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Journal of Virological Methods 179 (2012) 276– 280

Contents lists available at SciVerse ScienceDirect

Journal of Virological Methods

j ourna l ho me p ag e: www.elsev ier .com/ locate / jv i romet

hort communication

ransduction of striatum and cortex tissues by adeno-associated viral vectorsroduced by herpes simplex virus- and baculovirus-based methods

. Steve Zhanga,1, Eunmi Kimb,1, Slgirim Leeb, Ik-Sung Ahnb, Jae-Hyung Jangb,∗

Sangamo BioSciences Inc., Richmond, CA 94804, USADepartment of Chemical and Biomolecular Engineering, Yonsei University, Seoul 120-749, Republic of Korea

rticle history:eceived 11 August 2011eceived in revised form8 September 2011ccepted 4 October 2011vailable online 12 October 2011

eywords:deno-associated viruserpes simplex virus type Iaculovirusene delivery

a b s t r a c t

Recombinant adeno-associated virus (AAV) vectors can be engineered to carry genetic material encodingtherapeutic gene products that have demonstrated significant clinical promise. These viral vectors aretypically produced in mammalian cells by the transient transfection of two or three plasmids encodingthe AAV rep and cap genes, the adenovirus helper gene, and a gene of interest. Although this method canproduce high-quality AAV vectors when used with multiple purification protocols, one critical limita-tion is the difficulty in scaling-up manufacturing, which poses a significant hurdle to the broad clinicalutilization of AAV vectors. To address this challenge, recombinant herpes simplex virus type I (rHSV-1)-and recombinant baculovirus (rBac)-based methods have been established recently. These methods aremore amenable to large-scale production of AAV vectors than methods using the transient transfectionof mammalian cells. To investigate potential applications of AAV vectors produced by rHSV-1- or rBac-based platforms, the in vivo transduction of rHSV-1- or rBac-produced AAV serotype 2 (AAV2) vectors

ntracranial injection within the rat brain were examined by comparing them with vectors generated by the conventionaltransfection method. Injection of rHSV-1- or rBac-produced AAV vectors into rat striatum and cortextissues revealed no differences in cellular tropism (i.e., predominantly neuronal targeting) or anteropos-terior spread compared with AAV2 vectors produced by transient transfection. This report represents astep towards validating AAV vectors produced by the rHSV-1- and the rBac-based systems as promisingtools, especially for delivering therapeutic molecules to the central nervous system.

Adeno-associated virus (AAV), a parvovirus with a 4.7 kb single-tranded DNA genome containing two genes (rep and cap), has beenxplored in clinical trials for the treatment of a broad range ofiseases, including Parkinson’s disease (Fiandaca and Bankiewicz,010), hemophilia B (by delivering the Factor IX gene) (Kay et al.,000), and Leber’s congenital amaurosis (Bainbridge et al., 2008;aguire et al., 2008). AAV typically has the capability to deliver

enes to both dividing and non-dividing cells, such as muscleFisher et al., 1997), brain (Kaplitt et al., 1994), and retina (Flanneryt al., 1997) cells. AAV does not elicit toxic or immune responsesn animal models and does not suffer from rapid transcriptionalilencing, further enhancing its potential as a candidate for thereatments of a variety of human diseases.

One critical challenge for bringing AAV vectors into clinical tri-

ls involves manufacturing sufficient quantities of highly purifiedectors at acceptable costs. A conventional method used widely forroducing all AAV serotypes is the transient transfection of three

∗ Corresponding author. Tel.: +82 2 2123 2756; fax: +82 2 312 6401.E-mail address: j-jang@yonsei.ac.kr (J.-H. Jang).

1 Equal contribution.

166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2011.10.004

© 2011 Elsevier B.V. All rights reserved.

plasmids, including an AAV helper plasmid containing rep and capgenes in trans, the adenoviral helper gene plasmid required for vec-tor replication, and a plasmid carrying a gene of interest flanked byAAV inverted terminal repeats (ITRs) (Wright, 2009). The transienttransfection method, which has been shown to produce vectorscapable of efficient transduction of mammalian cells, is typicallyperformed by coprecipitation of the three plasmids using calciumphosphate (Graham and van der Eb, 1973), polycationic polymers(e.g., polyethylenimine) (Boussif et al., 1995), or cationic lipids(Felgner et al., 1987). This method can eliminate contaminationof the AAV stocks with a helper virus (e.g., adenovirus), which istypically required for AAV replication, during the process of pro-duction. It can also be adapted to produce AAV vectors of differentserotypes. However, a major limitation of this method is the dif-ficulty of scaling up transient transfections to produce sufficientvectors for large-scale clinical applications (Merten et al., 2005).

To address this limitation of the transient transfection method,large-scale viral vector production technologies based on use of

a recombinant herpes simplex virus (rHSV) type I (Conway et al.,1999; Wu et al., 2002) or a recombinant baculovirus (rBac) (Blissard,1996; Urabe et al., 2002) have been established. The rHSV-1platform is based on the use of a replication-defective rHSV-1

H.S. Zhang et al. / Journal of Virological Methods 179 (2012) 276– 280 277

Fig. 1. Characterization of each AAV vector produced by transfection of 293 cells and rBac- and rHSV-1-based methods. (A) Heparin column chromatograms are shown forthe comparison of the heparin affinities of the viral vectors. “Fraction virus eluted” (vertical axis) indicates the ratio of the infectious viral titer present in a designated elutiont ro trant ethodB -posit

ctaitabrnrsganctcfatettat

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ml(btc

o the infectious viral titer present in the solution prior to column loading. (B) In vitransient transfection of 293 cells, the rHSV-1-based method, and the rBac-based m16F10 cells (MOI 10,000), and CHO-K1 cells (MOI 10,000). The percentages of GFP

ontaining the AAV rep and cap genes and a cell line carryinghe recombinant AAV (rAAV) proviral genome, which is gener-ted by the stable transfection of a plasmid carrying the gene ofnterest. The rHSV-1 serves as a helper virus for rAAV replica-ion, resulting in viral yields (i.e., total viral quantities) that arepproximately two orders of magnitude higher than those acquiredy the conventional transfection method (Wu et al., 2002). TheBac platform, which is based on use of the Autographa califor-ica nuclear polyhedrosis virus, has also been adapted to produceAAV in invertebrate cells on a large scale (Urabe et al., 2002). Thistrategy involves engineering the AAV rep/cap genes and a trans-ene into three recombinant baculoviruses to generate a rep-rBac,

cap-rBac, and a transgene-rBac. Coinfection of these recombi-ant baculoviruses into insect cells (e.g., Spodoptera frugiperdaells) resulted in a 10-fold higher yield of AAV particles thanhat obtained with the transient transfection method using 293ell lines (Urabe et al., 2002). Both rHSV-1- and rBac-based plat-orms can be alternatives to the transient transfection method,nd they produce much larger quantities of AAV vectors, makinghem potentially more suitable for human gene therapy. How-ver, for AAV vectors produced by the rHSV or the rBac systemso be adopted widely, it is crucial to determine whether the vec-ors produced by these methods have predictable infectivity suchs tropism and vector spread, comparable to those of transientransfection.

The in vivo performances of the AAV vectors produced by theseifferent methods, which have not been evaluated previously in

single study, were assessed in the rat brain. Each AAV vector,ontaining AAV serotype 2 (AAV2) capsids and encoding green flu-rescence protein (GFP), was injected into the rat striatum andortex. These tissues are potential target regions for the delivery ofherapeutic molecules for the treatment of neurological disordersuch as Parkinson’s disease, Huntington’s disease, and stroke. Afterelivery of the AAV vectors generated by each production method,ellular tropism and the spread of the transgene expression werexamined. This study demonstrates the features of AAV2 vectorsanufactured by rHSV-1- and rBac-based production platforms,hich make them potentially suitable for applications in human

ene therapy.AAV serotype 2 vectors were produced by three different

ethods: (i) the transient transfection method using AAV 293 cellines (AAV2-293), (ii) a recombinant herpes simplex virus type I

rHSV-1)-based method (AAV2-rHSV-1), and (iii) a recombinantaculovirus (rBac)-based method (AAV2-rBac). For the transientransfection method, recombinant AAV2 (rAAV2) vectors carryingDNA encoding GFP were packaged and purified by CsCl-gradient

sduction abilities of the AAV2 vectors are indicated. AAV-GFP vectors produced by were used to infect HEK293T cells (genomic MOI: 10,000), HeLa cells (MOI 10,000),ive cells were quantified by flow cytometry.

ultracentrifugation followed by PD-10 (GE Healthcare Life Sci-ences, Pittsburgh, PA) desalting filtration, as described previously(Koerber et al., 2006; Maheshri et al., 2006). For rHSV-1- andrBac-induced packaging, the rAAV vectors carrying cDNA encodingGFP under a CMV promoter were purchased from Vector GeneTechnology Company (Beijing, China) and Virovek (Hayward, CA,USA). These companies have produced AAV vectors for previousstudies on rHSV-1-induced systems (Tian et al., 2011; Wu et al.,2002) and rBac-induced systems (Chen, 2008; Urabe et al., 2002).Viral vectors produced by the use of the rBac-induced system(i.e., those from Virovek) were purified by the same method asthose produced using the transient transfection method. Viralvectors generated using the rHSV-1-induced system (i.e., thosefrom the Vector Gene Technology Company) were purified usingion exchange column chromatography followed by filtrationthrough a molecular sieve (Sephacryl S-200 High Resolutioncolumn; Amersham Pharmacia Biotech, Piscataway, NJ). DNase-resistant genomic titers were determined by quantitative PCR(QPCR; Mini Opticon, Bio-Rad, Hercules, CA). As a result, thetransient transfection method produced the highest genomic titers(3.99 × 1012 ± 1.45 × 1012 vg (viral genomes)/mL), and the rHSV-1-and rBac-based methods produced 1.29 × 1012 ± 2.23 × 1011 vg/mLand 1.35 × 1012 ± 4.03 × 1011 vg/mL, respectively. Addition-ally, transduction of HEK293T cells lines by the transienttransfection-, rHSV 1-, and rBac-based methods, gener-ated 1.31 × 1010 ± 1.33 × 1010, 4.22 × 109 ± 2.53 × 109, and6.95 × 109 ± 8.67 × 109 transducing units (TU)/mL, respectively.There were no statistical differences in either the genomic titers ortransducing units among the viral vectors produced by the threedifferent methods (p > 0.05).

The heparin affinities of the AAV vectors produced by the dif-ferent packaging methods were assessed to determine whetherany of the AAV vectors could possibly use heparan sulfate as itsprimary receptor (Fig. 1A). Approximately 1011 purified genomicparticles of virus were loaded onto a 1 mL HiTrap heparin col-umn (GE Healthcare Life Sciences), which had been equilibratedwith Tris buffer (50 mM, pH 7.5) containing 150 mM NaCl. Elutionswere performed with a series of increasing NaCl concentrations(150–750 mM and 1 M), and infectious viral titers of the HEK293Tcells were determined by flow cytometry for each fraction. Similarto previous reports (Maheshri et al., 2006), all AAV vectors elutedin a sharp peak with 450–550 mM NaCl, indicating that none of the

production methods altered the ability of the AAV to bind heparin.

The capacity of each AAV vector to transduce culturedcells, including HEK293T, HeLa, B16F10, and CHO-K1 cells, wasassessed next (Fig. 1B). One day prior to infection, the cells

278 H.S. Zhang et al. / Journal of Virological Methods 179 (2012) 276– 280

Fig. 2. In vivo gene delivery and cellular tropism of GFP-expressing AAV2 vectors within the rat striatum. (A) Representative images of infections with AAV2 vectorsgenerated by transient transfection-, rBac-, and rHSV-1-based methods within the rat striatum show similar levels of GFP expression (green) at 3 weeks post-injection (scaleb g cell( (greel

w2eAtqCthAsoAtpi

ar: 180 �m). Representative images of cell types residing adjacent to GFP-expressinB) NeuN+ neurons (blue)/GFP+ cells (green), (C) GFAP+ astrocytes (red)/GFP+ cellsegend, the reader is referred to the web version of the article.)

ere seeded onto 24-well tissue culture plates at a density of0,000–30,000 cells/well, and the cells were infected on day 1 byach AAV vector at 104 genomic multiplicity of infection (MOI).t 48 h post-infection, the resulting transduction efficiencies (i.e.,

he fraction of cells that expressed GFP) of each rAAV vector wasuantified with a Becton Dickinson FACS Caliber (Yonsei Universityollege of Medicine Medical Research Center). For HEK293T infec-ion, AAV2 vectors produced from the rHSV-1 method exhibited theighest transduction efficiency (85.6%) compared with the otherAV2 vectors. The AAV2 vectors generated by the rBac-systemhowed the best performances in cellular transduction in severalther cell lines (HeLa, B16F10, and CHO-K1). However, all of the

AV vectors yielded over 50% transduction efficiencies in all cell

ypes, implying that the biological properties of the AAV2 vectorsroduced by the three helper systems are indistinguishable, which

s consistent with a previous study (Urabe et al., 2002).

s within the rat striatum demonstrate the equivalent tropism for each AAV2 vector:n) (scale bars: 50 �m). (For interpretation of the references to color in this figure

To investigate the in vivo performances of the AAV vectors pro-duced by the three different methods, the same number of genomicparticles of each AAV2 vector (3 �L of 1 × 109 vg/�L for a total of3 × 109 viral particles) were injected stereotaxically into the stria-tum (anteroposterior [AP], +0.2 mm; mediolateral [ML], ±3.5 mm;and dorsoventral [DV], −4.5 mm from the skull) and cortex ([AP],+2.7 mm; [ML], ±2.5 mm; and [DV], −0.5 mm from the skull) ofadult female Fischer 344 rats (150 g, 6 weeks old). Three weekspost-injection, robust GFP expression was observed in all animals,with a wide distribution around the injection sites in the stria-tum and the cortex (Figs. 2 and 3). GFP expression was detectedby a primary rabbit anti-GFP antibody (diluted 1:2000; Invitro-

gen, Carlsbad, CA). Primary mouse anti-neuron-specific nuclearprotein (NeuN, diluted 1:200; Chemicon International, Temecula,CA) and guinea pig anti-glial fibrillary acidic protein (GFAP, diluted1:1000; Advanced ImmunoChemical, Long Beach, CA) antibodies

H.S. Zhang et al. / Journal of Virological Methods 179 (2012) 276– 280 279

Fig. 3. In vivo gene delivery and cellular tropism of GFP-expressing AAV2 vectors within the rat cortex: (A) representative images of the rat cortex infected with AAV2 vectorsgenerated by transient transfection, the rHSV-1-based method, and the rBac-based method show similar levels of GFP expression (green) at 3 weeks post-injection (scaleb ithinn ale bar

w(idrpiidsc

AtuG

ar: 150 �m). Representative images of cell types adjacent to GFP-expressing cells weurons (blue)/GFP+ cells (green), (C) GFAP+ astrocytes (red)/GFP+ cells (green) (sceader is referred to the web version of the article.)

ere utilized to identify cell types such as neurons and glial cellsrepresented by astrocytes). The corresponding secondary antibod-es, which were labeled with Alexa Fluor 488, 546, and 633 andiluted 1:250, were used for detection. The sections containingegions exhibiting GFP expression were collected, and the antero-osterior spread of each AAV vector type that had been injected

nto the striatum and the cortex was quantified to evaluate then vivo performance of each AAV vector. As with in vitro trans-uction, AAV2 vectors produced by different methods exhibitedimilar performances following cranial delivery when assayed forell targeting and spread within the brain.

In both the striatum and the cortex, the cellular tropism of

AV2-rHSV-1 and AAV2-rBaculovirus vectors remained identical

o that of vectors produced by the transient transfection methodsing a 293 cell line (Figs. 2 and 3). Transduction (monitored byFP expression) primarily occurred in cells that stained positive

the rat cortex demonstrate the equivalent tropism for each AAV2 vector: (B) NeuN+

rs: 50 �m). (For interpretation of the references to color in this figure legend, the

for the neuronal marker NeuN, with minimal infection of glial cellsexpressing the astrocytic marker GFAP. These observations indi-cate that rAAV2 vectors created by the rHSV-1- and rBac-systemsare highly efficient in vivo, and their performances are not affectedsubstantially by these alternative AAV production methods. Thisis consistent with the result that rAAV2 replicated by the HSV-1 and baculovirus systems efficiently transduced photoreceptorcells in mice, which are typically permissive to rAAV2 producedby the transient transfection method. This indicates similar phys-ical and biological properties of the AAV vectors produced by thethree procedures (Urabe et al., 2002; Zhang et al., 1999).

Additionally, there were no statistical differences in viral infec-

tion spread along the parts of anteroposterior axis accessed byviral vector infection among the three different viral prepara-tions (Fig. 4). The gene delivery properties (anteroposterior spreadand cellular tropism) of the AAV2 vectors produced by transient

280 H.S. Zhang et al. / Journal of Virological Methods 179 (2012) 276– 280

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ig. 4. The anteroposterior spread of GFP expression within the rat striatum (A) andmong the three AAV2 vectors (p > 0.05). The AP spread is defined as the distance b

ransfection and the rHSV-1 and rBac systems are comparable inn vivo models, suggesting that the in vivo performances of theseiral vectors are not substantially affected by different viral pro-uction systems. Interestingly, free diffusion of each vector wasomewhat hindered in the cortex (∼500–1000 �m) relative to thetriatum (∼1000–2000 �m).

In conclusion, this study describes the in vivo performance ofAAV2 vectors produced by three different methods: (i) the tran-ient transfection of mammalian cells with three plasmids, (ii) theHSV-1 helper system, and (iii) the rBaculovirus helper platform.he rHSV-1- and rBaculovirus-based replication platforms did notnfluence either the in vitro or in vivo performances of each vec-or. Robust production methods for rAAV vectors will certainlynhance the applicability of AAV as a gene delivery vehicle, specifi-ally for human gene therapy applications, which typically requirearge amounts of viral vectors. Neuronal tropism of the AAV vectorsroduced by the rHSV-1 or the rBaculovirus system demonstrateshat these two platforms can be alternatives to the conventionalransfection approach, especially for investigating treatments foreurological disorders.

onflict of interest statement

The authors declare no conflict of interest.

cknowledgements

This work was supported by Advanced Biomass R&D Cen-er (ABC) of Korea (Grant No. 2010-0029734) and by theuman Resources Development of the Korea Institute of Energyechnology Evaluation and Planning (KETEP) grant funded byhe Korea government Ministry of Knowledge Economy (No.0104010100500).

eferences

ainbridge, J.W., Smith, A.J., Barker, S.S., Robbie, S., Henderson, R., Balaggan, K.,Viswanathan, A., Holder, G.E., Stockman, A., Tyler, N., Petersen-Jones, S., Bhat-tacharya, S.S., Thrasher, A.J., Fitzke, F.W., Carter, B.J., Rubin, G.S., Moore, A.T.,Ali, R.R., 2008. Effect of gene therapy on visual function in Leber’s congenitalamaurosis. N. Engl. J. Med. 358, 2231–2239.

lissard, G.W., 1996. Baculovirus–insect cell interactions. Cytotechnology 20, 73–93.oussif, O., Lezoualch, F., Zanta, M.A., Mergny, M.D., Scherman, D., Demeneix, B.,

Behr, J.P., 1995. A versatile vector for gene and oligonucleotide transfer intocells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U.S.A. 92,7297–7301.

x (B). The anteroposterior spread measurements indicate no significant differencesn the first and last tissue sections exhibiting GFP expression.

Chen, H., 2008. Intron splicing-mediated expression of AAV Rep and Cap genes andproduction of AAV vectors in insect cells. Mol. Ther. 16, 924–930.

Conway, J.E., Rhys, C.M., Zolotukhin, I., Zolotukhin, S., Muzyczka, N., Hayward, G.S.,Byrne, B.J., 1999. High-titer recombinant adeno-associated virus production uti-lizing a recombinant herpes simplex virus type I vector expressing AAV-2 Repand Cap. Gene Ther. 6, 986–993.

Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P.,Ringold, G.M., Danielsen, M., 1987. Lipofection: a highly efficient, lipid-mediatedDNA-transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84, 7413–7417.

Fiandaca, M.S., Bankiewicz, K.S., 2010. Gene therapy for Parkinson’s disease: fromnon-human primates to humans. Curr. Opin. Mol. Ther. 12, 519–529.

Fisher, K.J., Jooss, K., Alston, J., Yang, Y., Haecker, S.E., High, K., Pathak, R., Raper,S.E., Wilson, J.M., 1997. Recombinant adeno-associated virus for muscle directedgene therapy. Nat. Med. 3, 306–312.

Flannery, J.G., Zolotukhin, S., Vaquero, M.I., LaVail, M.M., Muzyczka, N., Hauswirth,W.W., 1997. Efficient photoreceptor-targeted gene expression in vivo by recom-binant adeno-associated virus. Proc. Natl. Acad. Sci. U.S.A. 94, 6916–6921.

Graham, F.L., van der Eb, A.J., 1973. A new technique for the assay of infectivity ofhuman adenovirus 5 DNA. Virology 52, 456–467.

Kaplitt, M.G., Leone, P., Samulski, R.J., Xiao, X., Pfaff, D.W., O’Malley, K.L., During,M.J., 1994. Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain. Nat. Genet. 8, 148–154.

Kay, M.A., Manno, C.S., Ragni, M.V., Larson, P.J., Couto, L.B., McClelland, A., Glader, B.,Chew, A.J., Tai, S.J., Herzog, R.W., Arruda, V., Johnson, F., Scallan, C., Skarsgard,E., Flake, A.W., High, K.A., 2000. Evidence for gene transfer and expression offactor IX in haemophilia B patients treated with an AAV vector. Nat. Genet. 24,257–261.

Koerber, J.T., Maheshri, N., Kaspar, B.K., Schaffer, D.V., 2006. Construction of diverseadeno-associated viral libraries for directed evolution of enhanced gene deliveryvehicles. Nat. Protoc. 1, 701–706.

Maguire, A.M., Simonelli, F., Pierce, E.A., Pugh Jr., E.N., Mingozzi, F., Bennicelli, J.,Banfi, S., Marshall, K.A., Testa, F., Surace, E.M., Rossi, S., Lyubarsky, A., Arruda, V.R.,Konkle, B., Stone, E., Sun, J., Jacobs, J., Dell’Osso, L., Hertle, R., Ma, J.X., Redmond,T.M., Zhu, X., Hauck, B., Zelenaia, O., Shindler, K.S., Maguire, M.G., Wright, J.F.,Volpe, N.J., McDonnell, J.W., Auricchio, A., High, K.A., Bennett, J., 2008. Safety andefficacy of gene transfer for Leber’s congenital amaurosis. N. Engl. J. Med. 358,2240–2248.

Maheshri, N., Koerber, J.T., Kaspar, B.K., Schaffer, D.V., 2006. Directed evolution ofadeno-associated virus yields enhanced gene delivery vectors. Nat. Biotechnol.24, 198–204.

Merten, O.W., Geny-Fiamma, C., Douar, A.M., 2005. Current issues in adeno-associated viral vector production. Gene Ther. 12 (Suppl. 1), S51–S61.

Tian, L., Yang, P., Lei, B., Shao, J., Wang, C., Xiang, Q., Wei, L., Peng, Z., Kijlstra, A., 2011.AAV2-mediated subretinal gene transfer of hIFN-alpha attenuates experimentalautoimmune uveoretinitis in mice. PLoS ONE 6, e19542.

Urabe, M., Ding, C., Kotin, R.M., 2002. Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum. Gene Ther. 13, 1935–1943.

Wright, J.F., 2009. Transient transfection methods for clinical adeno-associated viralvector production. Hum. Gene Ther. 20, 698–706.

Wu, Z., Wu, X., Cao, H., Dong, X., Wang, H., Hou, Y., 2002. A novel and highly efficientproduction system for recombinant adeno-associated virus vector. Sci. China C:

Life Sci. 45, 96–104.

Zhang, X., De Alwis, M., Hart, S.L., Fitzke, F.W., Inglis, S.C., Boursnell, M.E., Levinsky,R.J., Kinnon, C., Ali, R.R., Thrasher, A.J., 1999. High-titer recombinant adeno-associated virus production from replicating amplicons and herpes vectorsdeleted for glycoprotein H. Hum. Gene Ther. 10, 2527–2537.