Note: Descriptions are shown in the official language in which they were submitted.
CA 02227425 1998-03-27
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FIELD OF THE INVENTION
This invention relates to medical treatments and
composition and procedures useful therein. More specifically,
it relates to cell-based gene transfer systems for
administration to the pulmonary system of a mammalian patient.
BACKGROUND OF THE INVENTION
Cell-based gene transfer is a known, albeit relatively
new and experimental, technique for conducting gene therapy on
a patient. In this procedure, DNA sequences containing the
genes which it is desired to introduce into the patient's body
(the trans-gene) are prepared extracellularly, e.g. by using
enzymatic cleavage and subsequent recombination of DNA from
the patient's cells with insert DNA sequences. Mammalian
cells such as the patient's own cells are then cultured in
vit o so as to take up the transgene in an expressible form.
The trans-genes may be foreign to the mammalian cell, or
additional copies of genes already present in the cell, to
increase the amount of expression product of the gene. Then
the cells containing the trans-gene are introduced into the
patient, so that the gene may express the required gene
products in the body, for therapeutic purposes. The take-up
of the foreign gene by the cells in culture may be
accomplished by genetic engineering techniques, e.g. by
causing transfection of the cells with a virus containing the
DNA of the gene to be transferred, by cell fusion with cells
containing the required gene, by lipofection, by electro-
poration, or by other accepted means to obtain transfected
cells. This is sometimes followed by selective culturing of
the cells which have successfully taken up the transgene in an
expressible form, so that administration of the cells to the
patient can be limited to the transfected cells expressing the
trans-gene. In other cases, all of the cells subjected to the
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CA 02227425 1998-03-27
take-up process are administered.
This procedure has in the past required administration of
the cells containing the trans-gene directly to the body organ
requiring treatment with the expression product of the trans-
gene. Thus, transfected cells in an appropriate medium have
been directly injected into the liver or into the muscle
requiring the treatment, to enter the systemic circulation of
the organ requiring treatment.
Previous attempts to introduce such genetically modified
cells into the systemic circulation of a patient have
encountered a number of problems. For example, there is
difficulty in ensuring a sufficiently high assimilation of the
genetically modified cells by the specific organ or body part
where the gene expression product is required for best
therapeutic benefit. This lack of specificity leads to the
administration of excessive amounts of the genetically
modified cells, which is not only wasteful and expensive, but
also increases risks of side effects. In addition, many of
the transplanted genetically modified cells do not survive
when administered to the systemic circulation, since they
encounter relatively high arterial pressures. Infusion of
particulate materials, including cells, to other systemic
circulations such as the brain and the heart, may lead to
adverse consequences, i.e. ischemia and even infarction.
It is an object of the present invention to provide a
novel procedure of cell based gene transfer to mammals.
It is a further and more specific object of the invention
to provide novel uses and novel means of administration of
angiogenic factors in human patients.
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SUMMARY OF THE INVENTION
The present invention is based upon the discovery that
the pulmonary system of a mammal, including a human, offers a
potentially attractive means of introducing genetically
altered cells into the body, for purposes of gene therapy,
i.e. cell based gene transfer. The pulmonary system has a
number of unique features rendering it particularly suited to
a cell-based gene transfer. Thus, low arterial pressure and
high surface area with relatively low shear in the micro-
circulation of the lungs increase the chances of survival of
the transplanted cells. High oxygenation in the micro-
circulation of the ventilated lung also improves the viability
of the transplanted cells.
Moreover, the pulmonary circulation functions as a
natural filter, and is able to retain the infused cells
efficiently and effectively. This is in contra-distinction to
other systemic circulations, such as the brain and the heart,
where the infusion of particulate materials such as cells
could lead to the aforementioned adverse consequences. The
lung presents a massive vascular system. The high surface
area of the pulmonary endothelium allows the migration of the
transplanted cells trapped in the micro-circulation across the
endothelial layer to take up residence within the perivascular
space.
The pulmonary circulation, unlike any other circulation
in the body, receives the entire output of the heart.
Accordingly, it offers the greatest opportunity to release a
gene product into the circulation. This distinct property of
the lung is particularly useful for pulmonary gene therapy and
for the treatment of a systemic, rather than a pulmonary
disorder.
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(~ 'fl-~ ~ r o a
It is believed that the transfected cells become lodged
in the small artery-capillary transition regions of the
pulmonary circulation system, following simple intravenous
injection of the transfected cells to the patient. Products
administered intravenously by appropriate means move with the
circulation to the lungs and then to the heart. The
transfected cells administered according to the invention
appear to lodge in the small artery-capillary transition
regions of the circulatory system of the lungs, from where
they deliver expression products of the trans-genes, initially
to the lungs, making the process to the present invention
especially applicable to treatment of pulmonary disorders, and
thence to the general circulation for treatment of disorders
of other body organs.
Thus, according to a first aspect of the present
invention, there is provided a process of conducting gene
therapy in a mammalian patient, which comprises administering
to the pulmonary system of the patient, genetically modified
cells containing an expressible trans-gene which is capable of
expressing at least one gene product in the pulmonary
circulation after administration thereto.
A second aspect of the present invention is the treatment
of pulmonary hypertension. Primary pulmonary hypertension
(PPH) is associated with severe abnormalities in endothelial
function, which likely play a critical role in its
pathogenesis. The vasodilatory, anti-thrombotic and anti-
proliferative factor, nitric oxide (NO) has been demonstrated
to decrease pulmonary pressures in both experimental and
clinical situations. However, long-term viral-based methods
may cause significant local inflammation. Other, previous
attempts to treat PPH have involved the use of prostacyclin,
using continuous administration, but this is a difficult and
expensive procedure, liable to give rise to side effects.
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The present invention provides, from this second aspect,
a method of alleviating the symptoms of PPH which comprises
administering to the pulmonary system of a patient suffering
therefrom, at least one angiogenic factor, or a precursor or
genetic product capable of producing and releasing into the
pulmonary circulation at least one angiogenic factor.
An embodiment of this second aspect of the present
invention is the delivery to a patient suffering from PPH of
genetically modified cells containing a gene capable of
expressing in vivo at least one angiogenic factor, by a
process of cell-based gene transfer as described above. This
second aspect of invention, however, is not limited to any
specific form of administration, but pertains generally to the
use of angiogenic factors and precursors thereof which produce
angiogenic factors in situ, in treating or alleviating the
symptoms of PPH, delivered to t:he pulmonary circulation by any
suitable means.
Specific examples of useful angiogenic factors in the
present invention include nitric oxide synthase; vascular
endothelial growth factor (VEGF) in all of its various known
forms, i.e. VEGFI6s which is the commonest and is preferred for
use herein, VEGFzos, VEGF189 and VEGFIZ1; fibroblast growth factor
(FGF), angiopoietin-1, transforming growth factor -(3 (TGF-Vii),
and platelet derived growth factor (PDGF). DNA sequences
constituting the genes for these angiogenic factors are known,
and they can be prepared by the standard methods of
recombinant DNA technologies (for example enzymatic cleavage
and recombination of DNA), and introduced into mammalian
cells, in expressible form, by standard genetic engineering
techniques such as those mentioned above (viral transfection,
cell fusion, electroporation, lipofection, use of polycationic
proteins, etc).
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In addition, however, the angiogenic factors can be
administered directly to the patient, e.g. by direct infusion
of the angiogenic factor, into the vasculature intravenously.
They can also be administered to the patient by processes of
inhalation, whereby a replication-deficient recombinant virus
coding for the angiogenic factor is introduced into the
patient by inhalation in aerosol form, or by intravenous
injection of the DNA constituting the gene for the angiogenic
factor itself (although this is inefficient). Administration
methods as used in known treatments of cystic fibrosis can be
adopted.
Angiogenic factors such as those mentioned above have
previously been proposed for use as therapeutic substances in
treatment of vascular disease. It is not to be predicted from
this work, however, that such angiogenic factors would also be
useful in treatment of pulmonary hypertension. Whilst it is
not intended that the scope of the present invention should be
limited to any particular theory or mode of operation, it
appears that angiogenic growth factors may also have
properties in addition to their ability to induce new blood
vessel formation. These other properties apparently include
the ability to increase nitric oxide production and activity,
and/or decrease the production of endothelin-1, in the
pulmonary circulation, so as to improve the balance of
pulmonary cell nitric oxide in endothelin-1 production.
In preparing cells for transformation and subsequent
introduction into a patient's pulmonary system, it is
preferred to start with mammalian cells, obtained from the
eventual recipient. Thus, somatic cells are harvested from the
eventual recipient, e.g. by removal of a safenas vein and
culture of either smooth muscle cells or endothelial cells, or
the culture of cells from other readily available tissues
including adicytes from subcutaneous fat biopsies or dermal
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fibroblasts, etc. The culture methods are standard culture
techniques with special precautions for culturing of human
cells with the intent of re-implantation.
The somatic gene transfer in vitro to the recipient
cells, i.e. the genetic engineering, is performed by standard
and commercially available approaches to achieve gene
transfer, as outlined above. Preferably, the method includes
the use of poly cationic proteins (SUPERFECT*) available
commercially which enhances gene transfer. However, other
methods such as lipofection, electroporation, viral methods of
gene transfer including adeno and retro viruses, may be
employed. These methods and techniques are well known to
those skilled in the art, and are readily adapted for use in
the process of the present invention.
The re-introduction of the genetically engineered cells
into the pulmonary circulation can be accomplished by infusion
of cells either into a peripheral vein or a central vein, from
where they move with the circulation to the pulmonary system
as previously described. The infusion can be done either in a
bolus form i.e. injection of all the cells during a short
period of time, or it may be accomplished by a continuous
infusion of small numbers of cells over a long period of time,
or alternatively by administration of limited size boluses on
several occasions over a period of time.
EXAMPLE 1 - PULMONARY ARTERY EXPLANT CULTURE
Fisher 344 rats (Charles River Co.) were obtained at 6
weeks of age and were sacrificed by overdose with ketamine and
xylazine. The main pulmonary artery was excised and
transferred immediately into a phosphate-buffered saline (PBS)
solution containing 2% penicillamine and streptomycin (Gibco
BRL). The adventitia was carefully removed with sterile
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OO~t~-oQ. °u
forceps, the artery opened longitudinally and the endothelium
removed by abrasion of the intimal surface with a scalpel.
The vessel was cut into approximately 4 millimeter square
pieces which were placed intimal surface down on individual
fibronectin-coated (Sigma) tissue culture plates (Falcon).
The explants were then grown in Dulbecco's Modified Eagle
Media with 10% fetal calf serum (FCS) and 2% penicillamine and
streptomycin (all Gibco BRL), i:n a humidified environment with
95% 02 and 5% COz at 37°C, with the media being changed every
second day. Explants were passaged using 0.05% trypsin/EDTA
(Gibco BRL) once many cells of a thin, fusiform smooth muscle
cell phenotype could be clearly seen growing from the
pulmonary artery segment, at which time the remaining
explanted tissue was removed. The cells were then grown in
DMEM with 10 FCS and 2% penicillamine and streptomycin until
they were to be used in further experiments.
EXAMPLE 2 - ALPHA-ACTIN AND VON WILLEBRAND FACTOR FLUORESCENT
STAINING
To confirm their smooth muscle cell identity and rule out
endothelial cell contamination, cells at the third passage
were plated onto cover slips and grown until 70% confluent, at
which time they were fixed in acetone at room temperature for
10 minutes. The cells were incubated with FCS for 30 minutes
at 37°C to block non-specific bonding sites, and then with a
monoclonal anti-alpha-actin antibody (5 micrograms/millilitre)
(Boehringer Mannheim) and a rabbit-derived polyclonal anti-von
Willebrand Factor antibody (1:200 dilution) (Sigma) for 60
minutes at 37°C in a covered humidified chamber. Negative
control cover slips were incubated with PBS for the same
duration of time. The cover slips were then washed in PBS,
and incubated for 60 minutes at room temperature in a PBS
solution containing a Cy3-conjugated donkey anti-mouse IgG
antibody (1:200 dilution) (Jackson ImmunoResearch
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~D00
Laboratories), a fluorescein isothiocyanate (FITC)-conjugated
goat anti-rabbit IgG antibody (1:200) (Jackson ImmunoResearch
Laboratories), and Hoescht 33258 (Sigma), a fluorescent
nuclear counterstain. The cover slips were again washed with
PBS, and mounted using a 1:1 solution of PBS and gycerol.
Slides were examined using an Olympus BX50 epifluorescent
microscope with standard fluorescein, rhodamine and auto-
fluorescent emission and excitation filters. For each cover
slip the immunofluorescence for action, vWF, and for the
nuclear counterstain Hoescht was indicated as positive or
negative.
All of the explant derived cultures were found to be at
least 97% pure smooth muscle cell with very rare endothelial
contamination. This could be attributed to the vigorous
debridement of the endothelial lining during the initiation of
the explant, and early passaging with removal of the residual
explant material.
Fluorescent Cell Labeling - Cells between the fifth and
ninth passages were grown until 80% confluent and were then
labeled with the viable fluorop:hore, chloromethyl trimethyl
rhodamine (CMTMR, Molecular Probes Inc.). CMTMR affords a
very accurate method of detecting ex vivi labeled cells, as
the molecule undergoes irreversible esterification and
glucoronidation after passing into the cytoplasm of a cell and
thereby generates a membrane-impermeable final product. This
active fluorophore is then unable to diffuse from the original
labeled cell into adjacent cells or structures, and may be
detected in vivo for several months, according to the
manufacturer. The fluorescent probe was prepared by
dissolving the lyophilized product in dimethyl sulfoxide
(DMSO) to a concentration of 10 millimolar. This solution was
stored at -20°C, an diluted to a final concentration of 25
micromolar in serum-free DMEM immediately prior to use. Cells
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were exposed to the labeling agent for 45 minutes, and were
then washed with PBS twice and the regular media (DMEM with
10% FCS and 2% penicillin and streptomycin) replaced. The
cells were grown overnight and harvested 24 hours later for
injection into the internal jugular vein of recipient Fisher
344 rats. A series of in vitro experiments was also performed
by plating the cells on cover slips and the incubating them
with the fluorophore to determine the quality and duration of
fluorescence over time.
Immediately after incubation with the fluorophore CMTMR
at a concentration of 25 micromolar, 100% of cultured cells
were found to fluoresce intensely when examined under a
rhodamine filter. Cells were also examined 48 hours and 7
days after labeling, and despite numerous cell divisions 100%
of the cells present on the cover slip continued to fluoresce
brightly.
EXAMPLE 3 - EX VIVO CELL TRANSFECTION WITH THE CMV-(3Ga1
PLASMID
The vector CMV-(3Ga1 (Clontech Inc.), which contains the
beta-galactosidase gene under t:he control of the
cytomegalovirus enhancer/promoter sequence, was used as a
reporter gene to follow the course of in vivi transgene
expression. The plasmid DNA was introduced into a JM109 stain
of E. Coli via the heat-shock method of transformation, and
bacteria was cultured overnight in LB media containing 100
micrograms/millilitre of ampicillin. The plasmid was then
purified using an endotoxin-free purification kit according to
the manufacturer's instructions (Qiagen Endotoxin-Free Maxi
Kit) , producing plasmid DNA with an A26o/Azeo ratio of greater
than 1.75, and a concentration of at least 1.0
micrograms/microliter. To avoid the use of viral vectors and
simultaneously obtain significant in vitro transfection
CA 02227425 1998-03-27
efficiencies, the Superfect method of transfection was used.
This product is composed of charged polycations around which
the plasmid DNA coils in a manner similar to histone-genomic
DNA interactions. This Superfect-DNA complex then interacts
with cell surface receptors and is actively transported into
the cytoplasm, after which the ;plasmid DNA can translocate to
the nucleus. This technique allows the transfection reaction
to be performed in the presence of serum (an important
consideration in sensitive primary cell lines), and produces
no toxic metabolites.
Cells between the fifth and ninth passages were
trypsinized the day prior to transfection to obtain a density
of 5x105 cells/dish. The following day, 5 micrograms of
plasmid DNA was mixed with 300 microlitres of serum-free DMEM
in a sterile microcentrifuge tube. The plasmid-media solution
was then vortexed with 50 microlitres of Superfect
transfection agent (Qiagen), after which the tubes were
incubated for 10 minutes at room temperature. The
transfection mixture was then combined with 3 milliliters of
DMEM with 10% FCS and 2% penicillin and streptomycin and
applied to the culture dishes after the cells had been washed
with PBS. The solution was allowed to incubate at 37°C or 2
hours, and the cells were then washed with PBS twice and the
standard media replaced. The transfected cells were allowed
to grow overnight and were then harvested 24 hours later for
animal injection. For every series of transfection reactions
that were performed, one 100 millimeter dish of pulmonary
artery smooth muscle cells was stained in vitro, to provide an
estimate of the transfection efficiency of the total series.
In a total of 15 separate transfection reactions using
the pCMV-Gal plasmid, an average transfection efficiency of
11.4% was obtained with the primary pulmonary artery smooth
muscle cells. No staining was seen in mock transfected
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CA 02227425 1998-03-27
cultures.
EXAMPLE 4 - ANIMAL SURGERY
All animal procedures were approved by the Animal Care
S Committee of St. Michael's Hospital, Toronto, Canada. Six
week old Fisher 344 rats (Charles River Co.) were anesthetized
by intraperitoneal injection of xylazine (4.6
milligrams/kilogram) and ketamine (70 milligrams/kilogram),
and the cervical area shaved and cleaned with iodine and
ethanol. A midcervical incision was made with a scalpel and
the internal, external and common jugular veins identified.
Plastic tubing of 0.02 millimetres external diameter was
connected to a 23 gauge needle and flushed with sterile saline
(Baxter). Thus tubing was then used to cannulate the external
jugular vein and was introduced approximately 5 centimetres
into the vein to what was estimated to be the superior vena
caval level, and good blood return was confirmed.
Pulmonary artery smooth muscle cells which had been
labeled with the fluorophore CMTMR, transfected with the
plasmid vector CMV-(3Gal, or were being used as a negative
control were trypsinized, and centrifuged at 850 rpm for 5
minutes. The excise media was :removed and the pellet of cells
was resuspended in a total volume of 2 millilitres of
phosphate-buffered saline (PBS). A 100 microlitre aliquot of
these resuspended cells was then taken and counted on a
hemocytometer grid to determine the total number of cells
present per millilitre of PBS. The solution was then divided
into aliquots of approximately 500,000 cells and transferred
in a sterile manner to the animal care facility. These cells
were then resuspended by gentle vortexing and injected into
the animals via the external jugular vein catheter. The
solution was infused slowly over one to two minutes and the
catheter was then flushed again with sterile saline prior to
removal. The external jugular Vein was ligated, the incision
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~~~QO
closed with 3-0 interrupted absorbable sutures, and the
animals allowed to recover from surgery.
EXAMPLE 5 - DETECTION OF FLUORESCENTLY-LABELED CELLS IN TISSUE
At three time-points (48 hours, 7 days, and 14 days)
after surgery animals (n=6 for each time-point, and n=5 total
for the negative control) were sacrificed by anesthetic
overdose, and the chest cavity was opened. The pulmonary
artery and trachea were flushed with saline, and the right and
left lungs excised. Transverse slices were taken from the
basal, medial and apical segments of both lungs, and specimens
obtained from the liver, spleen, kidney and gastroenemius
muscle of certain animals. Tissue specimens were embedded in
OCT compound (Miles Laboratories) en face, and then flash
frozen in liquid nitrogen. Ten micron sections were cut from
these frozen blocs at 2 different tissue levels separated by
at least 200 microns, and these sections were then examined
under a fluorescent microscope 'using a rhodamine filter, and
the number of intensely fluorescing cells was counted in each
en face tissue specimen.
To provide an estimate of the total number of labeled
cells present within the entire lung, the total number of
fluorescent cells counted in each lung section was averaged
over the total number of sections counted. Since the total
height of the lung was known (having been measured at the time
of sacrifice), a mathematical approximation could be made of
the total number of cells present within the lung by
multiplying the average number of cells identified per section
by the height of the lung in 10 micron sections (i.e. a lung
2.5 centimeter in height is equivalent to 2500 sections) and
then dividing that number by the total number of
fluorescently-labeled cells injected. Given that each section
was 10 microns in thickness and that in vitro each pulmonary
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artery smooth muscle cell was approximately 20 microns in
diameter, it was assumed that each cell would appear in two
sections. Therefore, this number was divided by two for
correct of the presence of 1 cell in 2 sections, but was then
multiplied by two to account for the total number of cells
present in both lungs. This final number represent an
estimate of the percentage of original fluorescently-labeled
cells which survived until the time of sacrifice.
At 48 hours after intravenous infusion of the labeled
cells, approximately 45% could :be identified within the lung.
Most of these cells appeared to be lodged in either small
arterioles or in the capillary circulation at the alveolar
level. Seven days after cell delivery a significant decrease
in the total number of fluorescent cells identified was noted
(18% vs. 45%, P=0.001), and the location of the cells also
appeared to have changed. Many bright fluorescent signals
were not identified within the pulmonary parenchyma, or were
lodged within the wall of small vascular structures. At 14
days after injection, a similar number of cells could be
identified within the lung (P>0.05), and the cells appeared to
remain in approximately the same locations as seen at 7 days.
No brightly fluorescent signals were seen in any of the lungs
injected with non-labeled smooth muscle cells.
In the spleen, liver and skeletal muscle tissue that was
examined no fluorescent signals were identified. In 2 out or
4 kidneys examined at 48 hours following injection, irregular
fluorescent signals could be identified. None of these
appeared to conform to the shape of a whole cell, and were
presumed to present those cells that were sheared or destroyed
during cell injection or shortly thereafter.
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~OOD~
EXAMPLE 6 - DETECTION OF BETA-GALACTOSIDASE EXPRESSION IN
TIS UE
At three time-points after cell-based gene transfer (48
hours, 7 days, and 14 days), animals (n=5 for each time-point,
and n=4 total for the negative control) were sacrificed and
the chest opened. The pulmonary artery was flushed with
saline and the trachea was cannulated and flushed with 2%
paraformaldehyde until the lungs were well inflated.
Transverse slices were taken from the basal, medial and apical
segments of both lungs, and specimens obtained from the liver,
spleen, kidney and gastroenemius muscle of certain animals.
The specimens were incubated in 2% paraformaldehyde with 0.2%
glutaraldehyde for 1 hour, and then rinsed in PBS. The tissue
was then incubated for 18 hours at 37°C with a chromogen
solution containing 0.2% 5-bromo-4-chloro-3-indolyl-~-D-
galactoside (X-Gal, Boehringer Mannheim), 5 millimolar
potassium ferrocyanide (Sigma), 5 millimolar potassium
ferrocyanide (Sigma), 5 millimolar potassium ferrocyanide
(Sigma), and 2 millimolar magnesium chloride (Sigma), all
dissolved in phosphate buffered saline. The specimens were
then rinsed in PBS, embedded in OCT compound (Miles
Laboratories), cut into 10 micron sections, and counterstained
with neutral red.
The en face sections were examined microscopically, and
the number of intensely blue staining cells was determined.
As one dish of cells was used for in vitro staining to
determine the transfection efficiency for each reaction
series, an estimate of the percentage of cells that were
transfected with the reporter gene plasmid pCMV-Gal could be
made for every animal. Using this information and the
mathematical calculation described for approximating the
number of fluorescent cells present, an estimate could be made
of the total number of transfected cells remaining at the time
CA 02227425 1998-03-27
of animal sacrifice. For example, if 10 blue staining cells
were seen on average in the lung sections and the lung was
approximately 2.3 centimetres i:n height, a total of 23,000
cells (10x2300) were present in the lung. If the transfection
S efficiency was 15% for that reaction series, and a total of
500,000 cells was injected, then 75,000 cells should express
the transgene, and 30.6% (23,000=75,000) of the injected cells
would have been detected at this time point.
STATISTICAL ANALYSIS
Data are presented as means~standard error of the mean.
Differences in the number of fluorescently labeled cells or
transfected cells over time were analyzed by unpaired
tests. A value of P<0.05 was accepted to denote statistical
significance .
Following incubation with the X-Gal chromogen solution,
microscopic evidence of cell-based transgene expression could
be clearly seen at 48 hours with multiple intense blue
staining cells being seen throughout the pulmonary specimens.
Quantitatively, this represented approximately 37% of the
original transfected cells that were injected. As with the
fluorescently-labeled cells most of the beta-galactosidase
expressing cells appeared to be lodged within the
microvasculature at this time-point. By seven days after
injection, a significant decline in the number of cells that
could be identified was detected (18% vs. 37%, P=0.05), and
the intensity of staining also appeared to decrease. However,
the cells appeared to have either migrated into the pulmonary
parenchyma, or were forming a portion of a vascular wall.
Fourteen days after cell-based gene transfer no significant
decrease in the number of cells identified was noted, but the
intensity of beta-galactosidase staining had decreased again.
No evidence of beta-galactosidase expression was detected in
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~0~00
any of the lungs from animals (:n=4) injected with non-
transfected smooth muscle cells. At all three time points, no
evidence of pulmonary pathology, as determined by the presence
of an abnormal polymorphonuclear or lymphocytic infiltrate,
S septal thickening or alveolar destruction, could be detected.
In the spleen and skeletal muscle of animals injected
with transfected or non-transfected smooth muscle cells, no
blue staining cells could be identified. Liver and renal
specimens from animals injected with either transfected (n=5)
or non-transfected (n=3) smooth muscle cells would
occasionally show faint blue staining across the cut edge of
the tissue (n=2 for each group), but no intense staining was
seen at any time-point, and no staining was seen further than
one high power field into the tissue. Renal tissue from
animals injected with transfected smooth muscle cells would
rarely (n=1 out of 5 specimens) demonstrate beta-galactosidase
expression within the glomerulus or distal tubular cells, but
staining was very faint, and was thought to represent
endogenous beta-galactosidase activity.
EXAMPLE 7 - DEMONSTRATION OF CELL BASED GENE TRANSFER INTO THE
PULMONARY SYSTEM
Primary cultures of pulmonary artery smooth muscle cells
(SMCs) from Fisher 344 rats were labeled with a fluorescent,
membrane-impermeable dye (CMTMR) or transfected with the beta-
galactosidase reporter gene under the control of he MCV
enhancer/promoter (pCMV-a). Transfected or labeled SMC's (5 x
106 cells/animal) were delivered to syngeneic recipient rats by
injection into the jugular vein, the animals were sacrificed
at intervals 15 minutes to 2 weeks later, and the lungs
excised and examined.
At 15 minutes post transplantation, injected cells were
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CA 02227425 1998-03-27
~v
detected mainly in the lumen of small pulmonary arteries and
arterioles, and transgene expression persisted in situ for 14
days, with no evidence immune response and minimal attrition.
Using simple geometric assumptions, it was calculated that
approximately 45~16% of the transfected cells reintroduced
into the venous circulation could be identified in the lungs
after 1 hour. 25~9% at 24 hours, 13~6% at 1 week and 16~6% at
2 weeks (n=6-8 for each group).
These experiments demonstrate that ex-vivo transfection
of a patient's somatic cells and re-introduction of the
transfected cells offers an effective non-viral gene transfer
approach, at least for the treatment of pulmonary vascular
diseases.
EXAMPLE 8 - TREATMENT OF PRIMARY PULMONARY HYPERTENSION WITH
NITRIC OXIDE SYNTHASE INTRODUCED BY CELL BASED GENE TRANSFER
Pulmonary artery smooth muscle cells (SMC) were harvested
from Fisher 344 rats, and transfected in vitro with the full-
length coding sequence for eNOS under the control of the CMV
enhancer/promoter. 13 syngenetic rats were injected with 80
mg/kg of monocrotaline subcutaneously, and of these, 7 were
randomized to receive eNOS transfected SMC (5x105) via the
jugular vein. 28 days later right ventricular (RV) pressure
was measured by means of a Millar micro-tip catheter and
pulmonary histology examined.
ENDS gene transfer significantly reduced systolic RV
pressure from 52+/-6 mm Hg in control animals (monocrotaline
alone, n=6) to 33+/-7 in the eNOS treated animals (n=7,
p=0.001). Similarly, RV diastolic pressures were reduced from
15+/-7 mm Hg in the controls, to 4+/-3 in the eNOS treated
animals (p=0.0055). In addition, there was a significant
attenuation of the vascular hypertrophy and neomuscularization
18
CA 02227425 1998-03-27
t~ fl~ '
of small vessels in the animals treated with eNOS.
Cell-based gene transfer of the nitric oxide synthase to
the pulmonary vasculature is thus an effective treatment
strategy in the monocrotaline model of PPH. It offers a novel
approach with possibilities for human therapy.
EXAMPLE 9 - TREATMENT OF PRIMARY PULMONARY HYPERTENSION WITH
VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) BY CELL BASED GENE
TRANSFER
Pulmonary artery smooth muscle cells (SMC) were harvested
from Fisher 344 rats, and transfected in vitro with the full-
length coding sequence for VEGF:165 under the control of the MCV
enhancer/promoter. 15 syngeneic rates were injected with 80
mg/kg of monocrotaline subcutaneously, and of these 9 were
randomized to receive VEGF transfected SMC (5x105) via the
jugular vein. 28 days later right ventricular (RV) pressure
was measured by means of a Miller micro-tip catheter, right
ventricular weight/left ventricular weight (RV/LV) ratios
determined and pulmonary histology examined.
VEGF gene transfer significantly reduced systolic RV
pressure from 52+/-6 mm Hg in control animals (monocrotaline
alive, n=6) to 34+/-6 in the VEGF treated animals (n=9,
p=0.0001). Similarly, RV diastolic pressures were reduced
from 15+/-7 mm Hg in the controls, to 3.3+/-3.54 in the VEGF
treated animals (p=0.0013). The RV/LV ratio, an indicator of
RV hypertrophy, was reduced (0..33+/-0.058 vs 0.22+/-0.033 in
control and VEGF animals respectively, p=0.0008). Moreover,
there was a significant attenuation of the vascular
hypertrophy and neomuscularization of small vessels in the
animals treated with VEGF.
These results indicate that the cell-based gene transfer
19
CA 02227425 1998-03-27
of VEGF to the pulmonary vasculature is an effective treatment
in the monocrotaline model of PPH, and supports a novel
therapeutic role for this potent angiogenic factor.
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