell lysis and the effector:target ratio for CTL
.cells; and FIGURE 4 illustrates the results of a Western blot analysis.
Best Mode for CarrYin~ Out the Invention ~ ~
... ...-Thus,~in one embodiment,- the presentr--invention is used .to.treat diseases, such as.inherited diseases, systemic --diseases, diseases of the cardiovascular system,~ diseases of particular organs, or tumors by instilling normal or transformed cells or by transforming cells. . `: .-. , ; ; . . . : . -The.cells which may be instilled in the present methodinclude endothelium, smooth muscle, fibroblasts, monocytes, macrophages, and parenchymal cells. These cells may i a WO93/~51 2 1 1 2 3 7 ~ PCT/US92/05242 produce proteins which may have a therapeutic or diagnostic effect and which may be naturally occurring or ari~e from recombinant genetic material.
Referring now to the figures, wherein like reference numerals designate identical or corresponding parts throughout the several views, and more particularly to FIGURE 1 thereof, 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. In the figure, 5 is the wall of the blood vessel. The figure shows the catheter body 4 held in place by the inflation of inflatable balloon mean~
1 and 2. The section of the catheter body 4 situated between balloon means 1 and 2 is equipped with instillation port means 3. The catheter may be further eguipped with a , guidewire means 6. FIGURE ~ illustrates the use of a-~imilar 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 con~ain up to twelve individual instillation port means 3, with,five being illu~trated.~
, ,,,~ In the-case-of,delivery to an organ, the catheter'~may be.introduced into-the major-artery supplying the tissue.
Cells containing recombinant genes or vectors can be ,,, introduced through a-central instillation port-after 30,~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 r WO 93/~ ~51 ; r PCT/US92/05242 the vasculature using the double balloon catheter technique in the arterial circulation proximal to the target organ.
In this way, the recombinant genes may be secreted directly into the circulation which perfuse the involved tissue or may be synthesized directly within the organ.
In one embodiment, the therapeutic agents are secreted by vascular cells supplying specific organs affected by the disease. For example, ischemic cardiomyopathy may be treated by introducing angiogenic factors into the coronary circulation. This approach may also be used for peripheral, vascular or cerebrovascular diseases where angiogenic factors may improve circulation to the ~rain or other tissues. Diabetes mellitus may be treated by introduction of glucose-responsive insulin secreting cells in the portal circulation where the liver normally sees a higher insulin concentration than other tissues.
In addition to providing local concentrations of therapeutic agents, the present~imethod may also be used for delivsry of recombinant genes to pasenchymal tissues,-because high concentrations of viral vector and othervectors can be delivered to a specific circulation. Using this approach, deficiencies of organ-specific proteins may also be treated. For example, in the liver, ~-antitrypsin inhibitor deficiency or hyperchloresterolemia may be treatsd by introduction of ~-antitrypsin or the LDL
- ;r~eceptor~Lgen~e.~ In-addition,- this approach may~be-used for the treatment,of a-malignancy. Secretion of specific~
recombinant toxin genes into the circulation of `
inoperable-tumors:provides a therapeutic-effect. Examples 30 - include acoustic neuromas or certain hemangiomas~!which are otherwise unresurrectable. ~
. ~ . .
In clinical settings, these therapeutic recombinant wO 93/~Sl r PCT/US92/05242 -12.11237~i genes are introduced in cells supplying the circulation of the involved organ. Although the arterial and capillary circulations are the preferred locations for introduction of these cells, venous systems are also suitable.
In its application to the treatment of local vascular damage the present invention provides for the expression of proteins which ameliorate this condition in situ. In one embodiment, because vascular cells are found at these sites, they are used as carriers to convey the therapeutic agents.
The invention thus, in one of its aspects, relies on genetic alteration of endothelial and other vascular cells or somatic cell gene therapy, for transmitting therapeutic agents (i.e., proteins, growth factors) to the localized region of vessel injury. To successfully use gene transplantation in the cells, four requirements must be fulfilled. First, the gene which is to be implanted into the cell must be identified and isolated. Second, the gene to be expresced must be cloned and available for genetic manipulation. Third, the gene must be introduced into the cell in a form that will be expressed or functional.
Fourth, the genetically altered cells must be situated in the vascul~r region where it is needed.
s:~ In accordance with~--the~present invention the altered cells or appropriate vector may be surgically, -;-percutaneously, or intravenously introduced and attached to a section of a patiént's vessel wall. Alternatively, some of the cells existing on the patient's vessel wall are :-transformed with the desired genetic material or-by i 30 -directly applying the vector. In some instances, vascular cells which are not genetically modified can be introduced by these methods to repl~ce cells lost or damaged on the !
W093/000~1 PCr/US92/~ ~2 211237~ ` -12- ' vessel surface.
Any blood vessel may be treated in accordance with this invention, that is, arteries, veins, and capillaries.
These blood vessels may be in or near any organ in the human, or mammalian, body.
Introduction of normal or aeneticallv altered cells into a blood vessel:
This embodiment of the invention may be illustrated as follows:
I. Establishment of endothelial or other vascular cells in tissue culture.
Initially, a cell line is established and stored in liquid nitrogen. Prior to cryopreservation, an aliquot is taken for infection or transfection with a vector, viral or otherwise, containing the desired genetic material.
Endothelial or other vascular cells may be derived enzymatically from a segment of a blood vessel, using techniques previously described in J.W. Ford, et al., In yitro, 17, 40 (1981).~ The vessel is excised, inverted over a stainless steel rod and incubated in 0.1% trypsin in Ca++-and M ~ - free~Hank's~balanced~salt solution~(BSS) with 0.125% EDT~t~pH~8~for 10 min at 37C. ~
Cells (0.4 to 1.5 x 106) are collected by centrifugation and resuspended in medium l99 (GIBC0) containing 10%~fetal-bovine serum, endothelial cell growth -~upplement~(ECGS, Collaborative Research, Waltham, MA) at 25 ~g/ml, heparin at 15 U/ml, and gentamicin (50 ~g/ml).
Cell~ are added to a 75 cm2 tissue culture flask precoated WO93/~51 PCT/US92/05~2 21123rl 6 with gelatin (2 mg/ml in distilled water~. Cells are fed every second day in the above medium until they reach confluence.
After two weeks in culture, the ECGS and heparin may be omitted from the medium when culturing porcine endothelium. If vascular smooth muscle cells or fibroblasts are desired the heparin and ECGS can be omitted entirely from the culturing procedure. Aliquots of cells are stored in liquid nitrogen by resuspending to -approximately ~o6 cells in 0.5 ml of ice cold fetal calfserum on ice. An equal volume of ice cold fetal calf serum containing 10% DMS0 is added, and cells are transferred to a prechilled screw cap Corning freezing tube. These cells are transferred to a -70C freezer for 3 hours before long term storage in liquid nitrogen.
The cells are then infected with a vector containing the desired genetic materia}.-f , , ~ , . : ... ..
II. Introduction of cells expressing normal or exogenous`-proteins into the vasculature.
20A. Introduction of cells expressing relevant proteins by catheterization. i .:, . :,- . .. .
The patient is prepared for catheterization either by surgery or~percutaneously,:observing strict adherence to ~ terile technigues. -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 WO 93/00051 ~5 Pcr~usg2ios242 necessary, into the vessel (5) (Figure 1). This catheter means (4) is designed to introduce infected endothelial eells into a diserete region of the artery. The eatheter has a proximal and distal balloon means (2) and (1), S respeetively, (e.g., each balloon means`may be about 3 mm in length and about 4 mm in width), with a length of eatheter means between the balloons. The length of eatheter means between the balloons has a port means eonneeted to an instillation port means (3). When the proximal and distal balloons are inflated, a central space is ereated in the vessel, allowing for instillation of infeeted eells though the port.
A region of the blood vessel is identified by anatomieal landmarks and the proximal balloon means (2) is inflated to denude the endothelium by meehanical trauma (e.g., by foreeful passage of a partially inflated balloon eatheter within the vessel) or by meehanieal trauma in eombination with ~mall amounts of a proteolytie enzyme sueh as di~pase, trypsin, eollagenase, papain, pepsin, chyaotrypnin~;or eathepsin, or by ineubation with these proteolytie enzymes alone. In addition to proteolytie enzymes, lipo~es may be used. The region of the blood vessel may also be denuded by treatment with a mild detergent or the like, sueh as NP-40, Triton X100, deoxycholate, or 8DS.
The~d nudation eonditions are adjusted to aehieve - essentially eomplete loss-of endothelium for eell transfers or approximately 20 to 90%, preferably 50 to 75~, loss of eells from the-vessel wall for direet infeetion. In some instanees~eell removal may not be neeessary. The eatheter i8 then advaneediso that the instillation port means (3) is l~ plaeed in the region of denuded endothelium. Infeeted, ¦ transfeeted or normal eells are then instilled into the , : ~ ................. ..
wo 93/00051 2 1 12 3 7 ~ ~? `` `~ ~ Pcr/usg2io5~42 discrete section of artery over thirty minutes. If the blood vessel is perfusing an organ which can tolerate some ischemia, e.g., skeletal muscle, distal perfusion is not a major problem, but can be restored by an external shunt if necessary, or by using a catheter which`allows distal perfusion. After instillation of the infected endothelial cells, the balloon catheter is removed, and the arterial puncture site and local skin incision are repaired. If distal perfusion is necessary, an alternative catheter designed to allow distal perfusion may be used.
B. Introduction of recombinant genes directly into cells on the wall of a blood vessel or perfused by a specific circulation in vivo; infection or transfection of cells on the vessel wall and organs.
Surgical techniques are used as described above.
Instead of using infected cells, a high titer desired genetic material transducing viral vector (105 to 106 particles/ml) or DNA complexed to a delivery vector is`
directly instilled into the vessel wall using the double ` -balloon catheter technique. This vector is instilled in medium containing serum and polybrene (10 ~g/ml) to enhance the efficiency o~ 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 eva'cuated,~
25~ gently~washed'with phosphate-buffered~saline, and art'erial circulation i8 restored.-~`Similar`protocols are uséd for post operative recovery.
The vessel surface can be prepared by mechanical ~ denudation alone, in combination with small amounts'`of -proteolytic~enzymes such~as dispase, trypsin, collagenase or cathepsin, or by incubation with these proteolytic enzymes alone. The denudation conditions are adjusted to wo 93'~512 1 1 2 3 7 6 ; : PCT/US92/05242 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 s molecules such as polybrene (lo ~g/ml), poly-L-lysine, dextran sulfate, or any polycationic substance which is phyæiologically 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, ~ture, 314, 628 (1985); and-P.
Perez, R.W. Hoffman, J.A. Titus, and D.M. Segal, J. Exp.
~ed., 163, 166 (1986)) or biologically coupled using~hybrid hybridomas (U.D. Staerz and M.J. Bevan, Proc. ~atl. Acad.
Sci. USA, 83, 1453 (19~6); and C. Milstein and A.C. Cuello, ~ature, 305, 537 (1983)). After incubation in the central space of the cathe~ter for 0.2~-~to 2 hours or~more, the medium ~s evacuated,~gentl~ washed-with~phosphate~buffered;~
saline, and circulation restored- -~ ; Li. ,~
Using a different catheter design (see Figure 2), a different protocol for instillation can also be used. This i 30 second approachji~involves~the use of a single balloonimeans ¦ (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 i, i WO 93/000~;1 PCl/US92/05242 211237~
as described above and defective vector is introduced using similar adhesive molecules. In this instance, the use of a high pressure delivery system serves to optimize the interaction of vectors with cells in adjacent vascular tissue.
The present invention also provides for the use of growth factors delivered locally by catheter or systemically to enhance the efficiency of infection. In addition to retroviral vectors, herpes virus, adenovirus, or other viral vectors are suitable vectors for the present technique.
It is also possible to transform cells within an organ or tissue. Direct transformation of organ or tissue cells may be accomplished by one of two methods. In a first method a high pressure transfection is used. The high pressure will cause the vector to migrate through the blood vessel wall~ into the surrounding tissue. In a second method, injection into a capillary bed, optionally after - in~ury 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 Yessel a su~table time would be from O.Ol~to 12-hrs, preferably O.l to 6 hrs,~most preferably 0.2 to 2 hrs. Alternatively for high pressure instillation of vectors or cells,` shorter times might be preferred.
.
. . . .
Obtainina the cells used in this invention: -.
The term "genetic material" generally refers to DNA
which codes for a protein. This term also encompasses RNA
W093/~5l : PCT/US92/05~2 -when used with an RNA virus or other vector based on RNA.
Transformation is the process by which cells have incorporated an exogenous gene by direct infection, transfection or other means of uptake.
The term "vector" is well understood and is synonymous with the often-used phrase "cloning vehicle". A vector is non-chromosomal double-stranded DNA comprising an intact replicon such that the vector is replicated when placed within a unicellular organism, for example by a process of transformation. Viral vectors include retroviruses, adenoviruses, herpe~virus, papovirus, or otherwise modified naturally occurring viruses. Vector also means a formulation of DNA with a chemical or substance which allows uptake by cells.
In another embodiment the present invention provides for inhibiting the expression of a gene. Four approaches ~may be utilizsd to accomplish this goal. These include the use of antisense agents, either synthetic oligonucleotides which are complementary to the mRNA (Maher III, L.~. and Dolnick, 8.J. Arch. Biochem. Bio~hYs., 253, 214-220 (1987) and Zamecnik, P.C., et al., P~oc. 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.,-JScience,~229, 3~45-352,~(1985); Ççll, 36, 1077-1015 ~(198~ -In;addition, catalytic RNAs, called ribozymes, can specifically degrade RNA ~equences (Uhlenbeck, O.C., ature, 328, 596-600 (1987), Haseloff, J. and Gerlach, W.L., Nature, 334, 585-591 (1988)). The third approach involve~ n 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 WO g3/~51 2 1 1 2 3 7 6 PCT/US92/OS242 --19-- .,, ~ .
detail below.
The first approaches may be used to specifically eliminate transcripts in cells. The loss of transcript may be confirmed by Sl nuclease analysis, and expression of binding protein determined using a functional assay.
Single-stranded oligonucleotide analogues may be used to interfere with the processing or translation of the transcription factor mRNA. Briefly, synthetic oligonucleotides or thiol-derivative analogues (20-50 nucleotides) complementary to the coding strand of the target gene may be prepared. These antisense agents may be prepared against different regions of the mRNA. They are complementary to the 5' untranslated region, the translational initiation site and subsequent 20-50 base pairs, the central coding region, or the 3' untranslated region of the gene. The antisense agents may be incubated with cells transfected prior to activation. The efficacy of antigense competitors directed at different portions of the~essenger RNA may be compared to dete D lne~whether specific r~gions ~ay be moreS-effective in~preventing the expression of these genes.
RNA can also function in an autocatalytic fashion to cause autolysis or-to specifically degrade complementary ~NA seguence~ (Uhlenbeck, O.C., Nature,-328,~596-600 (198~), Haseloff, J. and Gerlach, W.L.,~Na~ure,~ 334,c ~
585-591 ~(1988), and Hutchins, C.J., et al, ~Lcleic Acids Res., 14, 3627-3640 (1986)).- ~he requirements-~for a successful RNA cleavage include a hammerhead structure with ;~ conserved RNA sequence at the region flanking this structure. ~Regions adjacent to this catalytic domain are made complementary to a specific RNA, thus targeting the ribozyme to specific cellular mRNAs. To inhibit the production of a specific target gene, the mRNA encoding - WO 93/00051~ r`.~ PCliUS92/OS242 this gene may be specifically degraded using ribozymes.
Briefly, any GUG sequence within the RNA transcript can serve as a target for degradation by the ribozyme. These may be identified by DNA sequence analysis and GUG sites S 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 ~pstream of the GUG site, the hammerhead structure and -20 base pairs of complementary sequence downstream of this site may be inserted at the relevant site in the cDNA. In this way, the ribozyme may be targeted to the same cellular compartment as the endogenous message. The ribozymes inserted downstream of specific enhancers, which give high level expression in specific cells may also be generated.
These plasmids may be introduced into relevant target cells us1ng electroporation and cotransfection with a neomycin res~stantcplasmid, pSV2-Neo-~or another selectable marker.
: .~The~expression of-these'~transcripts'may be confirmed by :.
Northern blot and Sl nuclease analysis. When confirmed, the expression of mRNA may:be evaluated by Sl nuclease protection to determine~whether expression of these ~ 25 tran~cripts reduces steady state levels of the target mRNA
:-~ and.~the~-genes~:which.. it:.regulates.- The level of';`protein may .
-~- a~lso;be examined.. ~ r~ ' ............................. ,l C .
: : Genes-may also be~inhibited~by preparing mutant . transcripts lacking-domains reguired for activation.
Briefly, after the domain has been identified,-a mutant form which is incapable of stimulating function is' '~ synthesized. This truncated gene product may be inserted downstream of the SV-40 enhancer in a plasmid containing the neomycin resistance gene (Nulligan, R. and Berg, P~, .
W093~0U05l 211237 6 PCI/US9~/~52~
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 Sl nuclease analysis and by immunoprecipitation. The function of the endogenous protein in these cells may be evaluated in two ways. First, the expression of the normal gene may be examined. Second, the known function of these proteins may be evaluated. In the event that this mutant intercellular interfering form is toxic to its host cell, it may be introduced on an inducible control element, such as metallothionein promoter. After the isolation of stable lines, cells may be incubated with Z~ or Cd to express this gene. Its effect on host cells can then be evaluated.
Another approach to the inactivation of specific genes is to overexpress recombinant proteins which antagonize the expression or function of other activities. For example, if one wished to decrea~e expression of TPA (e.g., in a clinical setting of disseminate thromboly~is), one could overexpress-plasminogen activator inhibitor.-~ -Advances in biochemistry and molecular biology in recent years have led to the construction of "recombinant"
vectors in which, for example, retroviruses and plasmids are ~ade to-contain exogenous RNA~or DNA, respectively. In 25~ particular~instances the recombinant-véctor can-`include ~-heterologou~RNA or DNA,~ by which is meant RNA or DNA that codes for a polypeptide ordinarily not produced by the organi~m susceptible to transformation by the recombinant vector.~ The~production of recombinant RNA and DNA vectors ~- 30 is well-under~tood-and need not be described in detail.
However,- a brief description of this process is included here for reference. -W093/ ~ 51 ' ~ PCTiUS92io5~2 For example, a retrovirus or a plasmid vector can be cleaved to provide linear RNA or DNA having ligatable termini. These termini are bound to exogenous RNA or DNA
having complementary like ligatable termini to provide a biologically functional recombinant RNA or D~A molecule having an intact replicon and a desired phenotypical property.
A variety of techniques are available for RNA and DNA
recombination in which adjoining ends of separate'RNA or DNA fragments are tailored to facilitate ligation.
The exogenous, i.e., donor, RNA or DNA used in the present invention is obtained from suitable cells. The vector is constructed using known techniques to obtain a transformed cell capable of in vivo expression of the therapeutic agent protein. The transformed cell is obtained by contacting a target cell with a RNA- or DNA-containing formulation permitting transfer and uptak~ of the RNA or DNA into the target cell.'~ Such formulations include, for example, retroviruses, plasmids, liposomal form~lations, or plasmids complexes with polycationic sU~stanceC such as poly-L-lysine, DEAC-dextran and targeting ligands.
- ~ The present-invention thus provides for the genetic alteration of-cells-as acmethod to transmit therapeutic'or'`'-;
25- L: i diagnostic agents to localized regions of the bl~od'vessel for local or systemic purposes. The range of reco~binant 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 . W093/00051 ~ PCI/USg2/05242 extended to genetic treatment of inherited disorders, or acquired diseases, localized or cystemic. The present invention may also be used to introduce normal cells to specific sites of cell loss, for example, to replace endothelium damaged during angioplasty or catheterization.
For example, in the treatment of ischemic diseases (thrombotic diseases), genetic material coding for tPA or modifications thereof, urokinase or streptokinase is used to transform the cells. In the treatment of ischemic organ (e.g., heart, kidney, bowel, liver, etc.) failure, genetic material coding for recollateralization agents, such as transforming growth factor ~ (TGF-~), transforming growth factor ~ (TGF-~), angiogenin, tumor necrosis factor ~, tumor necrosis factor ~, acidic fibroblast growth factor or basic fibroblast growth factor can be used. In the treatment of vasomotor diseases, genetic material coding for vasodilators or vasoconstrictors may be used. These include atrial natriuretic factor, platelet-derived growth factor or endothelin.. In the treatment of diabetes, genetic material coding for::insulin may be used.;-:^
.
The present invention can also be used in the treatment of malignancies by placing the transformed cells in proximity to the malignancy. In this application, genetic-material coding for diphtheria-tox~n, pertussis ~5 .-~-toxin,-.or~cholera~*oxin ma~be-used.
In one of;-its~embodiments,.the present.invention-provides for the therapy of mal~gnancy by either stimulating an immune response against tumor cells or - inhibiting tumor cell growth or metastasis.by genetic modification;in vivo. This approach differs from previous methods in which tumor cells are propagated, modified, and selected in vitro.
W093/~SI PCT/USg*OS~2
Technical Field The present invention relates-to the treatment of diseases by the site-specific instillation or transformation of cells and kits therefor. The present invention also relates to a method for modulating the immune ~ystem of an animal by the in vivo introduction of recombinant genes.
Backaround Art .. . . .
The effective treatment of many acquired and inherited diseases remains a major challenge to modern medicine. The ability to deliver therapeutic agents to specific sites n vivo would an asset in the treatment of, e.g., localized diseases.~ In addition the ability to cause a therapeutic agent to perfuse through the circulatory system would be effective~for the treatment of, e.g., inherited diseases and acquired diseases or cancers.
.
~- For,example, it would be-desirable to administer in a steadyvfashion an antitumor agent or toxin in close -proximity to a tumor. Similarly, it would be desirable to cause a-perfusion of, e.g., insulin in the blood of a , 30 person suffering from diabetes. However, for many WO93/~51 ~ 1 ' PCT/US92/05242 .. . .
therapeutic agents there is no satisfactory method of either site-specific or systemic administration.
In addition, for many diseases, it would be desirable to cause, either locally or systemically, the expression of a defective endogenous gene, the expression of a exogenous gene, or the suppression of an endogenous gene. Again, these remain unrealized goals.
In particular, the pathogenesis of atheroscle'rosis 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 man~fested by interference with the permeability barrier of the endothelium, alterations in the nonthromb'ogenic properties of the endothelial surface, and promotion of procoagulant properties of the endothelium. Monocytes migrate between endothelial cells, become active as ;~cavenger cells, and differentiate into macrophages.
;i Macrophages then-~ynthe~ize and-secrete growth factors including platelet derived growth factor (PDGF),' fibroblast growth factor (FGF), epidermal growth factor tEGF), and transforming growth factor~alpha (TGF-~). These growth factors sre extremely potent in stimulating the migration and proliferation of fibroblasts and smooth muscle cells in the atherosclerotic plaque. In addition, platelets may interact with the injured endothelial cell and the WO93/~51 2 1 1 2 3 7 6 s92~0s~2 -3- - ~
activated macrophage to potentiate the elaboration of growth factors and thrombus formation.
Two major problems in the clinical management of coronary artery disease include thrombus formation in acute myocardial ischemia and restenosis following coronary angioplasty (PTCA). Both involve common cellular events, including endothelial injury and release of potent growth factors by activated macrophages and platelets. Coronary angioplasty produces fracturing of the atherosclerotic plaque and removal of the endothelium. This vascular trauma promotes platelet aggregation and thrombus formation at the PTCA site. Further release of mitogens from platelets and macrophages, smooth muscle cell proliferation and monocyte infiltration result in restenosis.
Empiric therapy with antiplatelet drugs has not prevented this problem, which occurs in one-third of patients undergoing PTCA. A solution to restenosis is to prevent platelet aggregation, thrombus formation, and gmooth 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 ~ - dy~unction of endothelial cell~~ttachment,~Jand leading to -the expo~ure-of underlying macrophage foam cells.-~This permits the opportunity for circulating platelets to adhere, aggregate, and form thrombi.
-- The intravenous administration of thrombolytic agents, - such as tissue plasminogen activator (tPA) results in lysis of thrombus in approximately 70% of patients experiencing I an acute myocardial infarction. Nonetheless, approximately W093/~51 ~ PCT/US92/05~2 . ., . ", .
" ~ . ~ i ,, . I
30% of patients fail to reperfuse, and of those patients who undergo initial reperfusion of the infarct related artery, approximately 25% experience recurrent thrombosis within 24 hours. Therefore, an effective therapy for rethrombosis remains a major therapeutic challenge facing the medical community today.
As noted above, an effective therapy for rethrombosis is by far not the only major therapeutic challenge existing today. Others include the treatment of other ischemic conditions, including unstable angina, myocardial infarction or chronic tissue ischemia, or even the treatment of acquired and inherited diseases or cancers.
These might be treated by the effective administration of anticoagulants, vasodilatory, angiogenic, growth factors or growth inhibitors to a patient. Thus, there remains a strongly felt need for an effective therapy in all of these clinica} settings.
In addition, it is desirable to be able to modulate the i Dune system of an animal. In particular, much effort has been directed toward the development of vaccines to provide immunological protection from infection. However, the development of safe vaccines which can be readily administered to large numbers of patients is problematic, -and for many diseases,-such as, e.g., AIDS, no safe`and 25~ effective~vaccine-is as-yet ava~lable. Further, it is also ~sometimes~desirable toispecifically suppress an animals immune response to prevent rejection-of-a transplant.
Efforts to suppress transplant rejection have resulted in the development of drugs which result in a general suppression of the iDune response, rather than specific supression to transplantation antigens, and such drugs are not always effective. Thus, there remains a need for a method to modulate the i Dune system of an animal.
WOg3/H 051 2 1 1 2 3 7 6 PCriUsg~/05242 Disclosure of the Invention Accordingly, one object of the present invention is to provide a novel method for the site-specific administration of a therapeutic agent.
S It is another object of the present invention to provide a method for the perfusion of a therapeutic agent in the blood stream of a patient.
It is another object of the present invention to provide a method for causing the expression of an exogenous gene in a patient.
It is another object of the present invention to provide a method for causing the expression of a defective endogenous gene in a patient.
It is another object of the present invention to provide a method for suppressing the expression of an endogenous gene in a patient. ~ ` `
It is another object of the present invention to provide a method for site-specifically replacing damaged cells in a patient.
, . , ... ~ ... ... ; .~ , ,, . i ;
It is another object of^the present invention~toii provide a method for the treatment of a disease by causing either the site-specific administration of a therapeutic a~ent or the perfusion of a therapeutic agent in the bloodstream of a patient. -It is another ob~ect of the present invention to ; provide a method for the treatmént of a disease by causing either the expression of an exogenous gene, the expression 21123;~ 6 PCT/US92~0 ~ 2 ~, . .. . .
of a defective endogenous gene, or the suppression of the expression of an endogenous gene in a patient.
It is another object of the present invention to provide a method for the treatment of a disease by site-specifically replacing damaged cells in a patient.
It is another object of the present invention to provide a kit for site-specifically instilling normal or transformed cells in a patient.
It is another object of the present invention to provide a kit for site-specifically transforming cells in vivo.
It is another object of the present invention to provide a method for modulating the immune system of an animal.
, .
It is another object of the present invention to provide a method for modulating the immune system of an animal to sensitize the animal to a foreign molecule.
It is another object of the present invention to provide a method to stimulate the immune system of an anir~l-to reject proteins in order to protect against infection~byj~a microorganism or~ivirus.~ r~
~ -~ It is another object of the present invention to provide a method for modulating the immune system of an animal to tolerize the animal to a foreign molecule.
, It-is~another object of the present invention to provide a method for modulating the immune system of an animal to reduce the tendenoy to reject a transplant.
W093/~05l 2 1 1 2 3 7 6 PCT/US92/0~2 It is another object of the present invention to provide a novel kit for transforming cells by systemic administration in vivo.
These and other objects of this invention which will become apparent during the course of the following detailed description of the invention have been discovered by the inventors to be achieved by (a) a method which comprises either (i) site-specific instillation of either normal (untransformed) or transformed cells in a patient or (ii) site-specific transformation of cells in a patient and (b) a kit which contains a catheter for (i) site-specific instillation of either normal or transformed cells or (ii) site-specific transformation of cells.
Site-specific instillation of normal cells can be used to replace damaged cells, while instillation of transformed cells can be used to cause the expression of either a defective endogenous gene or an exogenous gene or the suppression of an endogenous gene product. Instillation of cells in the walls of the patient's blood vessels can be used to cause the steady perfusion of a therapeutic agent in the blood stream.
The inventors have-also discovered that by transforming cells of an animal, in vivo, it is possible to ~s~ modulate the animal's immune-system. In particular, by transforming cells of an animal, with a recombinant gene, by ~ite-specific or systemic administration it is possible to modulate the animal's immune system to sensitize the animal to the molecule for which the recombinant gene encodes. Alternatively, by transforming cells of an anima?
with a recombinant gene,~ specifically at a site which determines the specificity of the immune system, such as, e.g., the thymus, it is possible to modulate the immune W093/~51.. .~ PCT/USg2/05~2 2112~7 6 -8-system of an animal to suppress the immune response to the molecule encoded by the recombinant gene.
Brief Description of the Drawinas A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same become better understood by reference to the following detailed description when considered in connection with the accompanying figures, wherein:-FIGURES 1 and 2 il-ustrate the use of a catheter in accordance with the invention to surgically or percutaneously implant cells in a blood vessel or to transform ~n vivo cells present on the wall of a patient's blood vessel;
FIGURE 3 illustrates the relationship between the % of target cell lysis and the effector:target ratio for CTL
.cells; and FIGURE 4 illustrates the results of a Western blot analysis.
Best Mode for CarrYin~ Out the Invention ~ ~
... ...-Thus,~in one embodiment,- the presentr--invention is used .to.treat diseases, such as.inherited diseases, systemic --diseases, diseases of the cardiovascular system,~ diseases of particular organs, or tumors by instilling normal or transformed cells or by transforming cells. . `: .-. , ; ; . . . : . -The.cells which may be instilled in the present methodinclude endothelium, smooth muscle, fibroblasts, monocytes, macrophages, and parenchymal cells. These cells may i a WO93/~51 2 1 1 2 3 7 ~ PCT/US92/05242 produce proteins which may have a therapeutic or diagnostic effect and which may be naturally occurring or ari~e from recombinant genetic material.
Referring now to the figures, wherein like reference numerals designate identical or corresponding parts throughout the several views, and more particularly to FIGURE 1 thereof, 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. In the figure, 5 is the wall of the blood vessel. The figure shows the catheter body 4 held in place by the inflation of inflatable balloon mean~
1 and 2. The section of the catheter body 4 situated between balloon means 1 and 2 is equipped with instillation port means 3. The catheter may be further eguipped with a , guidewire means 6. FIGURE ~ illustrates the use of a-~imilar 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 con~ain up to twelve individual instillation port means 3, with,five being illu~trated.~
, ,,,~ In the-case-of,delivery to an organ, the catheter'~may be.introduced into-the major-artery supplying the tissue.
Cells containing recombinant genes or vectors can be ,,, introduced through a-central instillation port-after 30,~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 r WO 93/~ ~51 ; r PCT/US92/05242 the vasculature using the double balloon catheter technique in the arterial circulation proximal to the target organ.
In this way, the recombinant genes may be secreted directly into the circulation which perfuse the involved tissue or may be synthesized directly within the organ.
In one embodiment, the therapeutic agents are secreted by vascular cells supplying specific organs affected by the disease. For example, ischemic cardiomyopathy may be treated by introducing angiogenic factors into the coronary circulation. This approach may also be used for peripheral, vascular or cerebrovascular diseases where angiogenic factors may improve circulation to the ~rain or other tissues. Diabetes mellitus may be treated by introduction of glucose-responsive insulin secreting cells in the portal circulation where the liver normally sees a higher insulin concentration than other tissues.
In addition to providing local concentrations of therapeutic agents, the present~imethod may also be used for delivsry of recombinant genes to pasenchymal tissues,-because high concentrations of viral vector and othervectors can be delivered to a specific circulation. Using this approach, deficiencies of organ-specific proteins may also be treated. For example, in the liver, ~-antitrypsin inhibitor deficiency or hyperchloresterolemia may be treatsd by introduction of ~-antitrypsin or the LDL
- ;r~eceptor~Lgen~e.~ In-addition,- this approach may~be-used for the treatment,of a-malignancy. Secretion of specific~
recombinant toxin genes into the circulation of `
inoperable-tumors:provides a therapeutic-effect. Examples 30 - include acoustic neuromas or certain hemangiomas~!which are otherwise unresurrectable. ~
. ~ . .
In clinical settings, these therapeutic recombinant wO 93/~Sl r PCT/US92/05242 -12.11237~i genes are introduced in cells supplying the circulation of the involved organ. Although the arterial and capillary circulations are the preferred locations for introduction of these cells, venous systems are also suitable.
In its application to the treatment of local vascular damage the present invention provides for the expression of proteins which ameliorate this condition in situ. In one embodiment, because vascular cells are found at these sites, they are used as carriers to convey the therapeutic agents.
The invention thus, in one of its aspects, relies on genetic alteration of endothelial and other vascular cells or somatic cell gene therapy, for transmitting therapeutic agents (i.e., proteins, growth factors) to the localized region of vessel injury. To successfully use gene transplantation in the cells, four requirements must be fulfilled. First, the gene which is to be implanted into the cell must be identified and isolated. Second, the gene to be expresced must be cloned and available for genetic manipulation. Third, the gene must be introduced into the cell in a form that will be expressed or functional.
Fourth, the genetically altered cells must be situated in the vascul~r region where it is needed.
s:~ In accordance with~--the~present invention the altered cells or appropriate vector may be surgically, -;-percutaneously, or intravenously introduced and attached to a section of a patiént's vessel wall. Alternatively, some of the cells existing on the patient's vessel wall are :-transformed with the desired genetic material or-by i 30 -directly applying the vector. In some instances, vascular cells which are not genetically modified can be introduced by these methods to repl~ce cells lost or damaged on the !
W093/000~1 PCr/US92/~ ~2 211237~ ` -12- ' vessel surface.
Any blood vessel may be treated in accordance with this invention, that is, arteries, veins, and capillaries.
These blood vessels may be in or near any organ in the human, or mammalian, body.
Introduction of normal or aeneticallv altered cells into a blood vessel:
This embodiment of the invention may be illustrated as follows:
I. Establishment of endothelial or other vascular cells in tissue culture.
Initially, a cell line is established and stored in liquid nitrogen. Prior to cryopreservation, an aliquot is taken for infection or transfection with a vector, viral or otherwise, containing the desired genetic material.
Endothelial or other vascular cells may be derived enzymatically from a segment of a blood vessel, using techniques previously described in J.W. Ford, et al., In yitro, 17, 40 (1981).~ The vessel is excised, inverted over a stainless steel rod and incubated in 0.1% trypsin in Ca++-and M ~ - free~Hank's~balanced~salt solution~(BSS) with 0.125% EDT~t~pH~8~for 10 min at 37C. ~
Cells (0.4 to 1.5 x 106) are collected by centrifugation and resuspended in medium l99 (GIBC0) containing 10%~fetal-bovine serum, endothelial cell growth -~upplement~(ECGS, Collaborative Research, Waltham, MA) at 25 ~g/ml, heparin at 15 U/ml, and gentamicin (50 ~g/ml).
Cell~ are added to a 75 cm2 tissue culture flask precoated WO93/~51 PCT/US92/05~2 21123rl 6 with gelatin (2 mg/ml in distilled water~. Cells are fed every second day in the above medium until they reach confluence.
After two weeks in culture, the ECGS and heparin may be omitted from the medium when culturing porcine endothelium. If vascular smooth muscle cells or fibroblasts are desired the heparin and ECGS can be omitted entirely from the culturing procedure. Aliquots of cells are stored in liquid nitrogen by resuspending to -approximately ~o6 cells in 0.5 ml of ice cold fetal calfserum on ice. An equal volume of ice cold fetal calf serum containing 10% DMS0 is added, and cells are transferred to a prechilled screw cap Corning freezing tube. These cells are transferred to a -70C freezer for 3 hours before long term storage in liquid nitrogen.
The cells are then infected with a vector containing the desired genetic materia}.-f , , ~ , . : ... ..
II. Introduction of cells expressing normal or exogenous`-proteins into the vasculature.
20A. Introduction of cells expressing relevant proteins by catheterization. i .:, . :,- . .. .
The patient is prepared for catheterization either by surgery or~percutaneously,:observing strict adherence to ~ terile technigues. -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 WO 93/00051 ~5 Pcr~usg2ios242 necessary, into the vessel (5) (Figure 1). This catheter means (4) is designed to introduce infected endothelial eells into a diserete region of the artery. The eatheter has a proximal and distal balloon means (2) and (1), S respeetively, (e.g., each balloon means`may be about 3 mm in length and about 4 mm in width), with a length of eatheter means between the balloons. The length of eatheter means between the balloons has a port means eonneeted to an instillation port means (3). When the proximal and distal balloons are inflated, a central space is ereated in the vessel, allowing for instillation of infeeted eells though the port.
A region of the blood vessel is identified by anatomieal landmarks and the proximal balloon means (2) is inflated to denude the endothelium by meehanical trauma (e.g., by foreeful passage of a partially inflated balloon eatheter within the vessel) or by meehanieal trauma in eombination with ~mall amounts of a proteolytie enzyme sueh as di~pase, trypsin, eollagenase, papain, pepsin, chyaotrypnin~;or eathepsin, or by ineubation with these proteolytie enzymes alone. In addition to proteolytie enzymes, lipo~es may be used. The region of the blood vessel may also be denuded by treatment with a mild detergent or the like, sueh as NP-40, Triton X100, deoxycholate, or 8DS.
The~d nudation eonditions are adjusted to aehieve - essentially eomplete loss-of endothelium for eell transfers or approximately 20 to 90%, preferably 50 to 75~, loss of eells from the-vessel wall for direet infeetion. In some instanees~eell removal may not be neeessary. The eatheter i8 then advaneediso that the instillation port means (3) is l~ plaeed in the region of denuded endothelium. Infeeted, ¦ transfeeted or normal eells are then instilled into the , : ~ ................. ..
wo 93/00051 2 1 12 3 7 ~ ~? `` `~ ~ Pcr/usg2io5~42 discrete section of artery over thirty minutes. If the blood vessel is perfusing an organ which can tolerate some ischemia, e.g., skeletal muscle, distal perfusion is not a major problem, but can be restored by an external shunt if necessary, or by using a catheter which`allows distal perfusion. After instillation of the infected endothelial cells, the balloon catheter is removed, and the arterial puncture site and local skin incision are repaired. If distal perfusion is necessary, an alternative catheter designed to allow distal perfusion may be used.
B. Introduction of recombinant genes directly into cells on the wall of a blood vessel or perfused by a specific circulation in vivo; infection or transfection of cells on the vessel wall and organs.
Surgical techniques are used as described above.
Instead of using infected cells, a high titer desired genetic material transducing viral vector (105 to 106 particles/ml) or DNA complexed to a delivery vector is`
directly instilled into the vessel wall using the double ` -balloon catheter technique. This vector is instilled in medium containing serum and polybrene (10 ~g/ml) to enhance the efficiency o~ 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 eva'cuated,~
25~ gently~washed'with phosphate-buffered~saline, and art'erial circulation i8 restored.-~`Similar`protocols are uséd for post operative recovery.
The vessel surface can be prepared by mechanical ~ denudation alone, in combination with small amounts'`of -proteolytic~enzymes such~as dispase, trypsin, collagenase or cathepsin, or by incubation with these proteolytic enzymes alone. The denudation conditions are adjusted to wo 93'~512 1 1 2 3 7 6 ; : PCT/US92/05242 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 s molecules such as polybrene (lo ~g/ml), poly-L-lysine, dextran sulfate, or any polycationic substance which is phyæiologically 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, ~ture, 314, 628 (1985); and-P.
Perez, R.W. Hoffman, J.A. Titus, and D.M. Segal, J. Exp.
~ed., 163, 166 (1986)) or biologically coupled using~hybrid hybridomas (U.D. Staerz and M.J. Bevan, Proc. ~atl. Acad.
Sci. USA, 83, 1453 (19~6); and C. Milstein and A.C. Cuello, ~ature, 305, 537 (1983)). After incubation in the central space of the cathe~ter for 0.2~-~to 2 hours or~more, the medium ~s evacuated,~gentl~ washed-with~phosphate~buffered;~
saline, and circulation restored- -~ ; Li. ,~
Using a different catheter design (see Figure 2), a different protocol for instillation can also be used. This i 30 second approachji~involves~the use of a single balloonimeans ¦ (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 i, i WO 93/000~;1 PCl/US92/05242 211237~
as described above and defective vector is introduced using similar adhesive molecules. In this instance, the use of a high pressure delivery system serves to optimize the interaction of vectors with cells in adjacent vascular tissue.
The present invention also provides for the use of growth factors delivered locally by catheter or systemically to enhance the efficiency of infection. In addition to retroviral vectors, herpes virus, adenovirus, or other viral vectors are suitable vectors for the present technique.
It is also possible to transform cells within an organ or tissue. Direct transformation of organ or tissue cells may be accomplished by one of two methods. In a first method a high pressure transfection is used. The high pressure will cause the vector to migrate through the blood vessel wall~ into the surrounding tissue. In a second method, injection into a capillary bed, optionally after - in~ury 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 Yessel a su~table time would be from O.Ol~to 12-hrs, preferably O.l to 6 hrs,~most preferably 0.2 to 2 hrs. Alternatively for high pressure instillation of vectors or cells,` shorter times might be preferred.
.
. . . .
Obtainina the cells used in this invention: -.
The term "genetic material" generally refers to DNA
which codes for a protein. This term also encompasses RNA
W093/~5l : PCT/US92/05~2 -when used with an RNA virus or other vector based on RNA.
Transformation is the process by which cells have incorporated an exogenous gene by direct infection, transfection or other means of uptake.
The term "vector" is well understood and is synonymous with the often-used phrase "cloning vehicle". A vector is non-chromosomal double-stranded DNA comprising an intact replicon such that the vector is replicated when placed within a unicellular organism, for example by a process of transformation. Viral vectors include retroviruses, adenoviruses, herpe~virus, papovirus, or otherwise modified naturally occurring viruses. Vector also means a formulation of DNA with a chemical or substance which allows uptake by cells.
In another embodiment the present invention provides for inhibiting the expression of a gene. Four approaches ~may be utilizsd to accomplish this goal. These include the use of antisense agents, either synthetic oligonucleotides which are complementary to the mRNA (Maher III, L.~. and Dolnick, 8.J. Arch. Biochem. Bio~hYs., 253, 214-220 (1987) and Zamecnik, P.C., et al., P~oc. 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.,-JScience,~229, 3~45-352,~(1985); Ççll, 36, 1077-1015 ~(198~ -In;addition, catalytic RNAs, called ribozymes, can specifically degrade RNA ~equences (Uhlenbeck, O.C., ature, 328, 596-600 (1987), Haseloff, J. and Gerlach, W.L., Nature, 334, 585-591 (1988)). The third approach involve~ n 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 WO g3/~51 2 1 1 2 3 7 6 PCT/US92/OS242 --19-- .,, ~ .
detail below.
The first approaches may be used to specifically eliminate transcripts in cells. The loss of transcript may be confirmed by Sl nuclease analysis, and expression of binding protein determined using a functional assay.
Single-stranded oligonucleotide analogues may be used to interfere with the processing or translation of the transcription factor mRNA. Briefly, synthetic oligonucleotides or thiol-derivative analogues (20-50 nucleotides) complementary to the coding strand of the target gene may be prepared. These antisense agents may be prepared against different regions of the mRNA. They are complementary to the 5' untranslated region, the translational initiation site and subsequent 20-50 base pairs, the central coding region, or the 3' untranslated region of the gene. The antisense agents may be incubated with cells transfected prior to activation. The efficacy of antigense competitors directed at different portions of the~essenger RNA may be compared to dete D lne~whether specific r~gions ~ay be moreS-effective in~preventing the expression of these genes.
RNA can also function in an autocatalytic fashion to cause autolysis or-to specifically degrade complementary ~NA seguence~ (Uhlenbeck, O.C., Nature,-328,~596-600 (198~), Haseloff, J. and Gerlach, W.L.,~Na~ure,~ 334,c ~
585-591 ~(1988), and Hutchins, C.J., et al, ~Lcleic Acids Res., 14, 3627-3640 (1986)).- ~he requirements-~for a successful RNA cleavage include a hammerhead structure with ;~ conserved RNA sequence at the region flanking this structure. ~Regions adjacent to this catalytic domain are made complementary to a specific RNA, thus targeting the ribozyme to specific cellular mRNAs. To inhibit the production of a specific target gene, the mRNA encoding - WO 93/00051~ r`.~ PCliUS92/OS242 this gene may be specifically degraded using ribozymes.
Briefly, any GUG sequence within the RNA transcript can serve as a target for degradation by the ribozyme. These may be identified by DNA sequence analysis and GUG sites S 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 ~pstream of the GUG site, the hammerhead structure and -20 base pairs of complementary sequence downstream of this site may be inserted at the relevant site in the cDNA. In this way, the ribozyme may be targeted to the same cellular compartment as the endogenous message. The ribozymes inserted downstream of specific enhancers, which give high level expression in specific cells may also be generated.
These plasmids may be introduced into relevant target cells us1ng electroporation and cotransfection with a neomycin res~stantcplasmid, pSV2-Neo-~or another selectable marker.
: .~The~expression of-these'~transcripts'may be confirmed by :.
Northern blot and Sl nuclease analysis. When confirmed, the expression of mRNA may:be evaluated by Sl nuclease protection to determine~whether expression of these ~ 25 tran~cripts reduces steady state levels of the target mRNA
:-~ and.~the~-genes~:which.. it:.regulates.- The level of';`protein may .
-~- a~lso;be examined.. ~ r~ ' ............................. ,l C .
: : Genes-may also be~inhibited~by preparing mutant . transcripts lacking-domains reguired for activation.
Briefly, after the domain has been identified,-a mutant form which is incapable of stimulating function is' '~ synthesized. This truncated gene product may be inserted downstream of the SV-40 enhancer in a plasmid containing the neomycin resistance gene (Nulligan, R. and Berg, P~, .
W093~0U05l 211237 6 PCI/US9~/~52~
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 Sl nuclease analysis and by immunoprecipitation. The function of the endogenous protein in these cells may be evaluated in two ways. First, the expression of the normal gene may be examined. Second, the known function of these proteins may be evaluated. In the event that this mutant intercellular interfering form is toxic to its host cell, it may be introduced on an inducible control element, such as metallothionein promoter. After the isolation of stable lines, cells may be incubated with Z~ or Cd to express this gene. Its effect on host cells can then be evaluated.
Another approach to the inactivation of specific genes is to overexpress recombinant proteins which antagonize the expression or function of other activities. For example, if one wished to decrea~e expression of TPA (e.g., in a clinical setting of disseminate thromboly~is), one could overexpress-plasminogen activator inhibitor.-~ -Advances in biochemistry and molecular biology in recent years have led to the construction of "recombinant"
vectors in which, for example, retroviruses and plasmids are ~ade to-contain exogenous RNA~or DNA, respectively. In 25~ particular~instances the recombinant-véctor can-`include ~-heterologou~RNA or DNA,~ by which is meant RNA or DNA that codes for a polypeptide ordinarily not produced by the organi~m susceptible to transformation by the recombinant vector.~ The~production of recombinant RNA and DNA vectors ~- 30 is well-under~tood-and need not be described in detail.
However,- a brief description of this process is included here for reference. -W093/ ~ 51 ' ~ PCTiUS92io5~2 For example, a retrovirus or a plasmid vector can be cleaved to provide linear RNA or DNA having ligatable termini. These termini are bound to exogenous RNA or DNA
having complementary like ligatable termini to provide a biologically functional recombinant RNA or D~A molecule having an intact replicon and a desired phenotypical property.
A variety of techniques are available for RNA and DNA
recombination in which adjoining ends of separate'RNA or DNA fragments are tailored to facilitate ligation.
The exogenous, i.e., donor, RNA or DNA used in the present invention is obtained from suitable cells. The vector is constructed using known techniques to obtain a transformed cell capable of in vivo expression of the therapeutic agent protein. The transformed cell is obtained by contacting a target cell with a RNA- or DNA-containing formulation permitting transfer and uptak~ of the RNA or DNA into the target cell.'~ Such formulations include, for example, retroviruses, plasmids, liposomal form~lations, or plasmids complexes with polycationic sU~stanceC such as poly-L-lysine, DEAC-dextran and targeting ligands.
- ~ The present-invention thus provides for the genetic alteration of-cells-as acmethod to transmit therapeutic'or'`'-;
25- L: i diagnostic agents to localized regions of the bl~od'vessel for local or systemic purposes. The range of reco~binant 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 . W093/00051 ~ PCI/USg2/05242 extended to genetic treatment of inherited disorders, or acquired diseases, localized or cystemic. The present invention may also be used to introduce normal cells to specific sites of cell loss, for example, to replace endothelium damaged during angioplasty or catheterization.
For example, in the treatment of ischemic diseases (thrombotic diseases), genetic material coding for tPA or modifications thereof, urokinase or streptokinase is used to transform the cells. In the treatment of ischemic organ (e.g., heart, kidney, bowel, liver, etc.) failure, genetic material coding for recollateralization agents, such as transforming growth factor ~ (TGF-~), transforming growth factor ~ (TGF-~), angiogenin, tumor necrosis factor ~, tumor necrosis factor ~, acidic fibroblast growth factor or basic fibroblast growth factor can be used. In the treatment of vasomotor diseases, genetic material coding for vasodilators or vasoconstrictors may be used. These include atrial natriuretic factor, platelet-derived growth factor or endothelin.. In the treatment of diabetes, genetic material coding for::insulin may be used.;-:^
.
The present invention can also be used in the treatment of malignancies by placing the transformed cells in proximity to the malignancy. In this application, genetic-material coding for diphtheria-tox~n, pertussis ~5 .-~-toxin,-.or~cholera~*oxin ma~be-used.
In one of;-its~embodiments,.the present.invention-provides for the therapy of mal~gnancy by either stimulating an immune response against tumor cells or - inhibiting tumor cell growth or metastasis.by genetic modification;in vivo. This approach differs from previous methods in which tumor cells are propagated, modified, and selected in vitro.
W093/~SI PCT/USg*OS~2
2 11237 6 -24-In accordance with this embodiment, the present method is used to deliver a DNA sequence or an RNA sequence, including recombinant genes, to tumor cells ,in vivo with (1) retroviral or viral vectors as vehicles, (2) DNA or RNA/liposome complexes as vehicles, (3) chemical formulations containing the DNA or RNA sequence and coupled to a carrier molecule which facilitates delivery of the sequence to the targeted cells, or (4) by utilizing cell-mediated gene transfer to deliver genes to specific sites in vivo, e.g., by relying upon the use of vascular smooth muscle cells or endothelia cells which have been transduced in vitro as a vehicle to deliver the recombinant gene into the site of the tumor.
In an aspect of this embodiment, the present invention relies on the immune system to provide protection against cancer and play an important role as an adjuvant treatment for a malignancy. Immunotherapy has shown promise as an adjuvant-approach to the treatment of malignancies. Both cytolytic.T cells and lymphokines can~facilitate tumor cell de~truction, and strategies:to enhance tumor-regression by administration of cytokines or tumor infiltrating lymphocytes have shown efficacy in animal models and human trials. For example, it is known that lymphokine activated ,~ killer cells (LAK) and tumor infiltrating lymphocytes (TIL) ;.~.can,.lyse n~oplastic~cell- and~-produce partial--or complete tumor rejection. Expression-.~-of;cytokine~genes.~-in ma}ignant cellsrhas.l-also enhanced.tumor..regression..
,~. ,The:present invention provides a'novel gene transfe~
,~ approach. against:tumors by,.the.introduction of recombinant , ~' 30,,,.genes,jidirectly~~into.tumor.cells in-vivo,.where, by.~
- contrast, traditional gene transfer techniques have focused . on modification of tumor cells in vitro followed by transfer of the modified cells. The prior art approaches ,. .
~WO93/~51 2 112~7 ~ PCT/US92/05~2 are disadvantageous because they subject the cells to selection in different growth conditions from those which act in vivo, and because they also require that cell lines be established for each malignancy, thereby rendering adaptability to human disease considerably more difficult.
Genes which may be used with this embodiment include genes containing a DNA sequence (or the corresponding RNA
sequence may be used) encoding an intracellular, secreted, or cell surface molecule which is exogenous to the patient and which (1) is immunogenic to the patient, (2) induces rejection, regression, or both, of the tumor, or (3) is toxic to the cells of the tumor.
The vectors containing the DNA sequence (or the corresponding RNA sequence) which may be used in accordance with the invention may be an eukaryotic expression vector containing the DNA or the RNA sequence of interest.
Techniques for obtaining expression of exogenous DNA or RNA
sequences in a host are known. See, for example, Korman et al, Proc. Nat. Acad. Sci. C(USA~, (1987) 84:2150-2154,'which 20 i8 hereby incorporated by reference.
This vectorj as noted above, may be administered to the patient in a retroviral or other viral vector (i.e., à
-viral vector) ^vehicle, a DNA or RNA/liposome complex,'~or by utilizing cell-mediated gene tran~fer.~-Further,'-ithe;~
vector, when-present in non-viral form, may be administered as a DNA or RNA-sequence-containing chemical-formul~ation coupled to a carrier molecule which facilitates delivery to the host cell. Such carrier molecule would include an antibody specific to the cells to which the vector is being delivered or a molecule capable of interacting with a ' receptor associated with the target cells.
WO 93/OOOS1 . PCriUS92105242 2 1 ~ 26-~ - ~ .
Cell-mediated gene transfer may be used in accordance with the invention. In this mode, one relies upon the delivery of recombinant genes into living organisms by transfer of the genetic material into cells derived from the hcst and modification in cell culture, followed by the introduction of genetically altered cells into the host.
An illustrative packaging cell line which may be used in accordance with this embodiment is described in ~anos et al, Proc. Natl. Acad. Sci. (USA~ (1988) 85:6460, which is hereby incorporated by reference.
The DNA or RNA sequence encoding the molecule used in accordance with the invention may be administered to the patient, which may be human or a non-human animal, either locally or systemically. The systemic administration is preferably carried out using the non-viral DNA or RNA
chemical formulation coupled to a carrier molecule which facilitates delivery to the host cells. Any of the admini~trations may be performed by IV or IN injection or - ~ubcutaneous injection using any known means, or by the use of the catheter in accordance with the present invention.
, The retroviral vector vehicles used in accordance with the pre~ent invention comprise a viral particle derived from a naturaIly-occurring retrovirus which has been genetically altered to render it replication defective and to4~eYpr,e i~a;lrecombinant gene of intere~t in accordance ; 1,-w th the invention.- Once the virus delivers its genetic material~*o a cell, it does not generate additional infectious virus but does introduce exogenous~recombinant genes *o the cell.
; s In other viral vectors, the virus particle used is derived fro~ other naturally-occurring viruses which have been genetically ~ltered to render th-m replication !
WO93/~51 PCT/US92S0~2 defective and to express recombinant genes. Such viral vectors may be derived from adenovirus, papillomavirus, herpesvirus, parvovirus, etc.
The sequences of the present invention may also be administered as DNA or RNA/liposome complex. Such complexes comprise a mixture of fat particles, lipids, which bind to genetic material, DNA or RNA, providing a hydrophobic coat, allowing genetic material to be delivered into cells. This formulation provides a non-viral vector for gene transfer. Liposomes used in accordance with the invention may comprise DOPE (dioleyl phosphatidyl ethanol amine), CUDMEDA (N-(Scholestrum-3-~-ol 3-urethanyl)-N', N'-dimethylethylene diamine).
As noted above, other non-viral vectors may also be used in accordance with the present invention. These include chemical formulations of DNA or RNA coupled to a carries molecule (e.g., an antibody or a receptor ligand) which facilitates delivery to host cells for the purpose of altering-the biologic properties of the host cells. The term nchemical formulations" used herein refers to modifications of nucleic acids to allow coupling of the nucleic acid compounds to a protein or lipid, or derivative thereof, carrier molecule. ~Such carrier molecules include antibodies specific to~the host-~cells or~receptor ligands, -25 ,~,~,r ~i.e~ molecules~able-to;interact-~with receptors associated with the hogt cells. ;~
The molecules which may be used in accordance with this invention, include-the following: (1) genes encoding immune stimulants, such as Class I histocompatibility 30 - genes-, Class II histocompatibility genes. bacterial genes, including mycobacterial (PPD) genes and genes encoding heat shock proteins, viral glycoproteins encoding genes, W093/ ~ 51 . i PCTiUSg2/05242 including vesicular stomatitis virus G protein, influenza hemagglutinin, and herpes virus glycoprotein ~, minor histocompatibility antigens, foreign proteins, such as lysozyme or bovine serum albumin, and oncogenes, including EIA, P53 (mutants) and tax; (2) immune and growth stimulants/inhibitors, including inducers of differentiation, such as stimulants, including interleukin-2 (IL-2) IL-4, 3, 6 or 8, inhibitors/inducers of differentiation, such as TNF-~ or ~, TGF-~ (1, 2 or 3), IL-l, soluble growth factor receptors (PDGF, FGF -receptors), recombinant antibodies to growth factors or receptors, analogs of growth factors (PDGF, FGF), interferons (~, ~ or ~) and adhesion molecules; or (3) toxins or negative selectable markers, including thymiidine kinase, diphtheria toxin, pertussis toxin or drug-sensitive proteins.
The DNA/RNA sequence is preferably obtained from a source of the same species as the patient, but this is not absolutely reguired, and the present invention provides for -the use of DNA sequences-obtained from a source-of a~
species different from the patient in accordance with this embodiment. A preferred embodiment of the present invention, genes encoding immune stimulants and toxins or negative ~electable markers, oorresponding to (1) and (3) above,-iare~preferably~-se}ected--~from a`--species different -than~he;species~to~which-~the patient~belongs-~ ^For immune~;
and growth ~ti~ulants/inhibitors, corresponding to (2)~
above, in accordance with another preferred embodiment of the in~vention, one preferably employs a gene obtained from a species~-which is the same as the species of the patient.
In the u~e-of the present invention in-the treatment of AIDS, genetic material coding for soluble CD4 or derivatives thereof may be used. In the treatment of W093/~51.~ 2112 3 7 6 PCT/US92/05242 genetic diseases, for example, growth hormone deficiency, genetic material coding for the needed substance, for example, human growth hormone, is used. All of these genetic materials are readily available to one skilled in this art.
In another embodiment, the present invention provides a kit for treating a disease in a patient which contains a catheter and a solution which contains either an enzyme or a mild detergent, in which the catheter is adapted for insertion into a blood vessel and contains a main catheter body having a balloon element adapted to be inserted into said vessel and expansible against the walls of the blood vessel so as to hold the main catheter body in place in the blood vessel, and means carried by the main catheter body for delivering a solution into the blood vessel, and the solution which contains the enzyme or mild detergent is a physiologically acceptable solution. The solution may contain a.proteolytic enzyme,.such as dispase, trypsin, collagena~e, papain, pepsin, or chymotrypsin. In addition to proteolytic enzymes, liposes may be used. As a mild detergent, the solution may contain NP-40, Triton X100, deoxycholate, SDS or the like.
Alternatively, the kit may contain a physiological acceptable solution which-containsJ~an agent such as heparin, poly-L-lysine, polybrene, dextran sulfate, a -~c;.polycationic material, or bivalent antibodies. This . solution may also contain.vectors or cells (normal or:
transformed). In yet another embodiment the kit may . contain a-catheter and both~.a solution which contains an 30 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.
~WO93/~051 ;!~ PCTJUS92/05~2 211237~
The kit may contain a catheter with a ~ingle balloon and central distal perfusion port, together with acceptable solutions to allow introduction of cells in a specific , organ or vectors into a capillary bed or cells in a specific organ or tissue perfused by this capillary bed.
Alternatively, the kit may contain a main catheter body which has two spaced balloon elements adapted to be inserted in a blood vessel witb both being expansible against the walls of the blood vessel for providing a chamber in the blood vessel, and to hold the main catheter body in place. In this case, the means for delivering a solution into the chamber is situated in between the balloon elements. The kit may contain a catheter which possesses a plurality of port means for delivering the solution into the blood vessel.
Thus, the present invention represents a method for treating a disease in a.patient by causing a cell attached , onto.the walls.o,f:a vessel or the cells of an organ -' pe~fused..by.this vessel in the patient to express an ~' exogenous,therapeutic agent protein, wherein the protein treats the disease or may be useful for diagnostic purposes. The pr~sent method may be used to treat diseases, such.as an ischemic disease, a vasomotor disease, diabetes~ a,.malignancy,,~AIDS~.or,~a genetic-disease.;S~
c ~J ~.~ ~ ~ ~j ~ r~,~ t ~ ~d~ ;~S~ c~
~,The present method.,.may ~se exogenous-therapeutic~agent proteins,-,.;such as.tPA and.modifications thereof,~urokinase, streptokinase,,acidic~fibroblast growth factor, basic ,fibroblast,growthifactor,.tumor necrosis factor ~, tumor : ,,.necr~osis,:factor ~ ,transforming growth factor ~
~ 30 transforming growth factor ~, atrial natriuretic-factor, - platelet-derived growth factor, endothelian, insulin, -- diphtheria toxin, pertussis toxin, cholera toxin, soluble WOg3/~5l 2 1 1 2 3 7 6 PCT/US92ios242 :
CD4 and derivatives thereof, and growth hormone to treat .
diseases.
The present method may also use exogenous proteins of ~;
diagnostic value. For example, a marker protein, such as ~-galactosidase, may be used to monitor cell migration.
It is preferred, that the cells caused to express the exogenous therapeutic agent protein be endothelial cells.
Other features of the present invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.
The data reported below demonstrate the feasibility of endothelial cell transfer and gene transplantation; that endothelial cells may be stably implanted in situ on the arterial wall by catheterization and express a recombinant marker protein, ~-galactosidase, in vivo. : ;
Because atherogenesis in swine has similarities to humans, an inbred pig strain, the Yucatan minipig (Charles River Laboratories, Inc~, Wilmington, MA), was chosen as an animal ~odel (1). A primary endothelial cell line was established..from the internal jugular vein~of an 8~
.-fmonth-old female minipig.~ The endothelial-cell~identity of .~this line..was confirmed-in that theScells exhibited growth characteristics and morphology typical of porcine `~
endothelium:in tissue culture. Endothelial cells`also expressfreceptors 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
W0 93/00051 . . ` ~ ' PCI /I~JSg2io52~2 ., i ~ ~,i ; `
21123~6 -32-uptake.
Two independent ~-galactosidase-expressing endothelial lines were isolated following infection with a murine amphotropic ~-galactosidase-transducing'retroviral vector 5 (BAG), which is replication-defective and contains both ' ~-galactosidase and neomycin resistance genes (3). Cells containing this vector were selected for their ability to grow in the presence of G-418. Greater than 90% of selected cells synthesized ~-galactosidase by histochemical staining. The endothelial nature of these genetically altered cells was also confirmed by analysis of fluorescent ACLDL uptake. Infection by BAG retrovirus was further verified by Southern blot analysis which revealed the presence of intact.proviral DNA at approximately one copy per genome.
Endothelial cells derived from this inbred strain, being syngeneic, were applicable for study in more than one minipig, and were tested,in nine different experimental subjects. Under general anesthesia, the femoral and iliac arteries were exposed, and a catheter was introduced into the vessel (Figure 1).. Inti-al tissues of the arterial ,wall were denuded mechanically by forceful passage of a : partially inflated balloon catheter within the vessel. The artery was,,rinsQd with~heparinized saline and incubated . 25~ ,with~the,,neutr,al,~pr.otease,~-i,dispase (50 U/ml), which:Jremoved ,any r -aining luminal~:~endothelial~cells. Residual enzyme was rapidly.inactivated by ~2 globulin in plasma upon -deflating the catheterlbal'loons and allowing blood to flow"
th,ro,ugh the vessel segment. The cultured endothelial cells 30,s.which,expressed,~-galactosidase were introduced using a specially designed arterial catheter (USCI, Billerica, MA) that contained two balloons and a central instillation port (Figure 1).
WO93/OOOS1 2 1 1 2 3 7 ~ ~CT/US92/05~2 When these balloons were inflated, a protected space was created within the artery into which cells were instilled through the central port 3 (Figure 1). These endothelial cells, which expressed ~-galactosidase, were allowed to incubate for 30 minutes to facilitate their attachment to the denuded vessel. The catheter was then removed, the arterial branch ligated, and the incision closed.
Segments of the artery inoculated with ~-galactosidase- expressing endothelium were removed 2 to 4 weeks later. Gross examination of the arterial specimen after staining using the X-gal chromogen showed multiple areas of blue coloration, compared to an artery seeded with uninfected endothelium, indicative of ~-galactosidase activity. Light microscopy documented ~-galactosidase staining primarily in endothelial cells of the intima in experimentally seeded vessels.
In contrast, no evidence of similar staining was observed in control segments which had received endothelial cells containing no ~-galactosidase. ~-Galactosidase staining was occasionally evident in deeper intimal tissues, suggesting entrapment or migration of seeded endothelium within the previously injured ves~el wall~
Iocal ~hrombosis was observed in the first two experimental c~ubjects.- ~Thisicomplication was minimized`in ~ubsèquent ~tudies by administering acetylsalicylic acid prior to the endothelial cell transfer procedure and use of heparin - anticoagulation at the time of inoculation. In instances of thrombus-formation, ~-galactosidase staining was seen in endothelial cells extending from the vessel wall to the surface of~the thrombus.
, .
A ma~or concern of gene transplantation in vivo ~W093/~51 ~ PCT/US92/05242 2112376 -34_ 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 n vitro. Although defective viruses were used because of their high rate of infectivity and their stable integration into the host cell genome (4), this approach to gene transfer is adaptable to other viral vectors.
A second concern involves the longevity of expression of recombinant genes in vivo. Endothelial cell expression of ~-galactosidase appeared constant in vessels examined up to six weeks after introduction into the blood vessel in the present study.
These tests have demonstrated that genetically-altered endothelial cells can be introduced into the vascular wall of the Yucatan minipig by arterial catheterization. Thus, the-present method can be used for the localized biochemical treatment of vascular disease using genetically-altered endothelium as a vector.
A major complication of current interventions $or vascular disease, such as balloon angioplasty or insertion of a graft into a~di~eased vessel, is disruption of the atherosclerotic plaque and thrombus formation at sites of ~' , . . , , ., . . i, .
25~ 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 throm~osis.
This technique can also be used in other ischemic settings, including unstable angina or myocardial infarction. For instance, antithrombotic effects can be WO93/00051~ 2 1 1 2 3 7 6 PCr/US9~/05242 achieved by introducing cells expressing genes for tissue plasminogen activator or urokinase. This technology is also useful for the treatment of chronic tissue ischemia.
For example, elaboration of angiogenic or growth factors (7) to stimulate the formation of collateral vessels to severely ischemic tissue, such as the myocardium. Finally, somatic gene replacement for syætemic inherited diseases is feasible using modifications of this endothelial cell gene transfer technique.
Another aspect of the present invention relates a method for modulating the immune system of an animal by in vivo transformation of cells of the animal with a recombinant gene. The transformation may be carried out either in a non-site-specific or systemic manner or a site-specific manner. If the transformation is carried out in a systemic fashion or at sites other than those which confer specificity on the immune system, such as the thymus, then the immune system will be ~odulated to result in the animal being sensitized to the molecule for which the recombinant gene encodes.~ Alternatively, if the transformation i8~
carried out in a site-specific manner and is localized to a site which determines the specificity of the immune system, e.g., the thymus, the immune system will be modulated to result in the animal being tolerized to the molecule encoded by the recombinant gene. ~ c ~ - By the term sensitized, it`is meant that the immune system exhibits a stronger response to the molecule encoded by the DNA after in vivo transformation as compared to before transformation. By the term tolerized, it is meant that the immune system displays a reduced response to-the molecule encoded by the recombinant gene after transformation as compared to before transformation. Thus, one may modulate an immune system to provide either a .. , . ., , . , - - - ~ . , . . ~
W093/~051~ .'' '' PCT/US92~05i42 211237~ -36-resistance or a tolerance to the molecule encoded by the DNA.
Examples of molecules for which it may be desirable to provide a resistance to include: cell surface molecules, such as tumor antigens (carcinoembryonic antigen), protozoan antigens (pneumocystis), viral antigens (HIV
gpl20 and gpl60, ~. influenza antigen, and hepatitis B
surface antigen), Lyme disease antigen, Bacterial antigens, and transplantation antigens (Class I or II), ras or other oncogenes, including erb-A or neu; cytoplasmic proteins, such as the raf oncogene, src oncogene, and abl oncogene;
nuclear proteins, such as ElA oncogene, mutant p53 oncogene, tat, tax, rev, vpu, vpx, hepatitis core antigen, EBNA and viral genes; and secreted proteins, such as ~5 endotoxin, cholera toxin, TNF, and osteoclast activating factor.
. Examples of molecules for which it may be desirable to .: provide;a resisitance to include:/ cell surface molecules, such ~s growth--factor receptors,^~insiulin recéptors, thyroid hormone receptors,-transplantation antigens (class I or II), blood group antigens, and ~DL receptor; cytoplasmic proteins, such as cytochrome P450, galactosyl transferase, dystrophin, neomycin resistance gene, and bacterial heat shock protein; nuclear proteins,~such~as retinoblastoma and-tran~dominant rev; and secreted proteins, such as growth ~-hormone.ifor~;dwarfs,~insulin for diabetics',:'and'adenosine deaminase.~
It'-is.:to be`understood-that theinucleic acid, DNA, RNA,i or derivative *hereof,~in the~`recombinant gene-may be of any suitable origin. That~is-the nucleic acid may be . isolated~from a naturally occurring source or may be of synthetic origin.
W093/~51~ 2112 3 7 6 PCT/US92ios242 The recombinant gene may be introduced in the cells of the animal using any conventional vector. Such vectors include viral vectors, cationic lipids complexed to DNA or RNA (DNA or RNA/liposomes) and DNA or RNA complexes with S polycations, such as DEAE, dextran, and polybrene.
As noted above the recombinant gene can be introduced into cells in a site-specific manner to confer resistance to the molecule encoded by the recombinant gene. Suitable sites include, e.g., endothelial cells or reticuloendothelial cells in the vasculature or any specific tissue or organ. The form of the preparation containing the vector and recombinant gene used in the transformation will depend on the specific tissue to be transformed. Suitable preparations for transforming endothelial cells are described elsewhere in this specification. In addition, preparations suitable for oral or other means of administration (e.g., endoscopic) may be used to provide mucosal resistance. Such preparation could include detergents`,~gelatins, capsules or other delivery 20 ` vehicles to protect against degradation and énhance ~~
delivery to the mucosal surface, in addition to the vector and gene.
Alternatively, the recombinant gene may be introduced in a site`'specific'fashion to a site which determinës'`'the 'spécificity of the immuné system.` The thymus-is~such~a ~sité (sèé: A.~M.'~Posselt-et-'al-, Science, vol. 249, p.-l292 ' '(l990)). Thus,'by introducihg a recombinant~gene sité-specifically into the thymus, the immune system may be modulated to result in~à tolerance to the moleculë èncoded by the gene. In this way, transplant rejectionlmay bë~`' suppressed. The same prepàrations and techniques used to site-specifically transform tumors described above may be used to introduce the recombinant gene into the thymus.
WO 93/~H~ f ! ~ ~' PCT~US92/05242 Specifically, the transformation preparation may be injected directed into the thymus or tumor or into the vascular supply of the thymus or tumor.
The present method may be practiced on any animal, such as chickens or mammals such as cows, horses, cats, dogs, monkeys, lemurs or humans.
When the recombinant gene is introduced using a liposome, it is preferred to first determine in vi-tro the optimal values for the DNA: lipid ratios and the absolute concentrations of DNA and lipid as a function of cell death and transformation efficiency for the particular type of cell to be transformed and to use these values in the in ~ivo transformation. The in vitro determination of these values can be easily carried out using the techniques described in the Experimental Section of this specification.
.
Another aspect of the present invention relates to a kit for the in vivo systemic-introduction of a recombinant gene into cells of an animal. Such a kit would include approximately the optimal amount of a carrier, such as a lipid, and nucleic acid, and/or a means of delivery, e.g., an~endoscope or a syringe. The kit may also contain instructions for the administration of the transforming ~, . , ~ ~ . .. . .
p#paration.~ The carrier and nucleic~acid may-be free~e dried and may be packaged separately or premixed. The kit may also contain a solution to optimally reconstitute the complexes of the carrier and the nuclèic acid, which provide for efficient delivery to cells in vivo.- Such a - solution~may contain one or more ingredients, such as buffers, sugars, salts, proteins, and detergents.
Having generally described the invention, a further WO 93/00051 PCI/US92io5~42 understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.
Ex~erimental section:
A. Analysis of AcLDL receptor expression in normal and ~-galactosidase-transduced porcine endothelial cells.
Endothelial cell cultures derived from the Yucatan minipig, two sublines infected with BAG retrovirus or 3T3 fi~roblast controls were analyzed for expression of AcLDL
receptor using fluorescent labelled AcLDL.
Endothelial cells were derived from external jugular veins uæing the neutral protease dispase (8). Excised vein segments were filled with dispase (50 U/ml in Hanks' balanced salt solution) and incubated at 30C for 20 minute~. Endothelium obtained by this means was maintained in medium 199 (GIBC0, Grand Island, N.Y.) supplemented with fetal calf serum (10%), 50 ~g/ml endothelial cell growth supplement (ECGS) and heparin ~100 ~g/ml). These cells were infected with BAG retrovirus, and selected for resistance-to G-418. Cell cultures were incubated with (l,l'-dioctade~yl3,3,3',3'-tetramethylindocarbacyanine -perchlorate)~(Dil)~AcLDL (Biomedical Technologies,;^~
-- Stoughton,-MA) ~lO ~g/ml) for:4-6 hrs.-at 37C,~followed by three rinses with phosphate- buffered saline containing 0.5% glutaraldehyde. Cells were visualized by phase contrast and fluorescent microscopy. -~ .
B. Method of introduction of endothelial c~lls bycatheterization.
.- W0 93/OOOSI ~ PCI /USg2/05242 A double balloon catheter was used for instillation of endothelial cells. The catheter has a proximal and distal balloon, each 6 mm in length and 5 mm in width, with a 20 mm length between the balloons. The central section of the catheter has a 2 mm pore connected to an instillation port.
Proximal and distal balloon inflation isolates a central space, allowing for instillation of infected cells through the port into a discrete segment of the vessel. For a schematic representation of cell introduction by catheter, lo see Figures 1 and 2.
Animal care was carried out in accordance with "Principles of Laboratory Animal Care" and "Guide for the Care and Use of Laboratory Animals" (NIH publication No.
80-23, Revised 1978). Female Yucatan minipigs (80-100 kg) - 15 were anesthetized with pentobarbital (20 mg/kg), intubated, and mechanically ventilated. These ~ubjects underwent sterile surgical exposure of the iliac and femoral arteries. The distal femoral artery was punctured, and the double-balloon catheter was advanced by guidewire into the iliac ar,tery. The external iliac artery was identified;
the proximal balloon was partially inflated and passed proximally and distally so as to mechanically denude the endothelium. The catheter was then positioned with the central space located in the region of denuded endothelium, and both;,balloon~,were inflated. The denuded egment was irrigated~with~heparinized saline, and,-residual~-adherent cells were removed,by instillation of di~pase (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 sub~equently removed, and antegrade blood flow was re~tored. 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-W O 93/00051 2112 3 7 ~ PC~r/US92/05242 ~41-buffered saline, and another portion was mounted in a paraffin block for cectioning~ The presence of retroviral expressed ~-galactosidase was determined by a standard histochemical technique (19).
C. Analysis of endothelial cells in vitro and n vivo .
~ -Galactosidase activity was documented by histochemical staining in (A) primary endothelial- cells fro~ 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 histochemiaal staining. The enzymatic activity of the E. coli ~-galactosidase protein was u ed to identify infected endothelial cells n vitro and in viyo. The ~-galactosidase transducing Mo-MuLV
vector (2), (BAG) was kindly provided by Dr. Constance Cepko. This vector used the wild type Mo-MuLV LTR as a ~romoter for the ~-galactosidase gene. The simian ~irus 40 ~.(SV-40)- early promoter linked to the Tn5 neomycin- - ---25..re~istance gene provides resi tance to the drug G-418 and ~is inserted downstream of the ~-galac~osidase gene, providing a marker to select for retrovirus-con aining, : ~-galactosidase expressing cells. This defective retro~irus was prepared from fibroblast ~ am cells (3,10), ~0 -and maintained in Dulbecco's modified Eagle's medium (DMEM) and 10% calf serum. Cells were passaged twice weekly following trypsinization. The supernatant, with titers of WO 93/00051 `, `` ` ~ `: PCI'/USg2/05242 211237~ -42-104-105/ml G-418 resistant colonies, was added to endothelial cells at two-thirds confluence and incubated for 12 hours in DMEM with 10% calf serum at 370C in 5% Co2 in the presence of 8 ~g/ml of polybrene. Viral supernatants were removed, and cells maintained in medium 199 with 10% fetal calf serum, ECGS (50 ~g/ml), and endothelial cell conditioned medium (20%) for an additional 24 to 48 hours prior to selection in G-418 (0.7 pg/ml of a 50% racemic mixture). G-4 18 resistant cells were isolated and analyzed for ~-galactosidase expression using-a standard histochemical stain (9). Cells stably expressing the ~-galactosidase enzyme were maintained in continuous culture for use as needed. Frozen aliquo~s were stored in liquid nitrogen.
D. Immunotherapy of Malignancy by In Vivo Gene Transfer.
A retroviral vector which the H-2KS gene was prepared.
CT26 cells.were infected with this vector in.vitro, selected:for G418 resistance, and analyzed by fluorescence activated cell sorting (FACS). Transduced CT26 cells showed a higher mean fluorescence intensity than uninfected CT26 cells or CT26 infected with different retroviral vectors. When 106 CT26 cells which express H-2K~ were --injected.subcutaneously. into BALB/c mice (H-2d) sensitized to this~.antigen,-:~no~stumors.were observed over an.8-week --~period in-icontrast to~the.unmodified CT26.(H-2d)-tumor line which routinely.formed tumors at thiæ dose. The immune response to H-2K~ could therefore provide protection-against.CT26 cells.bearing this antigen. When CT26 H-2K~
and CT26 were co-inoculated, however,.tumor growth was observed, suggesting that H-2K~ conferred senstivity.only to modified cells..
WO93/0~51` 2112 3 7 6 PCT/US92/05242 To determine whether protective effects could be achieved by introduction of H-2K~ in growing CT26 tumors, the recombinant H-2K~ reporter or a ~-galactosidase gene was introduced into tumors either with a DNA/liposome or a retroviral vector. Tumor capsules (0.5-l cm diameter) were exposed surgically and multiple needle injections (2-lO) delivered to the parenchyma. With ~-galactosidase reporter plasmids, recombinant gene expression could be readily detected after intra-tumor injection of DNA/liposome or retroviral vectors.
In mice which received intra-tumor injections of the H-2K8 DNA/liposome comples or H-2Ke retroviral vector, the recombinant DNA was detected by PCR in the tumor and occasioinally in other tissues. When found in the other organs, no evidence of inflammation or ogran toxicity was detected pathologically. An immune response to the recombinant H-2K~ protein was evident in these animals, however. Lymphocytes derived from the H-2K8, but not ~-- galactoæida~e transduced tumors, demonstrated a cytolytic response to H-2K~, whether delivered by retroviral vectors or liposomes. Nore importantly, lymphocytes derived from the H-2K~, but not ~-galactosidase transduced animals, recognized and lysed unmodified CT26 cells, indicating that this stimulation induced immune reactivity against genetically unmodified tumor cells.
To as~ess the protective effect of the immune response -againgt H-2K8, tumor growth in vivo was quantitated. When animals received no prior sensitization to H-2K8, one o~
four tumors transduced with H-2K8 showed attenuation of tumor growth which was not complete. In contrast, no anti-tumor effect was seen in unmodified (n=4) or ~-galactosidase transduced controls (n=4). Because these tumors were large at the time of initial injection and W093/~5l ~ PCTiUS92/05~2 . ' .,' '. ' . ~ ' ''`' .
continued to grow as the primary immune response was generated, an attempt was made to optimize the anti-tumor response by pre-immunization of mice with irradiated CT26 H-2K~ tumor cells, and by earlier and/or more frequent injections of vector. Tumors were transduced on days 12 and 32 by intra-tumor injuection of H-2K~ or ~-galactosidase DNA/liposome vectors. Treatment with the H-2KB liposome complex improved survival and attenuated tumor growth, in contrast to ~-galactosidase transduced tumors where there was no difference in growth rate compared to the uninjected controls. Complete tumor regression was acheived in two mice by increasing the number of injections and by delivery of H-2K~ into tumors at an earlier stage.
This treatment was protective, since control animals showed continued tumor growth and did not survive beyond 35 days.
E. Modulation of the Immune System.
$he response to injection of cationic lipids and pla~mids was determined after in~ection intravenously into 8A$B/c mice (6-12 weeks). In the first experiments, a gene encoding the H-2K~ molecule was introduced by tail vein injection. Two to four weeks later, spleen cells were harvested and analyzed for their ability to mediate a cytolytic T cell response. When these cells were tested using 5~Cr target cells ~(CT26~cells expressing the-H-2X~
gene), significant cytolysis was observed which was not een in animals injected with the cointrol vector, ~-- galactosidase (See Figure 3)~. Up to 25% of target cells were lysed at effector: target ratios of 25:1.
- - Inr-addition to this specific cytolytic T cell -~-~ 3Q response, serologic or antibody responses to genes encoded - by expression vector plasmids have been examined. When a plasmnid encoding the gpl60 molecule of HIV is injected, an .
WO93/~51 PCTiUS92/05242 antibody response is elicited in treated mice. In contrast to control animals injected with cationic lipids containing ~-galactosidase, mice injected with cationic lipids with gp 160 plasmid showed an antibody response to the gpl60 and gpl20 form of this molecule by Westerm blot analysis (See Figure 4). These results demonstrate that systemic administration of cationic lipid/DNA complexes can be used successfully to induce cell-mediated and antibody-mediated immunity against foreign pathogens.
10F. Determination of Optimal Transfection Conditions.
(1) Plasmid Construction A plasmid containing the E. coli lacZ gene under the control of the Rous Sarcoma Virus LTS (RSV-~-gal) (Norton and Coffin, Mol. Cell. Biol., 5(2), 281-290, 1985) was used for transfection of porcine primary endothelial and HeLa cells. In addition, a plasmid containing the lacZ gene under the control of preproendothelin-l 5'-flanking DNA (-1410 to +83) (Wilson et al., Mol. Cell. Biol., 10~9), 4854-4862, 1990) was used for transfection of endothelial cells.
For in vivo toxicity analysis, the RSV-~-gal plasmid, and a plasmid derived from the PLJ vector containing the cDNA
encoding an H-2K8 mouse MHC class I gene were used.
~ (2)~Seli--Culture, Transfection Analysis,-and Toxicity - in Vitro ~
., ., . ~ .
25Primary endothèlial cells, derived from the Yucatan minipig (YPE-cells), were incubated with medium 199 (M199) cupplemented with 10% FBS, 2~M l-glutamine, 50 U/ml-- penicillin, and 5 ~g/ml streptomycin. HeLa cells were maintained in Dulbeccos Modified Eagles Medium (DNEM) 30supplemented with 5% FBS, 2mM 1-glutamine, 50 U/ml ~WO~g3/, ~ 51 !~ ', PCT/US92/05242 211237 ~ -46- ' penicillin and 5 ~g/ml streptomycin. The DNA liposome mixture was prepared with lipid concentrations of DOPE/DC-Chol between 2.5 and 25 ~M added to 0.2 ml of serum-free media or Ringer's lactate solution in polystyrene tubes.
After mixinq gently, the solution was allowed to stand at room temperature for 15-20 minutes. For transfection analysis, cells were grown in 60 mm tissue culture dishes at 75% confluency or greater. Cells were washed twice with serum-free media or lactated Ringers solution and then placed in 0.5 mls of the same media. The DNA liposome solution (0.2 ml) was then added slowly to the cells, with gentle mixing, with a final volume of 0.7 ml. This resulted in DNA concentrations between .7 and 7 ~g/ml (13-130 ~M), and lipid concentrations of 7-70 ~M. Transfection was allowed to proceed for 1-5 hours, after which the cells were placed ir. media supplemented as decribed above. At 24-48 hours after transfection the enzymatic activity of the E. coli ~-galactosidase protein was used to identify transfected cells by staining with the X-gal chromagen.
Toxicity in vitro was assessed by cytopathic effect or .
~rypan blue exclusion. , ~ ~
.
In an aspect of this embodiment, the present invention relies on the immune system to provide protection against cancer and play an important role as an adjuvant treatment for a malignancy. Immunotherapy has shown promise as an adjuvant-approach to the treatment of malignancies. Both cytolytic.T cells and lymphokines can~facilitate tumor cell de~truction, and strategies:to enhance tumor-regression by administration of cytokines or tumor infiltrating lymphocytes have shown efficacy in animal models and human trials. For example, it is known that lymphokine activated ,~ killer cells (LAK) and tumor infiltrating lymphocytes (TIL) ;.~.can,.lyse n~oplastic~cell- and~-produce partial--or complete tumor rejection. Expression-.~-of;cytokine~genes.~-in ma}ignant cellsrhas.l-also enhanced.tumor..regression..
,~. ,The:present invention provides a'novel gene transfe~
,~ approach. against:tumors by,.the.introduction of recombinant , ~' 30,,,.genes,jidirectly~~into.tumor.cells in-vivo,.where, by.~
- contrast, traditional gene transfer techniques have focused . on modification of tumor cells in vitro followed by transfer of the modified cells. The prior art approaches ,. .
~WO93/~51 2 112~7 ~ PCT/US92/05~2 are disadvantageous because they subject the cells to selection in different growth conditions from those which act in vivo, and because they also require that cell lines be established for each malignancy, thereby rendering adaptability to human disease considerably more difficult.
Genes which may be used with this embodiment include genes containing a DNA sequence (or the corresponding RNA
sequence may be used) encoding an intracellular, secreted, or cell surface molecule which is exogenous to the patient and which (1) is immunogenic to the patient, (2) induces rejection, regression, or both, of the tumor, or (3) is toxic to the cells of the tumor.
The vectors containing the DNA sequence (or the corresponding RNA sequence) which may be used in accordance with the invention may be an eukaryotic expression vector containing the DNA or the RNA sequence of interest.
Techniques for obtaining expression of exogenous DNA or RNA
sequences in a host are known. See, for example, Korman et al, Proc. Nat. Acad. Sci. C(USA~, (1987) 84:2150-2154,'which 20 i8 hereby incorporated by reference.
This vectorj as noted above, may be administered to the patient in a retroviral or other viral vector (i.e., à
-viral vector) ^vehicle, a DNA or RNA/liposome complex,'~or by utilizing cell-mediated gene tran~fer.~-Further,'-ithe;~
vector, when-present in non-viral form, may be administered as a DNA or RNA-sequence-containing chemical-formul~ation coupled to a carrier molecule which facilitates delivery to the host cell. Such carrier molecule would include an antibody specific to the cells to which the vector is being delivered or a molecule capable of interacting with a ' receptor associated with the target cells.
WO 93/OOOS1 . PCriUS92105242 2 1 ~ 26-~ - ~ .
Cell-mediated gene transfer may be used in accordance with the invention. In this mode, one relies upon the delivery of recombinant genes into living organisms by transfer of the genetic material into cells derived from the hcst and modification in cell culture, followed by the introduction of genetically altered cells into the host.
An illustrative packaging cell line which may be used in accordance with this embodiment is described in ~anos et al, Proc. Natl. Acad. Sci. (USA~ (1988) 85:6460, which is hereby incorporated by reference.
The DNA or RNA sequence encoding the molecule used in accordance with the invention may be administered to the patient, which may be human or a non-human animal, either locally or systemically. The systemic administration is preferably carried out using the non-viral DNA or RNA
chemical formulation coupled to a carrier molecule which facilitates delivery to the host cells. Any of the admini~trations may be performed by IV or IN injection or - ~ubcutaneous injection using any known means, or by the use of the catheter in accordance with the present invention.
, The retroviral vector vehicles used in accordance with the pre~ent invention comprise a viral particle derived from a naturaIly-occurring retrovirus which has been genetically altered to render it replication defective and to4~eYpr,e i~a;lrecombinant gene of intere~t in accordance ; 1,-w th the invention.- Once the virus delivers its genetic material~*o a cell, it does not generate additional infectious virus but does introduce exogenous~recombinant genes *o the cell.
; s In other viral vectors, the virus particle used is derived fro~ other naturally-occurring viruses which have been genetically ~ltered to render th-m replication !
WO93/~51 PCT/US92S0~2 defective and to express recombinant genes. Such viral vectors may be derived from adenovirus, papillomavirus, herpesvirus, parvovirus, etc.
The sequences of the present invention may also be administered as DNA or RNA/liposome complex. Such complexes comprise a mixture of fat particles, lipids, which bind to genetic material, DNA or RNA, providing a hydrophobic coat, allowing genetic material to be delivered into cells. This formulation provides a non-viral vector for gene transfer. Liposomes used in accordance with the invention may comprise DOPE (dioleyl phosphatidyl ethanol amine), CUDMEDA (N-(Scholestrum-3-~-ol 3-urethanyl)-N', N'-dimethylethylene diamine).
As noted above, other non-viral vectors may also be used in accordance with the present invention. These include chemical formulations of DNA or RNA coupled to a carries molecule (e.g., an antibody or a receptor ligand) which facilitates delivery to host cells for the purpose of altering-the biologic properties of the host cells. The term nchemical formulations" used herein refers to modifications of nucleic acids to allow coupling of the nucleic acid compounds to a protein or lipid, or derivative thereof, carrier molecule. ~Such carrier molecules include antibodies specific to~the host-~cells or~receptor ligands, -25 ,~,~,r ~i.e~ molecules~able-to;interact-~with receptors associated with the hogt cells. ;~
The molecules which may be used in accordance with this invention, include-the following: (1) genes encoding immune stimulants, such as Class I histocompatibility 30 - genes-, Class II histocompatibility genes. bacterial genes, including mycobacterial (PPD) genes and genes encoding heat shock proteins, viral glycoproteins encoding genes, W093/ ~ 51 . i PCTiUSg2/05242 including vesicular stomatitis virus G protein, influenza hemagglutinin, and herpes virus glycoprotein ~, minor histocompatibility antigens, foreign proteins, such as lysozyme or bovine serum albumin, and oncogenes, including EIA, P53 (mutants) and tax; (2) immune and growth stimulants/inhibitors, including inducers of differentiation, such as stimulants, including interleukin-2 (IL-2) IL-4, 3, 6 or 8, inhibitors/inducers of differentiation, such as TNF-~ or ~, TGF-~ (1, 2 or 3), IL-l, soluble growth factor receptors (PDGF, FGF -receptors), recombinant antibodies to growth factors or receptors, analogs of growth factors (PDGF, FGF), interferons (~, ~ or ~) and adhesion molecules; or (3) toxins or negative selectable markers, including thymiidine kinase, diphtheria toxin, pertussis toxin or drug-sensitive proteins.
The DNA/RNA sequence is preferably obtained from a source of the same species as the patient, but this is not absolutely reguired, and the present invention provides for -the use of DNA sequences-obtained from a source-of a~
species different from the patient in accordance with this embodiment. A preferred embodiment of the present invention, genes encoding immune stimulants and toxins or negative ~electable markers, oorresponding to (1) and (3) above,-iare~preferably~-se}ected--~from a`--species different -than~he;species~to~which-~the patient~belongs-~ ^For immune~;
and growth ~ti~ulants/inhibitors, corresponding to (2)~
above, in accordance with another preferred embodiment of the in~vention, one preferably employs a gene obtained from a species~-which is the same as the species of the patient.
In the u~e-of the present invention in-the treatment of AIDS, genetic material coding for soluble CD4 or derivatives thereof may be used. In the treatment of W093/~51.~ 2112 3 7 6 PCT/US92/05242 genetic diseases, for example, growth hormone deficiency, genetic material coding for the needed substance, for example, human growth hormone, is used. All of these genetic materials are readily available to one skilled in this art.
In another embodiment, the present invention provides a kit for treating a disease in a patient which contains a catheter and a solution which contains either an enzyme or a mild detergent, in which the catheter is adapted for insertion into a blood vessel and contains a main catheter body having a balloon element adapted to be inserted into said vessel and expansible against the walls of the blood vessel so as to hold the main catheter body in place in the blood vessel, and means carried by the main catheter body for delivering a solution into the blood vessel, and the solution which contains the enzyme or mild detergent is a physiologically acceptable solution. The solution may contain a.proteolytic enzyme,.such as dispase, trypsin, collagena~e, papain, pepsin, or chymotrypsin. In addition to proteolytic enzymes, liposes may be used. As a mild detergent, the solution may contain NP-40, Triton X100, deoxycholate, SDS or the like.
Alternatively, the kit may contain a physiological acceptable solution which-containsJ~an agent such as heparin, poly-L-lysine, polybrene, dextran sulfate, a -~c;.polycationic material, or bivalent antibodies. This . solution may also contain.vectors or cells (normal or:
transformed). In yet another embodiment the kit may . contain a-catheter and both~.a solution which contains an 30 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.
~WO93/~051 ;!~ PCTJUS92/05~2 211237~
The kit may contain a catheter with a ~ingle balloon and central distal perfusion port, together with acceptable solutions to allow introduction of cells in a specific , organ or vectors into a capillary bed or cells in a specific organ or tissue perfused by this capillary bed.
Alternatively, the kit may contain a main catheter body which has two spaced balloon elements adapted to be inserted in a blood vessel witb both being expansible against the walls of the blood vessel for providing a chamber in the blood vessel, and to hold the main catheter body in place. In this case, the means for delivering a solution into the chamber is situated in between the balloon elements. The kit may contain a catheter which possesses a plurality of port means for delivering the solution into the blood vessel.
Thus, the present invention represents a method for treating a disease in a.patient by causing a cell attached , onto.the walls.o,f:a vessel or the cells of an organ -' pe~fused..by.this vessel in the patient to express an ~' exogenous,therapeutic agent protein, wherein the protein treats the disease or may be useful for diagnostic purposes. The pr~sent method may be used to treat diseases, such.as an ischemic disease, a vasomotor disease, diabetes~ a,.malignancy,,~AIDS~.or,~a genetic-disease.;S~
c ~J ~.~ ~ ~ ~j ~ r~,~ t ~ ~d~ ;~S~ c~
~,The present method.,.may ~se exogenous-therapeutic~agent proteins,-,.;such as.tPA and.modifications thereof,~urokinase, streptokinase,,acidic~fibroblast growth factor, basic ,fibroblast,growthifactor,.tumor necrosis factor ~, tumor : ,,.necr~osis,:factor ~ ,transforming growth factor ~
~ 30 transforming growth factor ~, atrial natriuretic-factor, - platelet-derived growth factor, endothelian, insulin, -- diphtheria toxin, pertussis toxin, cholera toxin, soluble WOg3/~5l 2 1 1 2 3 7 6 PCT/US92ios242 :
CD4 and derivatives thereof, and growth hormone to treat .
diseases.
The present method may also use exogenous proteins of ~;
diagnostic value. For example, a marker protein, such as ~-galactosidase, may be used to monitor cell migration.
It is preferred, that the cells caused to express the exogenous therapeutic agent protein be endothelial cells.
Other features of the present invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.
The data reported below demonstrate the feasibility of endothelial cell transfer and gene transplantation; that endothelial cells may be stably implanted in situ on the arterial wall by catheterization and express a recombinant marker protein, ~-galactosidase, in vivo. : ;
Because atherogenesis in swine has similarities to humans, an inbred pig strain, the Yucatan minipig (Charles River Laboratories, Inc~, Wilmington, MA), was chosen as an animal ~odel (1). A primary endothelial cell line was established..from the internal jugular vein~of an 8~
.-fmonth-old female minipig.~ The endothelial-cell~identity of .~this line..was confirmed-in that theScells exhibited growth characteristics and morphology typical of porcine `~
endothelium:in tissue culture. Endothelial cells`also expressfreceptors 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
W0 93/00051 . . ` ~ ' PCI /I~JSg2io52~2 ., i ~ ~,i ; `
21123~6 -32-uptake.
Two independent ~-galactosidase-expressing endothelial lines were isolated following infection with a murine amphotropic ~-galactosidase-transducing'retroviral vector 5 (BAG), which is replication-defective and contains both ' ~-galactosidase and neomycin resistance genes (3). Cells containing this vector were selected for their ability to grow in the presence of G-418. Greater than 90% of selected cells synthesized ~-galactosidase by histochemical staining. The endothelial nature of these genetically altered cells was also confirmed by analysis of fluorescent ACLDL uptake. Infection by BAG retrovirus was further verified by Southern blot analysis which revealed the presence of intact.proviral DNA at approximately one copy per genome.
Endothelial cells derived from this inbred strain, being syngeneic, were applicable for study in more than one minipig, and were tested,in nine different experimental subjects. Under general anesthesia, the femoral and iliac arteries were exposed, and a catheter was introduced into the vessel (Figure 1).. Inti-al tissues of the arterial ,wall were denuded mechanically by forceful passage of a : partially inflated balloon catheter within the vessel. The artery was,,rinsQd with~heparinized saline and incubated . 25~ ,with~the,,neutr,al,~pr.otease,~-i,dispase (50 U/ml), which:Jremoved ,any r -aining luminal~:~endothelial~cells. Residual enzyme was rapidly.inactivated by ~2 globulin in plasma upon -deflating the catheterlbal'loons and allowing blood to flow"
th,ro,ugh the vessel segment. The cultured endothelial cells 30,s.which,expressed,~-galactosidase were introduced using a specially designed arterial catheter (USCI, Billerica, MA) that contained two balloons and a central instillation port (Figure 1).
WO93/OOOS1 2 1 1 2 3 7 ~ ~CT/US92/05~2 When these balloons were inflated, a protected space was created within the artery into which cells were instilled through the central port 3 (Figure 1). These endothelial cells, which expressed ~-galactosidase, were allowed to incubate for 30 minutes to facilitate their attachment to the denuded vessel. The catheter was then removed, the arterial branch ligated, and the incision closed.
Segments of the artery inoculated with ~-galactosidase- expressing endothelium were removed 2 to 4 weeks later. Gross examination of the arterial specimen after staining using the X-gal chromogen showed multiple areas of blue coloration, compared to an artery seeded with uninfected endothelium, indicative of ~-galactosidase activity. Light microscopy documented ~-galactosidase staining primarily in endothelial cells of the intima in experimentally seeded vessels.
In contrast, no evidence of similar staining was observed in control segments which had received endothelial cells containing no ~-galactosidase. ~-Galactosidase staining was occasionally evident in deeper intimal tissues, suggesting entrapment or migration of seeded endothelium within the previously injured ves~el wall~
Iocal ~hrombosis was observed in the first two experimental c~ubjects.- ~Thisicomplication was minimized`in ~ubsèquent ~tudies by administering acetylsalicylic acid prior to the endothelial cell transfer procedure and use of heparin - anticoagulation at the time of inoculation. In instances of thrombus-formation, ~-galactosidase staining was seen in endothelial cells extending from the vessel wall to the surface of~the thrombus.
, .
A ma~or concern of gene transplantation in vivo ~W093/~51 ~ PCT/US92/05242 2112376 -34_ 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 n vitro. Although defective viruses were used because of their high rate of infectivity and their stable integration into the host cell genome (4), this approach to gene transfer is adaptable to other viral vectors.
A second concern involves the longevity of expression of recombinant genes in vivo. Endothelial cell expression of ~-galactosidase appeared constant in vessels examined up to six weeks after introduction into the blood vessel in the present study.
These tests have demonstrated that genetically-altered endothelial cells can be introduced into the vascular wall of the Yucatan minipig by arterial catheterization. Thus, the-present method can be used for the localized biochemical treatment of vascular disease using genetically-altered endothelium as a vector.
A major complication of current interventions $or vascular disease, such as balloon angioplasty or insertion of a graft into a~di~eased vessel, is disruption of the atherosclerotic plaque and thrombus formation at sites of ~' , . . , , ., . . i, .
25~ 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 throm~osis.
This technique can also be used in other ischemic settings, including unstable angina or myocardial infarction. For instance, antithrombotic effects can be WO93/00051~ 2 1 1 2 3 7 6 PCr/US9~/05242 achieved by introducing cells expressing genes for tissue plasminogen activator or urokinase. This technology is also useful for the treatment of chronic tissue ischemia.
For example, elaboration of angiogenic or growth factors (7) to stimulate the formation of collateral vessels to severely ischemic tissue, such as the myocardium. Finally, somatic gene replacement for syætemic inherited diseases is feasible using modifications of this endothelial cell gene transfer technique.
Another aspect of the present invention relates a method for modulating the immune system of an animal by in vivo transformation of cells of the animal with a recombinant gene. The transformation may be carried out either in a non-site-specific or systemic manner or a site-specific manner. If the transformation is carried out in a systemic fashion or at sites other than those which confer specificity on the immune system, such as the thymus, then the immune system will be ~odulated to result in the animal being sensitized to the molecule for which the recombinant gene encodes.~ Alternatively, if the transformation i8~
carried out in a site-specific manner and is localized to a site which determines the specificity of the immune system, e.g., the thymus, the immune system will be modulated to result in the animal being tolerized to the molecule encoded by the recombinant gene. ~ c ~ - By the term sensitized, it`is meant that the immune system exhibits a stronger response to the molecule encoded by the DNA after in vivo transformation as compared to before transformation. By the term tolerized, it is meant that the immune system displays a reduced response to-the molecule encoded by the recombinant gene after transformation as compared to before transformation. Thus, one may modulate an immune system to provide either a .. , . ., , . , - - - ~ . , . . ~
W093/~051~ .'' '' PCT/US92~05i42 211237~ -36-resistance or a tolerance to the molecule encoded by the DNA.
Examples of molecules for which it may be desirable to provide a resistance to include: cell surface molecules, such as tumor antigens (carcinoembryonic antigen), protozoan antigens (pneumocystis), viral antigens (HIV
gpl20 and gpl60, ~. influenza antigen, and hepatitis B
surface antigen), Lyme disease antigen, Bacterial antigens, and transplantation antigens (Class I or II), ras or other oncogenes, including erb-A or neu; cytoplasmic proteins, such as the raf oncogene, src oncogene, and abl oncogene;
nuclear proteins, such as ElA oncogene, mutant p53 oncogene, tat, tax, rev, vpu, vpx, hepatitis core antigen, EBNA and viral genes; and secreted proteins, such as ~5 endotoxin, cholera toxin, TNF, and osteoclast activating factor.
. Examples of molecules for which it may be desirable to .: provide;a resisitance to include:/ cell surface molecules, such ~s growth--factor receptors,^~insiulin recéptors, thyroid hormone receptors,-transplantation antigens (class I or II), blood group antigens, and ~DL receptor; cytoplasmic proteins, such as cytochrome P450, galactosyl transferase, dystrophin, neomycin resistance gene, and bacterial heat shock protein; nuclear proteins,~such~as retinoblastoma and-tran~dominant rev; and secreted proteins, such as growth ~-hormone.ifor~;dwarfs,~insulin for diabetics',:'and'adenosine deaminase.~
It'-is.:to be`understood-that theinucleic acid, DNA, RNA,i or derivative *hereof,~in the~`recombinant gene-may be of any suitable origin. That~is-the nucleic acid may be . isolated~from a naturally occurring source or may be of synthetic origin.
W093/~51~ 2112 3 7 6 PCT/US92ios242 The recombinant gene may be introduced in the cells of the animal using any conventional vector. Such vectors include viral vectors, cationic lipids complexed to DNA or RNA (DNA or RNA/liposomes) and DNA or RNA complexes with S polycations, such as DEAE, dextran, and polybrene.
As noted above the recombinant gene can be introduced into cells in a site-specific manner to confer resistance to the molecule encoded by the recombinant gene. Suitable sites include, e.g., endothelial cells or reticuloendothelial cells in the vasculature or any specific tissue or organ. The form of the preparation containing the vector and recombinant gene used in the transformation will depend on the specific tissue to be transformed. Suitable preparations for transforming endothelial cells are described elsewhere in this specification. In addition, preparations suitable for oral or other means of administration (e.g., endoscopic) may be used to provide mucosal resistance. Such preparation could include detergents`,~gelatins, capsules or other delivery 20 ` vehicles to protect against degradation and énhance ~~
delivery to the mucosal surface, in addition to the vector and gene.
Alternatively, the recombinant gene may be introduced in a site`'specific'fashion to a site which determinës'`'the 'spécificity of the immuné system.` The thymus-is~such~a ~sité (sèé: A.~M.'~Posselt-et-'al-, Science, vol. 249, p.-l292 ' '(l990)). Thus,'by introducihg a recombinant~gene sité-specifically into the thymus, the immune system may be modulated to result in~à tolerance to the moleculë èncoded by the gene. In this way, transplant rejectionlmay bë~`' suppressed. The same prepàrations and techniques used to site-specifically transform tumors described above may be used to introduce the recombinant gene into the thymus.
WO 93/~H~ f ! ~ ~' PCT~US92/05242 Specifically, the transformation preparation may be injected directed into the thymus or tumor or into the vascular supply of the thymus or tumor.
The present method may be practiced on any animal, such as chickens or mammals such as cows, horses, cats, dogs, monkeys, lemurs or humans.
When the recombinant gene is introduced using a liposome, it is preferred to first determine in vi-tro the optimal values for the DNA: lipid ratios and the absolute concentrations of DNA and lipid as a function of cell death and transformation efficiency for the particular type of cell to be transformed and to use these values in the in ~ivo transformation. The in vitro determination of these values can be easily carried out using the techniques described in the Experimental Section of this specification.
.
Another aspect of the present invention relates to a kit for the in vivo systemic-introduction of a recombinant gene into cells of an animal. Such a kit would include approximately the optimal amount of a carrier, such as a lipid, and nucleic acid, and/or a means of delivery, e.g., an~endoscope or a syringe. The kit may also contain instructions for the administration of the transforming ~, . , ~ ~ . .. . .
p#paration.~ The carrier and nucleic~acid may-be free~e dried and may be packaged separately or premixed. The kit may also contain a solution to optimally reconstitute the complexes of the carrier and the nuclèic acid, which provide for efficient delivery to cells in vivo.- Such a - solution~may contain one or more ingredients, such as buffers, sugars, salts, proteins, and detergents.
Having generally described the invention, a further WO 93/00051 PCI/US92io5~42 understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.
Ex~erimental section:
A. Analysis of AcLDL receptor expression in normal and ~-galactosidase-transduced porcine endothelial cells.
Endothelial cell cultures derived from the Yucatan minipig, two sublines infected with BAG retrovirus or 3T3 fi~roblast controls were analyzed for expression of AcLDL
receptor using fluorescent labelled AcLDL.
Endothelial cells were derived from external jugular veins uæing the neutral protease dispase (8). Excised vein segments were filled with dispase (50 U/ml in Hanks' balanced salt solution) and incubated at 30C for 20 minute~. Endothelium obtained by this means was maintained in medium 199 (GIBC0, Grand Island, N.Y.) supplemented with fetal calf serum (10%), 50 ~g/ml endothelial cell growth supplement (ECGS) and heparin ~100 ~g/ml). These cells were infected with BAG retrovirus, and selected for resistance-to G-418. Cell cultures were incubated with (l,l'-dioctade~yl3,3,3',3'-tetramethylindocarbacyanine -perchlorate)~(Dil)~AcLDL (Biomedical Technologies,;^~
-- Stoughton,-MA) ~lO ~g/ml) for:4-6 hrs.-at 37C,~followed by three rinses with phosphate- buffered saline containing 0.5% glutaraldehyde. Cells were visualized by phase contrast and fluorescent microscopy. -~ .
B. Method of introduction of endothelial c~lls bycatheterization.
.- W0 93/OOOSI ~ PCI /USg2/05242 A double balloon catheter was used for instillation of endothelial cells. The catheter has a proximal and distal balloon, each 6 mm in length and 5 mm in width, with a 20 mm length between the balloons. The central section of the catheter has a 2 mm pore connected to an instillation port.
Proximal and distal balloon inflation isolates a central space, allowing for instillation of infected cells through the port into a discrete segment of the vessel. For a schematic representation of cell introduction by catheter, lo see Figures 1 and 2.
Animal care was carried out in accordance with "Principles of Laboratory Animal Care" and "Guide for the Care and Use of Laboratory Animals" (NIH publication No.
80-23, Revised 1978). Female Yucatan minipigs (80-100 kg) - 15 were anesthetized with pentobarbital (20 mg/kg), intubated, and mechanically ventilated. These ~ubjects underwent sterile surgical exposure of the iliac and femoral arteries. The distal femoral artery was punctured, and the double-balloon catheter was advanced by guidewire into the iliac ar,tery. The external iliac artery was identified;
the proximal balloon was partially inflated and passed proximally and distally so as to mechanically denude the endothelium. The catheter was then positioned with the central space located in the region of denuded endothelium, and both;,balloon~,were inflated. The denuded egment was irrigated~with~heparinized saline, and,-residual~-adherent cells were removed,by instillation of di~pase (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 sub~equently removed, and antegrade blood flow was re~tored. 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-W O 93/00051 2112 3 7 ~ PC~r/US92/05242 ~41-buffered saline, and another portion was mounted in a paraffin block for cectioning~ The presence of retroviral expressed ~-galactosidase was determined by a standard histochemical technique (19).
C. Analysis of endothelial cells in vitro and n vivo .
~ -Galactosidase activity was documented by histochemical staining in (A) primary endothelial- cells fro~ 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 histochemiaal staining. The enzymatic activity of the E. coli ~-galactosidase protein was u ed to identify infected endothelial cells n vitro and in viyo. The ~-galactosidase transducing Mo-MuLV
vector (2), (BAG) was kindly provided by Dr. Constance Cepko. This vector used the wild type Mo-MuLV LTR as a ~romoter for the ~-galactosidase gene. The simian ~irus 40 ~.(SV-40)- early promoter linked to the Tn5 neomycin- - ---25..re~istance gene provides resi tance to the drug G-418 and ~is inserted downstream of the ~-galac~osidase gene, providing a marker to select for retrovirus-con aining, : ~-galactosidase expressing cells. This defective retro~irus was prepared from fibroblast ~ am cells (3,10), ~0 -and maintained in Dulbecco's modified Eagle's medium (DMEM) and 10% calf serum. Cells were passaged twice weekly following trypsinization. The supernatant, with titers of WO 93/00051 `, `` ` ~ `: PCI'/USg2/05242 211237~ -42-104-105/ml G-418 resistant colonies, was added to endothelial cells at two-thirds confluence and incubated for 12 hours in DMEM with 10% calf serum at 370C in 5% Co2 in the presence of 8 ~g/ml of polybrene. Viral supernatants were removed, and cells maintained in medium 199 with 10% fetal calf serum, ECGS (50 ~g/ml), and endothelial cell conditioned medium (20%) for an additional 24 to 48 hours prior to selection in G-418 (0.7 pg/ml of a 50% racemic mixture). G-4 18 resistant cells were isolated and analyzed for ~-galactosidase expression using-a standard histochemical stain (9). Cells stably expressing the ~-galactosidase enzyme were maintained in continuous culture for use as needed. Frozen aliquo~s were stored in liquid nitrogen.
D. Immunotherapy of Malignancy by In Vivo Gene Transfer.
A retroviral vector which the H-2KS gene was prepared.
CT26 cells.were infected with this vector in.vitro, selected:for G418 resistance, and analyzed by fluorescence activated cell sorting (FACS). Transduced CT26 cells showed a higher mean fluorescence intensity than uninfected CT26 cells or CT26 infected with different retroviral vectors. When 106 CT26 cells which express H-2K~ were --injected.subcutaneously. into BALB/c mice (H-2d) sensitized to this~.antigen,-:~no~stumors.were observed over an.8-week --~period in-icontrast to~the.unmodified CT26.(H-2d)-tumor line which routinely.formed tumors at thiæ dose. The immune response to H-2K~ could therefore provide protection-against.CT26 cells.bearing this antigen. When CT26 H-2K~
and CT26 were co-inoculated, however,.tumor growth was observed, suggesting that H-2K~ conferred senstivity.only to modified cells..
WO93/0~51` 2112 3 7 6 PCT/US92/05242 To determine whether protective effects could be achieved by introduction of H-2K~ in growing CT26 tumors, the recombinant H-2K~ reporter or a ~-galactosidase gene was introduced into tumors either with a DNA/liposome or a retroviral vector. Tumor capsules (0.5-l cm diameter) were exposed surgically and multiple needle injections (2-lO) delivered to the parenchyma. With ~-galactosidase reporter plasmids, recombinant gene expression could be readily detected after intra-tumor injection of DNA/liposome or retroviral vectors.
In mice which received intra-tumor injections of the H-2K8 DNA/liposome comples or H-2Ke retroviral vector, the recombinant DNA was detected by PCR in the tumor and occasioinally in other tissues. When found in the other organs, no evidence of inflammation or ogran toxicity was detected pathologically. An immune response to the recombinant H-2K~ protein was evident in these animals, however. Lymphocytes derived from the H-2K8, but not ~-- galactoæida~e transduced tumors, demonstrated a cytolytic response to H-2K~, whether delivered by retroviral vectors or liposomes. Nore importantly, lymphocytes derived from the H-2K~, but not ~-galactosidase transduced animals, recognized and lysed unmodified CT26 cells, indicating that this stimulation induced immune reactivity against genetically unmodified tumor cells.
To as~ess the protective effect of the immune response -againgt H-2K8, tumor growth in vivo was quantitated. When animals received no prior sensitization to H-2K8, one o~
four tumors transduced with H-2K8 showed attenuation of tumor growth which was not complete. In contrast, no anti-tumor effect was seen in unmodified (n=4) or ~-galactosidase transduced controls (n=4). Because these tumors were large at the time of initial injection and W093/~5l ~ PCTiUS92/05~2 . ' .,' '. ' . ~ ' ''`' .
continued to grow as the primary immune response was generated, an attempt was made to optimize the anti-tumor response by pre-immunization of mice with irradiated CT26 H-2K~ tumor cells, and by earlier and/or more frequent injections of vector. Tumors were transduced on days 12 and 32 by intra-tumor injuection of H-2K~ or ~-galactosidase DNA/liposome vectors. Treatment with the H-2KB liposome complex improved survival and attenuated tumor growth, in contrast to ~-galactosidase transduced tumors where there was no difference in growth rate compared to the uninjected controls. Complete tumor regression was acheived in two mice by increasing the number of injections and by delivery of H-2K~ into tumors at an earlier stage.
This treatment was protective, since control animals showed continued tumor growth and did not survive beyond 35 days.
E. Modulation of the Immune System.
$he response to injection of cationic lipids and pla~mids was determined after in~ection intravenously into 8A$B/c mice (6-12 weeks). In the first experiments, a gene encoding the H-2K~ molecule was introduced by tail vein injection. Two to four weeks later, spleen cells were harvested and analyzed for their ability to mediate a cytolytic T cell response. When these cells were tested using 5~Cr target cells ~(CT26~cells expressing the-H-2X~
gene), significant cytolysis was observed which was not een in animals injected with the cointrol vector, ~-- galactosidase (See Figure 3)~. Up to 25% of target cells were lysed at effector: target ratios of 25:1.
- - Inr-addition to this specific cytolytic T cell -~-~ 3Q response, serologic or antibody responses to genes encoded - by expression vector plasmids have been examined. When a plasmnid encoding the gpl60 molecule of HIV is injected, an .
WO93/~51 PCTiUS92/05242 antibody response is elicited in treated mice. In contrast to control animals injected with cationic lipids containing ~-galactosidase, mice injected with cationic lipids with gp 160 plasmid showed an antibody response to the gpl60 and gpl20 form of this molecule by Westerm blot analysis (See Figure 4). These results demonstrate that systemic administration of cationic lipid/DNA complexes can be used successfully to induce cell-mediated and antibody-mediated immunity against foreign pathogens.
10F. Determination of Optimal Transfection Conditions.
(1) Plasmid Construction A plasmid containing the E. coli lacZ gene under the control of the Rous Sarcoma Virus LTS (RSV-~-gal) (Norton and Coffin, Mol. Cell. Biol., 5(2), 281-290, 1985) was used for transfection of porcine primary endothelial and HeLa cells. In addition, a plasmid containing the lacZ gene under the control of preproendothelin-l 5'-flanking DNA (-1410 to +83) (Wilson et al., Mol. Cell. Biol., 10~9), 4854-4862, 1990) was used for transfection of endothelial cells.
For in vivo toxicity analysis, the RSV-~-gal plasmid, and a plasmid derived from the PLJ vector containing the cDNA
encoding an H-2K8 mouse MHC class I gene were used.
~ (2)~Seli--Culture, Transfection Analysis,-and Toxicity - in Vitro ~
., ., . ~ .
25Primary endothèlial cells, derived from the Yucatan minipig (YPE-cells), were incubated with medium 199 (M199) cupplemented with 10% FBS, 2~M l-glutamine, 50 U/ml-- penicillin, and 5 ~g/ml streptomycin. HeLa cells were maintained in Dulbeccos Modified Eagles Medium (DNEM) 30supplemented with 5% FBS, 2mM 1-glutamine, 50 U/ml ~WO~g3/, ~ 51 !~ ', PCT/US92/05242 211237 ~ -46- ' penicillin and 5 ~g/ml streptomycin. The DNA liposome mixture was prepared with lipid concentrations of DOPE/DC-Chol between 2.5 and 25 ~M added to 0.2 ml of serum-free media or Ringer's lactate solution in polystyrene tubes.
After mixinq gently, the solution was allowed to stand at room temperature for 15-20 minutes. For transfection analysis, cells were grown in 60 mm tissue culture dishes at 75% confluency or greater. Cells were washed twice with serum-free media or lactated Ringers solution and then placed in 0.5 mls of the same media. The DNA liposome solution (0.2 ml) was then added slowly to the cells, with gentle mixing, with a final volume of 0.7 ml. This resulted in DNA concentrations between .7 and 7 ~g/ml (13-130 ~M), and lipid concentrations of 7-70 ~M. Transfection was allowed to proceed for 1-5 hours, after which the cells were placed ir. media supplemented as decribed above. At 24-48 hours after transfection the enzymatic activity of the E. coli ~-galactosidase protein was used to identify transfected cells by staining with the X-gal chromagen.
Toxicity in vitro was assessed by cytopathic effect or .
~rypan blue exclusion. , ~ ~
.
(3) Animal Studies For intravenous injections, the DNA/liposomes were prepared as described for the in vitro transfection ~tudies 25~_~ in ~0,.2,ml of serum-free~Ml99 or,lactated-Ringers solution.
After 15-20 min of incubation, the mixture was diluted,to 0.7 ml and 0.1 to 0.2 ml of this dilution was then injected immediately into the tail vein of adult, female BALB/c , mice. Blood was collected-,before injection and 9-11 days following injection,,and serum chemistries were examined.
At -2-3 weeks following injection, the liver, kidney, lung, heart, and brain were extracted for histologic and PCR DNA
amplific-tion analysis as described previously. Intratumor t WO93/~51 . 2 1 1 2 3 7 6 PCT/US92/052i2 injection of CT26 cells (Fearon et al., Cell, 60, 397-403, 1990) and analysis were also performed according to the previous protocols.
After 15-20 min of incubation, the mixture was diluted,to 0.7 ml and 0.1 to 0.2 ml of this dilution was then injected immediately into the tail vein of adult, female BALB/c , mice. Blood was collected-,before injection and 9-11 days following injection,,and serum chemistries were examined.
At -2-3 weeks following injection, the liver, kidney, lung, heart, and brain were extracted for histologic and PCR DNA
amplific-tion analysis as described previously. Intratumor t WO93/~51 . 2 1 1 2 3 7 6 PCT/US92/052i2 injection of CT26 cells (Fearon et al., Cell, 60, 397-403, 1990) and analysis were also performed according to the previous protocols.
(4) Results The optimal conditions for transfection and toxicity of DNA/liposomes were initially determined in vitro. To obtain maximal transfection without toxicity in vitro, the ratio of DNA to cationic lipid, the absolute concentration of DNA or lipids, and the conditions for mixture of DNA and cationic lipids.were studied. The cationic lipid preparation was a formulation of two compounds, which include dioleoyl phosphatidylethanolamine (DOPE) and cholesten-3-~-ol 3-urethanyl-N', N' dimethylethylene diamine (DC-chol). The transfection efficiencies of this reagent were equal to or greater than those of Lipofectin0 (BRL) in several cell lines in vitro. Endothelial cells, which are typically difficult to transfect, and HeLa cells, which can be transfected easily using a variety of-techniques, were examined by transfection in vitro.
.
To determine the.optimal conditions for transfection of endothelial cells, the lipid was initially used at different concentrations while the DNA concentration was held constant.;~Maximal transfection efficiency was seen using 0.-7~ gJml~DNA~(13 nM)-.and 21 ~N of DOPE/DCOChol`--lipid, with:a ~harp decline in the number of transfected .cells with higher or lower lipid concentrations. Next, the DNA.concentration was altered as the lipid concentration remained constant. This analysis revealed a similar - sensitivity to DNA concentration, with the number of -transfected cells decreasing significantly with increments of DNA concentration as low as 0.4 ~g/ml. These results indicate that the ratio of DNA to lipid is important for WO93/~S1 ~ PCT/US92/05242 maximum transfection efficiency, and that the absolute concentration of each component is also important in determining the efficiency of transfection. An incrase in DNA and lipid concentration beyond the optimal concentration of 0.7 ~g/ml DNA (13 nM) and 21 ~M of DOPE/DC-Chol reduced the number of viable cells and did not increase the transfection efficiency of the remaining viable cells. Lipid concentrations greater than 35 ~M
reduced the number of viable cells by 50% compared to the untransfected control, whereas the optimal concentration of 0.7 ~g/ml DNA (13 nM) ande 21 ~M of lipid had no effect on cell viability after 5 hours of incubation.
To compare the optimal concentrations of transfection in a different cell type, transfections were performed on HeLa cells. In this case, a slightly different optimal ratio of DNA and lipid were observed. Peak transfection efficiencies were obtained at the same lipid concentration as endothelial cells (2l ~g/ml) but varied less with small differences in DNA concentrations. DNA concentrations of 1.4-4.2 ~g/ml were equally effective. Again, when the ratio of DNA to lipid was maintained but the concentration of each was decreased three-fold, very few cells were transfected, illustrating that both the ratio of DNA to lipid and the absolute concentration of each component are important in maximizing the number of transfected cells.
If HeLa cells~were transfected at->80% confluence or -greater, there was no toxicity using up to 35 ~M of lipid.
When cells were transfected at a lower saturation density, however, cell viability was reduced dramatically with as little as 7 ~M of lipid~compared to the untransfected control cells. These results demonstrate that the optimal conditions for transfection and toxicity may differ somewhat depending on the cell line.
2i123~6 WO 93/OOO51 ``i~ PCI /US92/05242 Another variable in the preparation of liposomes was the composition of the solution used to generate complexes of the cationic lipids with DNA. Among several media solutions analyzed, no substantial difference was noted in transfection efficiency or toxicity with M199, McCoys, optiMEM~ or RPNI media. A significant improvement in transfection efficiency was observed, however, using ;~
standard Ringers lactate. The number of transfected cells increased more than 3-fold compared to the serum-free medium, although prolonged incubation ( > 2 hours~ resulted in a loss of cell viability in some cell types.
` ' ., ~ . - , ,' : `
..
' .
, ` :
w093/~w~ . PCT~US92/05~2 211237~ -s2-U.S. patent application serial nos. 07/724,509, filedon June 28, 1991, and 07/331,366, filed on March 31, 1989, are incorporated herein by reference.
Obviously, numerous modifications and variations of the present invention are possible in light of the above-teachings. It is therefore to be understood that. within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.
.
To determine the.optimal conditions for transfection of endothelial cells, the lipid was initially used at different concentrations while the DNA concentration was held constant.;~Maximal transfection efficiency was seen using 0.-7~ gJml~DNA~(13 nM)-.and 21 ~N of DOPE/DCOChol`--lipid, with:a ~harp decline in the number of transfected .cells with higher or lower lipid concentrations. Next, the DNA.concentration was altered as the lipid concentration remained constant. This analysis revealed a similar - sensitivity to DNA concentration, with the number of -transfected cells decreasing significantly with increments of DNA concentration as low as 0.4 ~g/ml. These results indicate that the ratio of DNA to lipid is important for WO93/~S1 ~ PCT/US92/05242 maximum transfection efficiency, and that the absolute concentration of each component is also important in determining the efficiency of transfection. An incrase in DNA and lipid concentration beyond the optimal concentration of 0.7 ~g/ml DNA (13 nM) and 21 ~M of DOPE/DC-Chol reduced the number of viable cells and did not increase the transfection efficiency of the remaining viable cells. Lipid concentrations greater than 35 ~M
reduced the number of viable cells by 50% compared to the untransfected control, whereas the optimal concentration of 0.7 ~g/ml DNA (13 nM) ande 21 ~M of lipid had no effect on cell viability after 5 hours of incubation.
To compare the optimal concentrations of transfection in a different cell type, transfections were performed on HeLa cells. In this case, a slightly different optimal ratio of DNA and lipid were observed. Peak transfection efficiencies were obtained at the same lipid concentration as endothelial cells (2l ~g/ml) but varied less with small differences in DNA concentrations. DNA concentrations of 1.4-4.2 ~g/ml were equally effective. Again, when the ratio of DNA to lipid was maintained but the concentration of each was decreased three-fold, very few cells were transfected, illustrating that both the ratio of DNA to lipid and the absolute concentration of each component are important in maximizing the number of transfected cells.
If HeLa cells~were transfected at->80% confluence or -greater, there was no toxicity using up to 35 ~M of lipid.
When cells were transfected at a lower saturation density, however, cell viability was reduced dramatically with as little as 7 ~M of lipid~compared to the untransfected control cells. These results demonstrate that the optimal conditions for transfection and toxicity may differ somewhat depending on the cell line.
2i123~6 WO 93/OOO51 ``i~ PCI /US92/05242 Another variable in the preparation of liposomes was the composition of the solution used to generate complexes of the cationic lipids with DNA. Among several media solutions analyzed, no substantial difference was noted in transfection efficiency or toxicity with M199, McCoys, optiMEM~ or RPNI media. A significant improvement in transfection efficiency was observed, however, using ;~
standard Ringers lactate. The number of transfected cells increased more than 3-fold compared to the serum-free medium, although prolonged incubation ( > 2 hours~ resulted in a loss of cell viability in some cell types.
` ' ., ~ . - , ,' : `
..
' .
, ` :
w093/~w~ . PCT~US92/05~2 211237~ -s2-U.S. patent application serial nos. 07/724,509, filedon June 28, 1991, and 07/331,366, filed on March 31, 1989, are incorporated herein by reference.
Obviously, numerous modifications and variations of the present invention are possible in light of the above-teachings. It is therefore to be understood that. within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.
Claims (40)
1. A kit for treating a disease in a patient in need thereof, comprising a catheter means and a solution which contains an enzyme or mild detergent, wherein:
(i) said catheter means is adapted for insertion into a blood vessel and comprises a main catheter body having means including a balloon element adapted to be inserted into said vessel and expansible against the walls of said vessel so as to hold said main catheter body in place in said vessel, and means carried by said main catheter body for delivering a solution into said blood vessel; and (ii) said solution is a physiologically acceptable solution.
(i) said catheter means is adapted for insertion into a blood vessel and comprises a main catheter body having means including a balloon element adapted to be inserted into said vessel and expansible against the walls of said vessel so as to hold said main catheter body in place in said vessel, and means carried by said main catheter body for delivering a solution into said blood vessel; and (ii) said solution is a physiologically acceptable solution.
2. The kit of Claim 1, wherein said solution contains, as said enzyme, at least one member selected from the group consisting of dispase, trypsin, collagenase, papain, pepsin, chymotrypsin, and lipases.
3. The kit of Claim 1, wherein said solution contains at least one member selected from the group consisting of NP-40, Triton X100, deoxycholate, and SDS.
4. The kit of Claim 1, wherein said main catheter body comprises means including two spaced balloon elements, adapted to be inserted in a blood vessel and both being expansible against the walls of the blood vessel, for providing a chamber in said blood vessel and so as to hold said main catheter body in place, and whereas said means for delivering a solution into said chamber is situated in between said balloon elements.
5. The kit of Claim 1, wherein said means for delivering said solution into said blood vessel comprises a plurality of pore means.
6. A kit for treating a disease in a patient in need thereof, comprising a catheter means and a physiologically acceptable solution, wherein:
(i) said catheter means is adapted for insertion into a blood vessel and comprises a main catheter body having means including a balloon element, adapted to be inserted in said blood vessel and being expansible against the walls of said vessel so as to hold said main catheter body in place, and means carried by said main catheter body for delivering a solution into said blood vessel;
(ii) said physiologically acceptable solution comprises at least one member selected from the group consisting of heparin, poly-L-lysine, polybrene, dextransulfate, a polycationic material, and bivalent antibodies.
(i) said catheter means is adapted for insertion into a blood vessel and comprises a main catheter body having means including a balloon element, adapted to be inserted in said blood vessel and being expansible against the walls of said vessel so as to hold said main catheter body in place, and means carried by said main catheter body for delivering a solution into said blood vessel;
(ii) said physiologically acceptable solution comprises at least one member selected from the group consisting of heparin, poly-L-lysine, polybrene, dextransulfate, a polycationic material, and bivalent antibodies.
7. The kit of Claim 6, wherein said physiologically acceptable solution further comprises DNA.
8. The kit of Claim 6, wherein said physiologically acceptable solution further comprises a growth factor.
9. A method for treating a disease in a patient in need thereof, comprising causing a cell attached onto the walls of a vessel or in an organ or tissue in said patient to express an exogenous therapeutic agent protein, wherein said protein treats said disease.
10. The method of Claim 9, wherein said disease is an ischemic disease, a vasomotor disease, diabetes, a malignancy, AIDS or a genetic disease.
11. The method of Claim 9, wherein said disease is a systemic disease.
12. The method of Claim 9, wherein said exogenous therapeutic agent protein is one member selected from the group consisting of tPA and modifications thereof, urokinase, streptokinase, acidic fibroblast growth factor, basic fibroblast growth factor, tumor necrosis factor .alpha., tumor necrosis factor .beta., transforming growth factor .alpha., transforming growth factor .beta., atrial natriuretic factor, platelet-derived growth factor, endothelian, insulin, diphtheria toxin, pertussis toxin, cholera toxin, soluble CD4 and derivatives thereof, and growth hormone.
13. The method of Claim 9, wherein said cell is selected from the group consisting of endothelial cells, vascular smooth muscle cells, fibroblasts, connective tissue cells, macrophages, monocytes, and parenchymal cells.
14. A method for treating a disease, comprising site-specifically instilling cells.
15. The method of Claim 14, wherein said cells are transformed cells.
16. The method of Claim 14, wherein said cells are normal cells.
17. A method for treating a disease in a patient, comprising site-specifically transforming cells in vivo.
18. The kit of Claim 1, further comprising means for causing a cell attached onto the walls of a vessel or in an organ or tissue in said patient to express an exogenous therapeutic agent proteins, wherein said means comprises a formulation for the transfer and uptake of RNA or DNA into said cell attached onto the walls of a vessel or in an organ or tissue in said patient.
19. The kit of Claim 18, wherein said formulation comprises a retrovirus, a plasmid, a liposomal formulation, or a plasmid complex with a polycationic substance.
20. The kit of Claim 18, wherein said formulation is a liposomal formulation.
21. The method of Claim 17, wherein said disease is a malignancy associated with a tumor, and wherein said cells are transformed with a DNA sequence or an RNA sequence encoding an intracellular, secreted, or cell surface molecule which is (1) exogenous to said patient and which is (2a) immunogenic to said patient, (2b) induces rejection, regression, or both of said tumor, or (2c) toxic to the cells of said tumor.
22. The method of Claim 21, wherein said molecule is immunogenic to said patient.
23. The method of Claim 21, wherein said molecule induces rejection, regression, or both, of said tumor.
24. The method of Claim 23, wherein said molecule is a growth stimulant or inhibitor.
25. The method of Claim 23, wherein said molecule is an inducer of cellular differentiation.
26. The method of Claim 21, wherein said molecule is toxic to the cells of said tumor.
27. The method of Claim 21, wherein said DNA or RNA
sequence is obtained from a species which is the same as said patient.
sequence is obtained from a species which is the same as said patient.
28. The method of Claim 21, wherein said DNA or RNA
sequence is obtained from a species different than the species of said patient.
sequence is obtained from a species different than the species of said patient.
29. The method of Claim 21, wherein said DNA or RNA
sequence is administered locally to said patient in a viral vector.
sequence is administered locally to said patient in a viral vector.
30. The method of Claim 21, wherein said DNA or RNA
sequence is administered locally to said patient as a DNA
or RNA/liposome complex.
sequence is administered locally to said patient as a DNA
or RNA/liposome complex.
31. The method of Claim 21, wherein said DNA or RNA
sequence is administered locally to said patient via a cell line.
sequence is administered locally to said patient via a cell line.
32. The method of Claim 21, wherein said DNA or RNA
sequence is administered to said patient systemically as a chemical formulation containing said DNA or RNA sequence coupled to a carrier molecule which facilitates delivery of said sequence to target cells in said patient.
sequence is administered to said patient systemically as a chemical formulation containing said DNA or RNA sequence coupled to a carrier molecule which facilitates delivery of said sequence to target cells in said patient.
33. The method of Claim 21, wherein said DNA or RNA
sequence is one member selected from the group consisting of Class I histocompatibility genes, Class II
histocompatibility genes, bacterial genes encoding peptides having immunostimulant properties, genes encoding viral glycoproteins having immunostimulant properties, genes encoding minor histocompatibility antigens, genes encoding lysozymes or bovine serum albumin, and oncogenes.
sequence is one member selected from the group consisting of Class I histocompatibility genes, Class II
histocompatibility genes, bacterial genes encoding peptides having immunostimulant properties, genes encoding viral glycoproteins having immunostimulant properties, genes encoding minor histocompatibility antigens, genes encoding lysozymes or bovine serum albumin, and oncogenes.
34. The method of Claim 21, wherein said DNA or RNA
sequence is one member selected from the group consisting of DNA or RNA sequences encoding interleukin-1, 2, 3, 4, 6 or 8, TNF-.alpha. or .beta., TGF-.beta. (1, 2 or 3), soluble growth factors, recombinant antibodies to growth factors or receptors or analogues thereof, interferon .alpha., .beta. or .gamma., and adhesion molecules.
sequence is one member selected from the group consisting of DNA or RNA sequences encoding interleukin-1, 2, 3, 4, 6 or 8, TNF-.alpha. or .beta., TGF-.beta. (1, 2 or 3), soluble growth factors, recombinant antibodies to growth factors or receptors or analogues thereof, interferon .alpha., .beta. or .gamma., and adhesion molecules.
35. The method of Claim 21, wherein said DNA or RNA
sequence is one member selected from the group consisting of DNA and RNA sequences encoding thymidine kinase, diphtheria toxin, pertussis toxin or drug-sensitive proteins.
sequence is one member selected from the group consisting of DNA and RNA sequences encoding thymidine kinase, diphtheria toxin, pertussis toxin or drug-sensitive proteins.
36. A method for modulating the immune system of an animal by introducing in vivo a recombinant gene into cells of said animal, using a nucleic acid and a delivery vehicle or viral vector.
37. The method of Claim 36, wherein said introducing is carried out site specifically and introduces said recombinant gene into said animal's thymus.
38. The method of Claim 36, wherein said introducing results in a stimulation of said animal's immune system against the molecule encoded by said gene.
39. The method of Claim 36, wherein said introducing is carried out by injection a composition comprising said recombinant gene directly into a tissue containing said cells.
40. The method of Claim 36, wherein said introducing is carried out to mucosal surfaces by oral administration or intralumenal administration of a composition comprising said recombinant gene.
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US72450991A | 1991-06-28 | 1991-06-28 | |
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US724,509 | 1991-07-26 | ||
US07/741,244 US5328470A (en) | 1989-03-31 | 1991-07-26 | Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor |
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CA2112376A1 true CA2112376A1 (en) | 1993-01-07 |
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CA002112376A Abandoned CA2112376A1 (en) | 1991-06-28 | 1992-06-26 | Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor |
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US (1) | US5328470A (en) |
EP (4) | EP1013287A3 (en) |
JP (3) | JP3712409B2 (en) |
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DK (1) | DK0591385T3 (en) |
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WO (1) | WO1993000051A1 (en) |
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- 1991-07-26 US US07/741,244 patent/US5328470A/en not_active Expired - Lifetime
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1992
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- 1992-06-26 EP EP02075756A patent/EP1213032B1/en not_active Expired - Lifetime
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2000
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EP0591385A4 (en) | 1996-11-27 |
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DE1213032T1 (en) | 2003-02-06 |
EP1013287A2 (en) | 2000-06-28 |
JP2002239010A (en) | 2002-08-27 |
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