Patent Application: US-29996507-A

Abstract:
a method of generating a mutant herpes simplex virus is disclosed , wherein the generated hsv genome comprises nucleic acid encoding a nucleic acid sequence of interest , the method comprising the steps of : i . providing a nucleic acid vector comprising nucleic acid encoding first and second site specific recombination sequences and a nucleic acid encoding a nucleic acid sequence of interest between said site specific recombination sequences ; ii . providing an hsv , the genome of which comprises third and fourth site specific recombination sequences ; iii . contacting said nucleic acid vector of with said hsv of together with one or more recombinase enzymes capable of catalysing site specific recombination between the site specific recombination sequences of said nucleic acid vector and said hsv ; iv . identifying hsv containing the nucleic acid sequence of interest , wherein steps i - iii are conducted in a cell - free system .

Description:
in a preferred embodiment the hsv site specific recombination sequences are located in the hsv genome so as to disrupt or flank dna sequence that includes part , or all , of the mrna or protein coding sequence of the icp34 . 5 gene . two copies of the icp34 . 5 gene are normally present in hsv , therefore the hsv genome may contain 4 copies of the site specific recombination sequences — one pair of site specific recombination sequences at each icp34 . 5 ( rl1 ) locus . one or more of the site specific recombination sequences may be located within the rl1 gene so as to disrupt the icp34 . 5 coding sequence and prevent expression of a functional icp34 . 5 protein . hsv icp34 . 5 null mutants , i . e . that are incapable of expressing functional icp34 . 5 protein , are known to be non - neurovirulent . accordingly , they provide the basis for developing safe and effective viral vectors which find application in gene therapy techniques . furthermore some hsv icp34 . 5 null mutants are capable of oncolysis and may be used in the treatment of tumours of all types . accordingly , non - neurovirulent hsv are provided according to the invention which contain a nucleic acid cassette that disrupts the mrna and / or protein coding sequence of each copy of the icp34 . 5 gene , thereby rendering the hsv incapable of producing functional icp34 . 5 protein and non - neurovirulent , wherein the nucleic acid cassette is formed by a dna sequence flanked by site specific recombination sequences . the dna sequence may comprise a marker sequence , optionally together with an operably linked regulatory sequence , e . g . promoter . the dna sequence may additionally , or alternatively , comprise dna encoding a non - hsv originating polypeptide , which may also be operably linked to a regulatory sequence , e . g . promoter . in other preferred embodiments , the hsv site specific recombination sequences are located in the hsv genome so as to disrupt and / or flank dna sequence that includes part , or all , of the mrna encoding , or protein coding , sequence of any selected hsv gene ( in one or both copies of the gene as appropriate ). examples of such hsv genes include thymidine kinase , ribonucleotide reductase , icp0 ( also called ie1 , ie110 , rl2 , vmw110 ), icp4 ( also called ie175 ) and icp27 ( also called ul54 ). replication and / or infection defective hsv may be provided by disrupting the icp4 and / or icp27 genes . fig7 a - e illustrates the process of creation of the non - neurovirulent hsv destination vector ( e ) containing the site specific recombination sites in the hsv genome so as to disrupt the rl1 gene sequence and render the hsv incapable of producing functional icp34 . 5 protein . in preferred embodiments this process of homologous recombination is conducted in cell culture . fig7 f - g illustrate the use of the hsv destination vector to incorporate a nucleotide sequence of interest ( e . g . gene of interest : goi ) from an entry vector containing corresponding site specific recombination sequences flanking the sequence of interest . in preferred embodiments this process of site specific recombination is conducted in a cell - free system using recombinase enzymes suitable for recombination between the selected site specific recombination sequences . the invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided . aspects and embodiments of the present invention will now be illustrated , by way of example , with reference to the accompanying figures . further aspects and embodiments will be apparent to those skilled in the art . all documents mentioned in this text are incorporated herein by reference . embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which : hsv1716gateway virus destination vector dna containing cmv - dsred ( cytomeaglovirus promoter — ds red ) nucleic acid in the icp34 . 5 ( rl1 ) locus and flanked by the attr site specific recombinantion sites is mixed with entry vector nucleic acid encoding a gene of interest ( goi ) and pgk - gfp ( phosphogylcerokinase promoter — green fluorescent protein ) flanked by attl site specific recombination sequences together with lr clonase ™ ii enzyme mix to catalyse site specific recombination . rl1 . del is the pgem - 3zf (−) plasmid ( promega ) into which has been cloned an hsv - 1 fragment ( 123459 - 129403 ) consisting of the rl1 ( icp34 . 5 gene ) gene and its flanking sequences . the 477 bp pflmi - bsteii fragment of the rl1 gene ( 125292 - 125769 ) has been removed and replaced with a multi - cloning site ( mcs ) to form rl1 . del . southern blot of viral dnas probed with an icp34 . 5 dna probe . dna from hsv17 + ( lane 1 ), hsv1716 ( lane 2 ), mock ( lane 3 ) or hsv1716gatered ( lanes 4 and 5 ) infected bhk cells was extracted 24 hours post infection using the wizard sv genomic dna kit ( promega , southampton , uk ) and digested overnight with bamhi . after separation on a 1 % agarose gel , dna was transferred to a nylon membrane and probed using the alui / rsai icp34 . 5 dna fragment from plasmid pgem34 . 5 ( mckie et al 1994 ). molecular sizes are indicated on the left hand side of the gel and the k , s and q fragments hybridizing with the icp34 . 5 probe from bamhi digested hsv - 1 17 + dna , their equivalents , k * and s *, from hsv1716 dna and the bands from hsv1716gatered resulting from the insertion of a novel bam hi site are indicated by arrows . fluorescent microscope images showing red or green viral plaques after various recombination reactions . panels a - c show results from 1 , 3 or 4 μg hsv1716gatered dna incubated with 7 , 5 , 4 μg respectively of the gateway entry plasmid pentr1a modified to express gfp . panels d and e show results from incubation of 5 μg hsv1716gatered dna or 8 μg modified pentr1a alone with the site specific recombination enzyme mix and panel f shows results from homologous recombination between 100 μg hsv1716gatered dna and 50 μg prl1 - del containing a gfp expression cassette . panels g and h show results from site specific recombination reactions between 3 μg hsv1716gatered dna and 5 μg modified pentr1a with 500 bp or 2000 bp inserts respectively . 1 % agarose gel showing products obtained by rl1 pcr using hsv - 1 17 + ( lane 1 ), hsv1716 ( lane 2 ) and hsv1716 variants isolated after site specific recombination using pentr1a - gfp alone ( lane 3 ) or pentr1a - gfp with additional 250 bp ( lane 4 ), 500 bp ( lane 5 ), 1500 bp ( lane 6 ), 2000 bp ( lane 7 ) dna inserts . lane m indicates the 2 log dna ladder with the sizes of the principal bands indicated on the left hand side and lane 8 is a negative control comprising mock infected bhk cell dna as template . fluorescent microscope images which demonstrate the ease with which novel recombinant hsv1716 variants can be identified after the site specific recombination reaction in vitro . panel a shows a red plaque produced by virus resulting from virus derived from the input hsv1716gatered dna , panel b shows a mixed yellow plaque produced from viruses derived from the input hsv1716gatered dna and the dna resulting from the site specific recombination reaction and panel c shows a green plaque produced by virus derived from the dna resulting from the site specific recombination reaction . schematic diagram illustrating creation of hsv destination vector and use in generating other mutant hsv . ( a ) plasmid rl1 - del comprising the rl1 gene sequence with inserted multi - cloning site ( mcs ); ( b ) plasmid sp73 containing the cmv - dsred sequence flanked by site specific recombination sequences ( attr ); ( c ) the attr - cmv - dsred cassette is excised from plasmid sp73 by restriction digestion and inserted at the mcs in plasmid rl1 - del to form a modified rl1 - del plasmid which is contacted with the selected hsv - 1 genome ( d ) under conditions in which homologous recombination between the rl1 sequence components may occur to form the hsv mutant hsv1716gatered ( e ) which is the non - neurovirulent ‘ destination vector ’ encoding the dsred fluorescence marker protein operably linked to the cmv promoter . the destination vector is contacted with a selected entry vector plasmid ( pent ) ( f ) containing the nucleotide sequence of interest ( goi ) flanked by site specific recombination sequences ( attl ) corresponding to those contained in the destination vector in the presence of recombinase enzymes to produce the desired non - neurovirulent hsv mutant ( hsv - 1716goi ) ( g ). 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 . a destination site was produced in the plasmid sp73 by ligating attr sites ( available from invitrogen , carlsbad , calif ., usa as a gateway ™ vector conversion system ) into the ecorv - digested and alkaline phosphatase treated vector . once inserted , the dna ( encoding chloramphenicol resistance and the ccdb gene ) between the attr sites was removed by restriction enzyme digestion and replaced with a cmv - dsred expression cassette ( clontech ). the attr sites and intervening cmv - dsred expression cassette was then excised from sp73 and ligated into the bglii site in the ‘ smart plasmid ’ rl1 - del ( fig2 ), used for the production of hsv icp34 . 5 null mutants by homologous recombination , and this was used to create a herpes simplex virus designated “ hsv1716gatered ” which contains the cmv - dsred expression cassette flanked by attr sites in the rl1 loci . hsv1716gatered dna was then isolated and used for site - specific recombination in vitro . rl1 - del is shown in fig2 and is previously described in pct / gb2004 / 04839 ( wo 2005 / 049844 ), incorporated herein in its entirety by reference . the plasmid pentr1a ( invitrogen , carlsbad , calif ., usa ) was used to create a modified entry vector , pentr1a - gfp with the dna between the attl sites in pentr1a removed by ecor1 digestion and replaced with the pgk - gfp ( phosphoglycerokinase promoter - green fluorescent protein ) expression cassette . although ecor1 removes the ccdb gene from pentr1a , a number of other restriction sites between the attl flanking sequences are retained and these can be used to insert the gene / dna sequence of interest to be cloned into hsv alongside the pgk - gfp expression cassette between the attl flanking sequences . hsv recombinants are then generated by incubating hsv1716gatered dna with the gene / dna sequence of interest in the pentr1a - gfp plasmid and the recombinase enzyme gateway ™ lr clonase ™ ii enzyme mix ( invitrogen , carlsbad , calif ., usa ) for 18 hours at room temperature ( fig1 ). the gateway ™ lr clonase ™ ii enzyme mix lr contains the bacteriophage lambda recombination proteins integrase ( int ) and excisionase ( xis ), the e . coli - encoded protein integration host factor ( 1hf ) and reaction buffer . gateway ™ lr clonase ™ ii enzyme mix catalyses the exchange of the dna between the attl sites of the entry vector with the dna between the attr sites in hsv1716gatered dna and was then destroyed by proteinase k digestion . the entire reaction mix was then added to 250 μl of serum free medium containing 10 μl lipofectamine 2000 and used to transfect 50 % confluent bhk cells . after 4 - 6 hours the cells are dmso - shocked and cultured at 37 ° c . for 48 - 72 hours . cells were then harvested by scraping and , after 1 minute sonication in a sonicator bath , sequential 10 - fold dilutions were plated out on vero cells . after 72 hours , the plates were inspected by fluorescence microscopy and green plaques picked for further rounds of plaque purification . the site - specific recombination reaction was very efficient and at least 75 % of viruses were gfp positive , meaning that only one further round of plaque picking is usually required for virus purification . purified gfp - positive virus was obtained from one round of plaque picking ( which takes 1 week ) following the site specific recombination reaction 4 in table 1 compared with 5 rounds of plaque picking (= 5 weeks ) that were required to isolate a gfp - positive virus from the homologous recombination reaction . 12 different hsv1716 variants have been isolated following site specific recombination between the gene of interest in pentr1a - gfp and hsv1716gatered dna and in each case , purified virus has been obtained after one round of plaque picking . this contrasts with at least 30 different hsv1716 variants generated by homologous recombination which have required a minimum of 5 but in some cases up to twelve rounds of plaque picking to isolate purified virus . the site specific recombination method described provides a simple , straightforward and convenient procedure for the generation of mutant hsv and avoids complex multi - step procedures . a single in vitro recombination reaction circumvents the need for in vivo recombination in bacteria or tissue culture cells . isolation of recombinant viruses is very rapid with stocks of the hsv mutant are usually produced within 3 - 4 weeks . compared to between 3 - 6 months using rl1 - del / homologous recombination . homologous recombination using rl1 - del / pgk - gfp with hsv1716gatered dna resulted in 10 - 20 % gfp - positive plaques compared to 75 % gfp - positive plaques with pentr1a - gfp site - specific recombination . additionally , any contaminating non - recombinant viruses can be readily observed by fluorescence microscopy . rl1 - del is a large plasmid ( circa . 8 kbp ) and cloning additional dna into it is technically difficult . the entry vector pentr1a - gfp is much smaller ( circa . 4 kbp ) and cloning additional genes / dna sequences into it is much easier . the lr clonase ™ reaction uses very small amounts of viral dna / entry plasmid . typically , 3 μg of viral dna and 5 μg entry vector is required for a site - specific recombination reaction compared to co - transfection of 50 μg viral dna with 100 μg or 200 μg rl1 - del for homologous recombination . the hsv1716gatered variant with gateway destination sites in the icp34 . 5 deleted region was created as follows . the gateway vector conversion system ( invitrogen paisley , uk ) provided dna with attr site - specific recombination sequences for insertion into a vector of choice and was ligated into the ecorv - digested and alkaline phosphatase treated plasmid sp73 ( promega ) to create sp73gate . once inserted in the plasmid , the dna between the attr sites , encoding chloramphenicol resistance and the ccdb gene , was removed by not1 / bstxi digestion , the vector backbone was then blunt - ended with klenow and alkaline phosphatase treated . the 1 . 3 kbp cmv - dsred expression cassette was excised from the plasmid pcmv - dsred - express ( bd biosciences , uk ) by aflii / nsii digestion , blunt ended by klenow and ligated into the sp73gate backbone to create the plasmid sp73gatered . the attr sites and intervening cmv - dsred expression cassette was then excised from sp73gatered by bglii / xhoi digestion , blunt - ended with klenow and ligated into the blunt - ended , alkaline phosphatase - treated bglii site in the plasmid rl1 - del , used for the production of hsv1716 variants by homologous recombination . rl1 - del ( fig2 ) is a cloning vector suitable for generating icp34 . 5 null hsv - 1 consisting of an hsv - 1 dna fragment containing the rl1 gene and its flanking sequences with the majority of the icp34 . 5 open reading frame 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 gene and incorporation of the desired transgene . 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 ). the 477 bp pflmi / bsteii fragment from the rl10rf ( 125292 - 125769 ) has been removed to inactivate the icp34 . 5 gene and replaced with a mcs . the resultant plasmid , rl1 - del / gatered was used to create hsv1716gatered which contained the cmv - dsred expression cassette , flanked by attr destination sites in the rl1 loci by homologous recombination . approximately 50 μg of rl1 - del / gatered were linearized by xmni digestion and were cotransfected into bhk cells with hsv - 1 17 + dna . rl1 - del / gatered and viral dna ( c100 μg ) were mixed with 20 μl lipofectamine 2000 ( invitrogen ) in 250 μl dmem / f12 ( invitrogen ) 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 gmem supplemented with 10 % newborn calf serum ( both invitrogen ) and left for 72 hours . cells were then scraped into the supernatant , sonicated in a sonicator bath for 2 minutes and stored at − 70 ° c . until required . serial dilutions were plated out on vero cells in 60 mm plates , individual red 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 hsv1716gatered were produced . the pentr1a was modified as follows . the dna between the attl sites in pentr1a was removed by ecori digestion and the resulting vector backbone was blunt - ended and alkaline phosphatase treated . a green fluorescent protein expression cassette was inserted into the pentr1a backbone by ligating it with the 1 . 3 kbp blunt - ended ecori / aflii fragment that contains the pgk promoter / gfp gene excised from the vector psnrg ( oligoengine , seattle , wash ., usa ). although ecori digestion removed the ccdb gene from pentr1a , a number of other restriction sites between the attl flanking sequences were retained for insertion of additional gene / dna sequences of interest to be cloned into hsv1716 alongside the pgk - gfp expression cassette . hsv1716gatered dna was obtained by phenol / chloroform extraction from 4 × t175 flasks 24 hours after infection with the hsv1716gatered virus and was resuspended in 1 ml nuclease free water . approximately 1 , 3 or 4 μg viral dna were mixed with 7 , 5 or 4 μg pentr1agfp and after overnight incubation with lr clonase the enzymes were inactivated by digestion with 1 μg proteinase k for 10 minutes at 37 ° c . the entire reaction mix ( 11 μl ) was added to 250 μl of serum free dmem / f12 ( invitrogen ) medium containing 10 μl lipofectamine 2000 and used to transfect 50 % confluent bhk cells in a 60 mm dish . after 4 - 6 hours the cells were dmso - shocked in 25 % dmso / pbs , washed and then cultured in 5 ml of gmem at 37 ° c . for 48 - 72 hours . control transfections comprising either 8 μg pentr1agfp or 5 μg hsv1716gatered dna incubated alone with the lr clonase mix were performed also and , for comparison with in vivo homologous recombination , 50 μg of the rl1 - del shuttle vector with an inserted pgk - gfp expression cassette were cotransfected with 100 μg hsv1716gatered dna . cells were then harvested by scraping into the medium and , after 1 minute sonication in a sonicator bath , 5 sequential 10 - fold dilutions were plated out on vero cells . after 72 hours , fluorescence microscopy was used to estimate the numbers of green and red plaques on each plate . for site specific recombination reactions using pentr1a - gfp with additional dna inserts , 5 μg plasmid were incubated with 3 μl hsv1716gatered dna . viral dna was prepared using a wizard sv genomic dna kit from bhk cells 24 hours after infection with the relevant viruses at 5 pfu / cell and 20 μl extracted dna was used as template for amplification by rl1 pcr . in addition to the viral dna , the 50 μl pcr mix contained 24m primer r13 , 14 μm primer f3 , 1 mm mg 2 + , 200 μm each of datp , dgtp , dctp , dttp , 200 μm deazagtp and 1 . 25u platinum pfx dna polymerase ( invitrogen ). the f3 primer sequence is caggcacggcccgatgaccgcctc ( seq id no . 5 ) corresponding to bases 125172 - 125195 and complementary to bases 1176 - 1199 of the hsv strain 17 + sequence . primer r13 sequence is ggccagacgccgaaaacg ( seq id no . 6 ), complementary to bases 126035 - 126052 and corresponding to bases 319 - 336 of the hsv strain 17 + sequence . primer f3 is positioned in the icp34 . 5 coding region towards the 3 ′- end which is still present in hsv1716 and primer r13 lies outside the icp34 . 5 orf within the a sequence . the conditions for pcr were 94 ° c . for 2 minutes then 35 cycles of 94 ° c . for 15 seconds , 72 ° c . for 1 minute and 72 ° c . for 1 minute with a final extension of 72 ° c . for 2 minutes . samples were then analysed on 1 % agarose gels . we have described a highly efficient and extremely rapid site specific recombination method for the production of second generation variants of oncolytic hsv1716 . using an hsv1716 variant in which gateway destination sequences were incorporated into the icp34 . 5 deleted region we were able to derive recombinant viruses by simply incubating the viral dna with the gene / dna sequence of interest cloned into a gateway entry plasmid and the relevant recombinase enzyme . as well as inserting the gene / dna sequence of interest , site specific recombination also replaced a dsred expression cassette with a green fluorescent protein expression cassette and , after the recombination reaction and transfection of the viral dna into bhk cells , novel recombinant viruses were readily isolated by a single round of plaque purification . we investigated the use of site - specific recombination in vitro for the production of hsv1716 variants and , most surprisingly , we were able to develop a rapid method which allowed exceedingly efficient production of second generation hsv1716 recombinants . an hsv1716 variant , hsv1716gatered , which contained a gateway ( invitrogen , paisley , uk ) destination site located within the icp34 . 5 deleted region was created using homologous recombination . the presence of the dsred expression cassettes in both of the rl1 loci of hsv1716 was confirmed by southern blotting ( fig3 ) using the icp34 . 5 - containing plasmid pgem34 . 5 ( mckie et al 1994 ). the icp34 . 5 probe hybridizes with three main fragments in bamhi - digested hsv - 1 17 + dna ( fig3 , lane 2 ), termed k , q and s which have sizes of 5 . 9 kbp , 3 . 4 kbp and 2 . 9 kbp respectively . the icp34 . 5 deletion in hsv1716 reduces the sizes of k and s to 5 . 2 kbp ( k *) and 2 . 2 kbp ( s *) respectively ( fig3 , lane 1 ) with band q unaffected . insertion of the dsred expression cassette by homologous recombination with concomitant deletion of the icp34 . 5 orf introduced an additional bamhi site and , consistent with its incorporation into both rl1 loci , the k and s fragments are no longer detected and are replaced with novel bands of approximately 3 . 5 kbp , 3 . 0 kbp , 1 . 9 kbp and 1 . 5 kbp ( fig3 , lanes 4 and 5 ). the latter two bands are only faintly visible in fig3 , lane 4 but are more clearly observed in the longer exposure shown in lane 5 . combined insertion of the 1 . 3 kbp dsred expression cassette with the icp34 . 5 deletion increases the sizes of k and s to 6 . 6 kbp and 3 . 6 kbp respectively in hsv1716gatered but the additional bamhi site within the insert dna results in cleavage to generate novel dna fragments whose sizes are approximately equivalent to those shown in fig3 , lanes 4 and 5 , with the 3 . 5 kbp / 3 . 0 kbp and 1 . 9 kbp / 1 . 5 kbp bands comprising the hsv1716gatered k and s respectively . the gateway entry plasmid pentr1a ( invitrogen ) was modified to create a vector , pentr1a - gfp , suitable for site specific recombination reactions with hsv1716gatered . initially , an hsv1716gfp recombinant was produced by incubating hsv1716gatered dna with the pentr1a - gfp plasmid and the lr clonase ii enzyme mix ( invitrogen ) for 18 hours at room temperature . in vitro site specific recombination between hsv1716gatered dna and pentr1a - gfp is manifest by conversion of dsred expressing viruses to viruses expressing gfp and this switch was readily monitored by fluorescent microscopy . site - specific recombination between 1 , 3 or 4 μg viral dna and 7 , 5 or 4 μg pentr1a - gfp respectively resulted in approximately 50 %, 80 % or 70 % conversion of dsred to gfp expressing viruses respectively , as shown by the ratios of green to red plaques in fig4 , a - c . this compares very favourably with approximately 5 % conversion using in vivo homologous recombination between hsv1716gatered dna and the rl1 shuttle plasmid with an inserted pgk - gfp expression cassette , only a single gfp - positive plaque is visible in fig4 f . only dsred expressing viruses were obtained after incubating 5 μg hsv1716gatered dna overnight with lr clonase ( fig4 d ) and no viruses were produced following overnight incubation of 8 μg pentr1a - gfp with lr clonase ( fig4 e ). therefore , the in vitro site - specific recombination reaction was very efficient and converted up to 80 % of viral dna from dsred to gfp expression . several gfp - positive plaques picked from the serially diluted plates were 100 % pure as fluorescent microscopy of bhk cells infected with the plaque - picked viruses showed no contaminating dsred expressing viruses ( data not shown ) and these were used for virus stock production . in contrast , isolation of a gfp - positive virus from the much less efficient homologous recombination required 6 consecutive rounds of plaque purification to remove all dsred expressing viruses . the plasmid pentr1a - gfp has additional cloning sites both upstream and downstream of the pgk - gfp expression cassette and , following ligation of 250 bp , 500 bp , 1500 bp and 2000 bp dna inserts into these sites , the efficiency of the site specific recombination reaction to generate hsv1716 recombinants with additional dna inserts was investigated . using 5 μg of plasmid with 3 μg hsv1716gatered dna , in vitro site specific recombination reactions resulted in 70 - 90 % conversion of dsred to gfp - expressing viruses as assessed by fluorescent microscopy ( fig4 g and h ). depending on the insert size , some variation in recombination efficiencies was observed with 250 bp ( not shown ) or 500 bp ( fig4 g ) inserts generating 80 - 90 % hsv1716 recombinants whereas recombinations with 1500 bp ( not shown ) and 2000 bp ( fig4 h ) dna inserts were slightly less efficient with approximately 70 - 80 % conversion . the upstream or downstream location of the insert relative to the pgk - gfp expression cassette had no influence on recombination efficiencies ( data not shown ). irrespective of the insert sizes , in most cases , a single plaque picked from the serially diluted plates was 100 % pure with no dsred virus contamination and was used for hsv1716 recombinant stock production . occasionally , a low level (& lt ; 1 %) of dsred - positive virus contamination was observed but this was easily removed by a further single round of plaque purification . the genotype of the purified hsv1716 recombinants was confirmed using pcr with viral dna as template and primers designed to amplify across the hsv1716 icp34 . 5 deletion ( fig5 ). pcr with hsv - 1 17 + dna as template generated an 850 bp band ( lane 1 ) whereas amplification from hsv1716 dna resulted in a 120 bp band ( lane 2 ); these sizes are consistent with the wild type and deleted icp34 . 5 gene . pcr amplification of viral dna from hsv1716 recombinants generated using pentr1a - gfp and pentr1a - gfp with 250 bp , 500 bp , 1500 bp or 2000 bp inserts produced 1500 bp ( lane 3 ), 1700 bp ( lane 4 ), 2000 bp ( lane 5 ), 3000 bp ( lane 7 ) or 3500 bp ( lane 8 ) bands respectively and again , these sizes correspond well with the additional insert plus the pgk - gfp expression cassette . only a weak 1500 bp band was detected when the pcr was performed using hsv1716gatered dna as template ( not shown ), most likely because the attr sites immediately adjacent to the rl1 primer sites in hsv1716gatered dna have a detrimental effect on pcr which , as clearly shown in fig5 , is negated following the sequence changes resulting from recombination with the attl sites in pentr1a - gfp . oncolytic hsv1716 has immense therapeutic potential in the treatment of many forms of cancer and its potent ability to destroy tumour cells will be enhanced by variants that encode additional cytotoxic agents . such agents include enzymes for the activation of prodrugs , transporters for the specific uptake of radiolabelled compounds , antisense / sirnas directed against oncogenes and expression cassettes for tumour suppressor genes . previously , to generate such variants , we have used rl1 shuttle vectors and homologous recombination which results in simultaneously icp34 . 5 gene ablation and transgene incorporation . however there are a number of technical problems associated with this approach , principally that homologous recombination uses cotransfection of plasmid with large amounts of viral dna and , since , at best , only 1 in 20 viruses produced are recombinants , purification from high levels of contaminating wild - type usually requires between 6 and 10 rounds of time - consuming plaque picking . typically , after homologous recombination , isolation of the relevant hsv1716 variant for stock production usually requires 4 - 6 weeks . our novel site specific recombination method provides a straightforward and convenient procedure , with a simple bench top incubation of viral and plasmid dna with recombinase enzymes , for the generation of hsv1716 variants and avoids complex multi - step and technically demanding protocols . a single in vitro recombination reaction circumvents the need for any in vivo recombination in bacteria or tissue culture cells and variants are typically isolated in 5 - 7 days . also the efficiency of the recombination reaction was not affected by a variety of differently sized dna inserts , the largest of which , 2000 bp , consisted of the expression cassette for a 50 kda protein which was readily detected by western blotting of virally infected cell extracts ( data not shown ). other advantages of the method include the use of very small amounts of viral dna / entry plasmid such that the amount of hsv1716gatered dna isolated from 4 × t175 flasks was sufficient for 350 recombination reactions , and easy detection of contaminating non - recombinant dsred viruses by fluorescence microscopy . the advantages of fluorescent microscopy for visualisation of viral products from the site specific recombination reaction are clearly demonstrated in fig6 . recently , terada et al ( 2006 ) described a rapid method to generate oncolytic hsv vectors , called hsv / quik , which used two components consisting of the required hsv genome cloned in a bac and a replication conditional shuttle plasmid which contained the dna to be incorporated into the hsv variant . in their system , the hsv genome / bac component comprised the icp34 . 5 deletion mutant mgh1 dna with two site specific recombination sequences , frt and loxp , both in the ul39 gene , and transformation of bacteria with this bac plus an appropriate shuttle plasmid resulted in frt - mediated exchange of the relevant dna sequence from the shuttle plasmid into the ul39 gene . after bac isolation , residual prokaryotic sequences were removed by transfection of vero cells with the bac dna plus a cre recombinase expression plasmid . after several days in culture , recombinant viruses were produced . although the whole procedure is rapid ( 7 - 10 days ), the system is a multi - step procedure that requires two in vivo recombination reactions with associated transformation / transfection and an intervening plasmid isolation . although our method also uses site specific recombination with hsv1716 variants generated in 5 - 7 days , a single step recombination in vitro dispenses with the need for in vivo bacterial and mammalian cell recombination reactions allowing our straightforward procedure to facilitate greatly the accelerated production of many 2 nd generation hsv1716 variants to screen for improved tumour killing . 1 . bl liu , m robinson , z - q han , r h branston , c english , preay , y mcgrath , s k thomas , m thornton , p bullock , c a love and r s coffin ; gene therapy ( 2003 ) 10 , 292 - 303 . 2 . terada , k ., wakimoto , h ., tyminski , e ., chiocca , e a and saeki , y . development of a rapid method to generate multiple hsv vectors and their in vivo evaluation using syngeneic mouse tumor models . gene therapy 2006 , 13 : 705 - 714 . 3 . maclean , a . r ., fareed , m u , robertson , l ., harland , j . and brown , s m ( 1991 ) herpes simplex virus type 1 deletion variants 1714 and 1716 pinpoint neurovirulence related sequences in glasgow strain 17 + between immediate early gene 1 and the “ a ” sequence . j . gen virol . 72 , 631 - 639 . 4 . brown , s m , harland , j ., maclean , a r , podlech , j . and clements , j b . 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