Patent Application: US-41692999-A

Abstract:
methods for induction of e2f - 1 related vascular smooth muscle cell death to limit vascular stenosis or restenosis , to regress atherosclerotic plaque and to prevent atherogenesis are disclosed . also disclosed is an adenovirus vector containing the e2f - 1 gene , and a method of transferring the gene to a vessel or graft . a method of limiting cell proliferation and / or reducing cell numbers includes transferring the e2f - 1 gene into vsmc to achieve overexpression of e2f - 1 gene product , which drives vascular cells into s - phase and thereby causes their subsequent death .

Description:
vascular restenosis , a major unresolved problem for percutaneous coronary revascularization procedures , has thus far been resistant to all therapeutic strategies . the present disclosure describes a treatment strategy for restenosis which is directed toward interference with a specific cellular event that leads to neointimal formation , with a specific goal of decreasing the neointimal volume through apoptosis of proliferating vsmc . the present invention reduces the number of vsmc at the site of vascular injury by inducing their coordinated death through e2f - 1 - induced apoptosis . this coordinated death of vsmc at the site of injury reduces neointimal formation by decreasing the number of vsmc available to migrate towards the lumen , to produce growth factors , and to produce extracellular matrix ( ohno et al ., 1995 , chang et al ., 1995 ). in contrast to the cell cycle arrest genes that limit vsmc growth by preventing entry of the cells into s - phase ( ohno et al ., 1995 , chang et al ., 1995 ), e2f - 1 gene transfer drives the vsmc from g1 into s - phase , ultimately resulting in the apoptotic programmed death of the transformed vsmc . extraction of mrna from ml - 1 cells was performed using the qiagen mrna isolation kit ( qiagen , valencia , calif .). first - strand cdna was synthesized using superscript ii rnase h - reverse transcriptase ( gibco brl , gaithersburg , md .). e2f - 1 sequences were prepared by polymerase chain reaction using rtth dna polymerase xl ( perkin - elmer ,) along with the following primers : 3 ′ e2f 1_antisense : 5 ′- ccaagccctgtcagaaatcca - 3 ′ antisense primer , seq id no : 2 ( primers were designed according to the e2f - 1 sequence obtained from gene bank accession number m96577 ). the polymerase chain reaction amplified fragment was cloned into pcr - script vector , and positive clones were identified and sequenced . double - stranded dna sequencing was performed in the university of texas medical school sequencing core facility . the recombinant e2f - 1 adenovirus ( ad . e2f - 1 ) contains the human cytomegalovirus promoter , e2f - 1 cdna , and bovine growth hormone polyadenylation signal in a mini - gene cassette inserted into the e1 - deleted region of modified adenovirus type 5 ( ad5 ). replication - defective adenovirus carrying the human e2f - 1 gene ( ad . e2f - 1 ) was generated by cotransfecting the pxcjl - 1 and pjm 17 plasmids , which were provided by dr . f . l . graham ( microbix biosystems , hamilton , ontario ), into 293 cells ( mcgrory et al ., 1988 ). viral stocks were propagated in 293 cells . replication - defective adenovirus containing no foreign gene ( ad . rr ) was a gift by dr . robert d . gerard ( leuven , belgium ). recombinant adenoviruses ( ad . e2f - 1 and ad . rr ) were plaque - purified and purity of ad . e2f - 1 was established by immunoblotting for e2f - 1 on vsmc infected with virions amplified from individual plaques . high titer adenovirus was purified from 293 cells with modification of a previously described procedure ( gomez - foix et al ., 1992 ). these modifications included a 30 - min digestion of precipitated virions with benzonase ( 100 u / ml , american international chemical , inc ., natick , mass .) and the addition of 2 sequential cscl ( density 1 . 34 mg / ml ) equilibrium centrifugation steps at 180 , 000 × g for 6 hours at 4 ° c . purified recombinant virions were suspended in sucrose ( 2 % w / vol ) and mgcl 2 ( 2 mm ) in pbs , desalted by sepharose cl4b exclusion chromatography ( pharmacia corporation , piscataway , n . j . ), supplemented with 5 % glycerol and stored at − 80 ° c . the concentration of infectious viral particles was determined in 293 cells by plaque assay as described in mcgrory et al ., 1988 . all viral preparations were tested for endotoxin using a limulus amebocyte lysate assay and were found to be endotoxin - free (& lt ; 0 . 125 eu / ml ). human vascular smooth muscle cells ( vsmc ) were obtained for these studies from dr . timothy scott - burden ( texas heart institute ). passage 2 human coronary vsmc were purchased from cascade biologics , inc . ( portland , oreg .). cells were grown in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) from gibco or in medium 231 from cascade biologics , inc . for cell growth experiments , vsmc were seeded in triplicates at a density of approximately 10 × 10 3 / cm 2 and were growth arrested for 60 hours in dmem supplemented with 0 . 1 % bovine serum albumin or in medium 231 . for infection of the vsmc , recombinant adenovirus was suspended in the growth arrest medium ( dmem with 0 . 1 % bovine serum albumin or medium 231 ). cells were then infected for six hours with either the replication - deficient adenovirus encoding the human e2f - 1 gene ( ad . e2f - 1 ), with a recombinant adenovirus carrying the identical cmv promoter but no transgene ( empty virus , or ad . rr ), or with the growth arrest medium used for suspending the virus ( mock control ). multiplicities of infections ( moi , number of infectious virions / number of cells ) were from 10 to 200 . after six hours , the virus suspension or control medium was removed , and cells were washed twice with dmem and fed with dmem supplemented with 10 % fetal bovine serum . cells were counted every 24 hours , using a coulter counter ( model z1 , coulter inc .). human vsmc were plated at approximately 3 . 5 × 10 5 cells / 60mm tissue culture dish for 7 day experiments and growth arrested for 60 hours as described above ( cell count was approximately 4 . 5 × 10 5 on the day of infection ). cell density was at approximately 6 × 10 5 / 60 mm plate or 7 . 6 × 10 5 / 100 mm plate on the day of infection for 4 - day experiments . cells were then infected for six hours at the indicated moi with ad . e2f - 1 , ad . rr ( empty control virus ), or mock control ( growth arrest medium ) as described above . following removal of the virus , cells were washed twice with dmem and growth - stimulated with dmem supplemented with 10 % fbs . at 24 - hour intervals , cells were harvested for cell cycle and apoptosis analyses by dna flow cytometry . each sample was collected by pooling together detached cells in medium , attached cells , and all pbs washes before and after trypsinization . samples were centrifuged for 10 min at 1800 rpm and then resuspended in 0 . 2 ml of pbs , followed by dropwise addition of 5 ml of ice - cold 85 % ethanol while vortexing gently . fixed cells were stored at − 200 ° c . until all samples were collected . on the day of analysis , samples were centrifuged at 2500 rpm for 10 min and the ethanol was decanted . cells were then washed once with pbs , centrifuged at 3000 rpm for 10 min . each sample was resuspended in 200 or 400 μl of 100 - μg / ml propidium iodide with 50 μg / ml rnase and incubated at 37 ° c . for 20 min . at least 4 × 10 3 cells were analyzed on the coulter epics ® profile instrument ( miami , fla .). histograms were stored and files analyzed using multicycle program from phoenix flow systems ( san diego , calif .). in order to assess whether e2f - 1 was expressed in the vsmc at a time preceding the appearance of cell changes typical of apoptosis , vsmc infected with ad . e2f - 1 , ad . rr , and mock control were immunostained beginning at the completion of the six - hour infection . a mouse monoclonal antibody reactive with human e2f - 1 was used as primary antibody ( santa cruz biotechnology , santa cruz , calif .). antibody binding was visualized with dab , using a biotinylated secondary antibody and a streptavidin - biotin - horseradish peroxidase kit ( vector , burlingham , calif .). pbs with 0 . 1 % triton x - 100 was used to suspend the antibodies and for all washing steps . vsmc were plated into chamber slides ( nalgen nunc intl ., naperville , ill .) and serum - deprived for 60 hours and infected for six hours with ad . e2f - 1 , ad . rr , or mock control , as described above . after six hours , the virus was removed and cells were washed twice with dmem then growth - stimulated in dmem supplemented with 10 % fbs . cells were washed twice with phosphate - buffered saline ( pbs ), fixed for 10 min in three parts methanol and one part acetone at − 20 ° c ., and immunostained for e2f - 1 . cells were counterstained with alcian blue / methyl green in pbs , dehydrated in ethanol and coverslipped with cytoseal 60 mounting medium ( stephens scientific , riverdale , n . j .). cell morphology was evaluated by combined epifluorescence and differential interference contrast ( dic ) microscopy . briefly , cells were passaged to glass slides and kept in serum - free medium for 60 hours prior to treatment . after incubation with ad . e2f - 1 , ad . rr or growth arrest medium alone , cells were stimulated by addition of dmem with 10 % fbs . thirty hours after infection , the cells were incubated in the dark at 37 ° c . with 10 μg / ml of hoechst 33342 and 4 -( 4 -( dimethylamino ) styryl )- n - methylpyridinium iodide ( daspmi ) for staining of nuclear dna and mitochondrial membranes , respectively . dna staining and mitochondrial staining were , respectively , detected with a dapi filter and a fitc filter set . all micrographs were digitally captured on a zeiss axioskop epifluorescence microscope using an optronics ( goleta , calif .) dei - 750 ccd color camera with adobe premiere software ( adobe systems , mountainview , calif . ), a targa 2000 video board ( truevision , inc ., santa clara , calif .) and a powerpc macintosh 9500 ( apple computer , cupertino , calif .). images were edited with adobe photoshop software ( adobe systems , mountainview , calif .). growth - arrested human coronary vsmc were incubated at moi 50 with ad . e2f - 1 , ad . rr , and mock control as described above and after various time periods processed for e2f - 1 immunohistochemistry . predominant nuclear e2f - 1 immunostaining was present as early as 6 hours after start of infection as illustrated in fig1 a , or immediately after the removal of the ad . e2f - 1 in the transduced vsmc , whereas no staining was seen by 20 hours in control virus or mock treated cells ( see fig1 b ). after demonstrating that e2f - 1 was overexpressed in vsmc , dna flow cytometry analysis of the cells was performed to verify that e2f - 1 transfer had promoted s - phase entry of quiescent vsmc during growth arrest initiated by prolonged serum deprivation . growth arrested cells were infected with ad . rr , mock control and ad . e2f - 1 at moi of 100 , and kept in serum - free medium for an additional four days . cells were harvested every 24 hours and processed for dna flow cytometry . despite prolonged serum deprivation , ad . e2f - 1 ( but not ad . rr or mock control ) promoted s - phase entry in the quiescent vsmc , as shown by the flow cytometry results illustrated in fig2 . in contrast , the percentage of vsmc in s - phase remained consistently below 5 % in the ad . rr and mock - treated cells . s - phase entry of e2f - 1 transduced coronary vsmc is associated with induction of apoptosis within 24 - 36 hours after gene transfer of e2f - 1 to vsmc , the induction of apoptosis was observed . apoptotic changes , which were not observed after infection with the null adenoviral vector , ad . rr , included membrane blebbing and loss of cytoplasmic membrane integrity , cell shrinkage and detachment , chromatin condensation , and loss of mitochondrial integrity ( see fig3 a - 3c and fig4 ). loss of mitochondrial membrane integrity was visualized with daspmi , a mitochondrial membrane specific dye , as diffuse fluorescence alternating with areas of membrane condensation . chromatin condensation and fragmentation were apparent as intensified fluorescence of nuclear fragments after staining with the intercalating dna dye , hoechst 33342 . the development of apoptosis induced by e2f - 1 expression occurred in a relatively short - time frame , as shown by video time lapse microscopy . fig4 frames 1 - 9 show in phase - contrast the changes observed in a single cell from a sample of coronary vsmc transduced with e2f - 1 ( moi 100 ). surface blebbing and loss of membrane integrity with extrusion of cellular contents were nearly complete within 2 hours after the first changes were observed ( 30 - 32 hours post - infection ). in order to evaluate time course and magnitude of apoptosis in coronary vsmc transduced with e2f - 1 , the cells were infected for six hours with ad . e2f - 1 , ad . rr , or mock control at moi 10 - 200 followed by growth - stimulation in 10 % fbs . the vsmc were harvested every 24 hours and subjected to dna flow cytometry , which showed a dose - dependent induction of apoptosis by ad . e2f - 1 , as reflected by the hypodiploid cell population in sub g 1 due to cleavage of dna . increases in the sub go fraction were observed with dosages of ad . e2f - 1 as low as moi 5 ( fig5 a ). after the single application of ad . e2f - 1 , induction of apoptosis involved a percentage of cells that appeared to increase over time . the sub g 1 fraction of vsmc peaked at 85 % on day 5 with ad . e2f - 1 moi 200 and at 17 % on day 3 when a moi of 10 was used , compared to respectively , 8 % and 3 %, with ad . rr ( fig5 b ). after observing that e2f - 1 transduced vsmc undergo apoptosis , the rate of cell death by apoptosis and the rate of cell proliferation were determined to ascertain if e2f - 1 gene transfer would inhibit net growth of cultured vsmc . growth curves were established for human coronary vsmc , infected with ad . e2f - 1 , ad . rr , or mock control . when cell growth was followed for seven days , it was observed that gene transfer of e2f - 1 reduced vsmc growth after infection at moi 10 and completely abolished growth after infection at moi of 100 and 200 ( fig6 ). an addition set of quiescent vsmc were infected for 6 hours at multiplicity of infection 100 with an adenovirus ( ad ) encoding human e2f - 1 or a control virus without foreign gene ( ad . rr ), followed by serum stimulation for 6 days . e2f - 1 expression , by immunohistochemistry , was observed within 6 hours after exposure to ad . e2f - 1 . cell counts were performed in conjunction with dna flow cytometry to estimate apoptosis . by 24 hours , ad . e2f - 1 induced apoptotic features , including chromatin condensation , membrane blebbing , and cell detachment . after 2 days , 31 . 2 ± 0 . 7 % ( mean ± sd ) of ad . e2f - 1 treated cells had undergone apoptosis , as indicated by the fraction of cells in sub g1 on dna flow cytometry , and this percentage increased to 43 . 6 ± 0 . 1 % on day 4 . in contrast , only 1 . 8 ± 0 . 6 % and 5 . 37 ± 0 . 4 % of ad . rr infected vsmc were in sub g1 phase at 2 and 4 days , respectively . on day 6 , the sub g1 fraction of ad . e2f - 1 infected vsmc was still higher than that of ad . rr infected cells ( 20 . 0 ± 5 . 4 versus 6 . 5 ± 1 . 3 , p & lt ; 0 . 001 ). of the surviving ( cycling ) vsmc , the percentage in s - phase , measured daily , ranged from 22 . 4 ± 2 . 9 to 30 . 1 ± 4 . 5 in ad . e2f - 1 treated cells , whereas the cells in s - phase decreased to 3 . 2 ± 0 . 3 % in the ad . rr infected cells . daily counts of serum - stimulated vsmc showed an increase in cell number ( in 103 cells / well , n = 3 ) from 265 ± 1 . 9 ( quiescence ) to 3 , 585 ± 395 six days after infection with ad . rr , whereas ad . e2f - 1 treated cells increased to only 312 ± 54 ( p & lt ; 0 . 001 compared to ad . rr ). thus , gene transfer of e2f - 1 to human vsmc promoted s - phase entry and apoptosis and thereby markedly suppressed serum - dependent growth . these in vitro studies establish that gene transfer of e2f - 1 induces death of vsmc by inducing apoptosis and thereby reduces the proliferating vsmc mass . induction of cell death will irreversibly eliminate the vsmc as a source of paracrine and autocrine growth factors and extracellular matrix , which significantly contribute to the pathogenesis of the restenotic lesion . in addition , any potential for intimal migration of vsmc is abrogated after the vsmc dies . under approved protocols , male new - zealand rabbits ( 12 months of age ) were fed for 28 days a 0 . 75 % cholesterol - enriched chow and exhibited severe hypercholesterolemia . after this period , the rabbits were anesthetized , underwent transfemoral carotid balloon angioplasty , followed by a 30 - min . local dwell - delivery of ad . e2f - 1 virus or control ( ad . rr ) virus to the site of carotid balloon injury . ad . rr was given to the balloon injured rabbits at 1 × 10 10 pfu / ml and ad . e2f - 1 was administered at 1 × 10 10 , 1 × 10 9 , and 3 × 10 8 pfu / ml . after balloon angioplasty and virus delivery , the animals were allowed to recover and were kept for 28 days after surgery on the cholesterol - enriched diet . twenty - eight days after surgery , the animals were sacrificed , and were pressure - perfused with neutral - buffered formaldehyde using standard techniques . this model closely corresponds to the atherosclerosis model of pollman and associates ( pollman et al ., 1998 ). the injured and uninjured carotid arteries were harvested . paraffin - embedded arterial segment sections were prepared every 2 - mm , and were stained with the verhoeft / van giessen ( elastic ) stain , followed by quantitative histomorphometry , using standard equipment . the administration of ad . e2f - 1 at 3 × 10 9 pfu / ml significantly reduced atherosclerotic lesion formation compared to control virus . in addition , the lesions in the e2f - 1 ( 3 × 10 9 pfu / ml ) treated animals appeared to be less lipid - rich . yet plasma cholesterol in ad . rr ( 1 × 10 10 pfu / ml ) treated rabbits was 820 ± 116 mg / dl and 988 ± 324 mg / dl in ad . e2f - 1 ( 3 × 10 9 pfu / ml ) treated animals . these experiments were repeated in male and female new - zealand rabbits as described below , where ad . rr and ad . e2f - 1 were administered at the same dosage of 3 × 10 9 pfu / ml . expression of adenovirally mediated foreign expression was demonstrated in balloon - injured carotid arteries by immunohistochemistry with antibodies recognizing e2f - 1 ( clone kh95 , santa cruz biotechnology , inc .). anesthesia was induced in rabbits of either sex with xylazine and ketamine and maintained with isofluorane in oxygen . a left femoral cut - down was performed . after insertion of a 5 - f sheet into the left femoral artery 150 units of unfractionated heparin , 150 units / kg , were given intravenously . then , a 5 - f balloon angioplasty catheter with a 20 × 2 . 5 mm balloon was introduced into the sheath and advanced over a 0 . 014 inch guide wire to the right common carotid artery . a carotid cut - down was performed and the position of the balloon was adjusted so that its center was at the level of the branching point of the common carotid artery . this internal carotid artery was tied off and marked the center of the balloon - injured segment . the balloon was inflated 5 times for 30 sec to 8 atm , with a 60 sec interval between inflations . the catheter , with the balloon deflated under suction , was withdrawn to the caudal ( proximal ) end of the injured carotid segment , which was ligated temporarily with umbilical tape in a fashion that left included in the isolated vascular segment the proximal catheter tip . then , the guide wire was removed from the animal . through the opening of the wire lumen at the catheter tip 10 ml of physiological saline solution was introduced into the injured carotid segment to remove the blood in the injured segment . then , the distal end of the injured carotid segment was temporarily ligated with a second umbilical tape . all remaining saline was removed from the isolated carotid segment and purified recombinant adenoviral vectors at 3 × 10 9 pfu / ml were introduced through the wire lumen of the angioplasty balloon catheter into the temporarily isolated lumen of injured carotid artery in an amount sufficient to barely distend the isolated segment . ad . rr , the control vector , and ad . e2f - 1 were administered in the same manner . the suspension containing the adenoviral vectors were left in situ for 30 min , followed by their removal through the catheter . the umbilical tape was released and the catheter was withdrawn from the animal and the umbilical tape was removed from the injured carotid artery to allow return of blood flow into the carotid artery . the femoral and carotid cut - down was repaired and the rabbits were allowed to recover . dalteparin , 60 units / kg ( fragmin , pharmacia & amp ; upjohn , kalamazoo , michigan ) was given every 12 hours for 2 doses , beginning 1 hour after angioplasty . following removal of the catheter from the animal , the animals were returned to their cages and sacrificed 4 and 28 days after surgery , respectively , to confirm local expression of e2f - 1 and to evaluate the site of injury for neointima formation . four days after surgery , some animals were sacrificed after pressure perfusion - fixation with 10 % buffered formaldehyde . the balloon - injured carotid arteries were harvested and arterial rings were processed and embedded in paraffin using standard procedures . following preparation of 5 μm sections , these were post - fixed for 15 minutes in 4 % formaldehyde . after exposure for 10 minutes to 3 % h 2 o 2 in methanol , the sections were blocked for 20 minutes in 2 % horse serum in pbs and exposed for 1 hour at room temperature to a monoclonal antibody to human e2f - 1 ( clone kh95 , santa cruz biotechnology , inc .) or cytomegalovirus ( dako , carpinteria , california ) as negative control . sections were exposed for 30 min to a biotinylated horse antimouse antibody ( vectorlabs ) and incubated for 30 minutes in streptavidin - biotin - horse radish peroxidase ( vectorlabs ). antibody binding was visualized by exposure to dab ( vector labs ) and sections were counterstained in 1 % alcian blue and 2 % methyl green ( vectorlabs ) or hematoxylin & amp ; eosin , using routine procedures . pbs was used for washes . these methods confirmed e2f - 1 expression in rabbit carotid arteries treated with ad - e2f - 1 for 30 min . twenty - eight days after surgery , the rabbits were sedated and anesthetized with xylazine and ketamine . a carotid cut - down was performed on the side previously injured . then , the animal was pressure perfusion - fixed with 10 %- buffered formaldehyde and the injured carotid segment and a short segment of the contralateral carotid artery were harvested into 10 %- buffered formaldehyde . the arteries were cut in about 2 mm rings and , using routine histology procedures , embedded in paraffin . several arterial rings were placed next to each other in each mold , facilitating later preparation and evaluation on sections from ( 2 - mm ) adjacent sampling sites of the same carotid segment . about 5 - 9 arterial rings were obtained from each injured carotid segment , allowing sampling of the same number of histomorphometric measurements per carotid segment . sections used for histomorphometric analysis were stained with a verhoeft - van gieson elastic stain and magnified images were captured using a axiophot microscope ( zeiss , west germany ) and lumina videocamera ( lumina , lefs systems , co .). images were processed with software from optima imaging analysis systems 4 . 1 . measurements were performed by a technician unaware of the treatment the rabbits had received . the results of this histomorphometric analysis is shown in fig7 and 8 and indicate the gene transfer of e2f - 1 at 3 × 10 9 pfu / ml markedly suppresses neointima / arteriosclerosis lesion formation in the ad . e2f - 1 treated rabbit arteries . the success of the e2f - 1 gene transfer to prevent primary atherosclerotic lesions in the rabbit model illustrates that gene transfer of e2f - 1 can prevent atheromatous and fibroproliferative lesion formation in vivo despite severe elevation of cholesterol levels . thus , e2f - 1 gene transfer to arteries and vein grafts can be used to prevent or significantly reduce atherogenesis and fibroproliferative disorders in arteries , veins , grafts , arteriovenous fistulas and stent grafts . while the preferred embodiment of the invention has been shown and described , modifications thereof can be made by one skilled in the art without departure from the spirit and teachings of the invention . the embodiment described herein is exemplary only and is not limiting . many variations and modifications of the invention disclosed herein are possible and are within the scope of the invention . accordingly , the scope of protection is not limited by the description set out above , but is only limited by the claims which follow , that scope including all equivalent subject matter of the claims . the patents and technical publications referred to herein are incorporated by reference to the extent that they provide any necessary methods and materials not specifically set forth herein . agah , r ., kirshenbaum , l . a ., abdellatif , m ., truong , l . d ., chakraborty , s ., michael , l . h ., schneider , m . d . adenoviral delivery of e2f - 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