Patent Application: US-51789800-A

Abstract:
the invention provides a nucleic acid delivery vehicle with or having been provided with at least a tissue tropism for fibroblast - like or macrophage - like cells , preferably synoviocytes . in one aspect the nucleic acid delivery vehicle is a virus capsid or a functional part , derivative and / or analogue thereof . preferably , the virus capsid is an adenovirus capsid . preferably , the adenovirus is a subgroup b adenovirus , preferably adenovirus 16 . preferably , the tissue tropism is provided by at least a tissue tropism determining part of an adenovirus fiber protein or a functional derivative and / or analogue thereof . the invention further presents methods for the treatment of diseases , preferably joint related diseases .

Description:
the invention is further explained by the use of the following illustrative , detailed examples . generation of adenovirus template clones lacking dna encoding for fiber . the fiber coding sequence of ad5 is located between nucleotides 31042 and 32787 . to remove the ad5 dna encoding fiber we started with construct pbr / ad . bam - ritr ( fig1 ; deposited under ecacc deposit p97082122 ). from this construct , first a ndei site was removed . for this purpose , pbr322 plasmid dna was digested with ndei after which protruding ends were filled using kienow enzyme . this pbr322 plasmid was then re - ligated , digested with ndei and transformed into e . coli dh5a . the obtained pbr / dndei plasmid was digested with scai and sali and the resulting 3198 bp vector fragment was ligated to the 15349 bp scai - sali fragment derived from pbr / ad . bamitr , resulting in plasmid pbr / ad . bam - ritrdndei which hence contained a unique ndei site . next a pcr was performed with oligonucleotides “ ny - up ” and “ ny - down ” ( fig2 ). during amplification , both a ndei and a nsii restriction site were introduced to facilitate cloning of the amplified fiber dnas . amplification consisted of 25 cycles of each 45 sec . at 94 ° c ., 1 min . at 60 ° c ., and 45 sec . at 72 ° c . the pcr reaction contained 25 pmol oligonucleotides ny - up or ny - down , 2 mm dntp , pcr buffer with 1 . 5 mm mgcl 2 , and 1 unit of elongase heat stable polymerase ( gibco , the netherlands ). one - tenth of the pcr product was run on an agarose gel which demonstrated that the expected dna fragment of ± 2200 bp was amplified . this pcr fragment was subsequently purified using geneclean kit system . ( bio101 inc .) then , both the construct pbri / ad . bam - ritrdndei as well as the pcr product were digested with restriction enzymes ndei and sbfi . the pcr fragment was subsequently cloned using t4 ligase enzyme into the ndei and sbfi sites thus generating pbr / ad . bamrdfib ( fig3 ). to enable amplification of the dnas encoding fiber protein derived from alternative serotypes degenerate oligonucleotides were synthesised . for this purpose , first known dna sequences encoding for fiber protein of alternative serotypes were aligned to identify conserved regions in both the tail region as well as the knob region of the fiber protein . from the alignment , which contained the nucleotide sequence of 19 different serotypes representing all 6 subgroups , ( degenerate ) oligonucleotides were synthesised ( see , table i ). also shown in table i is the combination of oligonucleotides used to amplify the dna encoding fiber protein of a specific serotype . the amplification reaction ( 50 ml ) contained 2 mm dntps , 25 pmol of each oligonucleotide , standard 1 × pcr buffer , 1 , 5 mm mgcl 2 , and 1 unit pwo heat stable polymerase ( boehringer mannheim ) per reaction . the cycler program contained 20 cycles , each consisting of 30 sec . 94 ° c ., 60 sec . 60 - 64 ° c ., and 120 sec . 72 ° c . one - tenth of the pcr product was run on an agarose gel to demonstrate that a dna fragment was amplified . of each different template , two independent pcr reactions were performed . all amplified fiber dnas as well as the vector ( pbr / ad . bamrdfib ) were digested with ndei and nsii . the digested dnas were subsequently run on a agarose gel after which the fragments were isolated from the gel and purified using the geneclean kit ( bio101 inc ). the pcr fragments were then cloned into the ndei and nsii sites of pbr / adbamrdfib , thus generating pbr / adbamrfibxx ( where xx stands for the serotype number of which the fiber dna was isolated ). the inserts generated by pcr were sequenced to confirm correct amplification . the obtained sequences of the different fiber genes are shown in fig4 . to enable efficient generation of chimaeric viruses an avrii fragment from the pbr / adbamrfib16 , pbr / adbamrfib28 , pbr / adbamrfib40 - l constructs was subcloned into the vector pbr / ad . bam - ritr . pac # 8 ( ecacc deposit # p97082121 ) replacing the corresponding sequences in this vector . pbr / ad . bam - ritr . pac # 8 has the same adenoviral insert as pbr / ad . bam - ritr but has a paci site near the ritr that enables the itr to be separated from the vector sequences . the construct pwe / ad . aflii - eco was generated as follows . pwe . pac was digested with clai and the 5 prime protruding ends were filled in with klenow enzyme . the dna was then digested with paci and isolate from agarose gel . pwe / afliiritr was digested with ecori and after treatment with klenow enzyme digested with paci . the large 24 kb . fragment containing the adenoviral sequences was isolated from agarose gel and ligated to the clai digested and blunted pwe . pac vector . use was made of the ligation express kit from clontech . after transformation of xl10 - gold cells from stratagene , clones were identified that contained the expected construct . pwe / ad . alfii - eco contains ad5 sequences from base pairs 3534 - 27336 . three constructs , pclipsal - luc ( fig5 ) digested with sali , pwe / ad . aflii - eco digested with paci and ecori and pbr / adbamr . pac / fibxx digested with bamhi and paci were transfected into adenovirus producer cells ( per . c6 , fallaux et al ., 1998 ). fig6 schematically depicts the method and fragments used to generate the chimaeric viruses . only pbr / ad . bamrfib12 was used without subcloning in the paci containing vector and therefore was not liberated from vector sequences using paci but was digested with clai which leaves approximately 160 bp of vector sequences attached to the right itr . furthermore , the pbr / ad . bamrfib12 and pbr / ad . bamrfib28 contain an internal bamhi site in the fiber sequences and were therefor digested with sali which cuts in the vector sequences flanking the bamhi site . for transfection , 2 mg of pclipsal - luc , and 4 mg of both pwe / ad . aflii - eco and pbr / adbamr . pac / fibxx were diluted in serum free dmem to 100 ml total volume . to this dna suspension 100 ml 2 . 5 × diluted lipofectamine ( gibco ) in serum - free medium was added . after 30 minutes at room temperature the dna - lipofectamine complex solution was added to 2 . 5 ml of serum - free dmem which was subsequently added to a t25 cm 2 tissue culture flask . this flask contained per . c6 cells that were seeded 24 - hours prior to transfection at a density of 1 × 10 6 cells / flask . two hours later , the dna - lipofectamine complex containing medium was diluted once by the addition of 2 . 5 ml dmem supplemented with 20 % foetal calf serum . again 24 hours later the medium was replaced by fresh dmem supplemented with 10 % foetal calf serum . cells were cultured for 6 - 8 days , subsequently harvested , and freeze / thawed 3 times . cellular debris was removed by centrifugation for 5 minutes at 3000 rpm room temperature . of the supernatant ( 12 . 5 ml ) 3 - 5 ml was used to infect again per . c6 cells ( t80 cm 2 tissue culture flasks ). this re - infection results in full cytopathogenic effect ( cpe ) after 5 - 6 days after which the adenovirus is harvested as described above . 10 ml of the above crude cell lysate was used to inoculate a 1 liter fermentor which contained 1 - 1 . 5 × 10 6 per . c6 cells / ml growing in suspension . three days after inoculation , the cells were harvested and pelleted by centrifugation for 10 min at 1750 rpm at room temperature ( rt ). adenovirus present in the pelleted cells was subsequently extracted and purified using the following downstream processing protocol . the pellet was dissolved in 50 ml 10 mm napo 4 − and frozen at − 20 ° c . after thawing at 37 ° c ., 5 . 6 ml deoxycholate ( 5 % w / v ) was added . the solution was mixed and incubated for 15 minutes at 37 ° c . to completely lyse the cells . after homogenising the solution , 1875 ml 1m mgcl 2 and 5 ml glycerol was added . after the addition of 375 ml dnase ( 10 mg / ml ) the solution was incubated for 30 minutes at 37 ° c . cell debris was removed by centrifugation at 1880 × g for 30 minutes at rt without brake . the supernatant was subsequently purified from proteins by extraction with freon ( 3 ×). the cleared supernatant was loaded on a 1m tris / hcl buffered caesium chloride block gradient ( range : 1 . 2 / 1 . 4 gr / ml ) and centrifuged at 21000 rpm for 2 . 5 hours at 10 ° c . the virus band is isolated after which a second purification using a 1m tris / hcl buffered continues gradient of 1 . 33 gr / ml of caesium chloride was performed . the virus was then centrifuged for 17 hours at 55000 rpm at 10c . the virus band is isolated and sucrose ( 50 % w / v ) is added to a final concentration of 1 %. excess caesium chloride is removed by dialysis ( three times 1 hr at rt ) in dialysis slides ( slide - a - lizer , cut off 10000 kda , pierce , usa ) against 1 . 5 ltr pbs supplemented with cacl 2 ( 0 . 9 mm ), mgcl 2 ( 0 . 5 mm ) and an increasing concentration of sucrose ( 1 , 2 , 5 %). after dialysis , the virus is removed from the slide - a - lizer after which it is aliquoted in portions of 25 and 100 ml upon which the virus is stored at − 85 ° c . to determine the number of virus particles per ml , 50 ml of the virus batch is run on an high pressure liquid chromatograph ( hplc ) as described by shabram et al . ( 1997 ) using a 300 - 600 mm nacl gradient . the virus titer of the chimaeric virus was found to be in the same range as the ad5 . clip . luc virus batch ( ad5 . clip . luc : 2 . 2 × 10 11 vp / ml ; ad5 . luc - fib16 : 3 . 1 × 10 12 vp / ml ). to investigate the biodistribution of the chimaeric adenovirus ad5 . luc - fib16 in comparison to ad5 based luciferase viruses , 1 × 10 10 particles of each of the virus batches were diluted to 1 ml with pbs and the virus was injected in the tail vein of adult male wag / rij rats ( 3 rats / virus ). forty - eight hours after the administration of the virus , the rats were sacrificed after which the liver , spleen , lung , kidney , heart and brain were dissected . these organs were subsequently mixed with 1 ml of lysis buffer ( 1 % triton x - 100 in pbs ) and minced for 30 seconds to obtain a protein lysate . the protein lysate was tested for luciferase activity and the protein concentration was determined . the results , shown in table ii , demonstrate that the ad5 is targeted for a large part to the liver and to the spleen , whereas the ad5 . luc - fib16 chimearic virus is not . this experiment shows that it is possible to circumvent the uptake of adenoviruses by the liver by making use of fibers of other serotypes . another batch of ad5 . luc - fib16 was made by using 10 ml crude extract to inoculate a 1 liter fermentor which contained 1 - 1 . 5 × 10 6 cells / ml per . c6 that were specifically adapted to grow in suspension . three days after inoculation , the cells were harvested and pelleted by centrifuging for 10 min at 1750 rpm at room temperature . the chimeric adenovirus present in the pelleted cells was subsequently extracted and purified using the following downstream processing protocol . the pellet was dissolved in 50 ml 10 mm napo 4 − and frozen at − 20 ° c . after thawing at 37 ° c ., 5 . 6 ml deoxycholate ( 5 % w / v ) was added after which the solution was homogenated . the solution was subsequently incubated for 15 minutes at 37 ° c . to completely crack the cells . after homogenising the solution , 1875 μl ( 1m ) mgcl 2 − was added and 5 ml 100 % glycerol . after the addition of 375 μl dnase ( 10 mg / ml ) the solution was incubated for 30 minutes at 37 ° c . cell debris was removed by centrifugation at 1880 × g for 30 minutes at room temperature without the brake on . the supernatant was subsequently purified from proteins by loading on 10 ml of freon . upon centrifugation for 15 minutes at 2000 rpm without brake at room temperature three bands are visible of which the upper band represents the adenovirus . this band was isolated by pipetting after which it was loaded on a tris / hcl ( 1m ) buffered caesium chloride block gradient ( range : 1 . 2 to 1 . 4 gr ./ ml ). upon centrifugation at 21000 rpm for 2 . 5 hours at 10 ° c ., the virus was purified from remaining protein and cell debris since the virus , in contrast to the other components , does not migrate into the 1 . 4 gr / ml caesium chloride solution . the virus band is isolated after which a second purification using a tris / hcl ( 1m ) buffered continues gradient of 1 . 33 gr ./ ml of caesium chloride is performed . after virus loading on top of this gradient , the virus is centrifuged for 17 hours at 55 . 000 rpm at 10 ° c . subsequently , the virus band is isolated and after the addition of 30 μl of sucrose ( 50 w / v ) excess caesium chloride is removed by three rounds of dialysis , each round comprising of 1 hour . for dialysis the virus is transferred to dialysis slides ( slide - a - lizer , cut off 10 . 000 kda , pierce , usa ). the buffers used for dialysis are pbs which are supplemented with an increasing concentration of sucrose ( round 1 to 3 : 30 ml , 60 ml , and 150 ml sucrose ( 50 % w / v ) l 1 . 5 liter pbs , all supplemented with 7 . 5 ml 2 % ( w / v ) camgcl 2 ). after dialysis , the virus is removed from the slide - a - lizer after which it is aliquoted in portions of 25 and 100 μl upon which the ad5 . luc - fib16 virus is stored at − 85 ° c . to determine the number of virus particles per milliliter , 50 μl of the virus batch is run on a high - pressure liquid chromatograph ( hplc ). the adenovirus is bound to the column ( anion exchange ) after which it is eluted using a nacl gradient ( range 300 - 600 mm ). by determination of the area under the virus peak the number of virus particles can be calculated . to determine the number of infectious units ( iu ) per ml present in a virus batch , titrations are performed on 911 cells . for this purpose , 4 × 10 4 911 cells are seeded per well of 96 - well plates in rows b , d , and f in a total volume of 100 μl per well . three hours after seeding the cells are attached to the plastic support after which the medium can be removed . to the cells a volume of 200 μl is added , in duplicate , containing different dilutions of virus ( range : 102 times diluted to 2 × 10 9 ). by screening for cpe the highest virus dilution which still renders cpe after 14 days is considered to contain at least one infectious unit . using this observation , together with the calculated amount of virus volume present in these wells renders the number of infectious units per ml of a given virus batch . chimeric viruses display differences in synoviocyte cell transduction infection of human synoviocytes in a first set of experiments , 50 . 000 synoviocytes ( derived from i individual ) were seeded in each well of a 24 - wells plate in a volume of 1 ml per well . twenty - four hours after seeding , the cells were washed with pbs after which 200 μl of dmem supplemented with 2 % fcs was added to the cells . this medium contained various amounts of virus ( a multiplicity of infection ( moi ) of 50 , 250 , 1250 , 2500 , 5000 , and 10000 vp / cell was used ). viruses were either ad5 . clip . luc or ad5 . luc - fib16 . two hours after addition of virus the medium was replaced by normal medium thus removing the non - bound virus ( each infection in duplicate ). again forty - eight hours later cells were washed and lysed by the addition of 100 μl lysis buffer after which luciferase transgene expression was monitored . in fig8 , results are shown of the luciferase transgene expression per microgram protein after infection of synoviocytes . these results show that the fiber 16 chimeric adenovirus infects synoviocytes significantly better , based on transgene expression , as compared to the control ad5 . the fold increase of the fiber 16 chimeric adenovirus over the control ad5 ranged , depending on the moi used , from 2 . 4 × ( moi 50 ) to 1052 × ( moi 10000 ). identical experiments demonstrated on average ( n = 4 ) at least a factor 100 difference in transgene expression between the ad5 and the fiber 16 chimeric adenovirus . in a second set of experiments , an equal number of virus particles was added to different concentrations of synoviocytes . this experiment was performed since it is possible that the efficiency of infection of these cells is dependent on the confluency of the synoviocyte cell layer . a highly confluent cell layer may mimic the in vivo situation better . for this purpose , synoviocytes were seeded at concentrations of 12 . 500 , 25 . 000 , 50 . 000 , and 100 . 000 cells per well of 24 - well plates ( in duplicate ). twenty - four hours later these cells were infected as described above with medium containing 2 . 5 × 10 8 virus particles . the result of the luciferase transgene expression determined 48 hours after a two hours infection procedure ( see , fig9 ) shows that the fiber 16 chimeric adenovirus renders a ± 1000 fold higher expression of luciferase and thus is clearly better suited to infect synoviocytes also when cells are 100 % confluent . in a third set of experiments , we determined the differences in the level of luciferase transgene expression versus the time of virus exposure . this experiment was performed to demonstrate that the binding kinetics of the fiber 16 chimeric adenovirus is different from that of the ad5 control virus . for this purpose , 15 . 000 synoviocytes were seeded in 24 - well plates in a volume of 1 ml . twenty - four hours later , cells were infected ( in triplicate ) with an moi of 50 , 500 , or 5 . 000 vp / cell infection was allowed to proceed either for two hours or for 20 hours . the results , shown in fig1 , demonstrate that binding kinetics and characteristics of the fiber 16 chimeric adenovirus is distinct from that of the control ad5 and that the fiber 16 chimeric adenovirus infects synoviocytes much more efficient as compared to the control ad5 virus . from the described results , it is clear that the fiber 16 chimeric virus is better suited to infect synoviocytes as compared to the ad5 . since it is known that ad5 requires the coxacki adenovirus receptor (“ car ”) and the integrins α v β3 and α v β5 for entry , we monitored expression of these molecules on synoviocytes using flow cytometry . for this purpose , 1 × 10 5 synoviocytes were transferred to tubes designed specifically for flow cytometry . cells were washed once with pbs / 0 . 5 % bsa after which the cells were pelleted by centrifugation for 5 minutes at 1750 rpm at room temperature . subsequently , 10 μl of a 100 times diluted α v β3 antibody ( mab 1961 , brunswick chemie , amsterdam , nl ), a 100 times diluted antibody α v β5 ( antibody ( mab 1976 , brunswick chemie , amsterdam , nl ), or 2000 times diluted car antibody ( a gift from dr . bergelson , harvard medical school , boston , usa ( hsu et al ., 1988 ) was added to the cell pellet after which the cells were incubated for 30 minutes at 4 ° c . in a dark environment . after this incubation , cells were washed twice with pbs / 0 . 5 % bsa and again pelleted by centrifugation for 5 minutes at 1750 rpm room temperature . to label the cells , 10 μl of rat - anti - mouse igg1 labelled with phycoerythrine ( pe ) was added to the cell pellet upon which the cells were incubated for 30 minutes at 4 ° c . in a dark environment . finally , the cells were washed twice with pbs / 0 . 5 % bsa and analysed on a flow cytometer . the results of this experiment are shown in table iii . these flow cytometric results demonstrate that synoviocytes do not express detectable levels of car , which may be at least one of the reasons that these cells are difficult to transduce with the ad5 . as a control for the experiments performed on synoviocytes , a549 and per . c6 cells were infected . these cell lines can be readily infected by ad5 . this experiment is performed to investigate whether the observed differences on the synoviocytes can indeed be attributed to differences in cell binding or that the differences are caused by differences in virus particle per infectious unit ratio . for this purpose , 10 5 a549 cells were seeded in 24 - well plates in a volume of 200 μl . two hours after seeding the medium was replaced by medium containing different amounts of particles of either ad5 . luc - fib16 or ad5 . clip . luc ( moi = 0 , 5 , 10 , 25 , 100 , 500 ). twenty - four hours after the addition of virus , the cells were washed once with pbs after which the cells were lysed by the addition of 100 μl lysis buffer to each well ( 1 % triton x - 100 in pbs ) after which transgene expression ( luciferase activity ) and the protein concentration was determined . subsequently , the luciferase activity per μg protein was calculated . these data , shown in table iv , demonstrate that when using a identical amount of virus particles , differences in transgene expression observed in relevant cell types is due to differences in binding and / or internalisation of the virus and not to the amount of virus used . a similar experiment was performed on per . c6 cells using ad5 and the fiber chimera fiber 16 . for this purpose , 1 per . c6 cells , were seeded in 24 - wells plates in a total volume of 100 μl . three hours after seeding , the medium was replaced by medium containing 10 6 particles of either ad5 . clip . luc or ad5 . luc - fib16 ( moi = 10 ). twenty - four hours after addition of the virus , cells were washed once with pbs after which 100 μl lysis buffer was added to the attached cells . the lysate was subsequently used to determine transgene expression ( luciferase activity ) and the protein concentration . the results , shown in table v , again demonstrate that the differences in infection efficiency as observed on synoviocytes , in favour of the fiber 16 chimeric adenovirus , are differences related to binding efficiency rather than to the amount of virus used . the adenoviral vectors used in this study contain the recombinant gene inserted into the e1 region of an ad type 5 mutant . the cytomegalovirus promoter (“ cmv ”) and the major late promoter (“ mlp ”) were used to drive gene expression in the constructs harbouring the lacz and luciferase marker genes . mlp was used to drive gene expression in the ad harbouring the tk gene . virus concentrations were determined by titration of the virus . ad were tested to contain no replication competent wild - type ad or e1a recombination . the adenoviral vectors ig . ad . cmv . lacz , ig . ad . mlp . lacz , ig . ad . cmv . luc , ig . ad . mlp . luc and ig . ad . mlp - i . tk and their production have been previously described in detail ( imler et al . ; vincent et al ., 1996 ). human synovium was obtained from patients with ra defined by ara - criteria 1987 ( arnett et al ., 1988 ) at the time of joint replacement surgery . synovial tissue was collected in sterile phosphate buffered saline ( pbs ). fat and connective tissue were discarded and tissue was incubated with 0 . 5 mg collagenase / ml for 2 h at 37 ec . cells were washed and seeded in 75 - cm 2 flasks in 10 ml of iscoves modified dulbeco &# 39 ; s medium ( imdm ) 17 % fetal calf serum ( fcs ). medium was refreshed twice a week . confluent cultures of adherent synoviocytes were passaged at a 1 : 2 ratio in 75 - cm 2 flasks . the cells were detached from the flasks with 1 . 5 ml 0 . 25 % trypsin - edta dissolved in pbs at room temperature . the day prior to infections , synovial cells were plated at a density of 100 , 000 per 25 - cm 2 bottle in reporter gene experiments or 5 , 000 per well ( 24 wells plate ) in tk experiments . cells were cultured in respectively 10 or 1 ml of imdm 17 % fcs . in the procedure of infection of synoviocytes , medium was replaced by the appropriate dose of modulated virus in imdm 17 % fcs . after 2 days of incubation the number of synoviocytes were counted in a negative control and in a sample incubated with virus concentration multiplicity of infection ( moi ) 100 . remaining samples were washed with pbs , fixed briefly with glutaraldehyde 0 . 25 %, washed with pbs ( 2 ×) and stained by immersion in 5 mm k 4 fe ( cn ) 6 , 5 mm k 3 fe ( cn ) 6 , 2 mm mgcl 2 in pbs containing 0 . 5 mg / ml of x - gal stain ( 5 - bromo - 4 - chloro - 3 - indolyl - 8 - d - galactopyranoside ; sigma chemical co ., st . louis , mo ., usa ). after four to six hours samples were washed twice and the reaction was stopped by glutaraldehyde 0 . 25 % percentage of infected cells was assessed by light microscopy after counting at least 300 cells ( magnification 10 × 40 ). after 3 days of incubation with ad . luc synoviocyte counts were made comparable to lacz experiment . remaining samples were washed with pbs and trypsinised briefly . synoviocytes were lysed using 200 : 1 lysis buffer . samples of 20 : 1 were analysed by luminometric methods . one day after incubation with ad . tk medium was replaced by imdm 40 % normal human serum (“ nhs ”). in half of the cultures 10 μg gcv ( 9 -[ 1 , 3 - dihydrate - 2 - propoxy ] methyl ] guanine , roche nederland bv , nl ) was added per ml medium . medium plus or minus gcv was refreshed on day 3 . cells counts were made 5 days after virus infection . in the tk - bystander killing experiment , one 75 - cm 2 flask with synoviocytes was trypsinised and divided over three flasks . two flasks were infected with respectively ig . ad . mlpi . tk or ig . ad . cmv . tk . one day later infected and non - infected cells were mixed according to scheme ( see , fig1 ). medium was replaced by imdm 40 % nhs plus or minus gcv . cell counts were made after 7 days . all animal protocols were approved by the medical ethical committee and performed according to institutional guidelines . 8 adult rhesus monkeys ( macaca mulatta ) suffering from cia ( bakker , 1992 ) were used for these experiments and held under d2 containment . before handling , monkeys were anaesthetised with a single intramuscular dose of approximately 1 ml of 85 - 90 % ketamine [ 100 : 1 / kg , 10 mg / ml ] ( asp pharma bv oudewater , nl ) and 10 - 15 % vetranquil . if an animal was experiencing severe pain it was given twice a day 0 . 06 mg burprenorfine ( temgesic - r , schering - plough bv , amstelveen , nl ). before intra - articular punction the area surrounding knees was shaved and rinsed with iodine . using sterile technique , respectively 1 ml or 0 . 1 ml of purified recombinant virus suspended in pbs was injected according to scheme into the intra - articular space of the knee or proximal interphalangeal joint ( pip ). beginning forty - eight hours after injection of the virus , monkey 7 and 8 received 10 mg / kg gcv infused in half an hour , daily for fourteen days . animals were killed by intracordial punction and bleeding . for summary of rhesus monkeys experiments see table vi . animals were monitored daily for general health , which included recording of behaviour , appetite and stool consistency . evaluation of biochemical parameters was performed on a number of days after virus administration ( see , table vi ). for this purpose , animals were sedated as described above , body weight and rectal temperature were measured and venous blood samples were collected [ clotted and sodium ethylenediamine tetra - acetic acid ( edta )- treated blood ]. analysis of the blood serum included electrolytes ( na , k , cl and bicarbonate ); kidney function ( urea , creatinine ) and liver function [ alkaline phosphatase , asparagine - aminotransferasc ( asat ), alanine - aminotransferase ( alat ); lactate dehydrogenate ( ldh ) and total bilirubin ]; total protein and albumin ; and haematological parameters ( red and white blood cell counts , differential count , platelet count , erythrocyte sedimentation rate ( esr ). in monkey 5 - 8 venous blood was drawn in clot tubes and analysed for the presence of antibodies against ad by complement fixation assay , according to routine procedures at the department of infectious diseases and immunology ( ssdz delft , nl ). faeces , urine and pharyngeal swabs were collected on different sampling days ( see , table vi ) and stored frozen . analysis consisted of culturing extracts on 293 cells ( growth of wild - type and recombinant virus ) and hep - 2 cells ( growth of wild - type virus )( bout et al ., 1994 ). a complete post - mortem necropsy and histopathological examination of aorta , axillary lymphnodes , bladder , colon , duodenum , hart , inguinal lymphnodes , lung , liver , iymphnodes of the lung hilus , spleen , left kidney , oesophagus , pancreas , thyroid gland , skeleton muscle , bone marrow , thymus , trachea , cervix / vagina and ovary orprostate and testis were performed . samples of these tissues were fixed in 10 % phosphate buffered formalin for routine histopathological analysis . in addition in monkey 1 - 5 snap frozen samples of axillary lymphnodes , hart , inguinal lymphnodes , liver , spleen , left kidney , lung , bladder , oesophagus , bone marrow and synovium injected joints and non - injected control joints were taken for luciferase assay ( sawchuk , 1996 ). joints were opened , coloured with x - gal staining solution ( roessler et al ., 1993 ; bout et al ., 1993 ) and post - fixed in formalin for at least 72 hours . joints were cut using a diamond saw , subsequently pieces were imbedded in plastic and 6 : slices were cut using a microtome . slices were stained with haematoxylin and eosin according to standard procedures at the pathological laboratory of leiden university hospital , the netherlands . synoviocytes were infected with modified ad using different reportergenes and different promoters . two days after infection of synoviocytes with ig . ad . cmv . lacz at moi 100 , 67 % cells were positive for x - gal , as evidenced by a microscopically visible blue colour of the cells . in synovial cell cultures a doses response relation was observed between the amount of virus added and gene expression of the reporter gene , both after infection with ig . ad . cmv . lacz and ig . ad . cmv . luc ( see , table xi and table xii ). when incubation time was prolonged to five days , 100 % of synoviocytes stained blue . gene expression after infection with ad constructs driven by the cmv promoter is higher than by ad constructs driven by the mlp - promoter . this difference is more prominent using lacz as a reporter gene (± 100 ×) than using luciferase as a reporter gene (± 10 ×). two days after infection with ig . ad . mlp . lacz at moi 100 , less than 1 % was positive for lacz . however , clear gene expression in a dose dependent fashion was observed if the luciferase reporter gene was used ( table xii ). to assess possible toxicity of high doses ad for synoviocytes , synoviocytes were cultured without virus or incubated with ad . lacz , ad . luc or ad . tk at moi 100 . cell counts of synoviocyte cultures after infection with modified ad at moi 100 showed no significant differences compared to non - infected cultures ( table vii ). students t - test for paired samples p & gt ; 0 . 2 . synoviocytes incubated with ig . ad . mlp . tk were cultured with or without 10 : mg / ml gcv . 99 percent cell killing was observed after infection of synoviocytes with ig . ad . cmv . tk and incubation with gcv , infection with ig . ad . mlp . tk led to 80 % cell killing ( see , fig1 ). after mixing 25 % transduced with 75 % untransduced synoviocytes , bystander killing was assessed . both in ig . ad . cmv . tk and ig . ad . mlp . tk experiments extensive cell killing was observed ( see , fig1 ). possibility and specificity of gene transfer to inflamed synovial tissue in - vivo : 36 joints ( 10 knees and 26 pip &# 39 ; s ) of 8 different monkeys were injected with different amounts of ig . ad . lacz , ig . ad . luc or ig . ad . mlp - i . tk . in the biodistribution experiments , a cmv promoter was chosen to allow maximum sensitivity in detection of reporter gene product . histological examination of articular and peri - articular tissues obtained 2 - 3 days after infection with ig . ad . cmv . lacz showed lacz expressing cells present in synovial villi as well as in the synovial tissue covering tendons , bone , articular cartilage and subsynovial adipose tissue ( fig1 ). the cells expressing lacz activity were synoviocytes as evidenced by typical location and morphologic appearance . the percentage of infected cells ranged from 5 to 70 %. joints injected with pbs and non - injected joints did not show any lacz positive cells . no infection of cartilage , bone , fat or muscle tissue was observed . if the less efficient mip promoter was used ( in monkey 5 and in pip 2 monkey 2 ) no lacz positive cells could be found . in monkey 4 and 5 increasing amounts of modified ad were injected in consecutive pip - joints in the monkeys . in the pip - joints of monkey 4 , injected with ig . ad . cmv . lacz , an obvient dose - response relation was observed in percentage of lacz expressing cells ( table viii ). in monkey 5 , injected with ig . ad . mlp . lacz , no lacz positive cells were observed in the synovium . to asses toxicity of the procedure , biodistribution of the virus was determined using ad harbouring the luciferase reportergene . luciferase - activity , measured by luminometric methods , indicates infection of the organ by ad . monkey 1 - 5 were injected by ad . cmv . luc or ig . ad . mlp . luc and were sacrificed 2 - 3 days after virus administration . specimens of synovial tissue were harvested . from the same biopsies histological confirmation was obtained to judge if the sample contained relevant tissue . in monkey 4 the samples contained mainly connective tissue and no synovial tissue . samples of above mentioned organs and joints were analysed using the luciferase assay . samples obtained from a non - treated monkey were used as a control . except for one sample ( cervix ) and two non - virus injected joints that had slightly elevated luciferase counts , only ig . ad . luc injected joints were positive in the luciferase assay ( table ix ). to assess shedding of the virus excreta were cultured during the first 3 days of the experiment in the faeces ( day 0 - 3 ) of monkey 5 ad could be cultured both on 293 - and hep - 2 cells . the throat swab of this monkey was positive on 293 cells on day 1 . in the other monkeys faeces , urine and throat swabs were negative in the ad culture assay . during the experiment ; monkeys 3 and 5 suffered from severe arthritis , which made climbing difficult and led to diminished appetite and weight loss . one monkey that suffered from severe arthritis had a slightly elevated body temperature up to 40 ° c . analyses on blood samples indicated increase in crp , thrombocytosis , hypalbuminaemia and anaemia related to the presence of arthritis symptoms . a small increase in ldh - levels was observed ( table x ). histopathological analysis showed synovitis , moderate chronic pleuritis , necrotizing dermatitis , mild - chronic enteritis and inguinal and axillary lymphadenopathy in all monkeys . in order to analyse local inflammation induced by the procedure of i . a . administration of ad , non - injected , saline - injected and ad - injected joints were compared by routine histopathological analysis . no significant differences were observed in synovial hyperplasia or lymphocyte infiltration . during the tk - experiments , monkeys were closely observed to detect any toxicity of the procedure . the behaviour of the monkeys , clinical observations , biochemical parameters and histopathological analysis did only show abnormalities as has been reported before in monkeys with cia . no additional toxicity was seen in suicide gene treated groups as compared to reporter gene treated groups . histopathological analysis revealed no differences except for multifocal mid - zonal and peripheral infiltrations with lymphocytes and plasma cells in the liver of monkey 6 with single hepatocellular necrosis . effectivity of suicide gene transfer to inflamed synovial tissue can be seen as local toxicity of the procedure . histopathological analysis of the injected joint revealed no differences in synovial hyperplasia or lymphocyte infiltration compared to control joints . joint circumference diminished 1 cm in knees injected with ig . ad . mlp - i . tk followed by gcv and 1 to 1 . 5 cm in non - injected knees . in monkeys 6 , 7 and 8 who were terminated 14 to 18 days after intra - articular injection , a turn in antibody titer from negative to positive was observed after day 5 - 7 . no viruses were cultured from the excreta . in order to facilitate blunt end cloning of the itr sequences , wild - type human adenovinis type 5 ( ad5 ) dna was treated with kienow enzyme in the presence of excess dntps . after inactivation of the klenow enzyme and purification by phenol / chloroform extraction followed by ethanol precipitation , the dna was digested with bamhi . this dna preparation was used without further purification in a ligation reaction with pbr322 derived vector dna prepared as follows : pbr322dna was digested with ecorv and bamhi , dephosphorylated by treatment with tsap enzyme ( life technologies ) and purified on lmp agarose gel ( seaplaque gtg ). after transformation into competent e . coli dh5α ( life techn .) and analysis of ampiciline resistant colonies , one clone was selected that showed a digestion pattern as expected for an insert extending from the bamhi site in ad5 to the right itr . sequence analysis of the cloning border at the right itr revealed that the most 3 ′ g residue of the itr was missing , the remainder of the itr was found to be correct . the missing g residue is complemented by the other itr during replication . pbr / ad . bam - ritr was digested with bamhi and sali . the vector fragment including the adenovirus insert was isolated in lmp agarose ( seaplaque gtg ) and ligated to a 4 . 8 kb sali - bamhi fragment obtained from wt ad5 dna and purified with the geneclean ii kit ( bio 101 , inc .). one clone was chosen and the integrity of the ad5 sequences was determined by restriction enzyme analysis . clone pbr / ad . sal - ritr contains adeno type 5 sequences from the sali site at bp 16746 up to and including the ritr ( missing the most 3 ′ g residue ). wt adeno type 5 dna was digested with cai and bamhi , and the 20 . 6 kb fragment was isolated from gel by electro - elution . pbr322 was digested with the same enzymes and purified from agarose gel by geneclean . both fragments were ligated and transformed into competent dh5α . the resulting clone pbr / ad . cla - bam was analysed by restriction enzyme digestion and shown to contain an insert with adenovirus sequences from bp 919 to 21566 . clone pbr / ad . cla - bam was linearised with ecori ( in pbr322 ) and partially digested with aflii . after heat inactivation of aflii for 20 ′ at 65 ° c . the fragment ends were filled in with klenow enzyme . the dna was then ligated to a blunt double stranded oligo linker containing a paci site ( 5 ′- aattgtc ttaattaa ccgcttaa - 3 ′) ( seq . i . d . no . 1 ). this linker was made by annealing the following two oligonucleotides : 5 ′- aattgtcttaattaaccgc - 3 ′ ( seq . i . d . no . 2 ) and 5 ′- aattgcggttaattaagac . 3 ′ ( seq . i . d . no . 3 ), followed by blunting with klenow enzyme . after precipitation of the ligated dna to change buffer , the ligations were digested with an excess paci enzyme to remove concatameres of the oligo . the 22016 bp partial fragment containing ad5 sequences from bp 3534 up to 21566 and the vector sequences , was isolated in lmp agarose ( seaplaque gtg ), religated and transformed into competent dh5α . one clone that was found to contain the paci site and that had retained the large adeno fragment was selected and sequenced at the 5 ′ end to verify correct insertion of the paci linker in the ( lost ) aflii site . to allow insertion of a paci site near the itr of ad5 in clone pbr / ad . bam - ritr about - 190 nucleotides were removed between the clai site in the pbr322 backbone and the start of the itr sequences . this was done as follows : pbr / ad . bam - ritr was digested with clai and treated with nuclease bal31 for varying lengths of time ( 2 ′, 5 ′, 10 ′ and 15 ′). the extend of nucleotide removal was followed by separate reactions on pbr322 dna ( also digested at the clai site ), using identical buffers and conditions . bal31 enzyme was inactivated by incubation at 75 ° c . for 10 ′, the dna was precipitated and resuspended in a smaller volume te buffer . to ensure blunt ends , dnas were further treated with t4 dna polymerase in the presence of excess dntps . after digestion of the ( control ) pbr322 dna with sali , satisfactory degradation (˜ 150 bp ) was observed in the samples treated for 10 ′ or 15 ′. the 10 ′ or 15 ′ treated pbr / ad . bam - ritr samples were then ligated to the above described blunted paci linkers ( see , pbr / ad . aflii - bam ). ligations were purified by precipitation , digested with excess paci and separated from the linkers on an lmp agarose gel . after religation , dnas were transformed into competent dh5α and colonies analysed . ten clones were selected that showed a deletion of approximately the desired length and these were further analysed by t - track sequencing ( t7 sequencing kit , pharmacia biotech ). two clones were found with the paci linker inserted just downstream of the ritr . after digestion with paci , clone # 2 has 28 bp and clone # 8 has 27 bp attached to the itr . cosmid vector pwe15 ( clontech ) was used to clone larger ad5 inserts . first , a linker containing a unique paci site was inserted in the ecori sites of pwe15 creating pwe . pac . to this end , the double stranded paci oligo as described for pbr / ad . aflii - bamhi was used but now with its ecori protruding ends . the following fragments were then isolated by electro - elution from agarose gel : pwe . pac digested with paci , pbr / aflii - bam digested with paci and bamhi and pbr / ad . bam - ritr # 2 digested with bamhi and paci . these fragments were ligated together and packaged using λ phage packaging extracts ( stratagene ) according to the manufacturer &# 39 ; s protocol . after infection into host bacteria , colonies were grown on plates and analysed for presence of the complete insert . pwe / ad . aflii - ritr contains all adenovirus type 5 sequences from bp 3534 ( aflii site ) up to and including the right itr ( missing the most 3 ′ g residue ). pwe . pac was digested with clai and 5 ′ protruding ends were filled using klenow enzyme . the dna was then digested with paci and isolated from agarose gel . pwe / aflii - ritr was digested with ecori and after treatment with klenow enzyme digested with paci . the large 24 kb fragment containing the adenoviral sequences was isolated from agarose gel and ligated to the clai - digested and blunted pwe . pac vector using the ligation express ™ kit from clontech . after transformation of ultracompetent xl10 - gold cells from stratagene , clones were identified that contained the expected insert . pwe / aflii - ecori contains ad5 sequences from bp 3534 - 27336 . adapter plasmid pmlptk ( epo patent application 95202213 ) was modified as follows : sv40 polya sequences were amplified with primer sv40 - 1 ( introduces a bamhi site ) and sv40 - 2 ( introduces a bglii site ). in addition , ad5 sequences present in this construct ( from nt . 2496 to nt . 2779 ; ad5 sequences nt . 3511 to 3794 ) were amplified with primers ad5 - 1 ( introduces a bglii site ) and ad5 - 2 . sv40 - 1 : 5 ′- ggg ggatcc gaacttgtttattgcagc - 3 ′ ( seq . i . d . no . 4 ) sv40 - 2 : 5 ′- ggg agatct agacatgataagatac - 3 ′ ( seq . i . d . no . 5 ) ad5 - 1 : 5 ′- ggg agatct gtactgaaatgtgtgggc - 3 ′ ( seq . i . d . no . 6 ) ad5 - 2 : 5 ′- ggaggctgcagtctccaacggcgt - 3 ′ ( seq . i . d . no . 7 ) both pcr fragments were digested with bglii and ligated . the ligation product was amplified with primers sv40 - 1 and ad5 - 2 and digested with bamhi and aflii . the digested fragment was then ligated into pmlp . tk predigested with the same enzymes . the resulting construct , named pmlpi . tk , contains a deletion in adenovirus e1 sequences from nt . 459 to nt . 3510 . pmlpi . tk was used to make a new vector in which nucleic acid molecules comprising specific promoter and gene sequences can be easily exchanged . first , a pcr fragment was generated from pzipδmo + pyf101 ( n − ) template dna ( described in published international patent application pct / nl96 / 00195 ) with the following primers : ltr - 1 : 5 ′- ctg tac gta cca gtg cac tgg cct agg cat gga aaa ata cat aac tg - 3 ′ ( seq . i . d . no . 8 ) and ltr - 2 : 5 ′- gcg gat cct tcg aac cat ggt aag ctt ggt acc gct agc gtt aac cgg gcg act cag tca atc g - 3 ′ ( seq . i . d . no . 9 ). pwo dna polymerase ( boehringer mannheim ) was used according to manufacturers protocol with the following temperature cycles : once 5 ′ at 95 ° c . ; 3 ′ at 55 ° c . ; and 1 ′ at 72 ° c ., and 30 cycles of 1 ′ at 95 ° c ., 1 ′ at 60 ° c ., 1 ′ at 72 ° c ., followed by once 10 ′ at 72 ° c . the pcr product was then digested with bamhi and ligated into pmlp10 ( levrero et al ., 1991 ; gene 101 , 195 - 202 ) digested with pvuii and bamhi , thereby generating vector pltr10 . this vector contains adenoviral sequences from bp 1 up to bp 454 followed by a promoter consisting of a part of the mo - mulv ltr having its wild - type enhancer sequences replaced by the enhancer from a mutant polyoma virus ( pyf101 ). the promoter fragment was designated l420 . sequencing confirmed correct amplification of the ltr fragment however the most 5 ′ bases in the pcr fragment were missing so that the pvuii site was not restored . next , the coding region of the murine hsa gene was inserted . pltr10 was digested with bstbi followed by klenow treatment and digestion with ncoi . the hsa gene was obtained by pcr amplification on puc18 - hsa ( kay et al ., 1990 ; j . immunol . 145 , 1952 - 1959 ) using the following primers : hsa1 , 5 ′- gcg cca cca tgg gca gag cga tgg tgg c - 3 ′ ( seq . i . d . no . 10 ), 5 ′- ctg tac gta cca gtg cac tgg cct agg cat gga aaa ata cat aac tg - 3 ′ ( seq . i . d . no . 11 ) and ltr - 2 : 5 ′- gcg gat cct tcg aac cat ggt aag ctt ggt acc gct agc gtt aac cgg gcg act cag tca atc g - 3 ′ ( seq . i . d . no . 12 ) and hsa2 , 5 ′- gtt aga tct aag ctt gtc gac atc gat cta cta aca gta gag atg tag aa - 3 ′ ( seq . i . d . no . 13 ) 5 ′- ctg tac gta cca gtg cac tgg cct agg cat gga aaa ata cat aac tg - 3 ′ ( seq . i . d . no . 14 ) and ltr - 2 : 5 ′- gcg gat cct tcg aac cat ggt aag ctt ggt acc gct agc gtt aac cgg gcg act cag tca atc g - 3 ′ ( seq . i . d . no . 15 ). the 269 bp amplified fragment was subcloned in a shuttle vector using the ncoi and bglii sites . sequencing confirmed incorporation of the correct coding sequence of the hsa gene , but with an extra tag insertion directly following the tag stop codon . the coding region of the hsa gene , including the tag duplication was then excised as a ncoi ( sticky )- sali ( blunt ) fragment and cloned into the 3 . 5 kb ncoi ( sticky )/ bstbi ( blunt ) fragment from pltr10 , resulting in pltr - hsa10 . finally , pltr - hsa10 was digested with ecori and bamhi after which the fragment containing the left itr , packaging signal , l420 promoter and hsa gene was inserted into vector pmlpi . tk digested with the same enzymes and thereby replacing the promoter and gene sequences . this resulted in the new adapter plasmid pad5 / l420 - hsa that contains convenient recognition sites for various restriction enzymes around the promoter and gene sequences . snabi and avrii can be combined with hpai , nhei , kpni , hindiii to exchange promoter sequences , while the latter sites can be combined with the clai or bamhi sites 3 ′ from hsa coding region to replace genes in this construct . another adapter plasmid that was designed to allow easy exchange of nucleic acid molecules was made by replacing the promoter , gene and polya sequences in pad5 / l420 - hsa with the cmv promoter , a multiple cloning site , an intron and a polya signal . for this purpose , pad5 / l420 - hsa was digested with avrii and bglii followed by treatment with klenow to obtain blunt ends . the 5 . 1 kb fragment with pbr322 vector and adenoviral sequences was isolated and ligated to a blunt 1570 bp fragment from pcdna1 / amp ( invitrogen ) obtained by digestion with hhai and avrii followed by treatment with t4 dna polymerase . this adapter plasmid was named pad5 / clip . to enable removal of vector sequences from the adenoviral fragment pad5 / clip was partially digested with ecori and the linear fragment was isolated . an oligo of the sequence 5 ′ ttaagtcgac - 3 ′ ( seq . i . d . no . 16 ) was annealed to itself resulting in a linker with a sali site and ecori overhang . the linker was ligated to the partially digested pad5 / clip vector and clones were selected that had the linker inserted in the ecori site 23 bp upstream of the left adenovirus itr in pad5 / clip resulting in pad5 / clipsal . the adapter plasmid pad5 / clip . lacz was generated as follows : the e . coli lacz gene was amplified from the plasmid pmlp . nlslacz ( ep 95 - 202 213 ) by pcr with the primers 5 ′ ggggtggccagggtacctctaggcttttgcaa ( seq . i . d . no . 17 ) and 5 ′ ggggggatccataaacaagttcagaatcc ( seq . i . d . no . 18 ). the pcr reaction was performed ex taq ( takara ) according to the suppliers protocol at the following amplification program : 5 minutes 94 ° c ., 1 cycle ; 45 seconds 94 ° c . and 30 seconds 60 ° c . and 2 minutes 72 ° c ., 5 cycles ; 45 seconds 94 ° c . and 30 seconds 65 ° c . and 2 minutes 72 ° c ., 25 cycles ; 10 minutes 72 ; 45 seconds 94 ° c . and 30 seconds 60 ° c . and 2 minutes 72 ° c ., 5 cycles , i cycle . the pcr product was subsequently digested with kpn1 and bamhi and the digested dna fragment was ligated into kpni / bamhi digested pcdna3 ( invitrogen ), giving rise to pcdna3 . nlslacz . next , the plasmid pad5 / clip was digested with spei . the large fragment containing part of the 5 ′ part cmv promoter and the adenoviral sequences was isolated . the plasmid pcdna3 . nlslacz was digested with spei and the fragment containing the 3 ′ part of the cmv promoter and the lacz gene was isolated . subsequently , the fragments were ligated , giving rise to pad / clip . lacz . the reconstitution of the cmv promoter was confirmed by restriction digestion . the adapter plasmid pad5 / clip . luc was generated as follows : the plasmid pcmv . luc ( ep patent application 95 - 202 213 ) was digested with hindiii and bamhi . the dna fragment containing the luciferase gene was isolated . the adapter plasmid pad5 / clip was digested with hindiii and bamhi , and the large fragment was isolated . next , the isolated dna fragments were ligated , giving rise to pad5 / clip . luc . the adapter pclipsal . luc was generated in the same way but using the adapter pclipsal digested with hiii and bamhi as vector fragment . likewise , the tk containing hiii - bamhi fragment from pcmv . tk ( ep patent application 95 - 202 213 ) was inserted in pclipsal to generate pad5 / clip . tk . the presence of the sali site just upstream of the left itr enables liberation of vector sequences from the adeno insert . removal of these vector sequences enhances frequency of vector generation during homologous recombination in per . c6 . to enable convenient generation of recombinant adenoviruses with a ad5 / ad16 chimeric fiber we cloned the chimeric fiber gene in the place of the ad5 fiber in the cosmid clone pwe / ad . aflii - ritr . the pbr / adbamrpac . fib16 constructs and the pbr / ad . aflii - bamhi construct were digested with bamhi and paci to free it from the pbr plasmid . they were isolated from gel and cleaned by using agarase ( boehringer ). the pwe . pac construct was digested with paci to linearize it and cleaned by phenol / chloroform . a three - point ligation was used in which the bamhi sites of the pbr / adbamrpac . fib16 constructs and the pbr / ad . aflii - bamhi construct are ligated together and the pwe . pac construct is ligated at the paci sites . the ligation mix consists out of the three constructs , t4 ligase , 5 mm atp and ligation buffer without peg . 1 - 4 μl of the ligation mixture , containing 0 . 1 - 1 . 0 μg of ligated dna , is added to the packaging extract . separately , 1 μl of the positive wild - type lambda dna control was packaged . the tubes were spun quickly and incubated at rt for maximum 2 hrs . respectively 500 μl sm buffer ( 5 . 8 g nacl , 2 . 0 g mgso 4 . 7h 2 o , 50 ml 1m tris - hcl ( ph 7 . 5 ), 5 ml 2 % ( w / v ) gelatine and deionised water up to 1 liter ) and 20 μl of chloroform was added to the packaging mixture to stop the reaction . the tube was spinned briefly to sediment the debris . the supernatant , which contains the phage , can now be stored at 4 ° c . up to 1 month . a bacterial glycerol stock of dh5α strain and vcs257 strain were streaked onto lb agar plates and incubated o / n at 37 ° c . the next day 10 ml lb medium supplemented with 10 mm mgso 4 and 0 . 2 % ( w / v ) maltose was inoculated with a single colony of each bacteria strain . this was grown at 37 ° c . until an od 600 value of maximum 1 . 0 is reached . the bacteria were then pelleted at 500 × g for 10 minutes . the pellet is resuspended into 5 ml of sterile 10 mm mgso 4 and diluted in 10 mm mgso 4 till an od 600 value of approximately 0 . 5 is reached . of the positive wild - type lambda phage control a 10 − 2 and a 10 − 4 dilution was made in sm buffer . of the other final packaged reactions a 10 − 1 and a 10 − 2 dilution was made in sm buffer . out of the 10 − 4 dilution of the positive control 10 μl was added to 200 μl of vcs257 host cells ( od 600 0 . 5 ). this is incubated for 15 minutes at 37 ° c ., 3 ml of lb top agar ( 0 . 7 % agarose in lb medium ) ( 50 ° c .) is added and immediately plated on a pre - warmed lb agar plate . out of the 10 − 1 and a 10 − 2 dilution of the other final packaged reactions 25 μl was added to 25 μl of dh5αa host cells ( od 600 0 . 5 ). this is incubated for 30 minutes at rt . respectively 200 μl lb medium is added and an incubation for 1 hr at 37 ° c . followed . the mixture is spun down shortly , the bacteria pellet is resuspended in 100 μml lb medium and plated on lb agar plates with the required amount of ampicillin . the plates are incubated o / n at 37 ° c . eventually the colonies are grown and the required dna is tested by restriction digestion . pwe / ad . aflii - ritrfib16 contains all adenovirus type 5 sequences except for the fiber coding region 3 ′ from the ndei site present in ad5 fiber , these sequences are replaced by fiber sequences from ad16 leaving the open reading frame intact . the adapter plasmids pad5 / clip . tk , pad5 / clip . lacz or pad5 / clip . luc were digested with sali to liberate the homologous adenovirus sequences and the left itr from the vector . pwe / ad . aflii - ritrfib16 was digested with paci . dna was then purified using phenol / chloroform extraction and etoh precipitation and redissolved in sterile transfection qualified water . four μgr of each construct was transfected into per . c6 cells in a t25 flask seeded one day before with 2 . 5 × 10 6 cells . at the occurrence of full cpe 6 - 8 days latercells were harvested in the medium and amplified by infection of 3 ml 3 × freeze - thawed cell lysate on fresh per . c6 cells . at full cpe cells were harvested by freeze - thawing and virus was purified by two rounds of plaque purification on per . c6 cells . in all cases plaques were positive for transgene expression and one was picked to generate seed stocks for production . infection of synoviocytes with recombinant adenoviral vectors in non - human primates suffering from collagen induced arthritis . the transducibility of arthritic synovium by chimeric adenoviruses carrying the lacz gene from e . coli , which codes for the enzyme β - galactosidase , was tested in vivo in a non - human primate model for ra . the rhesus monkey maccaca mulatta was injected 10 times subcutaneously with , in total , 5 mg bovine collagen type ii ( 10 mg / ml ) emulsified in an equal volume of complete freunds adjuvant . the animal developed a full blown collagen induced arthritis ( cia ) within a period of 8 weeks . subsequently , the left knee was injected intra - articularly with 1 * 10 11 virus particles ( vp ) ig . ad . clip . lacz . the right knee was injected with 1 * 10 11 vpig . ad . cliplacz . fib16 . the vectors were administered in a total volume of 1 ml diluens ( pbs supplemented with 5 % sucrose ). the site of entry was medially , just below the midpoint of the patella . the needle was introduced in a line towards the suprapatellar pouch . after passing the joint capsule , the vector was injected into the joint cavity . thereafter , the syringe and needle were removed from the joint . at day 3 post infection , the animal was sacrificed . the left elbow was injected with 1 ml diluens only and served as a negative control . the right elbow was left untreated . the knee joints and elbows were isolated and fixed in phosphate buffered 2 % paraformaldehyde / 0 . 25 % glutaraldehyde for 3 hours and washed 3 times with pbs , incubated over night in x - gal solution ( 5 mm k 4 fe ( cn ) 6 , 5 mm k 3 fe ( cn ) 6 , 2 mm mgcl 2 and 0 . 5 mg / ml 5 - bromo - 4 - chloro - 3 - indolyl - 8 - d - galactopyranoside ) and extensively washed with pbs . the hyperplastic synovial lining of the knee joint stained blue with ig . ad . clip . lacz . however , the knee ig . ad . cliplacz . fib16 injected with stained blue more intensely , showing that recombinant chimeric adenoviruses carrying the fiber of ad16 infects hyperplastic synovium more efficiently than recombinant adenoviruses carrying fiber of ad5 . detailed analysis of the transduced tissue confirmed that the number of positive nuclei in the pannus tissue of the ig . ad . cliplacz . fib16 treated joint was significantly higher than the number of positive nuclei found in the ig . ad . clip . lacz treated joints . stained nuclei were found several cell layers deep in the pannus tissue . no staining was found in chondrocytes of the cartilage layer or in the diluens or non - injected injected joint . these results show that hyperplastic synovium can be transduced more efficiently with chimeric recombinant ig . ad . cliplacz . fib16 vectors in vivo , as compared to ig . ad . clip . lacz vectors . moreover , the results show that the diseased tissue ( hyperplastic synovium ), but not the chondrocytes ( benign cells that are required for cartilage regeneration ) were at least preferably transduced by the recombinant adenoviral vectors . dose dependent transduction of synoviocytes with recombinant adenoviral vectors in non - human primates suffering from collagen induced arthritis . the transducibility of arthritic synovium by chimeric adenoviruses carrying the lacz gene was tested in a dose escalation study in vivo in the non - human primate model for ra as described above . a monkey suffering from cia was treated with increasing doses of ig . ad . clip . lacz or ig . ad . cliplacz . fib16 given intra - articularly in the proximal interphalangeal ( pip ) in a total volume of 0 . 1 ml . pip 2 to 5 were injected with increasing vector doses , ranging from 1 × 10 7 to 1 × 10 10 vp , in the left or right hand , for ig . ad . clip . lacz or ig . ad . cliplacz . fib16 respectively . as a control , 0 . 1 ml diluens was injected in pip 1 of both hands . after sacrifice on day 5 , the pip joints of the hands were fixed in 2 % paraformaldehyde / 0 . 25 % and stained with x - gal to monitor lacz expression , as described above . a positive correlation was observed between injected dose of lacz adenoviruses and the number of lacz expressing cells in the synovial tissue . moreover , the ig . ad . cliplacz . fib16 vector treated joints contained more lacz positive cells than the ig . ad . clip . lacz treated joints at the same vector dose , confirming that hyperplastic synovium is transduced more efficiently by chimeric recombinant ig . ad . cliplacz . fib16 vectors than by ig . ad . clip . lacz vectors in a relevant model for rheumatoid arthritis . microscopy of the injected joints confirmed that the cells expressing lacz activity were synoviocytes , as evidenced by typical location and morphologic appearance . killing of diseased synovium from patients suffering from ra infected with ig . ad . clip . tk and ig . ad . clip . tk . fib16 followed by treatment with gcv in vitro . synovium was isolated from patients suffering from ra as discussed above . the day prior to infection , 10 4 synovium cells were plated on a tissue culture dish . the next day , eight dishes with synovial cells were infected with either ig . ad . clip . tk or ig . ad . clip . tk . fib16 at an increasing m . o . i . of 1 , 10 , 100 and 1000 vp / cell or mock treated . four hours post infection , the infection medium was replaced by imdm containing 40 % normal human serum supplemented with or without 10 μg / ml gcv . at day 0 , 5 , 7 and 10 cells were counted . ig . ad . clip . tk . fib infected cells were killed in medium containing gcv more efficiently , especially at lower m . o . i .&# 39 ; s , than ig . ad . clip . tk infected cells , as determined by the decrease in the total cell numbers in these dishes . neither the mock treated cells , nor the infected cells in medium without gcv were killed , showing that killing was caused by the combination of ad . tk vectors and gcv . thus , hyperplastic synovium from patients suffering from ra is sensitive to infection with recombinant ig . ad vectors expressing tk in combination with treatment with the pro - drug gcv . moreover , killing of synoviocytes following ig . ad . clip . tk . fib16 infection was more efficient than killing after ig . ad . clip . tk infection in the presence of gcv . next , the bystander effect of the treatment was addressed . to that end , synovial cells were infected with ig . ad . clip . tk at an m . o . i . of 100 as described above . the next day , the infected cells were trypsinized and mixed with non - infected synovial cells from the same patient at the ratio of 1 : 4 ( 25 %) or 1 : 2 ( 50 %). as controls , non - infected ( 0 %) and non - mixed ( 100 %) cells were included in the experiment . the following 7 days , the cells were cultured in imdm supplemented with 40 % normal human serum and 10 μg / ml gcv and the total amount of cells per dish was determined . the synovial cells infected ( 100 %) with ig . ad . clip . tk were killed . moreover , the mixed cell populations in which only a percentage of the cells ( 50 % and 25 % respectively ) was infected with ig . ad . clip . tk were killed too . this shows that human synovium cells that are infected with recombinant ig . ad vectors expressing the tk gene have a substantial bystander effect following gcv treatment . killing of hyperplastic synovium after intra - articular injection of ig . ad . clip . tk and ig . ad . clip . tk . fib16 followed by gcv treatment in non - human primates suffering from collagen induced arthritis . a rhesus monkey was injected 10 times subcutaneously with , in total , 5 mg bovine collagen type ii ( 10 mg / ml ) emulsified in an equal volume of complete freunds adjuvant to induce cia . the animal developed a full - blown arthritis within a period of 8 weeks . subsequently , the left knee was injected intra - articularly with 1 × 10 11 vp ig . ad . clip . tk in a total volume of 1 ml diluens . the right knee was injected with 1 × 10 11 vp ig . ad . clip . tk . fib16 in a total volume of 1 ml diluens . the sites of entry were medially , just below the midpoint of the patella . the needle was introduced in a line towards the suprapatellar pouch . after the joint capsule was passed , the substances were injected into the knee joint . thereafter , syringes and needles were removed from the joints . the left elbow was injected with 1 ml diluens and served as a negative control . from day 2 to day 15 the monkey was treated daily intravenously with gcv , 10 mg / kg / day in 25 ml sterile water given in approximately 30 minutes . after sacrifice on day 18 the knees and elbows of the monkey were taken out for histopathological analysis . synovial biopsies of the knees were snap - frozen in liquid nitrogen and stored at & lt ;− 60 ° c . cleaving of genomic dna during apoptosis yields double - stranded low molecular weight nuclear dna fragments ( mono - and oligonucleosomes ) as well as single strand breaks (“ nicks ”) in high molecular weight dna . tunel ( tdt - mediated dutp nick end labeling ) is a method for enzymatic in situ labeling of apoptosis induced dna strand breaks . strand breaks in the dna can be identified by labeling the free 3 ′- oh termini of dna fragments with modified nucleotides in an enzymatic reaction . terminal deoxynucleotidyl transferase (“ tdt ”), which catalyses polymerization of nucleotides to the free 3 ′- oh termini of fragmented dna , is used as the polymerase . incorporated fluorescein - 12 - dutp is detected by anti - fluorescein antibody fab fragments from sheep , conjugated with horseradish peroxidase (“ pod ”). the procedure is extensively described by the supplier ( promega ). after substrate reaction , the labelled fragmented genomic dna were visualised under the light microscope . the synovial tissue from the elbow that was injected with diluens showed background tissue staining , indicating that some apoptosis has taken place in diseased synovium . in the negative control ( no tdt enzyme was added ) no staining could be observed . the positive control ( a sample that was treated with dnase to induce dna strand breaks before the tunel assay was started ) showed staining in all parts of the tissue . the sample from the ig . ad . clip . tk injected joint showed more stained cells than the synovial tissue sample of the diluens treated joint , indicating that more cells went into apoptosis due to the ig . ad . clip . tk - gcv treatment . most stained cells were found in the joint injected with ig . ad . clip . tk . fib16 . the staining was present both in the synovial membrane and in the subsynovial tissue , suggesting that the treatment is efficacious throughout the whole tissue sample . these data show that treatment with recad vectors expressing the tk gene followed by gcv treatment is a feasible method to perform non - surgical synovectomy in arthritic joints . in addition , these data show that recombinant adenoviral vectors containing fiber 16 are superior in transducing transgenes to synovial tissue . in each experiment , a total of 15 , 000 ra synoviocytes was seeded per well in 12 - well microtiter dishes . cells were infected with ad5 . luc ( batch no . ic020 - 032 ) or ad5 . fib16 . luc ( batch no . b204 - 130c ) at various m . o . i .&# 39 ; s , and incubated overnight . luciferase activity was measured after 72 hours . data are summarized in table xiii ( fig1 ). in each experiment , a total of 15 , 000 ra synoviocytes was seeded per well in 12 - well microtiter dishes . cells were infected with ad5 . lacz ( batch no . b269 - 186 ) or ad5 . fib16 . lacz ( batch nos . b204 - 120a and b204 - 120b ) at various m . o . i .&# 39 ; s , and incubated overnight . % of lacz - positive cells was determined after 72 hrs . data are summarized in table xiv and fig1 . in each experiment , a total of 15 , 000 ra synoviocytes was seeded per well in 12 - well microtiter dishes . cells were infected with ad5 . gfp ( batch no . b204 - 103 and b204 - 130d ) or ad5 . fib16 . gfp ( batch nos . b204 - 103 and ic054 - 024b ) at various m . o . i .&# 39 ; s , and incubated overnight . gfp activity was measured after 72 hrs , and % of gfp - positive cells was determined . data are summarized in tables xva and xvb and fig1 a and b . a panel of chimeric adenoviruses was tested for its infectivity on ra synoviocytes . the following chimeric adenoviruses were produced : ad5 . fib5 , 11 , 16 , 24 , 28 , 33 , 35 , 45 and 47 , each carrying a luciferase transgene . in each experiment , a total of 15 , 000 ra synoviocytes was seeded per well in 12 - well microtiter dishes . cells were infected the chimeric adenoviruses at various m . o . i .&# 39 ; s , and incubated overnight . luciferase activity was measured after 72 hrs . data are summarized in table xvi ; the graphic representation is in fig1 . three chimeric adenoviruses , each carrying a b - type fiber were tested for its infectivity on ra synoviocytes , in comparison to ad5 . the following chimeric adenoviruses were produced : ad5 . fib16 , 35 and 51 , each carrying a gfp transgene . in each experiment , a total of 50 , 000 ra synoviocytes was seeded per well in 12 - well microtiter dishes . cells were infected the chimeric adenoviruses at various m . o . i .&# 39 ; s , and incubated overnight . gfp activity was measured after 72 hrs , and % of gfp - positive cells was determined . data are summarized in tables xviia and b ; the graphic representation is in fig1 a and b . three chimeric adenoviruses , each carrying a b - type fiber were tested for its infectivity on ra synoviocytes , in comparison to ad5 . the following chimeric adenoviruses were produced : ad5 . fib11 , 16 and 35 , each carrying a luciferase transgene . in each experiment , a total of 15 , 000 ra synoviocytes was seeded per well in 12 - well microtiter dishes . cells were infected the chimeric adenoviruses at various m . o . i .&# 39 ; s , and incubated overnight . luciferase activity was measured after 72 hrs . data are summarized in table xviii and fig1 . comparison of infection of ra synoviocytes with ad5 and ad5 . fib16 in different patients synoviocyte cells were obtained from 6 different patients suffering from rheumatoid arthritis synoviocytes were infected with ad5 . lacz or ad5 . fib16 . lacz during 2 or 20 hours , and stained for lacz expression after 48 hours . numbers of blue cells were counted under the microscope . data are summarized in table xix , plotted in fig2 . dna encoding fiber protein derived from alternative adenovirus serotypes . ( bold letters represent ndei restriction site ( a - e ), nsii restriction site ( 1 - 7 , 8 ), or paci restriction site ( 7 ). 5 ′- ccc gtg tat cca tat gat gca gac aac gac cga cc - 3 ′ ( seq . i . d . no . 19 ) 5 ′- ccc gtc tac cca tat ggc tac gcg cgg - 3 ′ ( seq . i . d . no . 20 ) 5 ′- cck gts tac cca tat gaa gat gaa agc - 3 ′ ( seq . i . d . no . 21 ) 5 ′- ccc gtc tac cca tat gac acc tyc tca act c - 3 ′ ( seq . i . d . no . 22 ) 5 ′- ccc gtt tac cca tat gac cca ttt gac aca tca gac - 3 ′ ( seq . i . d . no . 23 ) 5 ″- ccg atg cat tta ttg ttg ggc tat ata gga - 3 ′ ( seq . i . d . no . 24 ) 5 ′- ccg atg cat tya ttc ttg ggc rat ata gga - 3 ′ ( seq . i . d . no . 25 ) 5 ′- ccg atg cat tta ttc ttg ggr aat gta wga aaa gga - 3 ′ ( seq . i . d . no . 26 ) 5 ′- ccg atg cat tca gtc atc ttc tct gat ata - 3 ′ ( seq . i . d . no . 27 ) 5 ′- ccg atg cat tta ttg ttc agt tat gta gca - 3 ′ ( seq . i . d . no . 28 ) 5 ′- gcc atg cat tta ttg ttc tgt tac ata aga - 3 ′ ( seq . i . d . no . 29 ) 5 ′ - ccg tta att aag ccc tta ttg ttc tgt tac ata aga a - 3 ′ ( seq . i . d . no . 30 ) 5 ′- ccg atg cat tca gtc atc ytc twt aat ata - 3 ′ ( seq . i . d . no . 31 ) values represent percentages of cells that express car or either one of m = male , f = female ; lh = left hand , rf = right foot , hh = both hands , ff = both feet ; ** = in all joint on left side 10 % triamcinolonhexacetonide 20 mg / ml is added . negative controls were carried out in duplos in 3 of 5 experiments . luciferase counts in organs as a measure of virus spread after intra - articular injection of ldh levels in monkey 4 ( cmv . lacz / cmv . luc ), 5 ( mlp . lacz / mlp . luc ), 6 ( mlp . tk ), 7 and 8 ( mlp . tk + gcv ). ldh - levels in monkey 6 , 7 and 8 are determined with an other test than monkey 4 and 5 . % of infected cells with three b - type fiber - modified viruses on ra arnberg n ., mei y . and wadell g ., 1997 . fiber genes of adenoviruses with tropism for the eye and the genital tract . virology 227 : 239 - 244 . arnett f c , edworthy s m , bloch d a , et al . 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