Note: Descriptions are shown in the official language in which they were submitted.
WOg3/~52~ PCT/US92/OS243
~ 1 211237~
Description
.Treatment of Diseases by Site-Specific
Instillation of Cells or Site-Speci_ic
Transformation of Cells and Kits Therefor
Technical Field
The present invention relates to the treatment of
diseases by the site-specific instillation or
transformation of cells and kits therefor.
.
Background_Art
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 n
~ivo would be an asset in the treatment of, e.g., localized
diseases. In addition the ability to cause a therapeutic
agent to perfuse through the circulatory system would be
effective for the treatment of, e.g., systemic diseases.
., . . :, . . . ... . .
For example, it would be desirable to administer in a
steady fashion an antitumor agent or toxin in close`
proximity to a tumor. Similarly, it would be desirable to
cause a perfusion of, e.g., insulin in the blood of a
person suffering from diabetes. However, for many
therapeutic agents there is no satisfactory method o~`
either-site-specific orrsystemic administration. ~ Yj~
In addition, for many diseases, it would be desirable
to cause, either-locally or systemically, the express-ion
of a defective endogenous gene, the expression-of a
exogenous gene, or the suppression of an endogenous gene.
Again, these remain unrealized goals.
In particular, the pathogenesis of atherosclerosis is
characterized by three fundamental biological processes.
These are: 1) proliferation of intimal smooth muscle cells
:
W093/~52 2 11237 ~ PCT~US92/05243'
z- ~ ~
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
chol~sterol, within cells as well as in surrounding
connective tissue.
Endothelial cell injury is an initiating event and is
manifested by interference with the permeability barrier of
the endothelium, alterations in the nonthrombogenic
lo properties of the endothelial surface, and promotion of
procoagulant properties of the endothelium. Monocytes
migrate between endothelial cells, become active as
scavenger cells, and differentiate into macrophages.
Macrophages then synthesize and secrete growth factors
including platelet derived growth factor (PDGF), fibroblast
growth factor (FGF), epidermal growth factor (EGF), and
transforming growth factor alpha (TGF-~). These growth
factorc are extremely potent in stimulating the migration
and proliferation of fibroblasts and smooth muscle cells in
the atherosclerotic plaque. In addition, platelets may
- interact with the injured endothelial cell and the
activated macrophage to potentiate the elaboration of
growth factors and thrombus formation.
-Two major problems in the clinical management of
coronary artery disease include thrombus formation in acute
myocardial ischemia and restenosis~following coronary
angioplasty (PTGA). 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,
W093/00052 PCT/USg2/05~3
- 211237~
--3--
Empiric therapy with antiplatelet drugs has not
pre~ented this problem, which occurs in one-third of
patients undergoing PTCA. A solution to restenosis is to
prevent platelet aggregation, thrombus formation, and
smooth muscle cell proliferation.
Thrombus formation is also a critical cellular event
in the transition from stable to unstable coronary
syndromes. The pathogenesis most likely involves acute
endothelial cell injury and or plaque rupture, promoting
dysfunction of endothelial cell attachment, and leading to
the exposure of underlying macrophage foam cells. This
permits the opportunity for circulating platelets to
adhere, aggregate, and form thrombi.
The intravenous administration of thrombolytic agents,
such as tissue plasminogen activator (tPA) results in lysis
of thrombus in approximately 70% of patients experienGing
an acute myocardial infarction. Nonethele~s, 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.
c~ As noted above, an effective'-therapy for réthrombosis'`
25 . is byifar 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 effe'ctive 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.
W,093!000S2-. " PCr/US92io5243 ' ~
~1i2~7 S
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.
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 p-esent 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
15 ~ 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.
, ~r;~,It~is-,another,fobje,ct of-the present invention to
~' ,, provide a Amethod,,f,o~ithe!treatment of a disease by causing ^
either;,the,site-specific administration of a therapeutic
agent or the perfusion of`a therapeutic agent`in the
bloodstream~;of~a patient. ,,
It is an,o,ther object of the present invention to
provide a method for the treatment of a disease by causing
either the expre~sion of an exogenous gene, the expression
of a defective endogenous gene, or the suppression of the
expression of an endogenous gene in a patient.
W093/~52- 2 1 1 '~ 3 7 '3~ ,
--5--
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 nor,mal or
transformed cells in a patient.
It is another object of the present invention to
provide a kit for site-specifically transforming-cells in
vivo.
These and other objects of this invention which will
become apparent during the course of the following detailed
description of the invention have been discovered by the
inventors to be achieved by (a) a method which comprises
either (i) site-specific instillation or e~ther normal
(untransformed) or transformed cells in a patient or (ii)
site-specific trans~ormation of cells in a patient and (b)
a kit which contains a catheter`for (i) site-specific
instillation of either normal or transformëdicells 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'bé used-to cause3~thé expression of
eithérSa~defective'~'endogenous-'gene~;or~an exogenousigène or
~- the~:~uppression 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 Desc~iption of the Drawinqs
. - . .
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
W093/~s2- ~ PCT/US92/05243
2112375 -6-
to the following detailed description when considered in
connection with the accompanying figures, wherein:
FIGURES 1 and 2 illustrate the use of a catheter
in accordance with the invention to surgically or
percutaneously implant cells in a blood vessel or to
transform in vivo cells present on the wall of a patient's
blood vessel.
Best Mode for Carryina Out the Invention
Thus, in one embodiment, the present invention is used
to treat diseases, such as inherited diseases, systemic
diseases, diseases of the cardiovascular system, diseases
of particular organs, or tumors by instilling normal or
transformed cells or by transforming cells.
The cells which may be instilled in the present method
include endothelium, smooth muscle, fibroblasts, monocytes,
macrophages, and parenchymal cells. These cells may
produce proteins which may have a therapeutic or diagnostic
effect and which may be~naturally occurring or arise from
recombinant genetic material.
Referring now to the figures, wherein like reference
r~numerals designate~identical or corresponding parts l "~ t
throughout~c-~the several ~iews, ï e~and more particu}arly 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. tn 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
WO,~g3! ~ 52~, 2112 3 7 5 PCT/US92/05243
--7--
between balloon means 1 and 2 is equipped with instillation
port means 3. The catheter may be further equipped with a
guidewire means 6. FIGURE 2 illustrates the use of a
similar catheter, distinguished from the catheter
illustrated in Figure 1 by the fact that it is equipped
with only a single inflatable balloon means ~ and a
plurality of instillation port means 3. This catheter may
contain up to twelve individual instillation port means 3,
with five being illustrated.
.
In the case of delivery to an organ, the catheter may
be introduced into the major artery supplying the tissue.
Cells containing recombinant genes or vectors can be
introduced through a central instillation port after
temporary occlusion of the arterial circulation~ In this
way, cells or vector DNA may be delivered to a large amount
of parenchymal tissue distributed through the capillary
circulation. Recombinant genes can also be introduced into
the vasculature using the double balloon catheter technique
in,the arterial circulation proximal to the target organ.
In this way, the recombinant genes may be secreted directly
into the circulation which perfuse the involved tissue or
may be synthesized directly within the organ.
In one embodiment, the therapeutic agents are secreted
by,va~cularicells-supp}ying specific organs affected by the
25 --,d~seaQe.~ ;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
~ti~ue~.~ Diabetes me}litus~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
W093/~2,, , ~ t, ~ PCTiUS92~05~3
2112375 `~ "`'
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, ~-antitrypsin
inhibitor deficiency or hyperchloresterolemia may be
treated by introduction of ~-antitrypsin or ~e LDL
receptor gene. In addition, this approach may be used for
lo the treatment of malignancy. Secretion of specific
recombinant toxin genes into the circulation of
inoperable-tumors provides a therapeutic effect. Examples
include acoustic neuromas or certain hemangiomas which are
otherwise unresectable.
In clinical settings, these therapeutic
recombinant genes are introduced in cells supplying the
circulation of the involved organ. Although the arterial
and cnpillary circulations are the preferred locations for
,,~ introduction of these:cells,.venous systems are also
20~ suitable-. , ..-~ "".r~
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
~ite~ they.are,;.used as;carriers~to~convey the therapeutic-
, The invention thus, in one of its aspects, relies ongenetic alteration of endothelial and other vascular cells
,,, -or ~s,o.,matic cell:gene therapy,,.:for.transmitting therapeutic
30, agents~,,(i.e., proteins,:gr,owth~: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
-W093/~52-. 2 1 1 ~. ~ 7: PCT/US92ios~3
_g_
to be expressed must be cloned an-d available for genetic
manipulation. Third, the gene must be introduced into the
cell in a form that will be expressed or functional.
Fourth, the genetically altered cells must be situated in
the vascular region where it is needed.
In accordance with the present invention the altered
cells or appropriate vector may be surgically,
percutaneously, or intravenously introduced and attached to
a section of a patient's vessel wall. Alternatively, some
of the cells existing on the patient's vessel wall are
transformed with the desired genetic material or by
directly applying the vector. In some instances, vascular
cells which are not genetically modified can be introduced
by these methods to replace cells lost or damaged on the
15 vessel surface. ;
Any blood vessel may ~e treated in accordance with
this invention; that is, arteries, veins, and capillaries.
The~e blood vessels may be in or near any organ in the
human, or-mammalian, body. - ~
.., .,~ . . . ~ .
ntroduction of normal or qenetically altered cells into a
blood vessel:
,,
- ~This embodiment of the invention may be illustrated as
. follows: ~.~ c:
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
~093/OH~2 ~ PCT/US92/052i3
2 ~ 7 r~ o-
enzymatical~y from a segment of a blood vessel, using
techniques previously described in J.W. Ford, et al., In
vitro, 17, 40 (1981). The vessel is excised, inverted over
a stainless steel rod and incubated in 0.1% trypsin in ca'+-
and Mg~+- free Hank's balanced salt solution (BSS) with
0.125% EDTA at pH 8 for 10 min at 370C.
Cells (0.4 to 1.5 x 106) are collected by
centrifugation and resuspended in medium 199 (GIBC0)
containing 10% fetal bovine serum, endothelial cell growth
supplement (ECGS, Collaborative Research, Waltham, MA) at
25 ~g/ml, heparin at 15 U/ml, and gentamicin (50 ~g/ml).
Cells are added to a 75 cm2 tissue culture flask precoated
with gelatin (2 mg/ml in distilled water). Cells are fed
every second day in the above medium until they reach
confluence.
After two weeks in culture, the ECGS and heparin
may.be omitted from the medium.when culturing porcine
endothelium. If ~ascular...smooth muscle cells or
fibroblaits are desired the heparin and ECGS can be omitted
entirely from the culturing procedure. Aliquots of aells
are stored in liquid nitrogen by resuspending to
approximately Io.6 cells in 0.5 ml of ice cold fetal calf
~erum on ice. An equal volume of ice cold fetal calf serum
-. containing 10% DNS0 i8 added, and:cells are transferred to
. 25 a prechilled screw cap Corning freezing tube~<iThese 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.material. ...:.
, : . . . . .
II. Introduction of cells expressing normal or exogenous
proteins into the vasculature.
A. Introduction of cells expre~sing relevant
W093/ ~ 5~ PCT/US92ios2i3
2 il~37 S '''''`''~ t'
proteins by catheterization.
The patient is prepared for catheterization either by
surgery or percutaneously, observing strict adherence to
sterile techniques. A cutdown procedure is performed over
the target blood vessel or a needle is inserted into the
target blood vessel after appropriate anesthesia. The
vessel (5) is punctured and a catheter, such as described
in U.S. Patent 4,636,195, which is hereby incorporated by
reference
(available from USCI, Billerica, MA) is advanced by
guidewire means (6) under fluoroscopic guidance, if
necessary, into the vessel (5) (Figure 1). This catheter
means (4) is designed to introduce infected endothelial
cells into a discrete region of the artery. The catheter
has a proximal and distal balloon means (2) and (1),
respectively, (e.g., each balloon means may be about 3 mm
in length and about 4 mm in width), with a length of
catheter means between the balloons. The length of
catheter means between the balloons has a port~means ~
connected to an instillation port means (3). When the
proximal ~nd distal balloons are inflated, a central space
is created in the vessel, allowing for instillation of
infected cells though the port.
,, .
A region of the blood vessel is identified by
anatom~cal~andmarks ~nd~the proximalSballoon means (2) is
inflated to denude~theiendothelium bylmechanical 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
30 î i- as dispase,;trypsin, coIlagenase, 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,
W093/~52 ~ PCT/US92/05243
21~23~S !;
-12-
deoxycholate, or SDS.
The denudation conditions are adjusted to achieve
essentially complete loss of endothelium for cell transfers
or approximately 20 to 90%, preferably 50 to 75%, loss of
cells from the vessel wall for direct infection. In some
instances cell removal may not be necessary. The catheter
is then advanced so that the instillation port means (3) is
placed in the region of denuded endothelium. Infected,
transfected or normal cells are then instilled into the
discrete section of artery over thirty minutes. If the
blood vessel is perfusing an organ which can tolerate some
ischemia, e.g., skeletal muscle, distal perfusion is not a
major problem, but can be restored by an external shunt if
necessary, or by using a catheter which allows distal
perfusion. After instillation of the infected endothelial
cells, the balloon catheter is removed, and the arterial
puncture site and local skin incision are repaired. If
distal perfusion is necessary, an alternative catheter
designed to allow distal perfusion may be used.
-- B.-~ Introduction of recombinant genes directly into
ceils 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.
~, ~r ~~ QA ~ t~ ?~
~ 2~SurgicalPtechni~ues are~used as?described-~above.~
-,In~tead~o~,using infected cells,~a high titer desired-
genetic material transducing viral-vector -(105:ito 106
particles/ml) or DNA complexed to a delivery vector is
r ~ qirectly-instilled~into the~vessel wall using the double
balloon catheter technique.;;;This vector is instilled in
medium containing serum and polybrene (10 ~glml) 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
WOg3/~52 PCT/US92/OS243
"` 21123~ ;
-13-
circulation is restored. Similar protocols are used for
post operative recovery.
The vessel surface can be prepared by mechanical
denudation alone, in combination with small amounts of
proteolytic enzymes such as dispase, trypsin, collagenase
or cathepsin, or by incubation with these proteolytic
enzymes alone. The denudation conditions are adjusted to
achieve the appropriate loss of cells from the vessel wall.
Viral vector or DNA-vector complex is instilled in
Dulbecco's modified Eagle's medium using purified virus or
complexes containing autologous serum, and adhesive
molecules such as polybrene (10 ~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 perfu~ed 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.
25 ~ Honsik, R.A. Reisfeld, and--H.J. Nuller-Eberhard,`~Proc.
~atl. Acad. Sci. USA, 83, 4479 (1986); U.D. Staerz,l:O.
Kanagawa, and M.J. Bevan, ~a~ure, 314, 628 (lg85); 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.
.
W093/~2 , ~ , PCT/USg2/osi43
2112375 ~` ` f
using a different catheter design (see Figure 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
lo interaction of vectors with cells in adjacent va~cular
tissue.
, The present invention also provides for the use of
growth factors delivered locally by catheter or
systemically to enhance the efficiency of infection. In
addition to retroviral vectors, herpes virus, adenovirus,
or other viral vectors are suitable vectors for the present
technique.
It is also possible to transform cells within an organ
,or tissue. Direct transformation of organ or tissue cells
may~be,accomplished by one of two methods.; In a first
method a high pressure transfection is used. The high
pressure will cause the vector to migrate through the blood
vessel walls into the surrounding tissue. In a second
method, injection into a capillary bed, optionally after
injury/,to allow leaking, gives rise-to direct infection of
the-surrounding tissues.~ "~
. ~ ~ ... .
~,~ m e 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.
Obtainina the cells used in this invention:
W093~52 ` 2 1 1 2 3 7 ~ PCT/US92/05243
-15-
The term "genetic material" generally refers to DNA
which codes for a protein. This phrase also encompasses
RNA when used with an RNA virus or other vector based on
RNA.
Transformation is the process by which cells have
incorporated an exogenous gene by direct infection,
transfection or other means of uptake.
The term "vector" is well understood and is synonymous
with the often-used phrase "cloning vehicle". A vector is
non-chromosomal double-stranded DNA comprising an intact
replicon such that the vector is replicated when placed
within a unicellular organism, for example by a process of
transformation. Viral vectors include retroviruses,
adenoviruses, herpesvirus, papovirus, or otherwise modified
naturally occurring viruses. Vector also means a
formulation of DNA with a chemical or substance which
allows uptake by cells.
:, . .................... , -~
- ~ In-another embodiment the present invention provides
for inhibiting the expression of a gene. Four approaches
may be utilized to accomplish this goal. These include the
use of antisense^agents, either synthetic oligonucleotides
which are complementary to the NRNA (Maher III, L.J. and
- Dolnick,~-8.J~f-Arch. Biochem. Biohys.~,-253, 214-220 (1987)
and~(Zamecni~, P.~C.Y,~;;et à`l.l, Proc.~Natl.-Aaad. 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
invol~es "intracellular immunization", where analogues of
intracellular proteins can interfere specifically with
their function (Friedman, A.D., Tri~zenberg, S.J. and
W093/~52 ~ J " ~ ~ ~ PCT/US92/05~3
211237~ -16-
McKnight, S.L., Nature, 335, 452-454 (1988)), described in
detail below.
The first approaches may be used to specifically
eliminate transcripts in cells. The loss of transcript may
S 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 aIld 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 dîfferent portions of
the mes~enger RNA may-be compare~ to determine:~whether
specific regions may be more effective in preventing the
expression of these genes.
RNA can also function in an autocatalytic fashion to
, ~ -cause autolysis or to-specifically,degrade complementary
RNA ~equences~(Uhlenbeck, O.C.,~ ture, 328,~596-600
,,-~(1987),,.Haseloff,~J. and,Gerlach, W.L., Nature, 334,~
585-591,(1988), and Hutchins,,C.J.,-et al~Aucleic Acids
-~es., 14, 3627-3640 (1986)). The requirements for a
successful,,~NA cleavage include,a hammerhead str~ucture 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.
W093/~52 . 2 11 2 ~ 7 5 PCTiUsg2/0s243
-17-
Briefly, any GUG sequence within the RNA transcript can .;
serve as a target for degradation by the ribozyme. These
may be identified by DNA seguence 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
lo 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
resiitant plasmid, pSV2-Neo or another selectable marker.
20 ~The expression of these transcripts may be confirmed by
:- - Northern blot:and Slrnuclease:analysis.` When confirmed,
.. the expression of mRNA may be evaIuated by Sl nuclease
protection to determine whether expression of these
transcripts reduces steady state lev~ls of the target mRNA
and the genes which it regulates. The level of protein may
:also be examined. ;~ *~
-t~,~t~ /..Genes.may also-be~inhibited by~preparing mutan*'
.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
WO93!~W~2 ~ PCT/US92/05~3
211237~
-18-
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
10 lines, cells may be incubated with Zn or Cd to express this
gene. Its effect on host cells can then be evaluated.
Another approach to the inactivation of specific genes
is to overexpress recombinant proteins which antagonize the
expression or function of other activities. For example,
if one wished to decrease expression of TPA (e.g., in a
clinical ~etting of disseminate thrombolysis~, one could
overexpress plasminogen activator inhibitor.
..
.,.Advances in,biochemistry and molecular biology in
~,.recent years ha.ve.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,. iThei~production..-of~recombinant RNA and DNA vectors
is well,understood,and need not be described in detail.
However, a brief description of this process is included
here for reference~
. . .For example,.a retrovirus or a plasmid vector can be
- cleaved to provide,linear RNA or DNA having ligatable
termini. These termini are bound to exogenous RNA or DNA
having complementary like ligatable termini to provide a
biologically functional recombinant RNA or DNA molecule
WO 93!00052 ~f PCI`/USg2/05243
^ 211237~ `
--19--
having an intact replicon and a desired phenotypical
property.
A variety of techniques are available for RNA and DNA
recombination in which adjoining endæ of separate RNA or
DNA fragments are tailored to facilitate ligation.
The exogenous, i.e., donor, RNA or DNA used in the
present invention is obtained from suitable cells. The
vector is constructed using known techniques to obtain a
transformed cell capable of in vivo expression of the
therapeutic agent protein. The transformed cell is
obtained by contacting a target cell with a RNA or DNA--
containing formulation permitting transfer and uptake of
the RNA or DNA into the target cell. Such formulations
include, for example, retrovi~uses, plasmids, liposomal
lS formulations, or plasmids complexes with polycationic
substances such as poly-L-lysine, DEAC-dextran and
targeting ligands.
The present invention thus provides for the genetic -
alteration of cells as a method to transmit therapeutic or
diagnostic agents to localized regions of the blood vessel
for local or systemic purposes. The range of recombinant
proteins which may be expressed in these cells is broad and
varied.- It includes gene transfer-using vectors--expressing
proteins as tPA~for the treatment of thrombosis and -
restenosisi, 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.
~VO 93~00052 ~e PCI'/US92iO5W3
2 112 3 7 S -20-
For example, in the treatment of ischemic di~eases
~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 a (TGF-~), transforming growth
factor s (TGF-~), angiogenin, tumor necrosis factor a,
tumor necrosis factor ~, acidic fibroblast growth factor or
lo 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. ~n the treatment of diabetes,
genetic material coding for insulin may be used.
The present invention can also be used in the
treatment of malignancies by placing the transformed cells
in proximity to the malignancy. In this application,
genetic material coding for diphtheria toxin, pertusæis
-toxin,-or-cholera;-toxin-may-be.;:used.
In the use of the present invention in the treatment
of ~IDS, genetic material coding for soluble CD4 or
derivatives thereof may be used. In the treatment of
genetic diseases~so~iexample~ growth.hormone deficiency,
genetic material coding for tbe needed substance,~for-
example~.human:growthrhormone~ is used...All of..these c-
; -genetic materials are readily available to one skilled in
this art.
-. --..:In.another-:embodiment, the present invention provides
a rkit 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
wo ~3!~N~2` ~ 3 7 ~ PCT/US92/OS~3
-21-
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
lo detergent, the solution may contain NP-40, Triton X100,
deoxycholate, SDS or the like.
Alternatively, the kit may contain a physiological
acceptable solution which contains an agent such as
heparin, poly-L-lysine, polybrene, dextran sulfate, a
polycationic material, or bivalent antibodies. This
solution may also contain vectors or cells (normal or
transformed). In yet another embodiment the kit may
contain a catheter and both a solution which contains an
enzyme or mild de~ergent and a~solution which contains an
agent such as heparin, poly-L-lysin~e, poIybrene, dextran
sulfate, a polycationic material or bivalent antibody and
which may optionally contain vectors or cells.
The kit may contain a catheter with a single balloon
- .and central.distal perfusion port, together with acceptable
25?~- .solutions;to allow introduction bf cel~ls in a specific
organ.or~vectors into a capillary bed or cells in a ~
specific organ or tissue perfused by this capillary bed.
- Alternatively, the kit may contain a main catheter
body which has two spaced balloon elements adapted to be
inserted in a blood vessel with both being expansible
against the walls of the blood vessel for providing a
chamber in the blood vessel, and to hold the main catheter
body in place. In this case, the means for delivering a
solution into the chamber is situated in between the
WO g3/00052 .. PCr/USg2/05243
211237~ 22-
balloon elements. The kit may contain a catheter which
possesses a plurality of port means for delivering the
solution into the blood vessel.
Thus, the present invention represents a method for
treating a disease in a patient by causing-a cell attached
onto the walls of a vessel or the cells of an organ
perfused by this vessel in the patient to express an
exogeneous therapeutic agent protein, wherein the protein
treats the disease or may be useful for diagnostic
purposes. The present method may be used to treat
diseases, such as an ischemic disease, a vasomotor disease,
diabetes, a malignancy, AIDS or a genetic disease. ~-
The present method may use exogeneous therapeutic
agent proteins, such as tPA and modifications thereof,
urokinase, streptokinase, acidic fibroblast growth factor,
basic fibroblast growth factor, tumor necrosis factor a,
tumor necrosis factor ~, transforming growth factor ~,
transforming growth factor ~, atrial natriuretic factor,
platelet-derived g~owth factor, endothelian, insulin,~ '~
diphtheria toxin, pertussis toxin, cholera toxin, soluble
CD4 and derivatives thereof, and growth hormone to treat
diseases.
; T;he present method may also use exogenous proteins of
dia~no~tic value. For example,~a marker protein,~ such as-
~
~-galatosodase, may be used to monitor cell migration.
- ~, - -:, ,. . .,, - , . ;
It is preferred, that the cells caused to express t~e
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.
W093/~52 2 1 1 2 3 7 ~ PCT/US92/05243
-23-
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 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 con~irmed in that the cells exhibited growth
characteristics and morphology typical of porcine
endothelium in tissue culture. Endothelial cells also
express receptors for the acetylated form of low density
lipoprotein (AcLDL), in contrast to fibroblasts and other
mesenchymal cells (2). When analyzed for ACLDL receptor
expression, greater than 99% of the cultured cells
~ contained this receptor, as judged by fluorescent ACLDL
uptake.
Two independent ~-galactosidase-expressing
endothelial lines were isolated following infection with a
murine amphotropic ~-galactosidase-transducing retroviral
vector (BAG)i,;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 ~elected 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 8AG retrovirus was
fur~her verified by Southern blot analysis which revealed
the presence of intact proviral DNA at approximately one
copy per genome.
x ~ ~
W093/00052 PCT/US92/05~3
., ~ :,,
-24-
211Z37~
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). 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 (S0 U/ml), which removed
any remaining luminal endothelial cells. Residual enzyme
was rapidly inactivated by ~2 globulin in plasma upon
qeflating the catheter balloons and allowing blood to flow
through the vessel segment. The cultured endothelial cells
lS which expressed ~-galactosidase were introduced using a
specially designed arterial catheter (USCI, Billerica, 14A)
that contained two balloons and a central instillation port
(Figure 1).
When these balloons were inflated, a protected space
was created within the artery into which cells wexe
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
25~ removed, the arterial branch ligated, and the incision
closed.- .-. . - .~
: Segments of the artery innoculated with
~-galactosidase-expressing endothelium were removed 2 to 4
weeks later. Gross examination of the arterial specimen
after staining using the X-gal~chromagen showed multiple
areas of blue coloration, compared to an artery seeded with
uninfected endothelium, indicative of ~-galactosidase
activity. Light microscopy documented ~-galactosidase
staining primarily in endothelial cells of the intima in
experimentally seeded vessels.
W093/~52 PCT~US92/05243
237S
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 se2ded
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, ~-galactosidase staining was seen in
endothelial cells extending from the vessel wall to the
surface of the thrombus.
A major concern of gene transplantation in vivo
relates to the production of replication-competent
retrovirus from genetically engineered cells. In these
tests, this potential problem has been minimized through
the use of a replication defective retrovirus. No helper
virus-was^detectable among these-lines after 20-passages iB
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.
2S ~, A second concernrinvolves 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.
3 0 ~ J c~ ~ ï ~ .
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
W093/~52- . , PCT/US92/05243
~. ....
2 1123~ S -26-
genetically-altered endothelium as a vector.
A major complication of current interventions for
vascular disease, such as balloon angioplasty or insertion
of a graft into a diseased vessel, is disruption of the
atherosclerotic plaque and thrombus formation at sites of
local tissue trauma (5). In part, this is mediated by
endothelial cell injury (6). The present data show that
genetically-altered endothelial cells can be introduced at
the time of intervention to minimize local thrombosis.
This technique can also be used in other ischemic
settings, including unstable angina or myocardial
infarction. For instance, antithrombotic effects can be
achieved by introducing cells expressing genes for tissue
plasminogen activator or urokinase. This technology is
also useful for the treatment of chronic tissue ischemia.
For example, elaboration of angiogenic or growth factors
(7) to stimulate the formation of collateral vessels to
-- severely,ischemic tissue, cuch as the myocardium. Finally,
;--- 80mBtiC gene replacement for systemic-inherited diseases is
feasible using modifications of this endothelial cell gene
transfer technique.
ExDerimental 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
fibroblast controls were analyzed for expression of AcLDL
re~eptor.using fluorescent labelled ACLDL.
- ~Endothelial cells were derived from external jugular
veins using the neutral protease dispase (8). Excised vein
segments were filled with dispase (50 U/ml in Hanks'
balanced salt solution) and incubated at 30C for 20
s
i
W093/~52 PCT/USg2/05243
--- 211237S
-27-
minutes. Endothelium obtained by this means was maintained
in medium 199 (GIBC0, Grand Island, N.Y.) supplemented with
fetal calf serum (10~), 50 mg/~l 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'-dioctadecyl3,3,3',3'-tetramethylindocarbacyanine
perchlorate) (Dil) ACLDL (Biomedical Technologies,
Stoughton, MA) (10 ~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 cells by
catheterization.
A double balloon catheter was used for instillation of
endothelial cell~. 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 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) ; 30 were anesthetized with pentobarbital (20 mg/kg), intubated,
and mechanically ventilated. These subjects~underwent
sterile surgical exposure ~of the iliac and femoral
arterie~. 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;
~ W093/~52 PCT/US92/05243
211237 ~ ~ -28- /
the proximal balloon was partially inflated and passed
~'J' 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 ells
10 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
4 glutaraldehyde for five minutes and stored in
15 phosphate-buffered saline, and another portion was mounted
in a paraffin block for sectioning. The presence of
retroviral expressed ~-galactosidase was determined by a
standard histochemical technique (19).
. ~
~- C. Analysis of endothelial cells in vi~E~ and n
20 v vo. ;
- ~-Galactosidase activity was documented by
histochemical staining in tA) primary endothelial cells
~ from the Yucatan minipig, (B) a subline derived by
- infection with the BAG retroviral vector, tC) a segment of
~; 25 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 crosssection of artery instilled with
endothelium infected with the BAG retroviral vector.
Endothelial cells in tissue culture were fixed in 0.5
glutaraldehyde prior to histochemical staining. The
enzymatic activity of the E. coli ~-galactosidase protein
was used to identify infected endothelial cells
in vitro and in vivo. The ~-galactosidase transducing
Mo-MuLV vector (2), (BAG) was kindly provided by Dr.
i
W093/~52 PCT/US92ios243
- 211237~ :
-29-
Constance Cepko. This vector used the wild type MoMuLV LTR
as a promoter for the ~-galactosidase gene. The simian
virus 40 (SV-40) early promoter linked to the Tn5 neomycin
resistance gene provides resistance to the drug G-418 and
is inserted downstream of the ~-galactosidase gene,
providing a marker to select for retrovirus-containing,
~-galactosidase expressing cells. This defective
retrovirus was prepared from fibroblast ~ am cells (3,10),
and maintained in Dulbecco's modified Eagle's medium (DMEM)
and 10% calf serum. Cells were passaged twice weekly
following trypsinization. The supernatant, with titers of
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 37C in 5% Co2
in the presence of 8 ~g/mi of polybrene. Viral supernatants
were removed, and cells maintained in medium 199 with 10%
fetal ca}f serum, ECGS (50 ~g/ml), and endothelial cell
conditioned medium (20%) for an additional 24 to 48 hours
prior to selection in G-418 (0.7 ~g/ml of a 50% racemic
mixture). G-418 resistant cells were isolated and analyzed
for ~-galactosidase expression using a standard
-- histochemical stain (9). Cells stably expressing the
~-galactosidase enzyme were maintained in continuous
culture for use~as needed. Frozen aliquots were stored in
liquid nitrogen.
., ;
W093/~52 . ~ PCTiUS92/05~`43
211237~ -32-
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.
