Patent Application: US-7298005-A

Abstract:
the invention relates to the use of gene therapy in the treatment of aseptic loosening of orthopaedic prostheses and discloses methods of refixing such prostheses without open revision surgery . in particular , it provides adenoviral vectors and prodrugs for simultaneous , separate or sequential use in the destruction of interface tissue allowing subsequent recementing of loose prostheses in a minimally invasive manner .

Description:
the following examples are meant to illustrate the invention and do not limit it in any way . persons of ordinary skill in the art will recognize modifications within the spirit and scope of the invention as set forth in the appended claims . the drug product , ctl102 injection , is a sterile , clear or virtually clear , aqueous liquid solution containing ctl102 virions at a nominal mean potency of 2 × 10 11 particles ml − 1 , buffered at ph 7 . 4 . cb1954 is formulated as a sterile solution in solvent ( n - methylpyrrolidone : polyethylene glycol , 2 : 7 v / v with 17 . 8 mg cb1954 ml − 1 ). just prior to use , the prodrug in solvent is diluted in sterile saline to a maximum final cb1954 concentration of 5 mg ml − 1 . to stabilise the prosthesis , low viscosity bone cement ( simplex ® p with tobramycin from howmedica inc , rutherford , n . j ., usa ) is used . this radiopaque bone cement is a mixture of a liquid monomer component ( 2 ml 97 . 4 % methylmethacrylate , 2 . 6 % n , n - dimethyl - p - toluidine , 75 ppm hydroquinone ) and a polymer powder ( 6 g polymethylmethacrylate , 30 g methylmethacrylate - styrene copolymer , 4 g barium sulphate , 1 g tobramycin sulphate ). the components are vacuum mixed ( 0 . 9 bar , 1 minute ) immediately before use . for arthrography , hexabrix 320 ( ioxaglate sodium meglumine , guerbet , roissy charles de gaulle cedex , france ) contrast medium is used . following careful flushing of the joint to remove synovial fluid and inflammatory exudate that may contain neutralizing anti - adenovirus antibodies , 3 × 10 9 pfu ctl102 is injected intra - articularly resulting in delivery of vector to cells throughout the periprosthetic space . after 48 hours , to allow transduction of target cells and expression of the nitroreductase transgene , cb1954 ( at a dosage of 24 mg m − 2 ) is injected intra - articularly . to assure free access of ctl 102 and cb 1954 to the periprosthetic space it is preferred that patients are selected who have an arthrogram that shows contrast medium around the prosthesis . it is likely , therefore , that patients will usually undergo three arthrographies ( one to assure access of contrast medium , one to inject the viral vector , and one to inject the cb 1954 prodrug ). in some circumstances after a number of days dead interface tissue may be removed by flushing or physical debridement , as appropriate . when the interface tissue is successfully diminished the prosthesis is refixated . to re - anchor the prosthesis to the bone , cement is injected in the periprosthetic space . for the flushing of the periprosthetic space and injection of the cement a number of holes are drilled through the bone into the periprosthetic space . this depends on the design of the prosthesis used . in many common designs , four is the minimum , because three holes are necessary for the femoral component to fixate in 3d space and one is necessary to fixate the acetabulum . as the bone biopsies are rather painful and the bone cannot be anaesthetised locally , these procedures are performed under general or spinal anaesthesia . ctl102 was constructed as described in djeha et al ( 2001 ) by homologous recombination in perc6 helper cells . the cells were transfected at 90 % confluence with an equimolar mixture of the transfer vector ptx0375 and the backbone vector pps1160 complexed with lipofectamine transfection reagent ( life technologies ). ptx0375 was constructed in two stages : ( i ) the cmv promoter / enhancer fused to the ntr gene was excised from ptx0340 as a 1 . 5 - kb bamhi - partial bgiii fragment and cloned into the unique bamhi site of psw107 , which is a pbluescript - based vector ( stratagene ) that contains the human b - globin ivs ii fused to the human complement 2 gene polyadenylation sequence adjacent to the bamhi site . a plasmid , ptx0374 , which contains the cmv . ntr fragment in the required orientation , was identified by pcr using the t3 primer ( 5 ′- attaaccctcac - taaag - 3 ′) ( seq id no : 1 ) which anneals to the cmv promoter / enhancer , and an ntr primer , ecn2 ( 5 ′ tctgctcggcctgttcc - 3 ′) ( seq id no : 2 ). ( ii ) the complete ntr expression cassette was excised from ptx0374 as a 2 . 5 - kb spei fragment and cloned into the unique spei site of the e1 - deleted adenovirus transfer vector pps1128 in the left - to - right orientation with respect to ad5 sequences . pps1128 is a puc19 - based plasmid that contains ad5 sequences from the left - hand itr to nucleotides ( nt .) 359 fused to nt 3525 - 10589 . pps1160 was constructed by pad linearisation of pps1128 , ligation with a paci - compatible adaptor ( 5 ′- tacatctagataat - 3 ′ ( seq id no : 3 )+ 5 ′- p - ttatctagat - gta - 3 ′) ( seq id no : 4 ) containing an xbai site , followed by xbai digestion to release a 7 - kb xbai fragment containing ad5 sequences 3524 - 10589 . this was then cloned into xbai - linearised pps1022 , a puc19 - based plasmid containing ad5 sequences from nt . 10589 to the right - hand itr but lacking nt 28592 to 30470 ( e3 region ). recombinants containing the fragment in the required orientation were identified by pcr using primers flanking the xbai site at 10589 ( rightward , 5 ′- tcgagtcaaatacgtagtcgt - 3 ′ ( seq id no : 5 ); leftward , 5 ′- tgtttccggaggaatttgcaa - 3 ′) ( seq id no : 6 ). a plasmid , pps1160 / 18 , was confirmed to contain a single copy of the xbai fragment ( pps1160 / 18 ) by hindiii and psti digestion . transfected perc6 cells were harvested following the appearance of extensive cpe ( about 7 - 9 days after transfection ) and recombinant virus released by three freeze - thaw cycles in infection medium ( dmem , 1 % fcs , 2 mm mgcl2 ). after two rounds of plaque purification on perc6 cells the viruses were grown to large scale and purified by cscl density centrifugation . banded virus was dialysed against an excess of storage buffer ( 10 mm tris , ph 7 . 4 , 140 mm nacl , 5 mm kcl , 0 . 6 mm na 2 hpo 4 , 0 . 9 mm cacl 2 , 0 . 5 mm mgcl 2 , and 5 % sucrose ), snap - frozen in aliquots in liquid nitrogen , and stored at − 280 ° c . particle concentrations were determined using the bca protein assay reagent ( pierce , rockford , ill .) and the conversion factor 1 mg / ml = 3 . 4 × 10 12 virus particles / ml . infectious titres were determined by plaque assay . genomic dna was isolated from banded adenovirus by digestion with proteinase k / sds , phenol - chloroform extraction , and ethanol precipitation and characterised by restriction digestion . in order to demonstrate the feasibility of using a virally delivered enzyme - prodrug system to kill interface cells , cells taken from two patients during revision surgery were cultured in vitro , incubated with ctl102 at a range of mois and subsequently exposed to cb1954 . cell viability was then determined using a metabolic activity assay . for all experiments described , interface cells were used . interface tissue was removed from the periprosthetic space during revision - surgery by an orthopedic surgeon and collected in sterile phosphate buffered saline ( pbs ). connective tissue and fat were removed thoroughly and the interface tissue was digested for at least two hours at 37 ° c . using collagenase 1a ( 1 mg / ml ; sigma , st louis , mo ., usa ). cells were then harvested by filtering the tissue / collagenase substance through a 200 μm filter ( npbi , emmer - compascuum , the netherlands ). the cells were cultured in 75 cm 2 flasks ( cellstar , greiner , alphen aan de rijn , the netherlands ) with iscove &# 39 ; s modified dulbecco &# 39 ; s medium ( imdm ; biowitthaker , verviers , belgium ), supplemented with glutamax ( gibcobrl , paisley , uk ), penicillin and streptomycin ( boehringer mannheim , germany ), and 10 % fetal calf serum ( fcs ; gibcobrl , paisley , uk ) at 37 ° c . and 5 % co 2 . before each experiment interface cells were detached from the flasks using 0 . 25 % trypsin ( gibcobrl , paisley , uk ). the cells were counted in a bürker counter and death cells were excluded by trypan blue . cells were seeded in a 96 wells - plate ( flat bottom ) at a density of 5 , 000 cells per well . cells were incubated overnight to allow attachment to the bottom . before each experiment the wells were washed twice with imdm . for the experiments passage 2 to 4 interface cells were used . light microscopy indicated that more than 95 % of the cells were interface cells . day 0 : interface cells from 2 patients were seeded at 5000 cells / well in imdm ( 10 % fcs ) in 96 wells plates , 100 μl per well . day 1 : cells were infected with ctl102 ( or diluent ) at 0 , 1 , 5 , 25 , 100 , 200 iu / cell in imdm ( 10 % fcs ), 50 μl per well . day 2 : cells were washed twice with in imdm ( 10 % fcs ), hereafter cells were incubated for 2 hr or 24 hr with cb1954 ( or vehicle ) at 0 , 0 . 1 , 0 . 5 , 1 , 5 and 50 μm in imdm ( 10 % fcs , 10 % hs ), 50 μl per well . day 2 / 3 : cells were washed once with imdm ( 10 % fcs ) and then incubated in imdm ( 10 % fcs , 10 % hs ), 5 μl per well . day 4 : photographs were taken . medium was refreshed with imdm ( 10 % fcs ), 10 μl wst reagent ( roche ) was added and the plates were incubated for 2 hr . as shown in fig2 a and 2b , virus and cb1954 - dose dependent killing was observed for cells from both patients . importantly , efficient ( 90 %) killing was observed with virus and cb1954 doses ( 200 virus pfu / cell and a cb1954 concentration of 50 μm ) that is readily achievable in the clinic . these results demonstrate that interface cells can be transduced by an hadv - 5 - vector and killed by the ntr / cb1954 approach . human adenovirus 5 is capable of infecting a broad range of dividing and non - dividing human cells including fibroblasts and macrophages ( djeha et al , 2001 ). killing of cells by gdept has been studied before in various cell lines , using various approaches . the ntr / cb1954 approach is attractive for clinical evaluation for several reasons : ( 1 ) it generates a toxic agent that can kill both dividing and non - dividing cells , ( 2 ) induction of cell death occurs by a p53 - independent mechanism , and ( 3 ) cb1954 is well - tolerated in man ( djeha et al , 2001 ). cell killing by the ntr / cb1954 approach has been proved effective in a variety of human cancer cells ( chung - faye et al , 2001 ; bilsland et al , 2003 , green et al , 2003 ; mcneish et al , 1998 ; shibata et al , 2002 ; weedon et al , 2000 ; wilson et al , 2002 ), but has not previously been studied in synovial or interface cells . the current study shows that interface cells can be effectively killed by the ntr / cb1954 approach . for the current study passage 2 to 4 interface cells were used . these passages were used to maximally reduce culture artefacts . on the one hand , in very low passages ( 0 and 1 ) there is a risk for presence of contaminating cells ( especially macrophages ), which decreases with higher passages . on the other hand , at higher passages the risk of substantial in vitro alteration / growth selection exists ( especially at passages higher than 4 ) ( zimmerman et al , 2001 ). in the current study , cultured interface cells of different patients were used . for the interpretation of the results the data of all patients were pooled . however , it must be noted that individual differences in transducibility were observed . the experiment outlined in example 3 confirmed that cultured interface cells are ad5 - infectable . however , when a cell is present within an intact tissue , access of the virus to the cell surface may be prevented , for instance by the extracellular matrix and by the low rate of virus diffusion through the extracellular space . in view of this , the infectability of fresh intact interface tissue was examined using a lacz - expressing adenovirus and xgai staining of lacz - expressing tissue . using this approach , a virus dose - dependent increase in gene expression was observed , with strong levels of gene expression with the two highest virus doses tested ( fig3 ). interface tissue ( li014 ) was obtained from a revision operation of the hip of a rheumatoid arthritis patient . the tissue was cut in 7 pieces and the pieces were put in 10 ml round bottom tubes . different concentrations of ad . cmv . lacz ( 0 , 3 . 6 × 10 4 , 3 . 6 × 10 5 , 3 . 6 × 10 6 , 3 . 6 × 10 7 , 3 . 6 × 10 8 , 3 . 6 × 10 9 pfu ) in 200 μl imdm / 10 % fcs were added . the tissues were incubated at 37 ° c . for 2 hours , the tubes were shaken every 10 to 15 minutes . hereafter 5 ml imdm / 10 % fcs was added and after an overnight incubation the tissues were rinsed 3 × with pbs and subsequently put in 5 ml xgai colouring solution and incubated for 3 . 5 hours at 37 ° c . the tissues were rinsed 3 × with pbs and fixed in 10 % formalin . the tissues with the highest added amounts of ad . cmv . lacz have areas of dark blue staining , which is evident down to an infection at 3 . 6 × 10 7 pfu ad . cmv . lacz . demonstrating that infection of cells in intact interface tissue is effective . embedded paraffin sections of the tissues were examined microscopically and the presence of stained , infected cells was confirmed . to test further the susceptibility of interface cells to human adenovirus 5 ( hadv - 5 )- based vectors , primary cultures of interface cells were exposed to the hadv - 5 vector ad . cmv . lacz . twenty - four hours post - infection the cells were stained with x - gal solution for β - galactosidase reporter gene expression . the transduction efficiency increased with increasing vector concentration . at 400 plaque forming units / cell the percentage of cells expressing the reporter gene was 88 % ( sd 4 . 0 ) ( fig4 ). thus hadv - 5 vectors can transduce interface cells . the ad . cmv . lacz ( van der eb et al , 2002 ) vector is identical to ctl102 , but the e . coli lacz gene replaces the ntr gene . to study the transducibility of interface cells by hadv - 5 , interface cells were infected with ad . cmv . lacz vector ( in concentrations of 0 , 25 , 50 , 100 , 200 , 400 pfu / cell ). twenty - four hours post infection the cells were washed twice with imdm , and cultured for two days . medium was refreshed each day . on day three , the monolayer cultures were washed twice with pbs and fixed with 0 . 2 % glutaraldehyde and 2 % formaldehyde in pbs for 10 minutes at 4 ° c . subsequently cells were washed twice with pbs and stained for β - galactosidase activity in 50 μl of reaction mix ( 1 mg / ml xgai ( eurogentec , seraing , belgium ), 5 mm potassium ferrocyanide , 5 mm potassium ferricyanide , 2 mm mgcl 2 in pbs ) for 2 hours at 37 ° c . the percentage of transduced cells was assessed by counting at least 100 interface cells , using light microscopy . all conditions were tested in duplicate . interface cells were seeded in 96 - wells plates . into each well 50 μl of imdm / 20 % fcs and 50 μl of a solution containing contrast medium and 0 . 9 % nacl in various concentrations ( 0 , 12 . 5 , 25 , and 50 % contrast medium ) were added . the contrast medium used was the low - osmolarity , nonionic dimer iotrolan ( isovist ; schering , berlin , germany ). after four hours of exposure to the contrast medium , the cells were washed twice and incubated in imdm / 10 % fcs . the cells were cultured for three more days , changing the culture medium every day . on day four , cell viability was determined with the wst - 1 cell viability assay kit ( roche , mannheim , germany ) according to the manufacturers protocol . interface cells were seeded in 96 - wells plates . after overnight incubation cells were infected with ad . cmv . lacz ( concentrations of 0 , 25 , 100 , and 200 pfu / cell ) in imdm / 20 % fcs , 50 μl per well . fifty μl iotrolan ( isovist ) in 0 . 9 % nacl was added in concentrations of 0 , 25 , 50 , and 100 %. ( when diluted in the culture medium these concentrations decreased to 0 , 12 . 5 , 25 , and 50 %.) four hours after infection , the cells were washed twice with imdm and incubated for the rest of the day in imdm / 10 % fcs at 37 ° c . and 5 % co 2 . the ad . cmv . lacz transduced cells were cultured for three days after removal of the vector and contrast medium . subsequently , the cells were fixed and stained for β - galactasidase activity . the transduction rate was assessed as described above . a univariate analysis of variance and spearman &# 39 ; s correlation was used to study the interaction between vector and prodrug and between vector and contrast medium and to study the effect of cb1954 on viability of the cells . a mann - whitney test for independent groups was performed to determine the difference in cell killing between the cells that were exposed to contrast medium and the non - exposed cells . in the experiment to study the effect of transient exposure to contrast medium on transduction of hadv - 5 - vector spearman &# 39 ; s correlation between contact time and viability and between delay time and viability was tested . for all statistical analyses p & lt ; 0 . 05 was the level of statistical significance . the toxicity of contrast medium ( iotrolan ) on interface cells was evaluated ( fig5 ). iotrolan does not affect the viability of the cells at any concentration ( p = 0 . 563 ). adding of contrast medium to the interface cells for four hours does not lead to killing of the cells . the effect of contrast medium ( iotrolan ) on hadv5 - transduction of interface cells was investigated with ad . cmv . lacz . transducibility of the cells increases with the concentration of hadv - 5 vector . however , the contrast medium has restraining influence on the transduction efficiency . with higher concentrations of iotrolan , the hadv - 5 vector concentration has less effect on gene transfer efficiency . at a contrast medium concentration of 50 % none of the cells were transduced ( fig6 ). the effect of iotrolan on the transduction is statistically significant ( p & lt ; 0 . 001 ). furthermore , differences between cells from different individuals ( n = 6 ) have been observed . to evaluate the effect of contrast medium on cell killing by ntr / cb1954 , the previously described experiment for the efficiency of cell killing was repeated in the presence of contrast medium . the results showed that , in the presence of contrast medium , cells are not killed by the ntr / cb1954 approach ( results not shown ). the presence of hexabrix 320 contrast medium also inhibited viral transduction ( data not shown ). in summary , the results from these experiments demonstrate the incompatibility of viral administration in combination with the administration of two commonly used contrast media . this incompatibility may be due to the presence of iodine within the contrast media . screening of all available contrast media may allow determination of a contrast medium compatible with viral transduction . the influence of transient exposure to contrast medium on the transduction of interface cells was investigated . interface cells were exposed to contrast medium for 0 to 120 minutes and the period between washing away of the contrast medium and performing the ntr / cb1954 cell killing approach was varied . cell killing was not correlated with contact time ( corr − 0 . 033 , p = 0 . 691 ) or length of period between washing away of the contrast medium and addition of the vector ( corr − 0 . 004 , p = 0 . 962 ). killing of cells not exposed to contrast medium and those transiently exposed was equivalent . in this study the influence of contrast medium on cell killing by ntr / cb1954 was investigated in view of future clinical studies . results show that the contrast medium does not seem to have any influence on the interface cells . however , transduction of the cells by an adenoviral vector , in the presence of contrast medium , is almost negligible . the adenoviral vector is inactivated by the presence of contrast medium . in a putative clinical study the viral vector will be injected in the joint space . normally , contrast medium is used to verify the position of the needle in the joint . the results of this study however show that the use of contrast medium in combination with a viral vector is dissuaded . thus , for a clinical study , we propose that alternative methods for the visualization of the needle should be employed such as injection of air to create an “ air - arthrogram ”. in conclusion , this example shows that interface cells can be killed by the ntr / cb1954 enzyme prodrug approach . data are available from the first two patients from a phase - 1 study of 12 patients with a loosened hip experiencing debilitating pain and significant comorbidity . on day 1 the vector was injected into the hip joint and the prodrug injected on day 3 , as described above . on day 10 three holes were drilled in the femur and one in the acetabulum . biopsies are taken from the periprosthetic space and low viscosity cement ( osteopal , biomet merck , sjöbo , sweden ) injected under fluoroscopic guidance . patient 1 is an 82 - year old female with loosening of both hip prostheses , classified asa iv ( mortality risk 20 . 3 %, american society of anesthesiologists physical status classification , saklad , 1941 ). there were no adverse effects from vector injection ( 3 × 10 9 particles ) and 24 hours post - injection there was no detectable virus shedding . twelve hours after prodrug injection the patient experienced nausea , ( who grade 1 ) which was known as a reaction to the prodrug . also hip pain increased , which was anticipated as the initial therapy is intended to cause more loosening . 16 ml of cement was injected into periprosthetic space ( see fig7 b ) indicating significant destruction of interface tissue creating a void into which cement could now be introduced . the patient was ambulated the day after surgery . at two and four weeks after cement injection the patient had no pain in the treated hip , and was still improving . the maximum walking distance had increased from 4 - 5 metres to 30 metres . subjective walking distance assessed by the patient ( 0 : 0 metres , 100 : unlimited walking distance ) increased from 4 to 66 . the patient &# 39 ; s pain score ( 0 : no pain , 100 : unbearable pain ) decreased from 81 preoperatively to 2 . in addition , she could now sleep on her side without pain , which she had been unable to do for four years . in terms of perceived dependency ( 0 : completely dependent on others , 100 : completely independent ) the score decreased from 95 to 54 . patient 2 is a 72 year old woman with loosening of her left hip prosthesis and an asa classification of ii ( mortality risk 2 . 8 %). again , there was no detectable virus shedding 24 hours after vector injection . 18 ml of cement was injected following a similar procedure ( fig8 b ). four weeks post - treatment the pain score had decreased from 43 to 22 ( probably reflecting the presence of a post - operative haematoma , requiring 4 - 5 weeks to resolve ). specifically hip joint - related pain disappeared . maximum walking distance increased from 500 to 2000 metres . by the 3 month follow - up , the haematoma had completely resolved and pain score had further decreased to 7 . the patient continues to improve in terms of walking performance and other activities . the current study is the first to use in vivo intra - articular adenoviral mediated gene transfer in a clinical setting . the preliminary results suggest that gene therapy and cement injection for hip prosthesis refixation is clinically feasible . all references cited herein are hereby incorporated by reference in their entireties . 1 . anderson w f ( 1998 ) human gene therapy . nature 392 : ( 6679 suppl ): 25 - 30 . 2 . bilsland , a . e ., et al . 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