Journal of General Virology (1998), 79, 125–131. Printed in Great Britain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

An efficient selection system for packaging herpes simplexvirus amplicons

Xiaoliu Zhang,1 Helen O’Shea,1 Clare Entwisle,1 Mike Boursnell,1 Stacey Efstathiou2

and Stephen Inglis1

1 Cantab Pharmaceuticals Research Ltd, 184 Cambridge Science Park, Milton Road, Cambridge CB4 4GN, UK2 Division of Virology, Department of Pathology, University of Cambridge, Cambridge, UK

Due to their simplicity and flexibility of genomicconstruction, herpes simplex virus (HSV) amplicon-based vectors are attractive vehicles for genedelivery. However, a significant problem faced in thegeneration of amplicon stocks is the low amplicon tohelper virus (A/H) ratio. In order to improve theproportion of amplicons generated, a selectionsystem for amplicon production was developed inwhich the HSV thymidine kinase (TK) gene is insertedinto an amplicon plasmid and an HSV mutant withboth TK and glycoprotein H (gH) genes deleted is

IntroductionHerpes simplex virus (HSV) has some unique features and

vectors derived from it have great promise in gene delivery.HSV has a very wide host range and cell tropism (Boothman etal., 1989 ; Roizman, 1990 ; Breakefield & DeLuca, 1991 ; Leib &Olivo, 1993). It has been shown that HSV can infect essentiallyevery cell type in most vertebrates which have been examined.In addition, the natural property of the virus to infect andestablish latent infection indefinitely in post-mitotic neuroneshas generated substantial interest in using it to delivertherapeutic genes to the nervous system. There are two typesof HSV-based vectors : foreign genes can either be insertedinto a backbone virus genome (recombinant virus) or into anHSV amplicon-based vector. The development of ampliconvectors originates from studies of naturally occurring defectiveviruses during high-multiplicity virus propagation (Frenkel,1981 ; Frenkel et al., 1982 ; Spate & Frenkel, 1982 ; Stow et al.,1983). A typical amplicon contains a copy of an HSVreplication origin (oriS) and a packaging signal sequencelocated in the repeated ‘a ’ sequence of the HSV genome(Vlazny & Frenkel, 1981 ; Spate & Frenkel, 1982 ; Stow, 1982 ;

Author for correspondence: Xiaoliu Zhang.

Fax ­44 1223 424115. e-mail xzhang!cantab.co.uk

used as a helper virus. Using a protocol in whichamplicon stocks are passaged 2–3 times in BHKcells of TKNand gHMgenotype in the presence ofselection medium containing methotrexate, stockpreparations with high A/H ratio (up to 5:1) andhigh amplicon titre ("1¬109 infectious units/ml)were generated. In vitro characterization demon-strated that a high level of biologically functionalproducts can be efficiently produced from theseamplicon constructs.

Kwong & Frenkel, 1984). Once introduced into suitable cellstogether with helper virus, the helper virus provides transreplication and packaging functions. The amplicon genome canthen be amplified, presumably by a rolling-circle mechanism,and packaged. These packaged amplicon-containing viralparticles are in effect defective virions, containing multiplecopies of the amplicon sequences in a concatemeric form, andare able to infect mammalian cells. Foreign genetic materialinserted into the amplicon plasmid can therefore be efficientlyintroduced into target cells by infection.

A major problem facing the development of amplicontechnology for gene delivery is the difficulty of generating astock with a high amplicon titre and sufficiently high ampliconto helper virus (A}H) ratio (Glorioso et al., 1992 ; Ho, 1994).Attempts to improve the efficiency of amplicon packaging,including procedures such as incorporating the SV40 DNAreplication origin into an amplicon construct and pre-repli-cating it in COS cells before packaging, have met with littlesuccess (Wu et al., 1995). Fraefel et al. (1996) have recentlyreported a procedure for generating helper virus-free ampliconstocks in which the whole HSV viral genome was fragmentedand cloned into a few cosmid vectors. By co-transfecting anamplicon plasmid with these HSV sequence-containing cos-mids, a stock free of detectable helper virus could be made.However, the amplicon titre in such preparations is low and the

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stock cannot be further passaged for expansion. More recently,a selection system based on inserting an essential gene fromHSV and using a mutant HSV with that essential gene deletedas helper virus, a so-called ‘piggyback ’ system, has beenreported for raising the A}H ratio (Pechan et al., 1996).

Here, we report the development of an alternative selectionsystem for amplicon production, in which the HSV thymidinekinase (TK) gene is inserted into an amplicon and a TK−

glycoprotein H (gH)− HSV mutant is used as a helper virus.Using a protocol in which the initial transfection}super-infection harvest was passaged in BHK TK− gH+ cells in thepresence of selection medium containing methotrexate, stockpreparations with high A}H ratio (up to 5 amplicon to 1 helpervirus) and high amplicon titres (more than 1¬10*) wereachieved.

Methods+ Viruses and cells. Vero cells [catalogue no. 88020401 ; EuropeanCollection of Animal Cell Cultures (ECACC), Porton Down, UK] werefrom a World Health Organization (WHO) cell line. BHK TK− cells werealso obtained from ECACC (catalogue no. 85011423). CR-1 and CR-2cells are derived from Vero cells and their establishment has beendescribed in detail in elsewhere (Boursnell et al., 1997). These cell linescarry the genes encoding the gH of HSV-1 (CR-1) and HSV-2 (CR-2),respectively. These cells therefore serve as complementing cells forgrowing the gH-deleted virus which, in non-complementing cells, is adisabled infectious single-cycle (DISC) virus. The BHK TK− gH+ cell linewas established by transforming the parental BHK TK− cells with aplasmid containing the HSV-1 gH gene (C. Entwisle and others,unpublished data). CR-1 and CR-2 cells were grown in DMEM mediumplus 10% FCS and BHK cells were grown in Glasgow Modified Eagle’sMedium (GMEM) supplemented with 5% tryptose broth and 10% FCS.

HSV-1 strain SC16 is a well-characterized clinical isolate with a lowpassage history (Hill et al., 1975). PS1, which was used in this study ashelper virus for generating amplicon stocks, is a deletion mutant derivedfrom HSV-1 strain SC16. The deletion covers the gH gene and part of theTK gene. (This virus was kindly supplied by P. Speck, NorthwesternUniversity Medical School, Chicago, USA). HSV-2 strain HG52 (a giftfrom Moira Brown, MRC Institute of Virology, Glasgow) is a strain withlow neurovirulence. A gH deletion mutant of this virus, dH2A, has beendescribed elsewhere (Boursnell et al., 1997). To make a recombinant DISCexpressing IL-2, a gene cassette containing a copy of the human IL-2 genedriven by the CMV immediate early promoter was inserted into the virusby direct ligation into a unique PacI site engineered into the gH locus (M.Boursnell and others, unpublished data). All the DISC virus stocks weregrown and titrated on CR-1 or CR-2 cells.

+ Construction of amplicon plasmids. The basic amplicon plasmidpW7TK (Fig. 1) was constructed by inserting an HSV-1 TK gene cassette(i.e. the 2 kb PvuII–BamHI fragment from HSV-1 strain 17 BamHI Qfragment) into the pW7 plasmid which contains an HSV-1 replicationorigin (oriS) and the packaging signal (Stow et al., 1983). The constructionof amplicon plasmids containing the human papillomavirus E6}7 fusedgene or human IL-2 (hIL-2) gene was carried out in two steps. Initially, theE6}7 or hIL-2 coding sequences were cloned into pRc}CMV (Invitrogen)so that the genes were driven by the CMV immediate early promoter.Then the cassettes containing the promoter, the gene and the poly(A)signal were cloned into the unique SapI site in pW7TK by blunt-endligation. For constructing the amplicon containing the green fluorescent

Fig. 1. Schematic diagram of the basic amplicon pW7TK. The cis-actingsequences from HSV-1, the ‘a ’ sequence in which the HSV packagingsignal is located, together with oriS, are required for amplicon genomeamplification and packaging during the co-passage with helper virus. Theprokaryotic sequences in the vector allow manipulation in bacteria. The TKgene and SapI restriction enzyme site are also labelled.

protein (GFP) gene, a cassette consisting of GFP driven by the CMVpromoter contained in the plasmid pEGFP-N1 (Clontech) was ligated intothe SapI site of pW7TK by blunt-end ligation.

+ Amplicon stock preparations. Initially, amplicon plasmid DNAwas transfected into CR-1 cells either by calcium phosphate-precipitationor by lipofectamine (Gibco-BRL). Briefly, cells were seeded 1 day beforetransfection in 5 cm Petri dishes. For the calcium phosphate precipitation,8 µg DNA was mixed with 0±5 ml HEPES buffered saline (HEBS) pH 7±05and 70 µl 2 M CaCl

#at room temperature for 30 min. The culture

medium was removed and the DNA precipitate was added to the cells.The cells were incubated at 37 °C for 40 min before the transfectionmixture was removed and replaced with 4 ml GMEM plus 5% FCS. Cellswere incubated at 37 °C for another 4 h before treatment with 1 ml 25%DMSO in HEBS for exactly 4 min. The DMSO solution was thenremoved and cells were washed with serum-free medium. Five mlGMEM plus 5% FCS was added and the cells were incubated at 37 °C foranother 16 h. The lipofectamine transfection was carried out according tothe supplier’s instructions. Briefly, 2 µg amplicon plasmid DNA wasadded to 200 µl sterile H

#O and 10 µl lipofectamine was diluted 20-fold

in sterile H#O. The DNA and lipofectamine solutions were mixed

together gently and left at room temperature for 30 min. Then 1±6 mlOptiMEM medium (supplied by the manufacturer, Gibco-BRL) wasadded to the DNA–lipofectamine mixture. Cells were rinsed with serum-free medium and the DNA–lipofectamine mixture was laid gently overthe cells. After incubation for 5 h at 37 °C, the transfection solution wasremoved and replaced with 5 ml GMEM plus 5% FCS. Cells wereincubated at 37 °C for another 16 h and then infected with 1 p.f.u. PS1per cell for another 24 h. Viruses were harvested and titrated by plaqueassay. This virus stock was further passaged in BHK TK− gH+ cells 2–3times. For each passage, cells were infected with 3–5 p.f.u. virus per cell(based on the titre of PS1) for 1 h. Then a selection medium containing0±6 µM methotrexate and 1¬TGAG (40¬TGAG: 0±6 mM thymidine,3±8 mM glycine, 9 mM adenosine, 1±9 mM guanosine) was added andthe infected cells cultured at 37 °C for 24–28 h before harvesting andtitration.

+ DNA extraction and analysis. Packaged virion DNA wasprepared from cell-culture supernatants harvested from infected cells asdescribed previously (Zhang et al., 1994). Briefly, viral particles were

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pelleted by centrifuging the supernatant at 27000 g for 2 h. The viralpellet was resuspended in 1 ml TE (10 mM Tris, 1 mM EDTA, pH 8±0).For releasing viral DNA from the viral particles, SDS was added to a finalconcentration of 0±1% and proteinase K to 50 µg}ml. The sample wasincubated at 37 °C for 4 h. Viral DNA was gently extracted three timeswith phenol–chloroform, followed by ethanol precipitation. DNA wasresuspended in TE and digested with the restriction enzyme AseI. Thedigested DNA was loaded onto a 0±8% agarose gel, which waselectrophoresed overnight. The gel was stained with ethidium bromideand photographs were taken with both Polaroid Film 667 (for hard-copy)and 665 (for negative film). The image in the negative film was scannedand quantified using a Personal Densitometer SI (Molecular Dynamics).

+ ELISA assay of hIL-2. Ninety-six well plates were coated withanti-hIL-2 monoclonal neutralizing antibody (R&D Systems) overnight at4 °C. The coated wells were then blocked with 300 µl 1% BSA in PBS at37 °C for 1 h. For preparing the standard IL-2 solution, 10 µl of 10 µg}mlstock IL-2 (R&D Systems) was added to 9±99 ml medium (MEM­2±5%FCS) to give 10 ng}ml IL-2 solution, From that solution a series oftwofold dilutions of IL-2 starting from 25 pg}ml up to 400 pg}ml,together with the diluted samples, were added to the wells. Plates wereincubated at 37 °C for 1 h. Biotinylated anti-human IL-2 antibody (R&DSystems, 75 µl) reconstituted in PBS to give 50 µg}ml, was added to eachwell and the plates incubated at 37 °C for 1 h. Then 15 µlStreptavidin–HOURP (Zymed HOURP, 1}1000 dilution) was added toeach well and the plates were incubated at 37 °C for 30 min. One hundredµl o-phenylenediamine (Sigma) was added and the reaction left at roomtemperature for 10 min before 50 µl 1 M sulphuric acid was added toall wells to stop the reaction. The result was read at 492 nm using theMulticalc program with Titertek Multiskan MCC}340, MKII.

ResultsRationale for the TK selection system

The basic amplicon plasmid pW7TK (Fig. 1), which wasused to construct all the other amplicon plasmids reported inthis study, contains in its genome the HSV-1 TK gene inaddition to the HSV-1 replication origin (oriS) and packagingsignal. Transcriptional units containing promoter, gene ofinterest and poly(A) were inserted into the unique restrictionenzyme SapI site as shown in Fig. 1. The helper virus PS1 is agenetically mutated HSV-1 and lacks both gH and TK genes(P. Speck and others, unpublished data). The rationale for theTK selection system for amplicon packaging is shown in Fig. 2.The initial stock prepared from transfection}super-infectionusually contains mainly helper virus. Further passage of thisstock at moderately high m.o.i., such as 5 p.f.u. per cell, hasthree outcomes : (1) cells infected by helper virus alone(normally the majority of cells), (2) a small percentage of cells(depending on the ratio of amplicon}helper virus in theinoculum) infected by both amplicon and helper virus, and (3)a very small proportion of cells infected by amplicon alone.Under methotrexate selection, the helper virus amplification inthe first category will be eliminated or heavily inhibited.Theoretically, only cells in the second category, which areinfected by both amplicon and helper virus, will support virusgrowth. So this system allows amplicon amplification butinhibits the growth of the majority of helper virus duringconsecutive passages.

Fig. 2. Schematic illustration of TK selection during amplicon packaging.The helper virus particles are labelled as H and the packaged ampliconparticles are labelled as A.

Fig. 3. Titration of the effect of methotrexate on helper virus growth in CR-1 (D) and BHK TK− gH+ (E) cells. Cells in 5 cm Petri dishes wereinfected with 1 p.f.u. helper virus PS1 per cell for 1 h. Medium containingincreasing amounts of methotrexate was added to the infected cells.Twenty-four hours after infection, virus was harvested and titrated. Thetotal amount of virus from each harvest, together with the amount of virusinoculated (input) are plotted against the concentration of methotrexate inthe medium.

Amplicon packaging in the selection medium for TK

The concentration of methotrexate used in the selectionmedia was initially determined by testing the inhibition ofhelper virus growth in both BHK TK− gH+ cells which lackendogenous TK, and in CR-1 cells which show endogenous TKexpression. Cells were infected at 1 p.f.u. helper virus PS1 percell for 1 h. Then medium containing increasing amounts ofmethotrexate was added. Virus was harvested 24 h afterinfection and titrated by plaque assay. The results in Fig. 3showed that methotrexate at a concentration up to 2±4 µM didnot obviously inhibit helper virus growth in CR-1 cells. On theother hand, it showed a dose response in inhibiting helper virus

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Fig. 4. Diagrammatic representation ofAseI digestion of packaged helper andamplicon genomes. (a) AseI cuts thehelper virus genome once in the junctionregion and therefore generates twofragments of 125 kb and 26 kb. (b) AseIcuts most of our amplicon constructsonce. Digestion of the packaged amplicongenome generates 10–15 copies(depending on the size of amplicongenome) of a much smaller (9 kb in thiscase) DNA fragment which can be easilyseparated from helper virus DNAfragments by ordinary agarose gelelectrophoresis.

Fig. 5. Estimation of A/H ratio by DNA analysis. Viruses harvested from the initial transfection/super-infection were furtherpassaged in BHK TK− gH+ cells in the medium with (a) or without (b) methotrexate, three times consecutively (panels P-1 toP-3). Virion DNA from each passage was extracted. The uncut and AseI-digested DNA was electrophoresed on a 0±8% agarosegel, together with lambda DNA digested with HindIII as marker. The gel was stained with ethidium bromide and photographed.

growth in BHK TK− gH+ cells. The input helper virus titre didnot show any increase when medium containing 0±6 µMmethotrexate was used, and showed a slight decrease whenhigher methotrexate concentrations were used. Methotrexateat a concentration of 0±6 µM was therefore used in theselection medium. The results also showed that CR-1 cellssupport helper virus growth almost one order of magnitudebetter than BHK TK− gH+ cells.

For generating HSV amplicon stocks, CR-1 cells wereinitially transfected with the DNA of amplicon plasmid pE6}7(which contains a fusion of the human papillomavirus type 16E6 and E7 genes), by calcium phosphate precipitation. Twenty-four hours later, cells were super-infected with 1 p.f.u. helpervirus PS1 per cell for 1 h. Viruses were harvested 24 h afterhelper virus infection. These initial stocks were then con-secutively passaged in BHK TK− gH+ cells at 5 p.f.u. helpervirus per cell, in medium with or without methotrexate. Helpervirus from each passage was titrated by plaque assay. Theamplicon titre was estimated by comparing the amounts of

DNA retrieved from packaged amplicon particles with thosefrom helper virus by gel electrophoresis. As illustrated in Fig.4, the restriction enzyme AseI cuts the helper virus genometwice, generating two major DNA fragments with sizes of125 kb and 26 kb, which account for 99% of the viral genome.AseI cuts most amplicon plasmids only once, generating amuch smaller DNA fragment (depending on the size of theindividual amplicon construct ; in the case of pE6}7 it is 9 kb),which can be easily separated from the viral DNA fragmentsby ordinary agarose gel electrophoresis. After ethidiumbromide staining of the gel, photographs were taken usingboth negative and positive films and the amount of DNA ineach DNA band shown in the negative film was quantified bydensitometer scanning, and used for amplicon titre estimation.If there are equal amounts of amplicon and helper virus, theywill contain equal numbers of genome copies and hence anequal mass of DNA. These equal masses of DNA will give thesame intensities of ethidium bromide staining regardless of thesize of the fragment after AseI digestion. So the ratio of

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Table 1. Estimation of A/H ratio and amplicon titre byDNA analysis during serial passages in selection medium

Passage A/H ratio*Amplicon

titre†

Transf.}infect. 0±001 5¬10%Passage 1 0±4 4¬10'Passage 2 4±8 8¬10(Passage 3 4±5 6¬10(

* Ratio of intensities of the DNA fragments representing ampliconand helper virus shown in Fig. 5 (a), quantified by densitometerscanning.† Estimated by multiplying the helper virus titres by the A}H ratio.

Table 2. Further expansion of amplicon stocks in CR-1cells

CR-1 cells in 150 cm# tissue culture flasks were infected with theindicated m.o.i. (based on helper virus titre) for 24 h. Viruses wereharvested and the helper virus and amplicon titres in the stocksdetermined as described in Table 1.

M.o.i. Helper titre Amplicon titre A/H ratio

1 1±5¬10) 2±25¬10) 1±52 8±0¬10) 2±4¬10* 3±03 1±1¬10) 3±3¬10) 3±0

amplicon to helper genomes can be estimated by comparingthe total staining intensity of the multiple copies of the 9 kbamplicon band (see Fig. 4) to the total staining intensity of thetwo helper virus bands. As the helper virus from each stock hasbeen titrated by plaque assay, the inferred amplicon titre (givenas infectious units, IU) can therefore be estimated. Fig. 5ashows the DNA fragment patterns from each passage duringthe TK selection. The harvest from the initial transfection}super-infection had barely detectable amplicon DNA. How-ever, the intensity of the amplicon DNA fragment increasedduring the serial passages. By the second passage, the bandrepresenting packaged amplicon was substantially strongerthan that from helper virus. The DNA bands were quantifiedby densitometer scanning. The amplicon titres estimated fromthe quantification, together with the titres of helper virus, areshown in Table 1. The results show that, during the serialpassages, as the A}H ratio increased, the estimated amplicontitres also increased steadily. So the total stock was expandedwhile the A}H ratio was improved during the selection. Theincrease in A}H ratio was more obvious at earlier passageswhen the inoculated stocks had a very low percentage ofamplicon and started to plateau out when the ratio was over

Fig. 6. Comparison of hIL-2 expression from amplicon and recombinantHSV. CR-1 cells were infected either with 3 IU pW7IL-2 per cell, with3 p.f.u. dH2H per cell or with helper virus PS1 only. Culture supernatantswere collected 4 h (+) or 20 h (*) after infection and the hIL-2 releasedinto the supernatant quantified by ELISA assay as described in Methods.

1 :1. On the other hand, the A}H ratio in the stocks frompassages carried out in the ordinary medium without metho-trexate did not improve and the DNA band representingamplicon remained invisible even at the third passage (Fig. 5b).

In order to further amplify the stock and improve amplicontitre, the harvest from the third passage was passaged again,but in CR-1 cells, which had been found to support virusgrowth more efficiently (Fig. 3). CR-1 cells were infected atdifferent m.o.i. and the viruses were harvested 24 h afterinfection and titrated. The A}H ratio was again estimated byDNA analysis. The results in Table 2 show that, although theA}H ratio dropped slightly, the amplicon titre increased morethan 100-fold and the estimated amplicon titre reached2±4¬10* IU}ml.

In order to verify the accuracy of amplicon titre estimationby DNA analysis, an amplicon plasmid containing the GFPgene was constructed and a stock was subsequently generated.The helper virus titre was again determined by plaque assay.The amplicon titre was estimated either by DNA analysis asmentioned above or by titration in Vero cells by countinggreen fluorescent cells under UV light. The amplicon titresobtained from these two different detection methods showeda discrepancy of less than twofold (data not shown).

In vitro gene expression from amplicon constructs

Gene expression from an amplicon construct containing thehIL-2 gene driven by the CMV immediate early promoter,pW7IL-2, was determined and compared with hIL-2 releasedfrom a recombinant DISC HSV-2 containing the identical hIL-

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2 gene cassette in the gH locus (dH2H). Infectious pW7IL-2amplicon stocks were prepared as described and the A}H ratiowas determined by DNA analysis to be 2 :1. CR-1 cells wereinfected with either 3 IU}cell of pW7IL-2 (in this case the m.o.i.for the helper virus was 1±5 p.f.u. per cell) or 3 p.f.u. per cell ofdH2H. Culture medium was collected at 4 h and 20 h afterinfection, respectively, and the hIL-2 released into the mediumwas quantified by an ELISA assay (detailed in Methods). Theresults in Fig. 6 show that, at 4 h after infection, pW7IL-2-infected cells had already produced a significant amount of hIL-2, while the hIL-2 released from the recombinant virus-infectedcells was undetectable by this method. By 20 h after infection,hIL-2 released from pW7IL-2-infected cells reached more than100 ng per 10' cells. This is almost 20-fold higher than thatfrom dH2H-infected cells. The hIL-2 produced from theseinfected cells has also been demonstrated to be biologicallyfunctional (data not shown).

DiscussionAs compared with recombinant HSV viruses, HSV ampli-

con-mediated gene delivery has some unique features. Ampli-con-based vectors are simple to construct, whereas thegeneration of recombinant HSV is time-consuming andtechnically challenging. Unlike recombinant viruses, ampliconinfection per se is not toxic to the target cells. Therefore genedelivery by HSV amplicons may be safer and also moresuitable for long-term gene expression (Geller et al., 1993 ;During et al., 1994 ; Smith et al., 1995). However, one of themajor problems facing amplicon technology is the greatvariation in amplicon to helper ratios generated in stockpreparations ; in general amplicons usually constitute the minorcomponent within stocks compared with helper virus. In thisstudy we have reported a TK selection system for ampliconstock preparation. Under this selection system, the amplicon tohelper virus ratio was efficiently elevated to between 1 :1 and5 :1 during serial passages starting with initial stocks whichcontained barely detectable amplicon particles. Subsequentpassage of stocks with high A}H ratio in CR-1 cells withoutmethotrexate selection generated high amplicon titre pre-parations with the elevated A}H ratio largely maintained.

A recent publication has described a similar ‘piggyback ’amplicon packaging system (Pechan et al., 1996). In thissystem, a gene essential for HSV replication, IE3, is cloned intoan amplicon, and a mutant virus deleted in the same essentialgene (d120) is used as helper virus. Whilst stocks with highA}H ratio were produced, fully replication competent rever-tants were also generated, albeit at low frequency. The helpervirus PS1 used in the current study is a double deletion mutant.In addition to the deletion of the TK gene, it also has the geneencoding gH deleted. The gH protein is an essential gly-coprotein which is required for virus entry and spread(Gompels & Minson, 1986 ; Forrester et al., 1992). In non-complementing cells, the gH deletion mutant can only undergo

a single round of replication and the progeny released are nolonger infectious (Desai et al., 1988 ; Forrester et al., 1992). Itmight be expected that the frequency of generation ofrevertants containing the TK gene during the current ampliconstock preparation will be similar to the frequency by which theIE3 gene is picked up by d120 in the ‘piggyback ’ system(Pechan et al., 1996). However, these TK-containing helperviruses will still lack gH and therefore the safety of thisamplicon-mediated gene delivery is maintained.

Finally, when gene expression from an amplicon constructcontaining the human IL-2 gene is compared with that from arecombinant HSV containing the identical gene cassette, theformer construct gives a much higher level of hIL-2 release intothe culture medium. This may be due to the unique property ofits multiple copy gene delivery (on average, each packagedamplicon viral particle contains 15 copies of the gene ofinterest, compared with only one copy contained in each ofthe recombinant viral particles).

We would like to thank Dr Nigel Stow for generously providing thepW7 amplicon plasmid and Dr P. Speck for kindly providing the helpervirus PS1. We also wish to thank Sharon Dumps and Sally Ogborne forcarrying out the ELISA assay for hIL-2.

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Received 7 July 1997; Accepted 1 September 1997

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