CA2227425A1 - Cell-based gene therapy - Google Patents

Abstract

Cell-based gene transfer is effected by administering transfected cells containing an expressible transgene into the pulmonary system of a patient, where the cells express and secrete expression products of the transgene directly into the pulmonary system. Also provided is the use of angiogenic factors in treatment of pulmonary hypertension.

Description

~~~00$~~58't 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 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.

D 0 0 0 -~-8~
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.

(~ '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.

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).

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 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.

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 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.

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 ~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 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 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 cultures.

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 ~~~QO
closed with 3-0 interrupted absorbable sutures, and the animals allowed to recover from surgery.

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 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.

~OOD~

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 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 ~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.

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 ~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.

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 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.

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 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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Claims (9)

1. A process of conducting gene therapy in a mammalian patient, which comprises administering to the pulmonary system of the mammalian patient genetically modified cells containing an expressible trans-gene which is capable of expressing at least one gene product in the patient's pulmonary circulation after administration thereto.

2. The process of claim 1 wherein the genetically modified cells are somatic cells obtained from the patient and modified by genetic engineering to introduce said expressible, trans-gene.

3. The process of claim 2 wherein the expressible trans-gene is a gene coding for an angiogenic factor.

4. The process of claim 3 wherein the expressible gene is a gene coding for an angiogenic factor selected from nitric oxide synthase, a vascular endothelial growth factor, fibroblast growth factor, angiopoietin-1, transforming growth factor-.beta. and platelet derived growth factor.

5. A process of alleviating the symptoms of pulmonary hypertension in a mammalian patient suffering therefrom, which comprises administering to the pulmonary circulation system of the patient genetically transformed somatic cells containing expressible genes coding for an angiogenic factor.

6. The process of claim 5 wherein the expressible genes code for nitric oxide synthase or vascular endothelial growth factor.

7. Use of an angiogenic factor in treatment of pulmonary hypertension.

8. Use according to claim 7 wherein the angiogenic factor is nitric oxide synthase, vascular endothelial growth factor, fibroblast growth factor, angiopoieten-1, transforming growth factor-.beta. or platelet derived growth factor.

9. Use according to claim 7 of an angiogenic factor selected from nitric oxide synthase and vascular endothelial growth factor, administered to the patient's pulmonary circulation system.