Identification of a replication-defective herpes simplex virus for recombinant adeno-associated...

THE JOURNAL OF GENE MEDICINE R E S E A R C H A R T I C L EJ Gene Med 2004; 6: 555–564.Published online 25 February 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.542

Identification of a replication-defective herpessimplex virus for recombinant adeno-associatedvirus type 2 (rAAV2) particle assembly using stableproducer cell lines

Estelle Toublanc1

Abdellatif Benraiss2

Delphine Bonnin2

Veronique Blouin1

Nicole Brument1

Nathalie Cartier2

Alberto L. Epstein3

Philippe Moullier1

Anna Salvetti1*

1INSERM U649, CHU Hotel-Dieu, 30Avenue Jean Monnet, 44035, Nantes,France2INSERM U561, Hopital SaintVincent de Paul, 82 Avenue DenfertRochereau, 75014 Paris, France3CNRS UMR5534, Universite ClaudeBernard Lyon I, 43 Boulevard du 11Novembre 1918, 69622 Villeurbanne,France

*Correspondence to: Anna Salvetti,INSERM U649, CHU Hotel-Dieu, 30Avenue Jean Monnet, 44035,Nantes, France. E-mail:[email protected]

Received: 29 August 2003Revised: 20 November 2003Accepted: 11 December 2003

Abstract

Background The development of stable producer cell lines for recombinantadeno-associated virus (rAAV) assembly is a strategy followed by many groupsto develop scalable production methods suitable for good manufacturingpractice (GMP) requirements. The major drawback of this method lies inthe requirement for replicating adenovirus (Ad) for rAAV assembly. In thepresent study, we analyzed the ability of several replication-defective herpessimplex type 1 (HSV-1) helper viruses to induce rAAV2 particle productionfrom stable producer cell lines.

Methods Several stable rAAV producer cell clones were infected with wild-type and replication-defective HSV strains and analyzed for rep-cap geneamplification, viral protein synthesis and rAAV titers achieved. In vivo analysisfollowing rAAV injection in the murine brain was also conducted to evaluatethe toxicity and biopotency of the rAAV stocks.

Results We demonstrated that an HSV strain mutated in the UL30polymerase gene could efficiently be used in this context, resulting in rAAVtiters similar to those measured with wild-type HSV or Ad. Importantly, withrespect to clinical developments, the use of this mutant resulted in rAAVstocks which were consistently devoid of contaminating HSV particles andfully active in vivo in the murine central nervous system with no detectabletoxicity.

Conclusions This study, together with our previous report describing a rAAVchromatography-based purification process, contributes to the definition ofan entirely scalable process for the generation of rAAV particles. Copyright 2004 John Wiley & Sons, Ltd.

Keywords AAV production; stable producer cell lines; replication-defective HSV

Introduction

The adeno-associated virus type 2 (AAV-2) is a human parvovirus whichrequires the presence of a helper virus such as adenovirus (Ad) or herpessimplex virus (HSV) to accomplish a complete and productive replicationcycle. The AAV-2 genome is composed of two open-reading frames, repand cap, encoding for the regulatory and structural proteins, respectively,

Copyright 2004 John Wiley & Sons, Ltd.

556 E. Toublanc et al.

flanked by 145-bp inverted terminal repeats (ITRs).These latter elements constitute the essential cis-actingsequences involved in viral DNA replication [1]. Thehelper effects provided by Ad during the AAV life cyclehave been the object of many studies, demonstrating thatthe factors involved were those encoded by the E1a, E1b,E4(orf6), and E2a genes, as well as the VA1 RNA [2].In contrast, few studies have been performed to analyzethe HSV-1 helper activity [3,4]. So far, four HSV-1 earlygenes, UL5, UL8, UL52, and UL29, have been identifiedas necessary for the helper effect, even though their roleduring the AAV life cycle remains unclear [5].

Several in vivo studies have demonstrated the uniqueproperties of recombinant AAV vectors (rAAV) forgene transfer, in terms of transduction efficiency andpersistence of transgene expression [6]. rAAV vectorsare derived from the wild-type virus by deleting the repand cap genes and replacing them with the transgeneexpression cassette. The most common procedure forrAAV assembly relies on transient transfection of cells withplasmids encoding for the vector, the rep-cap sequences,followed by infection with Ad. This method is convenientfor the production of small-scale rAAV stocks but it quicklybecomes labor-intensive and inefficient for the large-scalerAAV production necessary for preclinical studies in largeanimals.

To develop a reliable and efficient procedure for rAAVproduction, many groups have established stable producercell lines containing both the rep and cap genes and thevector integrated into their genome [7–9]. The studiesconducted on these cell lines have shown that their abilityto assemble rAAV particles upon wild-type Ad infectioncorrelated with the amplification of the integrated rep-capsequences leading to a high level of Rep and Cap proteinsynthesis [10–12]. This amplification phenomenon wasdependent on the presence of cellular polymerases, Repproteins and the Ad DNA binding protein (DBP) [13].

A major achievement for rAAV production by transienttransfection was reached by replacing an infectiousadenovirus with a plasmid encoding for the Ad genesrequired for the helper effect, thus eliminating thegeneration of contaminant Ad particles [14,15]. Similarly,for assembling rAAV particles from stable producer celllines, it would be preferable to use replication-defectivehelper viruses. However, because most producer cell linesare derived from HeLa or A549 cells, and because theAd E1a and E1b genes are essential for the helpereffect [16], the conventional �E1 replication-defective Adcannot be used in this context. Replication-defective Adstrains mutated in genes like those encoding for the DNApolymerase or the terminal protein have been described[17,18] but, in our hands, have proved difficult to grow.Consequently, an alternative perspective is to focus onthe use of other helper viruses and particularly HSV.Indeed, earlier studies have shown that this virus is ableto help wild-type AAV replication and assembly [5,19]. Inaddition, several HSV replication-defective mutants areavailable.

The aim of this study was to investigate the ability ofHSV-1 to promote rAAV assembly using stable HeLa-derived producer cell lines, and particularly: (i) todetermine if wild-type HSV-1 was able to support high-yield rAAV production following infection of stableproducer cell clones and to compare it with wild-typeAd, (ii) to identify a replication-defective HSV that couldlead to a similar rAAV production level, and (iii) toprecisely characterize the rAAV stocks produced usingHSV and evaluate their in vivo efficiency compared withrAAV particles produced using wild-type Ad.

Materials and methods

Cell lines and viruses

African green monkey kidney fibroblats (Vero), Vero-derived cell lines, 293, and HeLa cell lines weremaintained in Dulbecco’s modified Eagle’s medium(DMEM, Sigma, France) supplemented with 10% heat-inactivated foetal calf serum (FCS, Sigma) and 1% (v/v)penicillin/streptomycin (Life Technologies, France; 5000U/ml). HeRC32 cells containing one to three copies ofthe AAV2 rep-cap genome with the ITR deleted have beendescribed previously [10]. The rAAV stable producer celllines were derived from HeRC32 by co-transfecting arAAV2 plasmid with the pSV-Hygro construct, conferringresistance to hygromycin. Hygromycin-resistant cloneswere isolated and screened for the presence of rAAV2sequences by PCR and for their ability to assemble rAAVparticles upon wild-type Ad infection.

Wild-type adenovirus type 5 (Ad5, ATCC VR-5) wasproduced and titrated using standard procedures [20],and used at a multiplicity of infection (MOI) of 50on HeLa cells and 5 on 293 cells. Wild-type HSV-1 (HSV-1-F), HSV1-�ICP27 (HSV-1-KOS 5dl1.2), andHSV1-�ICP4 (HSV-1-17 Cgal delIE3) were producedrespectively on Vero cells or on the 7b complementingcell line [21] (provided by P. Marconi, University ofFerrara, Italy) containing both the ICP27 and ICP4 genes.HSV�UL30 (HP66) was provided by D. Coen (HarvardUniversity, Boston, MA, USA) [22] and grown on polB3cells expressing the HSV UL30 gene [23] (providedby C. Hwang, SUNY Health Science Center, Syracuse,NY, USA). HSV�UL9 (hr94) [24] was provided by S.Weller and grown on the complementing A26 cellsexpressing the UL9 gene (provided by A. Patel, MRCVirology Unit, Glasgow, UK). Wild-type and mutatedHSV strains were produced and titrated using standardprocedures [25]. Replication-defective HSV stocks weretitrated and checked for absence of contaminating wild-type HSV by plaque assay performed in parallel onVero and the respective complementing Vero cells. Thetiter is expressed as the number of plaque-forming units(pfu).

Copyright 2004 John Wiley & Sons, Ltd. J Gene Med 2004; 6: 555–564.

Replication-Defective HSV for rAAV Assembly 557

Plasmids and DNA probes

All recombinant AAV plasmids used to generate stable pro-ducer cell lines were derived from psub201 [26]. PlasmidpAAVLZ contained the nuclear-localized β-galactosidasegene (LacZ) driven by the CMV promoter, and followedby the bovine growth hormone (BGH) polyadenylationsignal (poly A). Plasmids pAAVhALD and pAAVhIDUAcontained the human adrenoleukodystrophy (hALD) andiduronate-sulfatase (IDUA) cDNA, respectively, insertedbetween the mouse phosphoglycerate kinase-1 (PGK) pro-moter and the SV40 poly A signal. The probes used were:the rep probe corresponding to a 1.4 kb fragment ofthe AAV2 rep gene derived from Pst I digestion of plas-mid pspRC [27] and the hALD probe corresponding toa 1.06 kb fragment of the hALD gene derived from NsiI/Hinc II digestion of plasmid pAAVhALD. Plasmid pSV-Hygro used for the establishment of stable producer celllines contained the hygromycin B gene expressed fromthe SV40 promoter and followed by the SV40 poly A.

rAAV2 production and purification

rAAV particles were produced by infecting stable producercell lines with either wild-type Ad5 (MOI = 50) or HSV-1 (wild-type or replication-defective at an MOI = 5).Adenovirus-infected cells were harvested 48 h later,whereas HSV-infected cells were collected 24 h later.Cell pellets were obtained following centrifugation at2000 rpm for 15 min (GR-412 rotor; Jouan, France) andrAAV2 particles were purified following two differentprocedures. The first relied on the use of iodixanolgradients and heparin columns [28], the second one onthe use of ion-exchange chromatography columns (FPLC)[29]. Both methods have been described previously [29].

Titration of rAAV2 stocks

rAAV2 was titrated by: (1) dot blot, to measure thenumber of genome-containing particles; (2) replicationcenter assay (RCA), to measure the number of transducingparticles in the presence of adenovirus co-infection (bothtechniques were performed as previously described [27]);and (3) by A20 ELISA to determine the total amount ofassembled capsids [30].

Detection of proteins by silver stainingand Western blot

For the detection of protein contaminants and capsidproteins by sodium dodecyl sufate/polyacrylamide gelelectrophoresis (SDS-PAGE) in the rAAV-purified stocks,viral particles (as measured by dot blot) were precipitatedwith 4 volumes of acetone at −20 ◦C, pelleted at15 000 rpm for 30 min and then resuspended in 5×Laemmli buffer. For the analysis of proteins in cell

lysates, cellular extracts were prepared by resuspendingthe cell pellet in 5× Laemmli buffer. After denaturationfor 5 min at 100 ◦C, samples were run for 2 h at 100 Von a 10% SDS/polyacrylamide gel. Total proteins werevisualized after silver staining of the gel according to themanufacturer’s instructions (silver staining kit; AmershamBiosciences, Europe). The detection of AAV2 capsid (VP)proteins by Western blot was performed as previouslydescribed using B1 antibody diluted at 1 : 20 [31]. Adproteins were detected using rabbit antiserum againstAd diluted at 1 : 500. The HSV ICP5 capsid protein wasdetected using the ab6508 monoclonal antibody (Abcam,UK) diluted at 1 : 3200.

Southern blot analysis

Extraction of total DNA was performed as describedpreviously [13]. For Southern blot analysis, DNA wasdigested with the appropriate enzyme, run on 1%agarose gel and transferred to a Hybond N+ membrane(Amersham Biosciences, Europe). The membrane washybridized to a fluorescein-labelled probe (Random Primelabelling kit; Amersham Biosciences) and then processedaccording to the manufacturer’s instructions (CDP-stardetection module; Amersham Biosciences).

In vivo analysis

Mice deficient for the adrenoleukodystrophy protein(ALDP) were from a local breeding colony originallyobtained from Kirby D. Smith (Baltimore, MD, USA) [32].Three-month-old mice were anaesthetized with Avertine(200 mg/kg; Aldrich, France) and 2 µl of viral suspension(4 × 108 DNA-containing particles) were injected into thecorpus collusum using a stereotaxic frame (David KopfInstruments, USA). The mice were sacrificed 6 weekslater, and the brains mounted in tissue-freezing medium(Leica Instruments GmbH, Germany) and stored at−80 ◦C.

All brains were serially sectioned on a cryostat as10 µm thick coronal sections. Immunofluorescence wasperformed as previously described [33]. Briefly, sectionswere rehydrated in phosphate-buffered saline (PBS), fixedin 4% formaldehyde for 10 min, and permeabilized with0.1% Triton X-100 in PBS for 10 min. Sections werewashed in PBS three times and incubated overnight at 4 ◦Cwith the following primary antibodies: rabbit anti-ALDP1693 [34] and mouse monoclonal anti-NeuN (Valbiotech,France). The next day, sections were washed in PBSand stained with donkey anti-rabbit Cy3-conjugated orgoat anti-mouse FITC-conjugated antibodies (Interchim,France). The slides were mounted in DAPI containingVectashield (Vector Laboratories, USA). ALDP-positiveneurons were counted under the microscope (Nikon),in one of every fourteen 10 µm thick sections (140 mmapart) using a 40× magnification throughout therostrocaudal extent of the cortex. Data are reported as the



mean of the percentage of NeuN/ALDP double positivecells among the NeuN-positive neurons.

Results

Comparison of rAAV2 assembly fromdifferent stable producer cell linesusing either wild-type HSV-1 orwild-type Ad5 as a helper

The first aim of this study was to determine if wild-typeHSV-1 was able to induce rAAV production using stableproducer cell lines and compare it with wild-type Ad5.Stable producer cell clones were derived from HeRC32cells by co-transfecting a plasmid encoding for the rAAVvector with the pSV-Hygro plasmid. Clones resistant to thedrug were isolated and characterized for the presence ofthe rAAV vector DNA by PCR and Southern blot analysis.The best rAAV producer cell clones were then selected bymeasuring the rAAV titer in the cell lysate by dot blot 48 hfollowing wild-type Ad5 infection.

Because the level of rAAV assembly can vary dependingon the vector, three different types of producer cell clones,containing the AAVLZ, AAAVhALD, or the AAVhIDUAvector, were used to evaluate the HSV-1 helper activity(Figure 1). For five out of six clones the use of wild-type HSV-1 resulted in a rAAV production level similarto that obtained using wild-type Ad5, frequently with aslight advantage for HSV-1. The only exception was cloneAAVLZ149 for which a much higher titer was obtained inthe presence of HSV-1 than using Ad5. Importantly, forboth helper viruses, no correlation was found betweenthe initial rAAV copy number and the level of rAAV

Figure 1. Comparison of wild-type Ad5 versus wild-type HSV-1helper viruses for rAAV2 production by different producer cellclones. Stable producer cell clones (106 cells, indicated by theclone number and the AAV vector name, at the bottom of eachcolumn) were infected with either wild-type HSV-1 (MOI = 5) orwild-type Ad5 (MOI = 50) particles. Cells were harvested 24 or48 h post-infection, respectively, and lysed by three freeze/thawcycles. The level of DNA-containing rAAV2 particles present inthe cell lysate was measured by dot blot (see ‘Material andmethods’). The results are expressed as the number of particlesproduced per cell. The mean of two different experiments ispresented. The rAAV copy number indicated for each clone wasmeasured by Southern blot

particles produced, suggesting that other factors such asthe integration site and the integration pattern of thevector plasmid might be more important. For all theclones tested, the maximal rAAV yield was obtained 24 hfollowing HSV-1 infection, whereas 48 h were required inthe presence of Ad5 (data not shown). Finally, increasingthe MOI of HSV above 5 pfu/cell did not result in ahigher rAAV titer. At this MOI, approximately 90% ofthe cells were infected (data not shown). These findingare consistent with previous studies indicating that HSV-1 could efficiently help wild-type AAV2 replication andassembly and that the AAV2 life cycle was more rapidthan using Ad5 as a helper [19].

These results indicated for the first time that wild-typeHSV-1 is also able to induce rAAV assembly from stableHeLa producer cell clones, containing both the rep-capgenes and the vector integrated into their genome, to alevel at least equal to that observed with wild-type Ad5.

Identification of a replication-defectiveHSV-1 mutant able to help rAAVassembly

We next wanted to find an HSV-1 mutant that had theability to help rAAV assembly as efficiently as the wild-type virus, but unable to replicate. On the basis of aprevious study regarding the HSV-1 helper effect onwild-type and recombinant AAV2 [5], different HSV-1strains were evaluated: (1) HSV�ICP4, mutated in theimmediate early IE3 gene, was used as a negative control.Indeed, the absence of the ICP4 gene product completelyprevented the synthesis of the HSV-1 early and late genesas well as viral DNA replication [35]; (2) HSV�ICP27,mutated in the immediate early IE2 gene. This mutantwas defined as replication-defective but able to induce thesynthesis of the HSV-1 early genes. In addition, in contrastto what was observed with the HSV�ICP4 virus, a lowbut detectable level of viral DNA is synthesized by theHSV�ICP27 mutant [36]; (3) HSV�UL30 and HSV�UL9mutated in the UL30 and UL9 genes encoding for theviral DNA polymerase and the origin binding protein,respectively. Both these mutants expressed all the HSV-1immediate and the other early genes, but were absolutelydefective for DNA replication [22,24].

All the HSV-1 mutants, as well as wild-type HSV-1 andAd5, were used individually to infect the AAVhALD15producer cells, which were then analyzed for: (i) the levelof rAAV particle production, measured by dot blot inthe cell lysate; (ii) the amplification of the endogenousrep-cap sequences, determined by Southern blot; (iii) thelevel of Rep and Cap protein synthesis; and (iv) the levelof rAAV replication, measured by Southern blot analysis.

The measurement of the rAAV titer indicated thatamong the mutants tested only HSV�UL30 and HSV�UL9were able to induce rAAV assembly at a level similar to thatobtained with wild-type HSV-1 (Figure 2A). As expected,the HSV�ICP4 mutant was unable to produce rAAV



Figure 2. Analysis of the helper activity of different replication-defective HSV-1 mutants. AAVhALD15 producer cells (106 cells) wereinfected in quadruplicate with either mutated HSV viruses or wild-type HSV-1 (MOI = 5). For comparison, cells were also infectedwith wild-type Ad5 (MOI = 50). Twenty-four or 48 h post-infection, for HSV- or Ad5-infected cells, respectively, the followinganalyses were performed: (A) titration of rAAVhALD particles by dot blot in cell lysate; (B) detection of rep gene amplification, bySouthern blot. The detected band corresponds to the 1.4 kb expected rep gene fragment. (C) Analysis of rAAV DNA replication.Total DNA was separated on a 1% agarose gel and hybridized with a probe corresponding to the human ALD gene. The three majorbands detected on the film correspond to the single-stranded (ss), monomer double-stranded (mRF) and dimer double-stranded(dRF) rAAV DNA. (D) Detection of Rep and VP proteins by Western blot in the cell lysates. The lane numbers in (B), (C), and(D) correspond to: non-infected cells, lane 1; cells infected with HSV�ICP4, lane 2; HSV�ICP27, lane 3; HSV�UL30, lane 4;HSV�UL9, lane 5; wild-type HSV-1, lane 6; wild-type Ad5, lane 7

particles at a detectable level. Surprisingly, HSV�ICP27was also inefficient.

The analysis conducted in parallel on the AAVhALD15cells indicated that the amount of rAAV particles measuredin the cell lysate correlated with the ability of each ofthese viruses to induce rep-cap gene amplification and,consequently, Rep and Cap protein synthesis (Figures 2Band 2D). Importantly, this result confirmed that, asreported for wild-type Ad5 [10,11], the ability of HSV-1to induce rAAV assembly also correlated with its capacityto induce rep-cap gene amplification. Similarly, the levelof rAAV assembly observed with each viral strain wasalso correlated with the amount of rAAV-replicative formsdetected (Figure 2C), further suggesting that the sameHSV factors are involved in rep-cap amplification andrAAV replication. The higher amount of single-strandedDNA observed using HSV versus Ad5 likely reflected thedifference in the number of full rAAV particles measuredusing these two helper viruses.

These results demonstrated that, at least on a smallscale (approx. 106 cells), HSV strains mutated in either

the UL9 of the UL30 gene can be efficiently used to inducerAAV production from stable producer cell lines. Becausea pure and high titer stock of HSV�UL9 was more difficultto obtain than for HSV�UL30, we focused on this lattermutant for the following analysis.

In vitro characterization of alarge-scale rAAVhALD vector stockproduced using HSV�UL30

To further substantiate the above observations, a moredetailed characterization was conducted on a medium-scale production of rAAVhALD particles performed withthe HSV�UL30 helper virus. For this, AAVhALD15 cells(approx. 3 × 108) were infected with either wild-typeHSV-1, Ad5, or HSV�UL30 and the rAAV particlespurified using two different methods. The rAAVhALDstocks obtained were then analyzed for the number ofphysical and infectious rAAV particles as well as for thepresence of contaminants, particularly those derived fromthe helper virus itself.



The measurement of the rAAV titers obtained indicatedthat, as observed on a small scale, the use of wild-typeHSV-1 or HSV�UL30 resulted in a titer which was similaror even superior to that observed using wild-type Ad5(Table 1). It should be noted that the same differencesin titer were also observed in the initial cell lysates (datanot shown) indicating that they were not due to thepurification method used. More importantly, the ratiosbetween total and DNA-containing particles as well asbetween DNA-containing and infectious particles werenot significantly changed according to the helper virusused (Table 1).

Because the choice of a helper virus may additionallyimpact on the purity of the rAAV stock, we further ana-lyzed the rAAVhALD preparations purified according totwo different methods for the presence of contaminat-ing proteins derived from the cells and the helper virus.Iodixanol/heparin purification was chosen because it rep-resents a standard method used in most vector cores [28].For comparison, we also purified rAAVhALD15 particlesusing a recently described ion-exchange chromatography(FPLC) procedure [29]. The analysis of the rAAV vectorstocks by SDS-PAGE followed by silver staining indicatedthat, as previously demonstrated [29], purification byiodixanol/heparin resulted in rAAV stocks contaminatedwith significant amounts of proteins, independently of thehelper virus used (Figure 3A). In contrast, purification by

Table 1. rAAV production using different helper viruses

Iodixanol/heparin

Tot. part.a Full part.b Inf. part.c Tot./Full Full/Inf. part

wtAd5 6.4 × 1011 1.3 × 1011 3.0 × 109 4.9 43wtHSV-1 7.0 × 1011 1.3 × 1011 3.0 × 109 5.4 43HSV�UL30 6.6 × 1011 1.3 × 1011 3.0 × 109 4.9 43

FPLC

wtAd5 1.2 × 1012 2.8 × 1011 1.8 × 109 4.3 155wtHSV-1 1.2 × 1013 3.8 × 1012 3.8 × 1010 3.2 102HSV�UL30 1.0 × 1013 2.5 × 1012 3.4 × 1010 4.2 76

rAAV particles were produced by infection of AAVhALD15 cells (approx.3 × 108) with the indicated helper virus and purified using eitheriodixanol/heparin or FPLC.aAs measured by A20 ELISA.bDNA-containing particles measured by dot blot.cAs measured by a replication center assay.

FPLC resulted in stocks where only the three VP proteinswere detected when up to 1010 particles were loaded onthe gel (Figure 3B). We next focused on the presence ofcontaminants derived from the helper virus itself. rAAVstocks were analyzed by Western blot using antibodiesdirected either against the adenovirus or the HSV majorcapsid protein (ICP5). This analysis indicated that whenwild-type Ad5 was used as a helper, bands correspondingto adenoviral proteins were detected, particularly in the

Figure 3. Silver staining and Western blot analysis of rAAVhALD stocks produced using either wild-type Ad5, wild-type HSV-1,or HSV�UL30. rAAVhALD particles were purified using two different procedures: (A) iodixanol/heparin [28]; (B) two-stepion-exchange chromatography [29]. Samples containing known amounts of rAAV particles (measured by dot blot), indicatedon the top of each lane, were resolved by SDS-PAGE and either silver-stained or transferred to a membrane and analyzed byWestern blot using an anti-VP antibody (B1)



stocks purified by iodixanol/heparin (Figure 4A). WhenrAAV was purified by FPLC a single adenoviral band wasdetected by Western blot confirming previous observa-tions [29]. This latter result, together with the absence ofdetection of adenoviral DNA by PCR (data not shown),suggest that this contaminant is due to the co-purificationof this unique adenoviral protein with rAAV particles. Sim-ilarly, the use of wild-type HSV-1 resulted in the presenceof contaminating HSV particles when rAAV was purifiedby iodixanol/heparin as shown by the detection of ICP5by Western blot (Figure 4B) and of HSV DNA by PCR(data not shown). This was not unexpected since HSV-1 particles also bind to heparan sulfate residues [37].Accordingly, no such contaminants were detected whenrAAV was purified by FPLC. In contrast, no HSV proteinswere detected when the replication-defective HSV�UL30virus was used, independently of the purification pro-cess used (Figure 4B). This latter result clearly illustratesthe advantage of using a replication-defective HSV strainversus a wild-type virus.

In vivo characterization of large-scalerAAVhALD vector stocks producedusing HSV�UL30

The above results indicated that rAAV particles producedwith the HSV�UL30 virus and purified by FPLC werefree of HSV particles and of other detectable proteincontaminants derived from the helper virus or thecells. To validate the use of this new helper virusfor rAAV assembly, rAAVhALD particles produced usingHSV�UL30 were stereotactically injected into the brainsof adult mice deficient for adrenoleukodystrophy (ALD)gene expression. Brains harvested 6 weeks followinginjection were analyzed for: (i) ALD protein expression,as detected by indirect immunofluorescence using anantibody directed against the human protein; and(ii) potential toxicity, evaluated by light microscopy ofhematoxylin and eosin stained tissues. As a control, these

analyses were similarly conducted using a rAAVhALDpreparation made using wild-type Ad5 as a helper, andpurified by FPLC.

The human ALD protein (hALDP) is a membrane ATP-binding transporter localized in the peroxisome [38].Accordingly, human ALDP staining was observed in theperiphery of the nuclei, most of which also stained forthe NeuN marker for neuronal cells (Figures 5A–5D).This result indicated that, as previously documentedby other groups [39,40], rAAV2 particles preferentiallytarget neurons, and further indicated that this specificitywas not affected by the helper virus used. The numberof transduced cells in each brain section was assessedand revealed that the difference observed between thetwo viral stocks was not significantly different (p > 0.05,n = 3; Figure 5G).

To look for potential toxicity induced by the viralsuspension, selected tissue sections were stained withhematoxylin and eosin to detect cell and tissue alterations.As illustrated in Figures 5E and 5F, no alterations wereobserved for either viral stocks, compared with non-injected tissues. Similarly, indirect immunofluorescenceperformed to detect the number of astrocytes and brainmacrophages (microglia) did not show any differencebetween non-injected and injected animals (data notshown). These results likely suggest that both viral stockshad similar biological properties.

Discussion

Several studies have previously documented the advan-tages linked to the use of stable rAAV producer cell linesespecially for the development of scalable production pro-cedures [9,41,42]. The major drawback of this methodremains the need for the use of wild-type adenovirusto induce rAAV production which replicates and ampli-fies along with rAAV. Indeed, most stable producer cellclones are derived from HeLa cells and thus the con-ventional �E1 Ad strains cannot be used in this context

Figure 4. Detection of Ad- or HSV-derived proteins in rAAVhALD stocks by Western blot. Known amounts of rAAVhALD particles(measured by dot blot), indicated at the top of each lane, were resolved by SDS-PAGE and incubated with rabbit anti-adenovirusserum or with the monoclonal anti-ICP5 mouse antibody. As a control, known amounts of wild-type Ad5 and HSV-1 particles(indicated at the top of the lane as the number of pfu) were similarly analyzed



[16]. An alternative procedure recently proposed by Qiaoet al. used an inducible rep-cap cell line based on 293cells and a replication-defective Ad [43]. However, in thismodel, the �E1 Ad virus used could still replicate, thusconstituting an important source of contamination.

As an alternative procedure with reasonable clinicalapplications, we focused on the use of other helperviruses and particularly on replication-defective viralstrains derived from HSV-1.

The first objective of the present study was toinvestigate the potential of wild-type HSV-1 for rAAVassembly using HeLa-derived stable producer cell lines.We demonstrated that wild-type HSV-1 was an efficienthelper for rAAV production using stable producer celllines. Compared with wild-type Ad5, a lower MOIof HSV-1 particles was required to infect 90–100%of the cells. Moreover, a maximal level of rAAVassembly was reached 24 h post-infection. Both of these

properties are of particular interest for large-scale rAAVproduction.

Our second objective was to identify a replication-defective HSV that could lead to an efficient rAAVproduction level without generating infectious andreplicative HSV particles. Our results showed thata replication-defective HSV-1 strain mutated in thepolymerase gene (UL30) could be used in this context,with a similar efficiency of rAAV titers achieved. Amongthe other HSV mutants tested, HSV�UL9 was alsoable to efficiently induce rAAV assembly. However, weexperienced difficulties in amplifying viral stocks of thisparticular mutant devoid of contamination with wild-type HSV. Surprisingly, the HSV�ICP27 virus provedinefficient for rAAV assembly in our study. As recentlydocumented by others [44], we observed that this HSVmutant was toxic for the HeLa cell used in this study(data not shown). This latter result was in contrast toa previous report indicating that an HSV virus mutatedin the ICP27 gene could be successfully used to producerAAV2 particles, even though a direct comparison witheither wild-type HSV or Ad5 was not reported [45].In that study, the rep and cap genes were integratedby homologous recombination in the thymidine kinaselocus of the HSV�ICP27 genome and, consequently, werebrought into the cells by infection. Accordingly, the rAAVyield obtained increased along with the HSV MOI usedfor infection (up to 10 pfu/cell) [45]. Furthermore, thislatter study was performed with a different HSV�ICP27mutant. Both these elements, i.e. the cell type and thedifferent viral strain, could explain their results.

Finally, our third objective was to precisely characterizethe rAAV stocks produced using HSV versus wild-type

Figure 5. In vivo analysis of rAAVhALD stocks produced usingeither wild-type Ad5 or HSV�UL30. rAAVhALD particles purifiedby FPLC were injected into the brain of ALD-deficient mice. Threemice were injected per group, each receiving 4 × 108 rAAVhALDparticles (as measured by dot blot). The mice were sacrificed6 weeks later and the brain tissue analyzed for expressionof human ALD, detected by indirect immunofluorescence. In(A)–(D), hALD appears red, nuclei stained by DAPI blue, andthose expressing the NeuN marker of neuronal cells green.Note that ALD is expressed in peroxisomes and, thus, thestaining is surrounding the nucleus. The right panels of (C) and(D) represent the two-color staining (NeuN plus ALd or ALDplus DAPI) of cells in the insets. Original magnification ×20(A, B), ×60 (C, D). Bars 50 µm. (E, F) Counterstaining ofbrain tissues with hematoxylin and eosin to detect potentiallesions. Original magnification ×10. Bars 100 µm. (G) The insetillustrates the site of rAAV injection and the region analyzedfor ALD expression. To minimize surgery-related lesions withinthe cortex, the virus was injected into the white matter (corpuscallosum, indicated by a red dot). The diffusion of the viralparticles from the injection point was evaluated by counting thenumber of hALD-expressing neurons in one of every fourteen10 µm thick coronal sections (indicated by black dots in theinset). The data are reported on the graph as the meanvalue (n = 3) of the number of ALD-expressing neurons overthe total number of neurons in each section. Vertical barsindicate the standard deviation value. The thick red arrowindicates the injection site. Cc: corpus callosum; Ctx: cortex;Ob: olfactory bulb



Ad in vitro and in vivo. The analysis of a medium-scale production of rAAV particles performed with theHSV�UL30 helper suggested that this mutant could beof particular interest when associated with a standardiodixanol/heparin purification since no HSV contaminantswere detected in this rAAV stock. However, additionalanalyses are required to definitively assess this point. Inaddition, the HSV�UL30 mutant generated rAAV particleswhich had similar in vitro and in vivo biological propertiesto particles produced using wild-type Ad5.

We previously described a scalable purification proce-dure based on the use of ion-exchange chromatographyresins [29]. The production procedure now described,based on the use of stable producer cell lines infectedwith a replication-defective helper virus, provides the firststep required for the development of an entirely scal-able rAAV production process that can satisfy future GMPrequirements. Future studies will be devoted to trans-pose these procedures to the production and purificationof other rAAV serotypes. We have already demonstratedthat our FPLC purification procedure can be used forthe purification of rAAV5 particles [29]. Development ofproducer cell lines able to assemble pseudo-typed rAAVparticles is also underway and should provide new toolsfor the production of other rAAV serotypes.

Acknowledgements

We would like to thank Juergen Kleinschmidt for providing B1antibodies, Gabrielle Kroner-Lux for performing A20 ELISA tests,Yan Cherel for histochemical staining of the brain sections, andIsabelle Raimbaud for technical assistance. We also thank MarkHaskins for critically reading the manuscript. This work wassupported by the Association Francaise contre les Myopathies(AFM), Vaincre les Maladies Lysosomales (VML), AssociationNantaise de Therapie Genique (ANTG), the Fondation pour laTherapie Genique en Pays de la Loire, and the INSERM.

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