Patent Application: US-201213417708-A

Abstract:
a herpes simplex virus is disclosed in which the herpes simplex virus genome comprises a nucleic acid sequence encoding an ing4 polypeptide .

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 . an hsv1716 variant expressing the tumour suppressor gene inhibitor of new growth 4 an hsv1716 variant expressing the ing4 tumour suppressor gene was constructed as follows . primers for the pct amplication of ing4 were designed using the human ing4 sequence deposited in the ncbi at ncbi . nlm . nih . gov under accession no . nm — 016162 gi : 38201669 ). the forward primer ga gaattcgcggccgcg atggctgcggggatgtatttg ( seq id no . 1 ) hybridises at the atg start codon of ing4 ( in bold ) and incorporates ecor1 and not1 restriction sites ( underlined ) upstream of the translation start site . the reverse primer ag tctagactcgag ctatttcttcttccgttcttggga ( seq id no . 2 ) hybridises 36 nucleotides downstream ( ing4 sequence in bold ) from the tag stop codon of ing4 and incorporates xho1 and xba1 sites ( underlined ). the c750 bp cdna encoding ing4 ( seq id no . 5 ) was successfully amplified from commercially available human liver and placental cdna libraries ( fig1 ) and , as a stronger band was obtained with the placental cdna ( lanes 2 and 3 ) library , this was used for ing4 cloning . the pcr product was ligated into the pgem - teasy vector and sequencing confirmed a 100 % match with nm016162 . the pcr product was then digested directly with ecor1 followed by xba1 and ligated into the likewise digested mammalian expression vector pcdna4 - myc - his ( invitrogen , paisley , uk ). positive clones were identified by bamhi digestion of miniprepped dna ( there is an internal ing4 bamhi site at nucleotide 608 ) and the ing4 expression cassette ( cmv - ie promoter from plasmid plus ing4 cdna ) was excised from a positive clone by nrui / xhoi digestion , blunt ended using klenow and cloned into the bglii digested , blunt ended and ciap treated rl1 shuttle vector prl1del / gfp used for the production of hsv1716 variants by homologous recombination . rl1del / gfp is a modified version of prl1 - del containing an expression cassette for gfp ( pgk - gfp ). rl1 - del is a promoterless cloning vector , suitable for generating icp34 . 5 null hsv - 1 . it contains a hsv - 1 fragment formerly consisting of the rl1 gene and its flanking sequences with the majority of the rl1 gene removed and replaced with a multi - cloning sequence ( mcs ). the transgene to be inserted into the rl1 loci is ligated into the mcs of rl1 - del and homologous recombination with hsv - 1 dna , driven by the rl1 flanking sequences , results in concomitant deletion of the icp34 . 5 open reading frame and incorporation of the appropriate transgene . to assist in plaque purification of recombinant viruses , the green fluorescent protein gene is also inserted into the mcs of rl1 - del . rl1 - del contains the hsv - 1 bamhi k dna fragment 123459 - 129403 which includes the rl1 gene and its flanking sequences cloned into the bamhi site of plasmid pgem - 3zf ( promega , southampton , uk ). the 477 bp pflmi / bsteii fragment from the rl1 orf ( 125292 - 125769 ) has been removed to inactivate the icp34 . 5 gene and replaced with a mcs providing various restriction enzymes sites including those for bglii , nrui and xhoi . to create rl1 - del / gfp , the 1 . 3 kbp blunt - ended ecori / aflii fragment that contains the pgk promoter / gfp gene was obtained by restriction digestion followed by klenow treatment from the vector psnrg ( oligoengine , seattle , wash ., usa ) and ligated into the rl1 - del vector cut with the restriction enzyme nrui then alkaline phosphatase treated . insertion of the ing4 expression cassette in prl1 - del / gfp was confirmed by restriction enzyme digests and 50 μg of plasmid were linearized by scai digestion and , after column clean - up using a gfx kit ( ge healthcare , little chalfont , uk ), were used in conjunction with hsv - 1 dna to cotransfect bhk cells . rl1 - del / gfp / ing4 and viral dna ( c100 ug ) were mixed with 20 μl lipofectamine 2000 in 250 μl dmem / f12 serum - free medium and added to a 60 mm plate which contained 50 % confluent bhk cells . after 4 hours of incubation at 37 ° c . the medium was removed and the cells shocked for exactly 4 minutes with 25 % dmso . after 3 washes with 5 ml culture medium the cells were returned to 37 ° c . with 5 ml bhk medium and left for 72 hours . cells were then scraped into the supernatant , the mixture sonicated in a sonicator bath for 2 minutes and stored at − 70 ° c . until required . serial dilutions were then plated out on vero cells in 60 mm dishes , individual green fluorescent plaques were picked , added to 1 ml culture medium and sonicated in a sonicator bath for 2 minutes before serial dilutions were again plated out on vero cells . plaque purification was repeated 6 times before stocks of hsv1716ing4 were produced . the presence of the ing4 expression cassettes in the rl1 loci of hsv1716 was confirmed by both southern blotting using the alui / rsai icp34 . 5 fragment from plasmid pgem34 . 5 and by pcr using primers which amplify across the icp34 . 5 deleted region of hsv1716 . to confirm expression of the inserted ing4 transgene confluent monolayers of bhk cells in 60 mm plates were infected with either 0 . 1 or 1 pfu / cell hsv1716ing4 and , after 24 hours , whole cells extracts were prepared by the addition of 0 . 2 ml sds page sample buffer . sds page / western blotting using an ing4 antibody ( abcam , cambridge , uk ) identified a c29 kda protein in the cells infected with 1 pfu / cell hsv1716ing 4 ( fig2 , lanes 1 and 2 ). a weaker band was observed in the cells infected with 0 . 1 pfu / cell hsv1716ing4 ( fig2 ; lanes 3 and 4 ) and this protein was absent from mock infected cells ( fig2 , lane 5 ). in an initial experiment , 6 mice with subcutaneous implants of the cp70 ovarian tumour cell line were injected intratumorally with 1 × 10 7 pfu hsv1716ing4 on days 1 and 3 and tumour growth and survival was monitored daily . control mice were either injected with hsv1790 , an hsv1716 variant expressing the enzyme nitroreductase , or were injected with a similar volume of pbs . survival data is shown in fig3 . of the 6 mice treated with hsv1716ing4 , 1 tumour showed complete regression , 4 tumours grew at a slower rate than the untreated tumours and 1 tumour became ulcerated and the mouse was removed from the analysis . using survival times ( as measured by when the tumour reached the upper acceptable limit ) to assess each of the treatments , animals treated with the ing 4 virus had an average survival of 23 . 5 days compared to animals treated with no virus or those treated with hsv1790 which had survival times of 11 or 18 days respectively . log rank comparison of the survival curves shown in fig3 demonstrated significant differences with a p value of 0 . 03 . note that no cb1954 was administered to mice infected with hsv1790 and , under these circumstances this virus is equivalent to hsv1716 . in a subsequent experiment , groups of 6 mice bearing subcutaneous implants of human scc a431 tumour cells were given injections on days 1 and 3 of either 1 × 10 7 pfu hsv1716ing4 or hsv1716 or an equivalent volume of pbs and tumour growth and survival were determined daily . fig4 shows the average tumour volumes and demonstrated that the group treated with hsv1716ing 4 had a smaller average tumour volume than either the group treated with no virus or with the parental hsv1716 . statistical analysis showed that the differences in tumour volumes between hsv1716ing4 and hsv1716 and between hsv1716ing4 and no virus at day 15 were significantly different with p = 0 . 03 and p = 0 . 007 respectively . fig5 shows the km survival graph for the tumour - bearing mice treated with no virus , hsv1716 or hsv1716ing 4 . mice treated with no virus had a median survival of 8 days , those treated with hsv1716 had a median survival of 12 days and those treated with hsv1716ing 4 had a median survival of 21 days . comparison of the fig5 curves by log rank analysis shows that the difference between them is significant with p = 0 . 004 thus clearly demonstrating in this tumour model that hsv1716ing4 significantly reduced tumour burdens and enhanced survival . groups of 10 mice with subcutaneous human scc a431 tumours were intravenously injected with pbs ( no virus ) or with 1 × 10 6 pfu hsv1716 or hsv1716ing4 on days 1 and 3 and tumour growth and survival was monitored daily . after intravenous injection hsv1716ing4 significantly reduced tumour growth compared to no virus controls or to unmodified hsv1716 ( fig6 ). on day 10 post injection , the mean tumour volume for hsv1716 - injected mice was 421 ± 55 mm 2 compared to 193 ± 152 mm 2 for hsv1716ing4 injected mice and , using student &# 39 ; s t test , this difference is highly significant with p = 0 . 0022 . similarly , on days 14 or 20 , the average tumour volumes for hsv1716 - injected mice were 1096 ± 402 mm 2 or 1253 ± 155 mm 2 respectively versus 476 ± 448 mm 2 or 776 ± 438 mm 2 respectively for mice receiving hsv1716ing4 and , using student &# 39 ; s t test , both these differences are significant with p = 0 . 0125 or 0 . 0162 respectively . mice receiving no virus or 1 × 10 6 pfu hsv1716 had median survivals of approximately 14 days , whereas mice receiving hsv1716ing4 had a median survival of 17 days with 4 / 10 mice surviving beyond day 20 by which time all the pbs - or hsv1716 - injected mice had been sacrificed . a number of tumours from each of the groups of mice were removed at sacrifice and , after mechanical homogenisation , virus in the tumour extract was titrated with the results presented in table 1 . virus was readily extracted from all three tumours taken from the mice which received intravenous hsv1716ing4 but was only detected in ⅓ mice which received hsv1716 and the amounts of virus detected in the hsv1716ing4 tumours were approximately 1000 - fold greater than the amount extracted from the hsv1716 tumour suggesting better replication of hsv1716ing4 than hsv1716 within the tumour . previous studies , albeit in immunocompetent rats , have shown that only 0 . 001 % of intravenously injected virus lodges in the tumour 24 hours after administration ( schellingerhout et al 1998 , 2000 ) and , assuming that this provides a reasonable approximation for scid mice , then , of the 2 × 10 6 pfu administered intravenously , only 20 pfu will reach the tumour initially . assuming that there is no difference in biodistribution between hsv1716 and hsv1716ing4 , the above data demonstrates that hsv1716ing4 is significantly better at replication within human a431 scc tumour implants than hsv1716 and that this enhanced propagation improves survival . one of the hsv1716ing4 mice survived until day 30 , by which time the tumour had stopped growing and was almost completely regressed . after sacrifice of this mouse , all its organs were harvested including residual tumour tissue and , following mechanical homogenisation and titration , no virus was found in any of these tissues suggesting complete recovery and viral clearance in this mouse . indeed , there was no evidence of viral toxicity in any of the mice which received hsv1716ing4 indicating that the virus retains the restricted replication competence of the parental hsv1716 with propagation limited to the actively dividing tumour cells . the above in vivo data suggests that hsv1716ing4 demonstrates enhanced oncolysis by replicating with higher efficiency than unmodified hsv1716 and this was confirmed in vitro using a variety of different cells lines infected at low multiplicities of infection . cell lines used were bhk , vero , a431 human scc , cp70 human ovarian tumour , mda - mb - 468 human breast adenocarcinoma , ovcar3 human ovarian carcinoma and huh7 human hepatocellular carcinoma and propagation of hsv1716ing4 in these lines was compared with wild - type hsv - 1 17 +, hsv1716 , hsv1716ing4 and hsv1716egfr ( an egfr - targeted hsv1716 variant that expresses a targeting moiety approximately the same molecular size as ing4 ). cells were plated out in 60 mm dishes and after 24 hours they were infected with approximately 100 pfu ( bhk only ) or 1000 pfu ( all other cell types ) hsv - 1 17 +, hsv1716 , hsv1716ing4 or hsv1716egfr . dilutions of each virus preparation for these infections were titrated on bhk cells to confirm accurately the amounts of input virus . each virus infection on each cell type was performed in quadruplicate at least . after 72 hours of infection , cells and medium were harvested , subjected to one freeze / thaw cycle (− 70 ° c .) and titrated on bhk cells . the results in the tables 2 - 8 below are reported in yields / input virion with statistical comparisons made using anova . for bhk cells , the high yields of virus / input virion for hsv - 1 17 +, hsv1716 and hsv1716ing4 are very similar indicating that these three viruses have similar replication efficiencies in this cell type and the yields , equivalent to approximately 5 × 10 6 viruses per input virion , are probably the maximum output achievable from a 100 pfu infection of bhk cells in this experiment . for hsv1716egfr yields / input virus were approximately 10 - fold less than for hsv - 1 17 +, hsv1716 and hsv1716ing4 suggesting that , in this experiment , this virus replicates less efficiently in bhk cells , see table 2 . in vero cells , hsv1716ing4 replicates more efficiently than hsv - 1 17 +, hsv1716 or hsv1716egfr with at least a 2 - fold increase in yield / input virion , see table 3 . comparisons of the yield / input virion for hsv1716ing4 with each of the yields / input virion for hsv - 1 17 +, hsv1716 or hsv1716egfr indicated that these differences are significant . anova analysis of the data in table 3 gives significant p values of p & lt ; 0 . 01 , p & lt ; 0 . 05 or p & lt ; 0 . 001 respectively for hsv1716ing4 compared to hsv - 1 17 +, hsv1716 or hsv1716egfr and , since all of these values are above the 95 % confidence limit , it can be concluded that the presence of the ing4 expression cassette in hsv1716ing4 improves virus replication in this cell type . in a separate experiment , 5 × 10 5 vero cells were plated out in 60 mm dish and were allowed to attach for 6 hours before being infected in duplicate at a multiplicity of infection of 1 pfu / cell with hsv - 1 17 +, hsv1716 , hsv1716ing4 or hsv1716egfr . after exactly 24 hours in culture , cells and medium were harvested , subjected to one freeze / thaw cycle (− 70 ° c .) and titrated on bhk cells . for hsv1716ing4 , the yields of virus / infected cell were approximately twice the yields of virus / infected cell for hsv - 1 17 +, hsv1716 or hsv1716egfr . at an input of 1 pfu / cell , hsv1716ing4 produced 140 / 250 virions / infected cell compared to 50 / 70 for hsv - 1 17 +, 80 / 88 for hsv1716 or 50 / 70 for hsv1716egfr . thus , during 24 hours of infection in vero cells ( equivalent to one round of virus replication ), hsv1716ing4 must replicate more efficiently than hsv - 1 17 +, hsv1716 or hsv1716egfr . for the human ovarian cancer cell line ovcar 3 cells , hsv1716ing4 replicates more efficiently than hsv - 1 17 +, hsv1716 or hsv1716egfr with a 2 - 4 - fold increase in yield / input virion , see table 4 . comparing the yield / input virion for hsv1716ing4 with each of the yields / input virion for hsv - 1 17 +, hsv1716 or hsv1716egfr indicates that the differences are significant . anova analysis of the data in table 4 gives p values all of p & lt ; 0 . 001 for hsv1716ing4 compared to each of hsv - 1 17 +, hsv1716 or hsv1716egfr and , since all of these are highly significant with greater than 99 . 9 % confidence limits , hsv1716ing4 must replicate more efficiently in this cell type . for the human squamous cell carcinoma cell line a431 , hsv1716ing4 replicates more efficiently than either hsv1716 or hsv1716egfr with a 100 - fold increase in yield / input virion compared to hsv1716 and 10 - fold increase compared to hsv1716egfr , see table 5 . statistical comparison of the yield / input virion for hsv1716ing4 with the yields / input virion for either hsv1716 or hsv1716egfr by anova gives p values of p & lt ; 0 . 05 for both indicating that the differences are significant with the greater than 95 % confidence limit indicating that , compared to the parental hsv1716 or the hsv1716 variant hsv1716egfr , hsv1716ing4 must replicate more efficiently in this cell type . the mean yield / input virion for hsv1716ing4 is greater than the mean yield / input virion for wild type hsv - 1 17 + but the difference is not significant ( p & gt ; 0 . 05 ). however , although hsv - 1 17 + has a higher mean yield / input virion than either hsv1716 of hsv1716egfr , neither of these differences is significant ( both p & gt ; 0 . 05 ). similarly , although the mean yield / input virion for hsv1716egfr is higher than the mean yield / input virion for hsv1716 , the difference is not significant ( p & gt ; 0 . 05 ). when the yields / input virion for hsv1716ing4 and hsv - 1 17 + are compared using the less stringent student &# 39 ; s t test , the differences are significant with p = 0 . 004 suggesting that hsv1716ing4 replicates more efficiently in a431 cells than hsv - 1 17 +. in the human breast adenocarcinoma cells , hsv1716ing4 replicates more efficiently than hsv - 1 17 +, hsv1716 or hsv1716egfr with an approximately 10 - fold increase in yield / input virion , see table 6 . for hsv1716ing4 , comparison of the yield / input virion with each of the yields / input virion for hsv - 1 17 +, hsv1716 or hsv1716egfr indicates that the differences are significant . anova analysis of the data in table 6 , gives significant p values , all of p & lt ; 0 . 001 , for hsv1716ing4 compared to each of hsv - 1 17 +, hsv1716 or hsv1716egfr and these are highly significant with greater than 99 . 9 % confidence limits indicating that hsv1716ing4 replicates more efficiently in this cell line . for the human ovarian cancer cell line cp70 , hsv1716ing4 replicates more efficiently than either hsv1716 or hsv1716egfr with a 3 - fold increase in yield / input virion compared to hsv1716 or hsv1716egfr , see table 7 . statistical comparison of the yield / input virion for hsv1716ing4 with the yields / input virion for either hsv1716 or hsv1716egfr by anova gives p values of p & lt ; 0 . 01 for both indicating that the differences are significant with the greater than 99 % confidence limit indicating that , compared to the parental hsv1716 or the hsv1716 variant hsv1716egfr , hsv1716ing4 must replicate more efficiently in this cell type . there was no significant difference between the yields / input virion for hsv1716 and hsv1716egfr ( p & gt ; 0 . 05 ). the mean yield / input virion for hsv1716ing4 was not significantly different from the mean yield / input virion for wild type hsv - 1 17 + ( p & gt ; 0 . 05 ). however , hsv - 1 17 + replicated more efficiently than either hsv1716 or hsv1716egfr and these differences were significant ( p & lt ; 0 . 01 ) indicating that hsv1716 / hsv1716egfr are impaired for replication in this cell type . importantly , expression of ing4 overcomes this impairment and returns the efficiency of replication to wild - type levels . in the human huh7 hepatocellular carcinoma cells , hsv1716ing4 replicates more efficiently than hsv - 1 17 +, hsv1716 or hsv1716egfr with an approximately 5 - fold increase in yield / input virion , see table 8 . for hsv1716ing4 , comparison of the yield / input virion with each of the yields / input virion for hsv - 1 17 +, hsv1716 or hsv1716egfr indicated that the differences are significant . anova analysis of the data in table 8 gives significant p values , all of p & lt ; 0 . 001 , for hsv1716ing4 compared to each of hsv - 1 17 +, hsv1716 or hsv1716egfr and , as these are highly significant with greater than 99 . 9 % confidence limits , hsv1716ing4 must replicate more efficiently in this cell line . a hallmark of the attenuated hsv1716 phenotype in vitro is the inability of the virus to replicate in nih 3t6 cells whereas these cells are fully permissive for hsv - 1 17 + replication . in duplicate , nih 3t6 cells in 60 mm plates were infected with 1000 pfu hsv - 1 17 +, hsv1716 , hsv1716ing4 and hsv1716egfr . after 72 hours of infection , cells and medium were harvested , subjected to one freeze / thaw cycle (− 70 ° c .) and titrated on bhk cells . no virus was detected following infection of 3t6 cells with hsv1716 , hsv1716ing4 or hsv1716egfr whereas the yields from duplicate hsv - 1 17 + infections of 3t6 cells were 2 . 0 × 10 6 / 3 . 0 × 10 6 pfu . therefore , the ability of ing4 expression to enhance the replication of hsv1716 is not achieved at the expense of its attenuated phenotype and ing4 activity within the infected cell is unable to overcome the replicative restrictions caused by deletion of the icp34 . 5 gene . to generate hsv1716ing4 , the ing4 cdna was initially cloned into the mammalian expression plasmid pcdna4 / myc - hisa and this vector was used to create bhk cell lines which constitutively express ing4 . bhk cells were transfected with 100 ug of the ing4 expression vector or with the empty pcdna4 / myc - hisa plasmid mixed with 10 ul lipofectamine 2000 ( invitrogen ) in 250 ul of serum free dmem / f12 medium . after 72 hours of transfection , cells were trypsinized and plated out with growth medium containing 1 mg / ml zeocin ( invitrogen ). cells were selected with the zeocin antibiotic for 2 - 3 weeks after which time individual clones were clearly visible . cells were trysinized and cloned by limiting dilutions in 24 - well plates . five clones of bhk / ing4 or bhk / pcdna4 were expanded and maintained in appropriate medium containing 0 . 5 mg / ml zeocin . each of the clones was plated out in 60 mm dishes in medium without zeocin and after 24 hours they were infected with approximately 10 pfu hsv - 1 17 + or hsv1716 . after 72 hours of infection , cells and medium were harvested , subjected to one freeze / thaw cycle (− 70 ° c .) and titrated on bhk cells . dilutions of each virus prepared for the infections were also titrated to confirm accurately the amounts of input virus and the results in the tables 9 and 10 below are reported in yields / input virion with statistical comparisons made using student &# 39 ; s t test . propagation of hsv - 1 17 + on bhk cells engineered by transfection / antibiotic selection to express constitutively ing4 is approximately 10 - fold more efficient when compared with propagation on zeocin - resistant bhk cells produced using the empty pcdna4 . myc - his vector . comparison of the means using student &# 39 ; s t test indicates that this difference is highly significant with p & lt ; 0 . 0001 indicating a greater than 99 . 99 % confidence limit that ing4 expression improves hsv - 1 17 + replication in bhk cells . propagation of hsv1716 on bhk cells engineered by transfection / antibiotic selection to express constitutively ing4 is approximately 10 - fold more efficient when compared with propagation on zeocin resistant bhk cells generated by transfection with the empty pcdna4 . myc - his vector . comparison of the means using student &# 39 ; s t test demonstrates that this difference is highly significant with p = 0 . 02 indicating a confidence limit of 98 % that ing4 expression improves hsv1716 replication in bhk cells . the in vivo data suggested that hsv1716ing4 replicated with higher efficiency than unmodified hsv1716 . this was initially confirmed in vitro using a variety of different cells lines infected at low multiplicities of infection , as described below . these experiments were followed up with the experiments described in example 2 . cell lines used were bhk , vero , 3t6 , a431 human scc , cp70 ovarian tumour , mda human breast carcinoma , ovcar3 human ovarian carcinoma and uvw human glioblastoma and they were infected principally with hsv1716 , hsv1716ing4 and hsv1716egfr . experiment 1 . cells were plated out in 60 mm dishes and after 24 hours they were infected with 1 , 10 or 100 pfu hsv1716 , hsv1716ing4 or hsv1716egfr . after 72 hours of infection , cells and medium were harvested , subjected to one freeze / thaw cycle (− 70 ° c .) and , as each of these viruses expresses gfp , a tcid method was used to estimate the amounts of virus in each sample . results are presented in tables 11 - 14 below . experiment 1 clearly demonstrates that in all cell types except 3t6 cells , hsv1716ing4 replicates more efficiently than hsv1716 resulting in higher yields of virus at inputs of 1 , 10 and 100 pfu . all three viruses used failed to replicate in 3t6 cells confirming their hsv1716 phenotype . experiment 2 . cells were plated out in 60 mm dishes and after 24 hours they were infected in duplicate with 5 or 20 pfu hsv1716 , hsv1716ing4 or hsv1716egfr . after 72 hours of infection , cells and medium were harvested , subjected to one freeze / thaw cycle (− 70 ° c .) and , as each of these viruses expresses gfp , a tcid method was used to estimate the amounts of virus in each sample . results are presented in tables 15 - 17 below . again , experiment 2 clearly demonstrates that in all cell types hsv1716ing4 replicates more efficiently than hsv1716 resulting in higher yields of progeny virus at inputs of 5 or 20 pfu . the enhancement to hsv1716ing4 propagation is more pronounced at 5 pfu virus especially in ovcar3 and a431 cells in which hsv1716ing4 yields are up to 100 - fold greater than hsv1716 . experiment 3 . each of the above cell types was seeded in a t175 flask and once confluent , the cells were infected with hsv - 1 17 +, hsv1716 , hsv1716ing4 or hsv1716egfr . after 96 hours in culture , virus was harvested from both detached cells and supernatant by high speed centrifugation and titrated on bhk cells . the dilutions used to infect the flasks were also titred on bhk cells to quantitate accurately the amount of input virus . for each cell type , total amounts of virus / flask , % yields from input and the % yield ratios compared to the hsv1716 % yield ratio are presented in table 18 . an hsv1716 variant expressing the candidate tumour suppressor gene ing4 shows significantly enhanced inhibition of tumour growth and prolonged survival times when compared with hsv1716 alone . indeed , although not compared directly in the above experiments , the survival times for cp70 tumour - bearing mice treated with hsv1716ing4 were similar to those obtained following treatment with the nitroreductase - expressing variant hsv1790 in conjunction with the prodrug cb1954 . the enhanced survival times in the two models is unexpected and surprising given the postulated activities of ing4 as a subunit of various complexes involved in regulating transcription as such a mode of action in the infected tumour cells would not be expected to have such a dramatic effect on cytotoxicity against the background of an hsv1716 lytic infection . thus , the virus itself efficiently kills dividing tumour cells more rapidly than any toxic effects resulting from ing4 overexpression . possibly , ing4 expression destroys tumour cells which are infected but are resistant to virus oncolysis but this seems unlikely as the proportion of these cells in any given tumour will not be sufficient to account for the enhanced tumour destruction seen with hsv1716ing4 . alternatively , ing4 expressed in the infected tumour cell results in the release of anti - angiogenesis agents such as il - 6 or il - 8 which may act locally on surrounding uninfected cells within the tumour to suppress angiogenesis leading to enhanced tumour destruction . thus , according to this hypothesis , ing4 expression will stimulate release of locally - acting messengers from the infected cell which will act upon adjacent uninfected cells resulting in suppressed expression of genes promoting angiogenesis leading to a reduction in tumour vascularization and enhanced tumour destruction . moreover , mice which received intratumoral injections of pbs into either cp70 human ovarian cancer cell or a431 scc implants had median survivals of 11 or 8 days respectively . oncolytic infection of tumour cells by hsv1716 extended median survival in both theses models to 18 or 12 days respectively . further extensions to survival times to 23 . 5 or 21 days occurred with hsv1716ing4 injections indicating that the ing4 expression significantly augments hsv1716 oncolytic potency . intravenous injection of hsv1716ing4 also reduced tumour growth and prolonged survival compared to intravenously injected hsv1716 and extraction and titration of virus from the tumour implants of sacrificed mice indicated a potential mode of action for the enhance tumour cell killing by hsv1716ing4 . at least 1000 - fold more viruses were extracted from tumours of mice intravenously injected with hsv1716ing4 compared to mice treated with unmodified hsv1716 suggesting that the expression of ing4 in the hsv1716 - infected cell improves the efficiency of virus replication resulting in augmented progeny production . thus , during hsv1716ing4 infection , the ing4 protein conditions the cell such that it provides an improved environment for hsv1716 replication . consequentially , more progeny virions are produced per tumour cell infected and this enhancement to oncolysis reduces tumour cell numbers and promotes survival . experiments in vitro with a panel of different cell lines confirmed that ing4 expression improves the efficiency of hsv1716 replication in a panel of different human tumour cell lines . bhk cells are routinely used for the growth of wild type hsv - 1 17 + and hsv1716 and its variants as the cell line is an excellent substrate for viral replication and high yields are always obtained . for hsv - 1 17 +, hsv1716 and hsv1716ing4 approximately 5 × 10 8 - 1 × 10 9 pfu were obtained by infecting a 60 mm plate with c100 pfu virus and there was little difference in the yields / input virion probably because this is the maximum output for bhk cells in this culture format . vero cells can also be used for hsv - 1 propagation although in this experiment the yields / input virion for hsv - 1 17 +, hsv1716 , hsv1716ing4 and hsv1716egfr were lower than for bhk cells . at low moi , hsv1716ing4 replicated with a 2 - fold higher efficiency in vero cells compared to hsv - 1 17 +, hsv1716 or hsv1716egfr and statistical comparison , using the stringent anova test , of hsv1716ing4 with each of these other viruses indicated that the differences were significant . no significant differences were detected in yields / input virion amongst hsv - 1 17 +, hsv1716 and hsv1716egfr . further , using a much higher moi of 1 pfu / cell to infect vero cells for one lytic cycle , hsv1716ing4 again replicated with twice the efficiency compared to hsv - 1 17 +, hsv1716 or hsv1716egfr indicating that the growth advantage conferred by ing4 expression occurs within a single lytic cycle . in preliminary in vitro experiments with a panel of human tumour cell lines ( cp70 human ovarian tumour , mda - mb - 468 human breast adenocarcinoma , ovcar3 human ovarian carcinoma , uvw human glioblastoma and a431 scc cells ), hsv1716ing4 demonstrated enhanced replication in each of these cell types compared to either hsv1716 or hsv1716egfr . in subsequent experiments using numbers of replicates that allowed for statistically meaningful comparisons , significantly increased yields / input virion for hsv1716ing4 compared to yields / input virion for hsv1716 or hsv1716egfr were obtained in cp70 human ovarian tumour , mda - mb - 468 human breast adenocarcinoma , ovcar3 human ovarian carcinoma , huh7 human hepatocellular carcinoma and a431 scc cells with the hsv1716ing4 output enhanced by 2 - fold , 10 - fold , 5 - fold , 5 - fold and 10 - 100 - fold respectively in these cell types . significant differences were also obtained between hsv1716ing4 and hsv - 1 17 + replication in mda - mb - 468 human breast adenocarcinoma , ovcar3 human ovarian carcinoma and huh7 human hepatocellular carcinoma cells but there was no difference in replication efficiency between these two viruses in cp70 human ovarian tumour cells . for a431 scc cells , hsv1716ing4 output was significantly increased by 100 - fold or 10 - fold compared to hsv1716 or hsv1716egfr respectively and , although using anova , the 2 - fold higher hsv1716ing4 yield / input virion was not significantly different from that of hsv - 1 17 +, the difference was significant when the means were compared using student &# 39 ; s t test . as with the mda - mb - 468 human breast adenocarcinoma , ovcar3 human ovarian carcinoma and huh7 human hepatocellular carcinoma cells , in a431 cells there was no significant differences in yields / input virion for hsv - 1 17 +, hsv1716 or hsv1716egfr . thus , in 5 / 5 human cancer cell lines hsv1716ing4 replication was significantly more efficient compared to the parental hsv1716 or the hsv1716 variant hsv1716egfr ( expressing a similarly sized protein targeting moiety ) and , compared to the wild - type hsv - 1 17 +, hsv1716ing4 replication was improved in ⅘ of these lines . further , when infected with either 10 pfu hsv - 1 17 + or 10 pfu hsv1716 , the yields / input virion were significantly enhanced 10 - fold in 5 different clones of bhk cells engineered by transfection / antibiotic selection to constitutively express ing4 compared to bhk cells which derived antibiotic resistance from transfection with the empty pcdna4 vector . such bhk cells expressing ing4 constitutively may provide an improved substrate for propagation of hsv1716 and its variants , especially those with dna inserts that restrict viral growth . ing4 expression during hsv - 1 17 +/ hsv1716 infection must act to improve the efficiency of virus replication within the cell resulting in a greater output of progeny virions per cell infected . importantly , this enhancement to replication efficiency by ing4 did not compromise the hsv1716 phenotype as , in the animal tumour models , hsv1716ing4 was non - toxic and the virus failed to replicate in vitro in the hsv1716 - resistant 3t6 cell line . ing4 may act directly on virus genes to improve their expression or interact directly with a viral protein to enhance its activity or , alternatively , it may act upon cellular genes / proteins to condition the cellular environment such that it is more amenable to virus replication . in vivo , this improvement to virus replication augments the oncolytic potency of hsv1716 resulting in more viruses for better tumour cell killing . additionally , the other recognised activities of ing4 , such as suppression of angiogenesis leading to a reduction in tumour vascularization , may also contribute to the enhanced oncolytic potency of hsv1716ing4 . 1 . gunduz , m ., nagatsuka , h ., demircan , k . et al ., ( 2005 ) gene 356 ; 109 - 117 . 2 . garkavtsev , i ., kozin , s v ., chernova , o . et al ., ( 2004 ) nature 428 ; 328 - 332 . 3 . doyon , y ., cayrou , c ., ullah , m . et al ., ( 2006 ) molecular cell 21 ; 51 - 64 . 4 . ozer , a ., wu , l c ., bruick , r k , ( 2005 ) pnas 102 ; 7481 - 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