U.S. patent application number 11/788728 was filed with the patent office on 2007-12-13 for treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor.
This patent application is currently assigned to The Regents of the University of Michigan. Invention is credited to Elizabeth G. Nabel, Gary J. Nabel.
Application Number | 20070287682 11/788728 |
Document ID | / |
Family ID | 26987727 |
Filed Date | 2007-12-13 |
United States Patent
Application |
20070287682 |
Kind Code |
A1 |
Nabel; Elizabeth G. ; et
al. |
December 13, 2007 |
Treatment of diseases by site-specific instillation of cells or
site-specific transformation of cells and kits therefor
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.
Inventors: |
Nabel; Elizabeth G.; (Ann
Arbor, MI) ; Nabel; Gary J.; (Ann Arbor, MI) |
Correspondence
Address: |
BRINKS HOFER GILSON & LIONE
P.O. BOX 10395
CHICAGO
IL
60610
US
|
Assignee: |
The Regents of the University of
Michigan
|
Family ID: |
26987727 |
Appl. No.: |
11/788728 |
Filed: |
April 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09458610 |
Dec 10, 1999 |
7226589 |
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11788728 |
Apr 20, 2007 |
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08889399 |
Jul 8, 1997 |
6297219 |
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09458610 |
Dec 10, 1999 |
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08376522 |
Jan 23, 1995 |
5698531 |
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08889399 |
Jul 8, 1997 |
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07724509 |
Jun 28, 1991 |
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08376522 |
Jan 23, 1995 |
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07331366 |
Mar 31, 1989 |
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07724509 |
Jun 28, 1991 |
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Current U.S.
Class: |
514/44A ; 536/21;
536/23.2 |
Current CPC
Class: |
A61K 47/6957 20170801;
A61B 17/22 20130101; A61K 31/70 20130101; A61L 29/16 20130101; A61L
2300/252 20130101; A61B 2017/22038 20130101; A61B 2017/22054
20130101; A61L 2300/258 20130101; A61K 39/00 20130101; A61B
2017/22082 20130101; A61L 2300/626 20130101; A61B 2017/22084
20130101; A61K 9/0024 20130101; A61L 2300/64 20130101; A61K 48/00
20130101 |
Class at
Publication: |
514/044 ;
536/021; 536/023.2 |
International
Class: |
A61K 31/70 20060101
A61K031/70; C07H 21/00 20060101 C07H021/00; C08B 37/02 20060101
C08B037/02 |
Claims
1-17. (canceled)
18. A kit for treating a disease in a patient, comprising: a
catheter comprising a body and balloon element adapted to be
inserted into a blood vessel of said patient and being expansible
against the walls of said vessel so as to hold said catheter body
in place; and a physiologically acceptable solution comprising
cells, wherein said catheter body includes means for delivering
said composition into said blood vessel.
19. The kit of claim 18, wherein said catheter body comprises two
spaced balloon elements, adapted to be inserted in a blood vessel
and both being expansible against the wall of the blood vessel, for
providing a chamber in said blood vessel and so as to hold said
body in place, and where the means for delivering the composition
into the blood vessel is situated in between the balloon
elements.
20. The kit of claim 18, wherein the cells are normal
(untransformed) cells.
21. The kit of claim 18, wherein the cells are transformed
cells.
22. The kit of claim 18, wherein the means for delivering the
composition into the blood vessel comprises a plurality of
pores.
23. The kit of claim 18, wherein the number of pores is 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, or 12.
24. The kit of claim 18, wherein the cells are endothelial cells,
smooth muscle cells, fibroblasts, monocytes, macrophages, or
parenchymal cells.
25. The kit of claim 18, wherein the cells produce a protein which
has a therapeutic or diagnostic effect in the patient.
26. The kit of claim 18, wherein the cells produce an angiogenic
factor.
27. The kit of claim 18, wherein the cells are glucose-responsive
insulin-secreting cells.
28. The kit of claim 18, wherein the cells produce a toxin.
29. The kit of claim 18, wherein the composition is stored in
liquid nitrogen.
30. The kit of claim 1, wherein the catheter is a catheter as
described in U.S. Pat. No. 4,636,195.
31. The kit of claim 18, wherein the kit further comprises a
guidewire.
32. The kit of claim 18, wherein the composition further comprises
heparin, poly-L-lysine, polybrene, dextran sulfate, a polycationic
material, or a bivalent antibody.
33. The kit of claim 21, wherein the cell comprises a nucleic acid
sequence encoding tPA, urokinase, streptokinase, acidic fibroblast
growth factor, basic fibroblast growth factor, tumor necrosis
factor .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, a growth hormone, a marker protein, or derivatives
thereof.
34. A kit for treating a disease in a patient in need thereof,
comprising: a) a medical device for insertion into a blood vessel;
and b) an antisense agent which is complementary to a DNA or mRNA
encoded by a gene in said patient.
35. The kit of claim 34, wherein the antisense agent is
complementary to the 5' untranslated region of the mRNA.
36. The kit of claim 34, wherein the antisense agent is
complementary to the coding region of the mRNA.
37. The kit of claim 34, wherein the antisense agent is
complementary to the 3' untranslated region of the mRNA.
38. The kit of claim 34, wherein the antisense agent is a synthetic
oligonucleotide.
39. The kit of claim 34, wherein the antisense agent is antisense
DNA.
40. The kit of claim 34, wherein the medical device is a
catheter.
41. The kit of claim 34, wherein the medical device is a syringe
and needle.
42. The kit of claim 34, wherein the gene encodes an angiogenic
factor.
43. The kit of claim 34, wherein the antisense agent is in
solution.
44. The kit of claim 34, wherein the antisense agent is a plasmid
which expresses the revise complement of the gene.
45. The kit of claim 34, wherein the gene encodes a protein that
induces angiogenesis.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to the treatment of diseases
by the site-specific instillation or transformation of cells and
kits therefor.
[0003] 2. Discussion of the Background
[0004] The effective treatment of many systemic and inherited
diseases remains a major challenge to modern medicine. The ability
to deliver therapeutic agents to specific sites in vivo would be an
asset in the treatment of, e.g., localized diseases. In addition
the ability to cause a therapeutic agent to perfuse through the
circulatory system would be effective for the treatment of, e.g.,
systemic diseases.
[0005] For example, it would be desirable to administer in a steady
fashion an antitumor agent or toxin in close proximity to a tumor.
Similarly, it would be desirable to cause a perfusion of, e.g.,
insulin in the blood of a person suffering from diabetes. However,
for many therapeutic agents there is no satisfactory method of
either site-specific or systemic administration.
[0006] 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.
[0007] In particular, the pathogenesis of atherosclerosis is
characterized by three fundamental biological processes. These are:
1) proliferation of intimal smooth muscle cells together with
accumulated macrophages; 2) formation by the proliferated smooth
muscle cells of large amounts of connective tissue matrix; and 3)
accumulation of lipid, principally in the form of cholesterol
esters and free cholesterol, within cells as well as in
surrounding-connective tissue.
[0008] Endothelial cell injury is an initiating event and is
manifested by interference with the permeability barrier of the
endothelium, alterations in the non-thrombogenic properties of the
endothelial surface, and promotion of procoagulant properties of
the endothelium. Monocytes migrate between endothelial cells,
become active as scavenger cells, and differentiate into
macrophages.
[0009] Macrophages then synthesize and secrete growth factors
including platelet derived growth factor (PDGF), fibroblast growth
factor (FGF), epidermal growth factor (EGF), and transforming
growth factor alpha (TGF-.alpha.). These growth factors are
extremely potent in stimulating the migration and proliferation of
fibroblasts and smooth muscle cells in the atherosclerotic plaque.
In addition, platelets may interact with the injured endothelial
cell and the activated macrophage to potentiate the elaboration of
growth factors and thrombus formation.
[0010] 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.
[0011] Empiric therapy with antiplatelet drugs has not prevented
this problem, which occurs in one-third of patients undergoing
PTCA. A solution to restenosis is to prevent platelet aggregation,
thrombus formation, and smooth muscle cell proliferation.
[0012] Thrombus formation is also a critical cellular event in the
transition from stable to unstable coronary syndromes. The
pathogenesis most likely involves acute endothelial cell injury and
or plaque rupture, promoting dysjunction of endothelial cell
attachment, and leading to the exposure of underlying macrophage
foam cells. This permits the opportunity for circulating platelets
to adhere, aggregate, and form thrombi.
[0013] The intravenous administration of thrombolytic agents, such
as tissue plasminogen activator (tPA) results in lysis of thrombus
in approximately 70% of patients experiencing an acute myocardial
infarction. Nonetheless, approximately 30% of patients fail to
reperfuse, and of those patients who undergo initial reperfusion of
the infarct related artery, approximately 25% experience recurrent
thrombosis within 24 hours. Therefore, an effective therapy for
rethrombosis remains a major therapeutic challenge facing the
medical community today.
[0014] As noted above, an effective therapy for rethrombosis is by
far not the only major therapeutic challenge existing today. Others
include the treatment of other ischemic conditions, including
unstable angina, myocardial infarction or chronic tissue ischemia;
or even the treatment of systemic and inherited diseases or
cancers. These might be treated by the effective administration of
anticoagulants, vasodilatory, angiogenic, growth factors or growth
inhibitors to a patient. Thus, there remains a strongly felt need
for an effective therapy in all of these clinical settings.
SUMMARY OF THE INVENTION
[0015] Accordingly, one object of the present invention is to
provide a novel method for the site-specific administration of a
therapeutic agent.
[0016] 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.
[0017] It is another object of the present invention to provide a
method for causing the expression of an exogenous gene in a
patient.
[0018] It is another object of the present invention to provide a
method for causing the expression of a defective endogenous gene in
a patient.
[0019] It is another object of the present invention to provide a
method for suppressing the expression of an endogenous gene in a
patient.
[0020] It is another object of the present invention to provide a
method for site-specifically replacing damaged cells in a
patient.
[0021] It is another object of the present invention to provide a
method for the treatment of a disease by causing either the
site-specific administration of a therapeutic agent or the
perfusion of a therapeutic agent in the bloodstream of a
patient.
[0022] It is another object of the present invention to provide a
method for the treatment of a disease by causing either the
expression of an exogenous gene, the expression of a defective
endogenous gene, or the suppression of the expression of an
endogenous gene in a patient.
[0023] 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.
[0024] It is another object of the present invention to provide a
kit for site-specifically instilling normal or transformed cells in
a patient.
[0025] It is another object of the present invention to provide a
kit for site-specifically transforming cells in vivo.
[0026] These and other objects of this invention which will become
apparent during the course of the following detailed description of
the invention have been discovered by the inventors to be achieved
by (a) a method which comprises either (i) site-specific
instillation or either normal (untransformed) or transformed cells
in a patient or (ii) site-specific transformation of cells in a
patient and (b) a kit which contains a catheter for (i)
site-specific instillation of either normal or transformed cells or
(ii) site-specific transformation of cells.
[0027] Site-specific instillation of normal cells can be used to
replace damaged cells, while instillation of transformed cells can
be used to cause the expression of either a defective endogenous
gene or an exogenous gene or the suppression of an endogenous gene
product. Instillation of cells in the walls of the patient's blood
vessels can be used to cause the steady perfusion of a therapeutic
agent in the blood stream.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] 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:
[0029] FIGS. 1 and 2 illustrate the use of a catheter in accordance
with the invention to surgically or percutaneously implant cells in
a blood vessel or to transform in vivo cells present on the wall of
a patient's blood vessel.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0030] Thus, in one embodiment, the present invention is used to
treat diseases, such as inherited diseases, systemic diseases,
diseases of the cardiovascular system, diseases of particular
organs, or tumors by instilling normal or transformed cells or by
transforming cells.
[0031] The cells which may be instilled in the present method
include endothelium, smooth muscle, fibroblasts, monocytes,
macrophages, and parenchymal cells. These cells may produce
proteins which may have a therapeutic or diagnostic effect and
which may be naturally occurring or arise from recombinant genetic
material.
[0032] Referring now to the figures, wherein like reference
numerals designate identical or corresponding parts throughout the
several views, and more particularly to FIG. 1 thereof, this figure
illustrates the practice of the present invention with a catheter
having a design as disclosed in U.S. Pat. No. 4,636,195, which is
hereby incorporated by reference. This catheter may be used to
provide normal or genetically altered cells on the walls of a
vessel or to introduce vectors for the local transformation of
cells. In the figure, 5 is the wall of the blood vessel. The figure
shows the catheter body 4 held in place by the inflation of
inflatable balloon means 1 and 2. The section of the catheter body
4 situated between balloon means 1 and 2 is equipped with
instillation port means 3. The catheter may be further equipped
with a guidewire means 6. FIG. 2 illustrates the use of a similar
catheter, distinguished from the catheter illustrated in FIG. 1 by
the fact that it is equipped with only a single inflatable balloon
means 2 and a plurality of instillation port means 3. This catheter
may contain up to twelve individual instillation port means 3, with
five being illustrated.
[0033] In the case of delivery to an organ, the catheter may be
introduced into the major artery supplying the tissue. Cells
containing recombinant genes or vectors can be introduced through a
central instillation port after temporary occlusion of the arterial
circulation. In this way, cells or vector DNA may be delivered to a
large amount of parenchymal tissue distributed through the
capillary circulation. Recombinant genes can also be introduced
into the vasculature using the double balloon catheter technique in
the arterial circulation proximal to the target organ. In this way,
the recombinant genes may be secreted directly into the circulation
which perfuse the involved tissue or may be synthesized directly
within the organ.
[0034] In one embodiment, the therapeutic agents are secreted by
vascular cells supplying specific organs affected by the disease.
For example, ischemic cardiomyopathy may be treated by introducing
angiogenic factors into the coronary circulation. This approach may
also be used for peripheral vascular or cerebrovascular diseases
where angiogenic factors may improve circulation to the brain or
other tissues. Diabetes mellitus may be treated by introduction of
glucose-responsive insulin secreting cells in the portal
circulation where the liver normally sees a higher insulin
concentration than other tissues.
[0035] In addition to providing local concentrations of therapeutic
agents, the present method may also be used for delivery of
recombinant genes to parenchymal tissues, because high
concentrations of viral vector and other vectors can be delivered
to a specific circulation. Using this approach, deficiencies of
organ-specific proteins may also be treated. For example, in the
liver, .alpha.-antitrypsin inhibitor deficiency or
hyperchloresterolemia may be treated by introduction of
.alpha.-antitrypsin or the LDL receptor gene. In addition, this
approach may be used for the treatment of malignancy. Secretion of
specific recombinant toxin genes into the circulation of inoperable
tumors provides a therapeutic effect. Examples include acoustic
neuromas or certain hemangiomas which are otherwise
unresectable.
[0036] In clinical settings, these therapeutic recombinant genes
are introduced in cells supplying the circulation of the involved
organ. Although the arterial and capillary circulations are the
preferred locations for introduction of these cells, venous systems
are also suitable.
[0037] 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.
[0038] The invention thus, in one of its aspects, relies on genetic
alteration of endothelial and other vascular cells or somatic cell
gene therapy, for transmitting therapeutic agents (i.e., proteins,
growth factors) to the localized region of vessel injury. To
successfully use gene transplantation in the cells, four
requirements must be fulfilled. First, the gene which is to be
implanted into the cell must be identified and isolated. Second,
the gene to be expressed must be cloned and available for genetic
manipulation. Third, the gene must be introduced into the cell in a
form that will be expressed or functional. Fourth, the genetically
altered cells must be situated in the vascular region where it is
needed.
[0039] In accordance with the present invention the altered cells
or appropriate vector may be surgically, percutaneously, or
intravenously introduced and attached to a section of a patient's
vessel wall. Alternatively, some of the cells existing on the
patient's vessel wall are transformed with the desired genetic
material or by directly applying the vector. In some instances,
vascular cells which are not genetically modified can be introduced
by these methods to replace cells lost or damaged on the vessel
surface.
[0040] Any blood vessel may be treated in accordance with this
invention; that is, arteries, veins, and capillaries. These blood
vessels may be in or near any organ in the human, or mammalian,
body.
Introduction of Normal or Genetically Altered Cells into a Blood
Vessel:
[0041] This embodiment of the invention may be illustrated as
follows:
I. Establishment of Endothelial or Other Vascular Cells in Tissue
Culture.
[0042] 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.
[0043] Endothelial or other vascular cells may be derived
enzymatically from a segment of a blood vessel, using techniques
previously described in J. W. Ford, et al., In Vitro, 17, 40
(1981). The vessel is excised, inverted over a stainless steel rod
and incubated in 0.1% trypsin in Ca.sup.++- and Mg.sup.++-free
Hank's balanced salt solution (BSS) with 0.125% EDTA at pH 8 for 10
min at 37.degree. C.
[0044] Cells (0.4 to 1.5.times.10.sup.6) are collected by
centrifugation and resuspended in medium 199 (GIBCO) containing 10%
fetal bovine serum, endothelial cell growth supplement (ECGS,
Collaborative Research, Waltham, Mass.) at 25 .mu.g/ml, heparin at
15 U/ml, and gentamicin (50 .mu.g/ml). Cells are added to a 75
cm.sup.2 tissue culture flask precoated with gelatin (2 mg/ml in
distilled water). Cells are fed every second day in the above
medium until they reach confluence.
[0045] 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 10.sup.6 cells in 0.5 ml of ice cold fetal calf serum
on ice. An equal volume of ice cold fetal calf serum containing 10%
DMSO is added, and cells are transferred to a prechilled screw cap
Corning freezing tube. These cells are transferred to a -70.degree.
C. freezer for 3 hours before long term storage in liquid
nitrogen.
[0046] The cells are then infected with a vector containing the
desired genetic material.
II. Introduction of cells expressing normal or exogenous proteins
into the vasculature.
[0047] A. Introduction of Cells Expressing Relevant Proteins by
Catheterization.
[0048] The patient is prepared for catheterization either by
surgery or percutaneously, observing strict adherence to sterile
techniques. A cutdown procedure is performed over the target blood
vessel or a needle is inserted into the target blood vessel after
appropriate anesthesia. The vessel (5) is punctured and a catheter,
such as described in U.S. Pat. No. 4,636,195, which is hereby
incorporated by reference (available from USCI, Billerica, Mass.)
is advanced by guidewire means (6) under fluoroscopic guidance, if
necessary, into the vessel (5) (FIG. 1). This catheter means (4) is
designed to introduce infected endothelial cells into a discrete
region of the artery. The catheter has a proximal and distal
balloon means (2) and (1), respectively, (e.g., each balloon means
may be about 3 mm in length and about 4 mm in width), with a length
of catheter means between the balloons. The length of catheter
means between the balloons has a port means connected to an
instillation port means (3). When the proximal and distal balloons
are inflated, a central space is created in the vessel, allowing
for instillation of infected cells though the port.
[0049] A region of the blood vessel is identified by anatomical
landmarks and the proximal balloon means (2) is inflated to denude
the endothelium by mechanical trauma (e.g., by forceful passage of
a partially inflated balloon catheter within the vessel) or by
mechanical trauma in combination with small amounts of a
proteolytic enzyme such as dispase, trypsin, collagenase, papain,
pepsin, chymotrypsin or cathepsin, or by incubation with these
proteolytic enzymes alone. In addition to proteolytic enzymes,
lipases may be used. The region of the blood vessel may also be
denuded by treatment with a mild detergent or the like, such as
NP-40, Triton X100, deoxycholate, or SDS.
[0050] The denudation conditions are adjusted to achieve
essentially complete loss of endothelium for cell transfers or
approximately 20 to 90%, preferably 50 to 75%, loss of cells from
the vessel wall for direct infection. In some instances cell
removal may not be necessary. The catheter is then advanced so that
the instillation port means (3) is placed in the region of denuded
endothelium. Infected, transfected or normal cells are then
instilled into the discrete section of artery over thirty minutes.
If the blood vessel is perfusing an organ which can tolerate some
ischemia, e.g., skeletal muscle, distal perfusion is not a major
problem, but can be restored by an external shunt if necessary, or
by using a catheter which allows distal perfusion. After
instillation of the infected endothelial cells, the balloon
catheter is removed, and the arterial puncture site and local skin
incision are repaired. If distal perfusion is necessary, an
alternative catheter designed to allow distal perfusion may be
used.
[0051] 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.
[0052] 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 .mu.g/ml) to
enhance the efficiency of infection. After incubation in the dead
space created by the catheter for an adequate period of time (0.2
to 2 hours or greater), this medium is evacuated, gently washed
with phosphate-buffered saline, and arterial circulation is
restored. Similar protocols are used for post operative
recovery.
[0053] The vessel surface can be prepared by mechanical denudation
alone, in combination with small amounts of proteolytic enzymes
such as dispase, trypsin, collagenase or cathepsin, or by
incubation with these proteolytic enzymes alone. The denudation
conditions are adjusted to achieve the appropriate loss of cells
from the vessel wall.
[0054] Viral vector or DNA-vector complex is instilled in
Dulbecco's modified Eagle's medium using purified virus or
complexes containing autologous serum, and adhesive molecules such
as polybrene (10 .mu.g/ml), poly-L-lysine, dextran sulfate, or any
polycationic substance which is physiologically suitable, or a
hybrid antibody directed against the envelope glycoprotein of the
virus or the vector and the relevant target in the vessel wall or
in the tissue perfused by the vessel to enhance the efficiency of
infection by increasing adhesion of viral particles to the relevant
target cells. The hybrid antibody directed against the envelope
glycoprotein of the virus or the vector and the relevant target
cell can be made by one of two methods. Antibodies directed against
different epitopes can be chemically crosslinked (G. Jung, C. J.
Honsik, R. A. Reisfeld, and H. J. Muller-Eberhard, Proc. Natl.
Acad. Sci. USA, 83, 4479 (1986); U. D. Staerz, O. Kanagawa, and M.
J. Bevan, Nature, 314, 628 (1985); and P. Perez, R. W. Hoffman, J.
A. Titus, and D. M. Segal, J. Exp. Med., 163, 166 (1986)) or
biologically coupled using hybrid hybridomas (U. D. Staerz and M.
J. Bevan, Proc. Natl. Acad. Sci. USA, 83, 1453 (1986); and C.
Milstein and A. C. Cuello, Nature, 305, 537 (1983)). After
incubation in the central space of the catheter for 0.2 to 2 hours
or more, the medium is evacuated, gently washed with phosphate
buffered saline, and circulation restored.
[0055] Using a different catheter design (See FIG. 2), a different
protocol for instillation can also be used. This second approach
involves the use of a single balloon means (2) catheter with
multiple port means (3) which allow for high pressure delivery of
the retrovirus into partially denuded arterial segments. The vessel
surface is prepared as described above and defective vector is
introduced using similar adhesive molecules. In this instance, the
use of a high pressure delivery system serves to optimize the
interaction of vectors with cells in adjacent vascular tissue.
[0056] The present invention also provides for the use of growth
factors delivered locally by catheter or systemically to enhance
the efficiency of infection.
[0057] In addition to retroviral vectors, herpes virus, adenovirus,
or other viral vectors are suitable vectors for the present
technique.
[0058] It is also possible to transform cells within an organ or
tissue. Direct transformation of organ or tissue cells may be
accomplished by one of two methods. In a first method a high
pressure transfection is used. The high pressure will cause the
vector to migrate through the blood vessel walls into the
surrounding tissue. In a second method, injection into a capillary
bed, optionally after injury to allow leaking, gives rise to direct
infection of the surrounding tissues.
[0059] The time required for the instillation of the vectors or
cells will depend on the particular aspect of the invention being
employed. Thus, for instilling cells or vectors in a blood vessel a
suitable time would be from 0.01 to 12 hrs, preferably 0.1 to 6
hrs, most preferably 0.2 to 2 hrs. Alternatively for high pressure
instillation of vectors or cells, shorter times might be
preferred.
Obtaining the Cells Used in this Invention:
[0060] The term "genetic material" generally refers to DNA which
codes for a protein. This phrase also encompasses RNA when used
with an RNA virus or other vector based on RNA.
[0061] Transformation is the process by which cells have
incorporated an exogenous gene by direct infection, transfection or
other means of uptake.
[0062] The term "vector" is well understood and is synonymous with
the often-used phrase "cloning vehicle". A vector is
non-chromosomal double-stranded DNA comprising an intact replicon
such that the vector is replicated when placed within a unicellular
organism, for example by a process of transformation. Viral vectors
include retroviruses, adenoviruses, herpesvirus, papovirus, or
otherwise modified naturally occurring viruses. Vector also means a
formulation of DNA with a chemical or substance which allows uptake
by cells.
[0063] In another embodiment the present invention provides for
inhibiting the expression of a gene. Four approaches may be
utilized to accomplish this goal. These include the use of
antisense agents, either synthetic oligonucleotides which are
complementary to the mRNA (Maher III, L. J. and Dolnick, B. J.
Arch. Biochem. Biophys., 253, 214-220 (1987) and (Zamecnik, P. C.,
et al., Proc. Natl. Acad. Sci., 83, 4143-4146 (1986)), or the use
of plasmids expressing the reverse complement of this gene (Izant,
J. H. and Weintraub, H., Science, 229, 345-352, (1985); Cell, 36,
1077-1015 (1984)). In addition, catalytic RNAs, called ribozymes,
can specifically degrade RNA sequences (Uhlenbeck, O. C., Nature,
328, 596-600 (1987), Haseloff, J. and Gerlach, W. L., Nature, 334,
585-591 (1988)). The third approach involves "intracellular
immunization", where analogues of intracellular proteins can
interfere specifically with their function (Friedman, A. D.,
Triezenberg, S. J. and McKnight, S. L., Nature, 335, 452-454
(1988)), described in detail below.
[0064] The first approaches may be used to specifically eliminate
transcripts in cells. The loss of transcript may be confirmed by S1
nuclease analysis, and expression of binding protein determined
using a functional assay. Single-stranded oligonucleotide analogues
may be used to interfere with the processing or translation of the
transcription factor mRNA. Briefly, synthetic oligonucleotides or
thiol-derivative analogues (20-50 nucleotides) complementary to the
coding strand of the target gene may be prepared. These antisense
agents may be prepared against different regions of the mRNA. They
are complementary to the 5' untranslated region, the translational
initiation site and subsequent 20-50 base pairs, the central coding
region, or the 3' untranslated region of the gene. The antisense
agents may be incubated with cells transfected prior to activation.
The efficacy of antisense competitors directed at different
portions of the messenger RNA may be compared to determine whether
specific regions may be more effective in preventing the expression
of these genes.
[0065] RNA can also function in an autocatalytic fashion to cause
autolysis or to specifically degrade complementary RNA sequences
(Uhlenbeck, O. C., Nature, 328, 596-600 (1987), Haseloff, J. and
Gerlach, W. L., Nature, 334, 585-591 (1988), and Hutchins, C. J.,
et al, Nucleic Acids Res., 14, 3627-3640 (1986)). The requirements
for a successful RNA cleavage include a hammerhead structure with
conserved RNA sequence at the region flanking this structure.
Regions adjacent to this catalytic domain are made complementary to
a specific RNA, thus targeting the ribozyme to specific cellular
mRNAs. To inhibit the production of a specific target gene, the
mRNA encoding this gene may be specifically degraded using
ribozymes. Briefly, any GUG sequence within the RNA transcript can
serve as a target for degradation by the ribozyme. These may be
identified by DNA sequence analysis and GUG sites spanning the RNA
transcript may be used for specific degradation. Sites in the 5'
untranslated region, in the coding region, and in the 3'
untranslated region may be targeted to determine whether one region
is more efficient in degrading this transcript. Synthetic
oligonucleotides encoding 20 base pairs of complementary sequence
upstream of the GUG site, the hammerhead structure and -20 base
pairs of complementary sequence downstream of this site may be
inserted at the relevant site in the cDNA. In this way, the
ribozyme may be targeted to the same cellular compartment as the
endogenous message. The ribozymes inserted downstream of specific
enhancers, which give high level expression in specific cells may
also be generated. These plasmids may be introduced into relevant
target cells using electroporation and cotransfection with a
neomycin resistant plasmid, pSV2-Neo or another selectable marker.
The expression of these transcripts may be confirmed by Northern
blot and S1 nuclease analysis. When confirmed, the expression of
mRNA may be evaluated by S1 nuclease protection to determine
whether expression of these transcripts reduces steady state levels
of the target mRNA and the genes which it regulates. The level of
protein may also be examined.
[0066] Genes may also be inhibited by preparing mutant transcripts
lacking domains required for activation. Briefly, after the domain
has been identified, a mutant form which is incapable of
stimulating function is synthesized. This truncated gene product
may be inserted downstream of the SV-40 enhancer in a plasmid
containing the neomycin resistance gene (Mulligan, R. and Berg, P.,
Science, 209, 1422-1427 (1980) (in a separate transcription unit).
This plasmid may be introduced into cells and selected using G418.
The presence of the mutant form of this gene will be confirmed by
S1 nuclease analysis and by immunoprecipitation. The function of
the endogenous protein in these cells may be evaluated in two ways.
First, the expression of the normal gene may be examined. Second,
the known function of these proteins may be evaluated. In the event
that this mutant intercellular interfering form is toxic to its
host cell, it may be introduced on an inducible control element,
such as metallothionein promoter. After the isolation of stable
lines, cells may be incubated with Zn or Cd to express this gene.
Its effect on host cells can then be evaluated.
[0067] Another approach to the inactivation of specific genes is to
overexpress recombinant proteins which antagonize the expression or
function of other activities. For example, if one wished to
decrease expression of TPA (e.g., in a clinical setting of
disseminate thrombolysis), one could overexpress plasminogen
activator inhibitor.
[0068] Advances in biochemistry and molecular biology in recent
years have led to the construction of "recombinant" vectors in
which, for example, retroviruses and plasmids are made to contain
exogenous RNA or DNA, respectively. In particular instances the
recombinant vector can include heterologous RNA or DNA, by which is
meant RNA or DNA that codes for a polypeptide ordinarily not
produced by the organism susceptible to transformation by the
recombinant vector. The production of recombinant RNA and DNA
vectors is well understood and need not be described in detail.
However, a brief description of this process is included here for
reference.
[0069] For example, a retrovirus or a plasmid vector can be cleaved
to provide linear RNA or DNA having ligatable termini. These
termini are bound to exogenous RNA or DNA having complementary like
ligatable termini to provide a biologically functional recombinant
RNA or DNA molecule having an intact replicon and a desired
phenotypical property.
[0070] 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.
[0071] The exogenous, i.e., donor, RNA or DNA used in the present
invention is obtained from suitable cells. The vector is
constructed using known techniques to obtain a transformed cell
capable of in vivo expression of the therapeutic agent protein. The
transformed cell is obtained by contacting a target cell with a RNA
or DNA-containing formulation permitting transfer and uptake of the
RNA or DNA into the target cell. Such formulations include, for
example, retroviruses, plasmids, liposomal formulations, or
plasmids complexes with polycationic substances such as
poly-L-lysine, DEAC-dextran and targeting ligands.
[0072] The present invention thus provides for the genetic
alteration of cells as a method to transmit therapeutic or
diagnostic agents to localized regions of the blood vessel for
local or systemic purposes. The range of recombinant proteins which
may be expressed in these cells is broad and varied. It includes
gene transfer using vectors expressing such proteins as tPA for the
treatment of thrombosis and restenosis, angiogenesis or growth
factors for the purpose of revascularization, and vasoactive
factors to alleviate vasoconstriction or vasospasm. This technique
can also be extended to genetic treatment of inherited disorders,
or acquired diseases, localized or systemic. The present invention
may also be used to introduce normal cells to specific sites of
cell loss, for example, to replace endothelium damaged during
angioplasty or catheterization.
[0073] 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
.alpha. (TGF-.alpha.), transforming growth factor .beta.
(TGF-.beta.), angiogenin, tumor necrosis factor .alpha., tumor
necrosis factor .alpha., 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.
[0074] The present invention can also be used in the treatment of
malignancies by placing the transformed cells in proximity to the
malignancy. In this application, genetic material coding for
diphtheria toxin, pertussis toxin, or cholera toxin may be
used.
[0075] In the use of the present invention in the treatment of
AIDS, genetic material coding for soluble CD4 or derivatives
thereof may be used. In the treatment of genetic diseases, for
example, growth hormone deficiency, genetic material coding for the
needed substance, for example, human growth hormone, is used. All
of these genetic materials are readily available to one skilled in
this art.
[0076] In another embodiment, the present invention provides a kit
for treating a disease in a patient which contains a catheter and a
solution which contains either an enzyme or a mild detergent, in
which the catheter is adapted for insertion into a blood vessel and
contains a main catheter body having a balloon element adapted to
be inserted into said vessel and expansible against the walls of
the blood vessel so as to hold the main catheter body in place in
the blood vessel, and means carried by the main catheter body for
delivering a solution into the blood vessel, and the solution which
contains the enzyme or mild detergent is a physiologically
acceptable solution. The solution may contain a proteolytic enzyme,
such as dispase, trypsin, collagenase, papain, pepsin, or
chymotrypsin. In addition to proteolytic enzymes, lipases may be
used. As a mild detergent, the solution may contain NP-40, Triton
X100, deoxycholate, SDS or the like.
[0077] Alternatively, the kit may contain a physiological
acceptable solution which contains an agent such as heparin,
poly-L-lysine, polybrene, dextran sulfate, a polycationic material,
or bivalent antibodies. This solution may also contain vectors or
cells (normal or transformed). In yet another embodiment the kit
may contain a catheter and both a solution which contains an enzyme
or mild detergent and a solution which contains an agent such as
heparin, poly-L-lysine, polybrene, dextran sulfate, a polycationic
material or bivalent antibody and which may optionally contain
vectors or cells.
[0078] The kit may contain a catheter with a single balloon and
central distal perfusion port, together with acceptable solutions
to allow introduction of cells in a specific organ or vectors into
a capillary bed or cells in a specific organ or tissue perfused by
this capillary bed.
[0079] Alternatively, the kit may contain a main catheter body
which has two spaced balloon elements adapted to be inserted in a
blood vessel with both being expansible against the walls of the
blood vessel for providing a chamber in the blood vessel, and to
hold the main catheter body in place. In this case, the means for
delivering a solution into the chamber is situated in between the
balloon elements. The kit may contain a catheter which possesses a
plurality of port means for delivering the solution into the blood
vessel.
[0080] Thus, the present invention represents a method for treating
a disease in a patient by causing a cell attached onto the walls of
a vessel or the cells of an organ perfused by this vessel in the
patient to express an exogeneous therapeutic agent protein, wherein
the protein treats the disease or may be useful for diagnostic
purposes. The present method may be used to treat diseases, such as
an ischemic disease, a vasomotor disease, diabetes, a malignancy,
AIDS or a genetic disease.
[0081] The present method may use exogeneous therapeutic agent
proteins, such as tPA and modifications thereof, urokinase,
streptokinase, acidic fibroblast growth factor, basic fibroblast
growth factor, tumor necrosis factor .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 to treat diseases.
[0082] The present method may also use exogenous proteins of
diagnostic value. For example, a marker protein, such as
.beta.-galatosodase, may be used to monitor cell migration.
[0083] It is preferred, that the cells caused to express the
exogenous therapeutic agent protein be endothelial cells.
[0084] 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.
[0085] 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,
.beta.-galactosidase, in vivo.
[0086] Because atherogenesis in swine has similarities to humans,
an inbred pig strain, the Yucatan minipig (Charles River
Laboratories, Inc., Wilmington, Mass.), was chosen as an animal
model (1). A primary endothelial cell line was established from the
internal jugular vein of an 8 month-old female minipig. The
endothelial cell identity of this line was confirmed in that the
cells exhibited growth characteristics and morphology typical of
porcine endothelium in tissue culture. Endothelial cells also
express receptors for the acetylated form of low density
lipoprotein (AcLDL), in contrast to fibroblasts and other
mesenchymal cells (2). When analyzed for AcLDL receptor expression,
greater than 99% of the cultured cells contained this receptor, as
judged by fluorescent AcLDL uptake.
[0087] Two independent .beta.-galactosidase-expressing endothelial
lines were isolated following infection with a murine amphotropic
s-galactosidase-transducing retroviral vector (BAG), which is
replication-defective and contains both .beta.-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 .beta.-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.
[0088] Endothelial cells derived from this inbred strain, being
syngeneic, were applicable for study in more than one minipig, and
were tested in nine different experimental subjects. Under general
anesthesia, the femoral and iliac arteries were exposed, and a
catheter was introduced into the vessel (FIG. 1). Intimal tissues
of the arterial wall were denuded mechanically by forceful passage
of a partially inflated balloon catheter within the vessel. The
artery was rinsed with heparinized saline and incubated with the
neutral protease, dispase (50 U/ml), which removed any remaining
luminal endothelial cells. Residual enzyme was rapidly inactivated
by .alpha.2 globulin in plasma upon deflating the catheter balloons
and allowing blood to flow through the vessel segment. The cultured
endothelial cells which expressed .beta.-galactosidase were
introduced using a specially designed arterial catheter (USCI,
Billerica, Mass.) that contained two balloons and a central
instillation port (FIG. 1).
[0089] When these balloons were inflated, a protected space was
created within the artery into which cells were instilled through
the central port 3 (FIG. 1). These endothelial cells, which
expressed .beta.-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.
[0090] Segments of the artery innoculated with
.beta.-galactosidase-expressing endothelium were removed 2 to 4
weeks later. Gross examination of the arterial specimen after
staining using the X-gal chromagen showed multiple areas of blue
coloration, compared to an artery seeded with uninfected
endothelium, indicative of .beta.-galactosidase activity. Light
microscopy documented .beta.-galactosidase staining primarily in
endothelial cells of the intima in experimentally seeded
vessels.
[0091] In contrast, no evidence of similar staining was observed in
control segments which had received endothelial cells containing no
.beta.-galactosidase. .beta.-Galactosidase staining was
occasionally-evident in deeper intimal tissues, suggesting
entrapment or migration of seeded endothelium within the previously
injured vessel wall. Local thrombosis was observed in the first two
experimental subjects. This complication was minimized in
subsequent studies by administering acetylsalicylic acid prior to
the endothelial cell transfer procedure and use of heparin
anticoagulation at the time of innoculation. In instances of
thrombus formation, .beta.-galactosidase staining was seen in
endothelial cells extending from the vessel wall to the surface of
the thrombus.
[0092] A major concern of gene transplantation in vivo relates to
the production of replication-competent retrovirus from genetically
engineered cells. In these tests, this potential problem has been
minimized through the use of a replication defective retrovirus. No
helper virus was detectable among these lines after 20 passages in
vitro. Although defective viruses were used because of their high
rate of infectivity and their stable integration into the host cell
genome (4), this approach to gene transfer is adaptable to other
viral vectors.
[0093] A second concern involves the longevity of expression of
recombinant genes in vivo. Endothelial cell expression of
.beta.-galactosidase appeared constant in vessels examined up to
six weeks after introduction into the blood vessel in the present
study.
[0094] 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.
[0095] A major complication of current interventions for vascular
disease, such as balloon angioplasty or insertion of a graft into a
diseased vessel, is disruption of the atherosclerotic plaque and
thrombus formation at sites of local tissue trauma (5). In part,
this is mediated by endothelial cell injury (6). The present data
show that genetically-altered endothelial cells can be introduced
at the time of intervention to minimize local thrombosis.
[0096] This technique can also be used in other ischemic settings,
including unstable angina or myocardial infarction. For instance,
antithrombotic effects can be achieved by introducing cells
expressing genes for tissue plasminogen activator or urokinase.
This technology is also useful for the treatment of chronic tissue
ischemia. For example, elaboration of angiogenic or growth factors
(7) to stimulate the formation of collateral vessels to severely
ischemic tissue, such as the myocardium. Finally, somatic gene
replacement for systemic inherited diseases is feasible using
modifications of this endothelial cell gene transfer technique.
Experimental Section:
[0097] A. Analysis of AcLDL Receptor Expression in Normal and
.beta.-Galactosidase-Transduced Porcine Endothelial Cells.
[0098] Endothelial cell cultures derived from the Yucatan minipig,
two sublines infected with BAG retrovirus or 3T3 fibroblast
controls were analyzed for expression of AcLDL receptor using
fluorescent labelled AcLDL.
[0099] Endothelial cells were derived from external jugular veins
using the neutral protease dispase (8). Excised vein segments were
filled with dispase (50 U/ml in Hanks' balanced salt solution) and
incubated at 30.degree. C. for 20 minutes. Endothelium obtained by
this means was maintained in medium 199 (GIBCO, Grand Island, N.Y.)
supplemented with fetal calf serum (10%), 50 .mu.g/ml endothelial
cell growth supplement (ECGS) and heparin (100 .mu.g/ml). These
cells were infected with BAG retrovirus, and selected for
resistance to G-418. Cell cultures were incubated with
(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbacyanine
perchlorate) (Dil) AcLDL (Biomedical Technologies, Stoughton,
Mass.) (10 .mu.g/ml) for 4-6 hrs. at 37.degree. C., followed by
three rinses with phosphate-buffered saline containing 0.5%
glutaraldehyde. Cells were visualized by phase contrast and
fluorescent microscopy.
[0100] B. Method of Introduction of Endothelial Cells by
Catheterization.
[0101] A double balloon catheter was used for instillation of
endothelial cells. The catheter has a proximal and distal balloon,
each 6 mm in length and 5 mm in width, with a 20 mm length between
the balloons. The central section of the catheter has a 2 mm pore
connected to an instillation port. Proximal and distal balloon
inflation isolates a central space, allowing for instillation of
infected cells through the port into a discrete segment of the
vessel. For a schematic representation of cell introduction by
catheter, see FIGS. 1 and 2.
[0102] Animal care was carried out in accordance with "Principles
of Laboratory Animal Care" and "Guide for the Care and Use of
Laboratory Animals" (NIH publication No. 80-23, Revised 1978).
Female Yucatan minipigs (80-100 kg) were anesthetized with
pentobarbital (20 mg/kg), intubated, and mechanically ventilated.
These subjects underwent sterile surgical exposure of the iliac and
femoral arteries. The distal femoral artery was punctured, and the
double-balloon catheter was advanced by guidewire into the iliac
artery. The external iliac artery was identified; the proximal
balloon was partially inflated and passed proximally and distally
so as to mechanically denude the endothelium. The catheter was then
positioned with the central space located in the region of denuded
endothelium, and both balloons were inflated. The denuded segment
was irrigated with heparinized saline, and residual adherent cells
were removed by instillation of dispase (20 U/ml) for 10 min. The
denuded vessel was further irrigated with a heparin solution and
the BAG-infected endothelial cells were instilled for 30 min. The
balloon catheter was subsequently removed, and antegrade blood flow
was restored. The vessel segments were excised 2 to 4 weeks later.
A portion of the artery was placed in 0.5% glutaraldehyde for five
minutes and stored in phosphate-buffered saline, and another
portion was mounted in a paraffin block for sectioning. The
presence of retroviral expressed s-galactosidase was determined by
a standard histochemical technique (19).
[0103] C. Analysis of Endothelial Cells In Vitro and In Vivo.
[0104] .beta.-Galactosidase activity was documented by
histochemical staining in (A) primary endothelial cells from the
Yucatan minipig, (B) a subline derived by infection with the BAG
retroviral vector, (C) a segment of normal control artery, (D) a
segment of artery instilled with endothelium infected with the BAG
retroviral vector, (E) microscopic cross-section of normal control
artery, and (F) microscopic cross-section of artery instilled with
endothelium infected with the BAG retroviral vector.
[0105] Endothelial cells in tissue culture were fixed in 0.5%
glutaraldehyde prior to histochemical staining. The enzymatic
activity of the E. coli .beta.-galactosidase protein was used to
identify infected endothelial cells in vitro and in vivo. The
.beta.-galactosidase transducing Mo-MuLV vector (2), (BAG) was
kindly provided by Dr. Constance Cepko. This vector used the wild
type Mo-MuLV LTR as a promoter for the .beta.-galactosidase gene.
The simian virus 40 (SV-40) early promoter linked to the Tn5
neomycin resistance gene provides resistance to the drug G-418 and
is inserted downstream of the s-galactosidase gene, providing a
marker to select for retrovirus-containing, .beta.-galactosidase
expressing cells. This defective retrovirus was prepared from
fibroblast .phi. am cells (3,10), and maintained in Dulbecco's
modified Eagle's medium (DMEM) and 10% calf serum. Cells were
passaged twice weekly following trypsinization. The supernatant,
with titers of 10.sup.4-10.sup.5/ml G-418 resistant colonies, was
added to endothelial cells at two-thirds confluence and incubated
for 12 hours in DMEM with 10% calf serum at 37.degree. C. in 5%
CO.sub.2 in the presence of 8 .mu.g/ml of polybrene. Viral
supernatants were removed, and cells maintained in medium 199 with
10% fetal calf serum, ECGS (50 .mu.g/ml), and endothelial cell
conditioned medium (20%) for an additional 24 to 48 hours prior to
selection in G-418 (0.7 .mu.g/ml of a 50% racemic mixture). G-418
resistant cells were isolated and analyzed for .beta.-galactosidase
expression using a standard histochemical stain (9). Cells stably
expressing the .beta.-galactosidase enzyme were maintained in
continuous culture for use as needed. Frozen aliquots were stored
in liquid nitrogen.
PUBLICATIONS CITED
[0106] 1. J. S: Reitman, R. W. Mahley, D. L. Fry, Atherosclerosis
43, 119 (1982). [0107] 2. R. E. Pitos, T. L. Innerarity, J. N.
Weinstein, R. W. Mahley, Arteriosclerosis 1, 177 (1981); T. J. C.
Van Berkel, J. F. Kruijt FEBS Lett. 132, 61 (1981); J. C. Voyta, P.
A. Netland, D. P. Via, E. P. Zetter, J. Cell. Biol., 99, 81A
(abstr.) (1984); J. M. Wilson, D. E. Johnston, D. M. Jefferson, R.
C. Mulligan, Proc. Natl. Acad. Sci. U.S.A., 84, 4421 (1988). [0108]
3. J. Price, D. Turner, C. Cepko, Proc. Natl. Acad. Sci. U.S.A.,
84, 156 (1987). [0109] 4. R. Mann, R. C. Mulligan, D. Baltimore,
Cell 33, 153 (1983); C. L. Cepko, B. E. Roberts, R. C. Mulligan,
Cell 37, 1053 (1984); M. A. Eglitis, W. F. Anderson, Biotechniques
6, 608 (1988). [0110] 5. S. G. Ellis, G. S. Roubin, S. B. King, J.
S. Douglas, W. S. Weintraub et al., Circulation 77, 372 (1988); L.
Schwartz, M. G. Bourassa, J. Lesperance, H. E. Aldrige, F. Kazim,
et al., N. Engl. J. Med. 318, 1714 (1988). [0111] 6. P. C. Block,
R. K. Myler, S. Stertzer, J. T. Fallon, N. Engl. J. Med. 305, 382
(1981); P. M. Steele, J. H. Chesebro, A. W. Stanson, Circ. Res. 57,
105 (1985); J. R. Wilentz, T. A. Sanborn, C. C. Handenschild, C. R.
Valeri, T. J. Ryan, D. P. Faxon, Circulation 75, 636 (1987); W.
McBride, R. A. Lange, L. D. Hillis, N. Engl. J. Med. 318, 1734
(1988). [0112] 7. J. Folkmah, M. Klagsbrun, Science 235, 442
(1987); S. J. Leibovich, P. J. Polyerini, H. Michael Shepard, D. M.
Wiseman, V. Shively, N. Nuseir, Nature 329, 630 (1987); J. Folkman,
M. Klagsbrun, Nature 329, 671 (1987). [0113] 8. T. Matsumura, T.
Yamanka, S. Hashizume, Y. Irie, K. Nitta, Japan. J. Exp. Med. 45,
377 (1975); D. G. S. Thilo, S. Muller-Kusel, D. Heinrich, I.
Kauffer, E. Weiss, Artery, 8, 25a (1980). [0114] 9. A. M.
Dannenberg, M. Suga, in Methods for Studying Mononuclear
Phagocytes, D. O. Adams, P. J. Edelson, H. S. Koren, Eds. (Academic
Press, New York, 1981), pp 375-395. [0115] 10. R. D. Cone, R. C.
Mulligan, Proc. Natl. Acad. Sci. U.S.A. 81, 6349 (1984).
[0116] 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.
* * * * *