Patent Application: US-6535006-A

Abstract:
the use of herpes simplex virus in the treatment of tumour by extratumoural administration of said hsv , and the use of hsv in treatment of tumour by combination therapy with a pharmaceutical wherein the hsv and / or pharmaceutical is administered at an extratumoural location , is disclosed .

Description:
specific details of the best mode contemplated by the inventors for carrying out the invention are set forth below , by way of example . it will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details . nude mice were subcutaneously injected with ˜ 20 × 10 6 uvw tumour cells ( a glioma cell line ) at 60 % confluency per mouse to generate uvw xenografts in the right flank . the mice were inspected regularly for xenograft formation . once the xenografts had reached approximately 5 mm × 5 mm the mice were injected intravenously with 10 7 pfu hsv 1790 via tail vein injection . in these experiments hsv 1790 was used as an exemplary hsv - 1 strain 17 icp34 . 5 null mutant . no prodrug activatable by ntr was administered . one mouse was sacrificed at day 1 post injection and one at day 7 and the organs harvested . the following tissues were collected : tumour , blood , liver , lung , spleen , heart , kidney , gut , brain and skin . half of each sample was flash frozen in ln2 and processed for polymerase chain reaction ( pcr ) analysis , the other half was fixed in neutral buffered formalin for use in immunohistochemistry . sections were stained with an hsv polyclonal antibody ( dako hsv type 1 polyclonal { cat . no . b0144 } and secondary antibody vectastain elite rabbit igg kit { cat . no . pk6101 }) and counterstained with haematoxylin . the sections were observed by light microscopy . positive staining was indicated by a dark brown colour and negative staining by a blue colour . background staining was present in some samples and showed variation between tissue types , for example muscle and cardiac cells exhibited a light brown background stain . the background staining observed was separate and entirely distinguishable from the dark brown colour indicative of positive hsv staining . the results of the immunohistochemical analysis are shown in fig1 - 31 . dna and rna was isolated from the flash frozen samples . the rna was used to make cdna using the improm reverse transcriptase kit ( promega ). the cdna obtained was then used for pcr . the following pcrs were performed : hs13 ( acg acg acg tcc gac ggc ga ; [ seq id no . 1 ]) and hs14 ( gtg ctg gtg ctg gac gac ac [ seq id no . 2 ]) these primers anneal to hsv - 1 sequence co - ordinates 93536 - 93555 and 93813 - 93794 ( complementary ) which lie within the ul42 region of the genome . this region codes for a sub - unit of the viral dna polymerase — the dna polymerase accessory protein . the resulting pcr product is 278 base - pairs in length and can be visualised by agarose gel electrophoresis . the reaction conditions used were a 94 ° c . ‘ hot - start ’ for 2 minutes followed by 34 cycles of { 94 ° c . for 15 seconds ( denaturation ); 72 ° c . for 1 min ( annealing ); 72 ° c . for 1 minute ( extension )} and a final extension step at 72 ° c . for 2 minutes . pcr utilised primers from the nitroreductase ( ntr ) enzyme from e . coli b genomic dna . sequence information for e . coli ntr can be found at the ncbi database ( http :// www . ncbi . nlm . nih . gov /) under accession numbers ba000007 ( gi : 47118301 )— e . coli complete genome sequence — and bab34039 ( gi : 13360074 )— nitroreductase sequence information . upstream primer 5 ′- ctttcacattgagtcattatgg - 3 ′( seq id no . 3 ); and downstream primer 5 ′- ttacacttcggttaaggtgatg - 3 ′ ( seq id no . 4 ) were used , based on those of clark et al 15 . following initial denaturation at 94 ° c . for two minutes , pcr conditions were 95 ° c . for 30s ( denaturating ), 55 ° c . for 30s ( annealing ), and 72 ° c . for 60s ( extension ), for 32 cycles . following initial denaturation at 94 ° c . for two minutes , pcr conditions were 95 ° c . for 30s ( denaturating ) 57 ° c . annealing temperature and 72 ° c . ( extension ) for 60s for 30 cycles . the results of the pcr analysis , as visualised by agarose gel electrophoresis , are contained in tables 1 and 2 . the pcr results are consistent for both dna and rna . whilst the dna pcr results may be used to confirm the presence of the hsv in a particular tissue , rna pcr provides an indication of virus activity in that tissue . in particular , the rna results indicate whether the hsv is actively replicating and thus provides information regarding the existence of a pathogenic infection of the cells of a given tissue . this information may be used in conjunction with the immunohistochemical analysis for corroboration of the results . a weak positive signal is present in the tumour samples on day 1 increasing to a strong positive signal by day 7 . this is consistent for both hsv and ntr pcr for both rna and dna and indicates the presence and accumulation of replicating hsv in tumour tissue . the pcr observations are reflected by the immunohistochemical analysis . fig1 shows positive staining for hsv confirming the presence of hsv in tumour tissue at day 1 . fig2 and 3 show regions of hsv positive staining . in particular , fig3 shows an island of positive hsv staining in which the cells show the classic appearance of hsv infection . the cells are large , multi - nucleated and necrotic . by day 7 ( fig4 ) the areas of hsv staining within tumour tissue are larger and more widespread compared to day 1 . fig7 shows an increased magnification view of area e of fig4 . the tissue in this region is highly necrotic and cell debris from dead cells is present . in spleen tissue , the dna pcr results at day 1 are positive for hsv , but not ntr . the day 1 rna pcr result was negative for both hsv and ntr indicating that any hsv present is not actively replicating . at day 7 no positive result was obtained either by dna or rna pcr of spleen tissue . the immunohistochemical results for spleen tissue are shown in fig1 ( day 1 ) and fig2 ( day 7 ). in fig1 some positive hsv staining is present to the right of the line indicated . by day 7 ( fig2 ) this has decreased and most of the cells are negative for hsv . these results are consistent with the pcr data that show a loss of hsv in spleen between day 1 and day 7 . in view of the fact that the rna pcr result is negative for spleen tissue at both day 1 and day 7 ( indicating that hsv in the spleen are not replicating ), this result is most likely attributable to spleenic filtering of the blood . the dna pcr data indicates weak positive results in blood , skin , gut , heart and liver . these results are weak and are not consistently repeated . moreover , they are not borne out by the immunohistochemical analysis except for some possible weak positive staining for hsv in skin as shown in fig2 . of course , the rna pcr results are negative for blood , skin , gut , liver and spleen . given that the hsv is administered intravenously in the tail vein , the presence of some hsv particles in various tissues owing to the circulation of the hsv particles in the blood is not surprising . importantly , the rna pcr results show that hsv replication activity , which may lead to lysis and a therapeutic effect , is exclusively limited to tumour cells by day 7 . the rna pcr data indicates a medium positive result in heart tissue at day 1 . however , in contrast to the results in tumour tissue this result is not consistent for both hsv and ntr , disappears by day 7 and is not borne out by the immunohistochemical analysis which does not show any positive staining for hsv . changes in tumour size were not followed in these experiments but preliminary survival data indicates a median survival time of 7 days in the absence of virus and 14 days where virus was administered intravenously . this data supports the ability of the hsv to not only target the tumour , but to treat the tumour and improve survival time . the results demonstrate that non - neurovirulent hsv - 1 mutants of strain 17 may be administered at a site on the body that is distal to a tumour requiring treatment such that the hsv accumulates in the tumour . the results support an increasing accumulation of hsv in the tumour over time and indicate that the hsv may self - target the tumour . hsv rna production is exclusively limited to tumour cells by day 7 and supports the accumulation of hsv in tumour tissue by exploiting the ability of the oncolytic hsv to selectively replicate in dividing tumour cells . the immunohistochemical analysis provides further support for both hsv infection of tumour cells and necrosis of tumour cells and is consistent with a mechanism of lytic hsv replication in tumour tissue . hsv1790 is a second generation oncolytic virus generated by inserting the bacterial enzyme nitroreductase ( ntr ) into the oncolytic virus hsv 1716 , under the control of the cmv ie promoter . ntr converts the inactive prodrug cb1954 into an active alkylating agent which has an anti - tumor effect . the purpose of this study was to determine the anti - tumor efficacy of the combination of hsv1790 and cb 1954 in vitro and in vivo , and to explore the efficacy of this combination after systemic ( intravenous ) administration of hsv1790 . in vitro , cells which are known to be non permissive for hsv 1790 replication were used in order to distinguish between an oncolytic effect due to viral replication , and cell death due to activated prodrug . in vivo , intratumoural administration of hsv1790 ( 10 5 - 10 9 pfu ) with , or without administration of cb1954 ( max 80 mg / kg ) by the intraperitoneal route was performed on mouse xenograft models of a2780 , cp70 and a431 cell lines , and tumor volume measured regularly . tumor and organ distribution of hsv1790 following intravenous administration was determined by immunohistochemistry and by analysis of dna and rna from harvested tumor tissues and organs . administration of hsv 1790 , followed by cb1954 , enhanced tumor cell killing in 3t6 cells compared to hsv1790 alone . in vivo , the combination of intra - tumoral administration of hsv1790 , followed by intraperitoneal cb1954 , enhanced tumor reduction and improved survival compared to administration of virus alone . following systemic administration of hsv1790 , viral replication is detected in tumor tissue , but not in normal organs . hsv1790 , when used in combination with cb1954 , can enhance tumor cell killing in vitro and enhance tumor reduction and survival in vivo without toxicity in normal tissues and organs . cancer is a genetic disease , and the hallmarks of individual cancer cells are mutations in genes related to growth control , apoptosis , immortality and also functional aberrations that support the ability of cancer cells to invade and metastasize ( 1 ). genetic therapies in cancer are designed to produce one of several types of outcome . firstly , the genetic material introduced into the host or tumor may result directly in cancer cell death , for example by the intratumoral administration of a replication competent virus . secondly , the genes introduced into the host or tumor cells are expressed , and can induce an immune response directed against the tumor ( 2 ). thirdly , the gene product may be toxic to the tumor cell or may activate a subsequently administered drug into a cytotoxic agent that results in cancer cell death . this approach is frequently described as ‘ gene directed enzyme pro - drug therapy ’ ( gdept ) or ‘ suicide gene therapy ’ ( 3 ). herpes simplex virus type 1 ( hsv - 1 ) has a number of pertinent characteristics that support its use in cancer therapy . it infects a broad range of cell types , it is cytolytic by nature ( the life cycle of the virus results in host destruction ), and it has a well characterized and , in the case of glasgow strain 17 +, a fully sequenced genome ( 4 ). furthermore , its large genome ( 152 kb ) contains non essential genes that can be replaced by therapeutic transgenes of up to 30 kb ( 5 ). hsv1716 is a selectively replication competent mutant of the hsv - 1 in which both copies of the rl1 gene has been deleted ( 6 ). the rl1 gene encodes the protein icp34 . 5 , which is a specific determinant of virulence ( 7 ). icp34 . 5 functions by complexing with proliferating cell nuclear antigen ( pcna ), which is involved in dna replication and repair ( 8 ). in most tumor cells , pcna levels are high , and icp34 . 5 is not required for productive hsv replication . in contrast , in normal , terminally differentiated cells , pcna levels are low and icp34 . 5 is required to recruit any available pcna to initiate virus replication . thus hsv1716 replicates in actively dividing but not terminally differentiated cells ( 9 ), and has an antitumor effect in vitro in a range of tumor cell types including gliomas ( 10 ). hsv 1716 has demonstrated selective tumor cell killing , with minimal toxicity , and its administration has resulted in improved survival in a number of xenograft tumor models in mice , including glioma ( 1 l ), melanoma ( 12 - 14 ), mesothelioma ( 15 ), ovarian ( 161 , lung ( 17 , 18 ) and breast ( 19 ) carcinomas . clinical trials of intra - lesional administration of hsv1716 in patients with glioma , melanoma and squamous cell carcinoma of the head and neck have been performed ( 20 - 23 ) and have demonstrated the safety of this approach , and with evidence that the virus is capable of directly destroying human tumor cells while leaving normal cells intact . one potential limitation of the intra - lesional administration of hsv1716 for the treatment of human tumors is that there is heterogeneity of cell type and growth state within a tumor , and consequently not all cells within a tumor will be permissive for lytic replication by hsv1716 . one strategy to overcome this limitation is to combine the oncolytic effects of hsv1716 with a gene - directed enzyme prodrug therapy approach . a number of enzyme pro - drug systems have been proposed for cancer gene therapy ( 24 ), including the e . coli nitroreductase ( ntr ) with the pro - drug cb1954 ( 25 - 27 ). cb1954 [ 5 ( aziridin - 1 - yl )- 2 , 4 - dinitrobenzamide ] is a mono - functional alkylating agent that is poorly metabolized in human cells and thus has low toxicity . the enzyme , ntr , converts the inactive cb 1954 pro - drug into its active form , which is a functional cytotoxic alkylating agent that introduces poorly repaired inter - strand cross - links into dna and these lesions kill cells regardless of their cell cycle state ( 28 ). in addition , the active metabolite is diffusible and membrane permeable — this results in an efficient bystander effect ( 29 , 30 ). we have generated and characterized a second generation virus ( hsv1790 ) which contains the e . coli nitroreductase gene inserted into the rl1 locus of the hsv1716 genome . as the virus should only replicate and produce ntr in tumor cells , toxicity to normal cells should be avoided . in this manuscript , we report the generation of this second generation virus , and demonstrate that the combination of hsv 1790 and the prodrug cb 1954 has enhanced tumor cell killing in vitro , and results in improved tumor reduction and survival in vivo compared to the oncolytic effect of hsv 1790 alone . another potential drawback in the application of genetic therapies in cancer medicine is that administration of the genetic therapeutic usually requires direct injection into the patient &# 39 ; s tumor . consequently many of the clinical trials of these therapies have been restricted to patients with localized tumors that are accessible by direct injection or by injection under radiological guidance or post operatively . however , patients with advanced cancer invariably have metastatic disease or tumors that are inaccessible for direct injection . in this manuscript we report safety and efficacy data after systemic ( intravenous ) administration of hsv1790 in athymic mice bearing human tumor xenografts . we demonstrate that the virus selectively locates to tumor tissues , replicates and produces ntr within tumors without affecting other organs . furthermore , tumour reduction , and enhanced survival , is observed in vivo in tumor bearing mice following pro - drug administration . these studies indicate that systemic administration of hsv1790 and cb1954 should be explored further in human clinical trials . the plasmid pps949 , containing the ntr gene downstream of the cmv ie promoter ( pcmv - ntr ) in a plncx ( clonetech ) backbone , was a kind donation from professor lawrence young ( university of birmingham ). the pcmv - ntr fragment was excised from pps949 and cloned into bglii digested , cip treated rli -. dires - gfp . clones were screened for the pcmv - ntr insert using bgliixhoi restriction enzyme analysis and one clone was found to contain the insert in the correct orientation ( data not shown ). the plasmid rli . dcmv - ntr - gfp was digested with scai which cuts in a region outwith the flanking sequences and the pcmv - ntr - ires - gfp - polya fragment . bw cells were co - transfected with the linearised plasmid and hsv17 + dna . recombinant virus was identified using gfp fluorescence and several green plaques were plaque purified as described in ( 31 ). to ensure that recombination had taken place in the correct location and that the endogenous copy of the rl1 gene had been replaced by the pcmv - ntr - ires - gfp - poly a cassette , dna from hsv1790 was purified , digested with bamhi and analysed by southern blot ( data not shown ). fig3 shows a schematic representation of hsv1716 / cmv / ntr ( designated hsv 1790 and referred to as such hereafter ) genome and relevant fragment sizes expected from bamhi digestion of hsv 1790 dna . bhk ( baby hamster kidney 21 clone 13 ) cells and mouse embryo 3t6 cells were obtained from the european collection of cell culture ( ecacc ). bhk cells were grown in eagle &# 39 ; s medium supplemented with 10 % newborn calf serum and 10 % ( v / v ) tryptose phosphate broth . this will be referred to subsequently as etc 10 . for virus titrations and plaque purification emc10 ( eagle &# 39 ; s medium containing 1 . 5 % methylcellulose and 10 % newborn calf serum ) was used to overlay the cells . the ovarian cell lines a2780 and cp70 ( obtained from ecacc ) were cultured in rpmi 1640 medium supplemented with 10 % fetal calf serum . cell cultures were incubated in a humidified atmosphere of 5 % co 2 / 95 % o 2 at 37 ° c . the squamous cell carcinoma cell line a431 , cervical carcinoma c33a and the glioma line u373mg were all obtained from the american tissue culture collection (- atcc ) and were cultured in dmem containing 10 % fetal calf serum and incubated in a humidified atmosphere of 10 % co 2 / 90 % o 2 at 37 ° c . all cultures were tested for mycoplasma using venorgem ® mycoplasma pcr detection kit ( cambio , cambridge , uk ). titration of virus stocks was carried out as previously described in ( 32 ). western blotting was carried out using standard techniques . for ntr detection a rabbit polyclonal anti - ntr antibody was kindly provided by professor lawrence young ( university of birmingham , uk ) and used at a concentration of 1 / 1000 . the secondary antibody , goat anti - rabbit igg peroxidase conjugate ( sigma ) was also used at 1 / 1000 . in vivo tumor reduction and biodistribution studies . female 6 - 8 week old athymic nude mice ( charles river labs ) were maintained under specific pathogen free conditions and in isolation after virus injection . actively growing a2780 , cp70 and a431 cells were harvested and resuspended in phosphate buffered saline ( pbs ). cells ( 1 × 10 7 for a2780 and a431 , 5 × 10 6 cp70 per mouse ) were injected into the flanks of athymic mice . the mice were examined regularly for tumor growth . when the mean tumor diameters were approximately 5 mm , the animals were randomized into groups . hsv 1790 or hsv1716 or pbs ( maximum volume 100 μl ) was administered by direct intratumoral injection . virus was diluted in pbs + 10 % fetal calf serum and kept at − 70 ° c . until use . cb1954 , resuspended in archais oil with 10 % acetone was then administered by intraperitoneal injection ( maximum volume 100 μl , maximum dose 3 × 80 mg / kg ) at least 48 hrs after viral injection . for general examination of toxicity , animals were weighed regularly and tumor volumes were calculated from caliper measurements ( volume = d 3 × π / 6 ). for statistical analysis , unpaired student &# 39 ; s t - tests were used . p values of & lt ; 0 . 05 were considered significant . all animal experimentation was performed according to united kingdom home office regulations and ukccr guidelines adhered to at all times . organs and xenograft tumor samples were removed immediately from individual mice after sacrifice and fixed in neutral buffered formalin ( nbf ) for at least 24 hrs before embedding in paraffin using standard procedures , sections were prepared for immunohistochemistry by standard protocols . briefly , paraffin embedded sections were dewaxed , dehydrated and endogenous peroxidase was quenched . non - specific binding was reduced with 10 % normal goat serum before sections were incubated with primary antibody overnight at 4 ° c . ( hsv - 1 polyclonal , dako 1 : 1000 ). after incubation with biotinylated secondary antibody ( vector anti - rabbit elite kit 1 : 500 ) and avidin biotin complex ( abc ) solution ( elite kit , vector laboratories ) color was developed using diaminobenzidine ( dab ) ( vector labs ) as the chromogen . the slides were counterstained , dehydrated , mounted and visualized using a light microscope . hsv - 1 infected liver sections were used as a positive control , omission of primary antibody and tumor sections from mice uninfected with virus constituted the negative controls . tumor tissue and mouse internal organs were collected from individual mice at time of sacrifice and frozen at − 70 ° c . a small (& lt ; 0 . 5 g ) tissue sample was resuspended in buffer and the cells were disrupted with a retsch mm200 homogenizer . rna was extracted using the promega ( sv ) rna extraction kit following the manufacturers recommended procedure ( promega , southampton , uk ). tissue (& lt ; 0 . 5 g ) samples were homogenized using the retsch mm200 homogenizer and dna extracted using the nucleon st dna extraction kit ( thistle scientific ltd , glasgow , uk ) using the manufacturers instructions . reverse transcription was preformed using improm reverse transcriptase kit ( promega , southampton , uk ) using the random hexanucleotide primers under manufacturers recommended conditions . pcrs were carried out with ready - mix ( abgene , surrey , uk ) and 1 μl of rt reaction mix in a 20 μl reaction volume . for hsv pcr the primers hs 13 ( acg acg acg tcc gac ggc ga ) [ seq id no . 7 ] and hs 14 ( gtg ctg gtg ctg gag gac ac ) ( 34 ) [ seq id no . 8 ] were used . these anneal to hsv - 1 sequence co - ordinates 93536 - 93555 and 93813 - 93794 ( complementary ) which lie within the ul42 region of the genome . this region codes for a sub - unit of the viral dna polymerase — the dna polymerase accessory protein . the resulting pcr product is 278 base - pairs in length and was visualized by agarose gel electrophoresis . the reaction conditions used are a 94 ° c . ‘ hot - start ’ for 2 minutes followed by 34 cycles of { 94 ° c . for 15 seconds ( denaturation ); 72 ° c . for 1 min ( annealing ); 72 ° c . for 1 minute ( extension )} and a final extension step at 72 ° c . for 2 minutes . for ntr : the pcr used the primers sequence of the nitroreductase ( ntr ) enzyme from e . coli b genomic dna . upstream primer 5 - tttcacattgagtcattatgg - 3 [ seq id no . 9 ] and downstream primer 5 - ttacacttcggttaaggtgatg - 3 [ seq id no . 10 ] were used ( 35 ). following initial denaturation at 94 ° c . for two minutes , pcr conditions were 95 ° c . for 30s , 55 ° c . for 30s and 72 ° c . for 60s , for 32 cycles . expression of ntr in cell lines infected with hsv1790 . to demonstrate that hsv1790 expresses the ntr protein a western blot was performed using a polyclonal ntr antibody . although the recombinant virus strongly expressed gfp ( observed during the plaque purification process ) indicating that it should express ntr , this was not conclusive proof . four cell lines — bhk , c8161 , vm and 3t6 were infected with hsv1790 and protein expression analysed by western blotting , using an ntr - specific antibody . fig3 shows that the 24 kda ntr protein was expressed in all the cell lines infected with hsv1790 . even though the virus does not replicate efficiently in confluent 3t6 cells , reasonably strong expression of ntr was detected demonstrating that productive replication of the virus is not necessary for ntr expression in infected cells . enhanced cell kill in vitro in hsv1790 infected cells treated with cb1954 . previous experiments had shown the replication kinetics of the virus to be identical to that of the parental strain hsv 1716 indicating no alteration in replication potential due to the insertion of the ntr gene ( paul dunn , phd thesis , university of glasgow , 2003 ). to determine whether ntr expression would result in enhanced cell killing after hsv1790 infection and addition of the prodrug cb1954 , cytotoxicity assays were performed in 3t6 cells , a cell line in which the hsv did not replicate efficiently . as almost no hsv1790 replication occurs in 3t6 cells infected at a low moi (& gt ; 0 . 1 ), any significant cell death observed in the cells infected with hsv1790 after cb1954 administration would be due to ntr expression in the cells , and the subsequent activation of cb1954 . the addition of 50 pm cb1954 alone had previously been shown to cause less than 5 % cell death -( data not shown ). the effects of hsv1790 infection , with or without 50 pm cb1954 , were examined and the results shown in fig3 and 35 . five days after treatment , 75 % of the cells treated with 10 plaque forming units ( pfu )/ cell hsv1790 + cb1954 were dead compared to only 2 % in those treated with hsv1790 only ( fig3 and 35 ). it can therefore be concluded that almost 70 % of the cell death observed in the hsv1790 infected cells was due to ntr converting cb1954 to its toxic form and not from cell lysis from viral replication . no formal toxicity studies were performed . however in preliminary experiments , the dose of hsv1790 , administered by a single intratumoral injection , was escalated in groups of mice bearing a2780 human tumour xenografts ( n = 3 ) to determine the acceptable dose for subsequent experiments , based on toxicity . a dose of 1 × 10 9 pfu hsv1790 by intratumoural injection was not tolerated well ; the mice lost more than 10 % of their body weight and had to be sacrificed . at doses of 1 × 10 8 and below , the treatment was well tolerated and the mice did not show any signs of ill health or adverse effects . to determine the efficacy of hsv1790 + cb1954 in treating established subcutaneous human tumour xenografts , serial tumour volume measurement were taken regularly after administration of hsv1790 alone , hsv1790 + cb1954 or no treatment . administration of cb1954 alone was not performed as previous experiments had shown that cb1954 administration as a single agent has no anti - tumoral effect ( data not shown ). two doses of virus were administered as giving multiple intratumoral injections appears to be more effective than the administration of the same total dose on one occasion ( 36 ). cb1954 treatment was commenced 48 hrs after the last virus injection ( max 80 mg / kg on 3 occasions ). previous experiments in vitro ( data not shown ) and ( 36 ) suggested that administering the prodrug too soon after viral administration kills the cells in which the virus is replicating , effectively thereby reducing the number of infectious virus particles within the tumor . administration of hsv1790 and cb1954 had no effect on body weight ( data not shown ) and there were no signs of toxicity in the mice . in mice bearing a2780 ( fig3 a ) and a431 ( fig3 c ) xenografts , there was a marked reduction in tumor volume when the xenografts are treated with hsv1790 + cb1954 compared to hsv 1790 alone . for mice bearing a431 tumours , administration of hsv1790 + cb1954 resulted in significantly smaller tumour volumes ( p = 0 . 03 ) and significantly longer median survival ( p & lt ; 0 . 05 ) ( fig3 c and f ) than in the mice treated with hsv1790 alone . in mice bearing cp70 tumor xenografts , administration of hsv1790 reduced tumor volume compared to control mice ( fig3 b ) but the addition of prodrug had no further anti - tumor effect . to explore the tumor and organ distribution of hsv1790 after systemic ( intravenous ) administration , 1 × 10 7 pfu of hsv1790 was administered by tail vein injection to athymic nude mice bearing established uvw and a431 tumour xenografts overlying their right hind flank . mice were sacrificed either on day 1 or day 7 post injection and the tumors , blood and major organs ( brain , heart , lung , liver , spleen , intestine , kidney and skin ) were collected . tissue from these organs was analysed by pcr and immunohistochemistry for the presence of hsv1790 . immunohistochemical staining with an anti - hsv antibody revealed active replication of the virus within the xenograft tumours ( fig3 a - f ) necrosis was also widespread in the areas of positive staining ( fig3 f ). there was no indication of positive staining in any other organ ( fig3 g - j ) with the exception of skin ; in which some positive staining was visible in cells below the dermal and epidermal layer ( fig3 k ). dna and rna , extracted from the organs and tumours were also analysed by pcr , to determine presence of virus and the replication of the virus ( fig3 ). both viral dna and rna are detected in the tumour tissue at day 1 post i . v injection at a low level . by day 7 post i . v injection the pcr band intensity has increased , demonstrating that hsv is replicating within the tumor tissue . furthermore ntr dna and rna is detectable in the tumor demonstrating that hsv1790 is replicating within the tumor and is also producing the ntr protein . a high level of viral dna is seen in the spleen at day 1 . however there is no replicating virus detectable by rt - pcr . this suggests that the virus is not actively replicating and the positive pcr result is likely to be due to spleenic clearance of the virus from the circulating blood . tumor growth inhibition in athymic nude mice after administration of hsv1790 by intravenous injection . to determine whether hsv1790 replication within tumors seen after intravenous injection has any anti - tumor effects , mice with a431 xenografts were randomly allocated into groups : ( a ) 2 × ( 1 × 10 6 ) pfu of hsv1790 injected i . v ( intravenous ) at days 1 and 3 followed by 20 mg / kg injection cb1954 i . p ( intraperitoneal ) daily for 5 days ; ( b ) 2 × ( 1 × 10 6 ) pfu of hsv1790 injected i . v at days 1 and 3 ; ( c ) no virus injections ( injection of pbs only ). fig3 show that hsv1790 either alone or in combination with cb1954 results in significantly prolonged survival of tumor bearing mice . we have previously described the safety and potential efficacy of herpes simplex virus therapy using the selectively replication competent mutant hsv1716 both in vivo ( 11 - 19 ) and in clinical studies ( 20 - 23 ). however , it is anticipated that hsv1716 although able to infect all cells will not lyrically replicate in all cells within tumors due , predominantly , to the heterogeneity of the cell state within a tumor mass . to overcome this , we have generated hsv1790 , a second generation herpes simplex virus derived from the icp34 . 5 null mutant hsv1716 and in which the e . coli ntr gene has been inserted under the control of the cmv ie promoter . western blot analyses demonstrated that the ntr protein was expressed in all four cell lines tested following infection with hsv1790 . furthermore , the addition of the prodrug cb1954 to 3t6 cells infected with hsv1790 resulted in a significantly enhanced cell kill compared to infection with hsv1790 alone . administration of the prodrug cb1954 to athymic mice , bearing tumor xenografts of either a431 or a2780 cells , 48 hours after intratumoral administration of hsv1790 resulted in a marked reduction of tumor volumes , and also resulted in significantly improved survival for mice bearing a431 tumors , compared to administration of hsv1790 without administration of cb1954 . in contrast , administration of cb1954 following administration of hsv1790 to mice bearing cp70 tumor xenografts had no additional anti - tumor effect compared to administration of hsv1790 alone . cp70 is a derivative of the ovarian carcinoma cell line a2780 , and has a drug - resistant phenotype due to the loss of mlh1 , a key protein involved in mismatch repair ( 37 , 38 ). these cells display resistance to a variety of dna damaging agents and alkylating agents . cp70 has a higher tolerance of cb1954 than the parental a2780 , with ic 50 values in vitro of 29 μm and 60 μm respectively ( 39 ). expression of ntr in these cells results in similar - fold sensitisation to the prodrug , with the value for a2780 remaining approximately half of that for cp70 cells ( 39 ). as the active form of cb 1954 is a bi - functional alkylating agent , it is possible that the intratumoral activation of cb 1954 to its active form by ntr expression , following hsv1790 administration , is insufficient to overcome the drug resistant phenotype of these cp70 cells . expression of a prodrug activating enzyme early in the virus replication cycle risks killing the virus ( 40 , 41 ). in addition , the insertion of a transgene could theoretically worsen virus efficacy in vivo . however , neither of these potential drawbacks is likely with hsv1790 based on the results reported in this manuscript . another potential drawback of combining an oncolytic virus with a ‘ suicide - gene therapy ’ approach is that if the virus alone can induce efficient cytopathic effects , then the transgene may not be expressed in target cells prior to the cell death , and there may be no additional therapeutic benefit to the combined approach . again , the data presented in this manuscript suggests that the second generation virus hsv1790 , when combined with the addition of the prodrug , has an enhanced anti - tumor efficacy compared to hsv1716 . most human clinical trials of genetic therapies for cancer , including those using oncolytic viruses , continue to use direct intra - tumoral injection as the route of administration of the therapeutic virus . however , as most patients have metastatic disease , the preferred route of delivery of oncolytic viruses is through intravenous administration . intravenous administration of viruses can cause significant systemic side effects , due to the acute release of cytokines . these symptoms can be effectively minimised by appropriate pre - radiation ( 42 - 44 ) and intravenous administration of newcastle disease virus ( 42 , 43 ) and onyx 015 ( 44 , 45 ) have been well tolerated in initial clinical studies with minimal toxicity . our results demonstrate replication of hsv1790 and expression of the ntr transgene within tumor tissue following intravenous administration , but with no evidence of viral replication in normal organ tissues . however , as athymic nude mice have a compromised immune system , the effect of a normal immune system on viral toxicity and efficacy is unknown . viral infection of tumors can attribute an anti - tumor immune response which is beneficial ( 13 , 46 , and 47 ). in contrast , the host &# 39 ; s immune system has been shown to neutralise virus and inhibit oncolytic activity ( 48 ). however prior immunity to hsv does not appear to significantly impair the therapeutic efficacy of herpes simplex therapy in immunocompetent models ( 49 , 50 ). further studies of systemic administration hsv 1790 in immunocompetent models are required . in conclusion , we have demonstrated that the combination of the oncolytic herpes simplex virus hsv1716 with a ‘ suicide gene therapy ’ approach can enhance anti - tumor efficacy in comparison with the effects of the oncolytic virus alone . intravenous administration of hsv1790 results in expression of the ntr transgene in tumor tissues , but not in normal organs , with efficient anti - tumor efficacy following administration of prodrug . as the prototype oncolytic virus hsv1716 has been shown to be totally non - toxic in human patients and has recently entered a phase iii trial , the results presented in this paper lead us to the conclusion that clinical studies of hsv1790 and cb1954 are warranted in patients with otherwise refractory tumors . 1 . bl liu , m robinson , z - q han , rh branston , c english , preay , y mcgrath , sk thomas , m thornton , p bullock , c a love and rs coffin ; 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