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Controlled delivery of glial cell line-derived neurotrophic factor by a singletetracycline-inducible AAV vector

A. Chtarto a,b,1, X. Yang a,b,1, O. Bockstael a,b, C. Melas a,b, D. Blum a,b,2, E. Lehtonen a,b,L. Abeloos a, J.-M. Jaspar c, M. Levivier a, J. Brotchi a, T. Velu b, L. Tenenbaum a,b,⁎

a Laboratory of Experimental Neurosurgery, Multidisciplinary Research Institute (IRIBHM) Université Libre de Bruxelles, Hôpital Erasme, Belgiumb Research Unit in Biotherapy and Oncology, Multidisciplinary Research Institute (IRIBHM) Université Libre de Bruxelles, Hôpital Erasme, Belgium

c Biosource, Nivelles, Belgium

Received 2 November 2006; accepted 30 November 2006Available online 16 January 2007

Abstract

An autoregulated tetracycline-inducible recombinant adeno-associated viral vector (rAAV-pTetbidiON) utilizing the rtTAM2 reversetetracycline transactivator (rAAV-rtTAM2) was used to conditionally express the human GDNF cDNA. Doxycycline, a tetracycline analog,induced a time- and dose-dependent release of GDNF in vitro in human glioma cells infected with rAAV-rtTAM2 serotype 2 virus. Introducing theWoodchuck hepatitis virus posttranscriptional regulatory element (WPRE) downstream to the rtTAM2 coding sequence, resulted in a more rapidinduction and a higher basal expression level. In vivo, 8 weeks after a single injection of the rAAV-rtTAM2-GDNF vector encapsidated into AAVserotype 1 capsids in the rat striatum, the GDNF protein level was 60 pg/mg tissue in doxycycline-treated animals whereas in untreated animals, itwas undistinguishable from the endogenous level (∼4 pg/mg tissue). However, a residual GDNF expression in the uninduced animals wasevidenced by a sensitive immunohistochemical staining. As compared to rAAV1-rtTAM2-GDNF, the rAAV1-rtTAM2-WPRE-GDNF vectorexpressed a similar concentration of GDNF in the induced state (with doxycycline) but a basal level (without doxycycline) ∼2.5-fold higher thanthe endogenous striatal level.

As a proof for biological activity, for both vectors, downregulation of tyrosine hydroxylase was evidenced in dopaminergic terminals ofdoxycycline-treated but not untreated animals.

In conclusion, the rAAV1-rtTAM2 vector which expressed biologically relevant doses of GDNF in the striatum in response to doxycyclinewith a basal level undistinguishable from the endogenous striatal level, as measured by quantitative ELISA assay, constitutes an interesting tool forlocal conditional transgenesis.© 2006 Elsevier Inc. All rights reserved.

Keywords: Glial cell line-derived neurotrophic factor; Tetracycline-inducible; Parkinson; Striatum; Adeno-associated virus; Serotype 1; Tyrosine hydroxylase;Downregulation

Introduction

Glial cell line-derived neurotrophic factor (GDNF) is apotent neurotrophic factor which has demonstrated neuropro-tective and neurorestorative effects in a variety of rodent and

primate models of Parkinson's disease (Björklund et al., 2000;Kordower et al., 2000; Choi-Lundberg et al., 1997; Bilang-Bleuelet al., 1997; Eslamboli et al., 2003). GDNF is a member of thetransforming growth factor-β superfamily (Lin et al., 1994) and isexpressed at high levels in the developing striatum (Stromberget al., 1993). Response to GDNF requires the presence ofglycosylphosphatidyl-inositol-linked proteins (GFR-αs) (Jinget al., 1997). GFR-αs form a complex with the membranereceptor Ret thereby inducing receptor tyrosine autophosphoryla-tion (Trupp et al., 1998) and activation of signal transductionfinally leading to inhibition of apoptosis (Trupp et al., 1999).Consistently, GDNF has been shown to counteract dopaminergic

Experimental Neurology 204 (2007) 387–399www.elsevier.com/locate/yexnr

⁎ Corresponding author. Laboratory of Experimental Neurosurgery, Multi-disciplinary Research Institute (IRIBHM) Université Libre de Bruxelles, HôpitalErasme, Belgium. Fax: +32 2 5554655.

E-mail address: litenenb@ulb.ac.be (L. Tenenbaum).1 These authors equally contributed to this work.2 Present address: INSERM U815, Jean-Pierre Aubert Research Center,

Université de Lille 2, Lille, France.

0014-4886/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.expneurol.2006.11.014

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cell death induced by various insults such as medial forebrainbundle axotomy (Tseng et al., 1997), 6-hydroxydopamineinjection (Mandel et al., 1999) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment (Kordower et al., 2000).GDNF is also able to promote morphological differentiation ofdeveloping dopaminergic neurons (Costantini and Isacson,2000; Widmer et al., 2000), to enhance dopamine metabolismin neonatal (Beck et al., 1996) and adult rat brains (Hudson et al.,1995; Hebert et al., 1996; Martin et al., 1996) and to increaseexcitability of dopamine neurons (Xu and Dluzen, 2000). Allthese effects are presumably relevant for the therapeuticpotential of GDNF in lesioned animals (Kirik et al., 2000;Kordower et al., 2000).

However, GDNF delivery in healthy brain increases spon-taneous as well as amphetamine-induced locomotor activityand, at high doses, reduces food consumption (Martin et al.,1996; Kirik et al., 2000). Furthermore, chronic uncontrolledGDNF delivery results in downregulation of tyrosine hydro-xylase expression, the rate-limiting enzyme of dopaminebiosynthesis (Hagg, 1998; Georgievska et al., 2002, 2004a)thereby resulting in a negative feed-back that could potentiallycounterbalance the trophic effect of GDNF on dopaminemetabolism. Notably, in a Phase I clinical trial, intracerebro-ventricular delivery of high doses of recombinant GDNF hasproduced serious behavioral and cognitive side effects in at leastone patient (Kordower et al., 1999; Nutt et al., 2003). WhenGDNF was administered intraparenchymally, side effects didnot appear and clinical benefits have been reported (Gill et al.,2003). However, the positive outcome of this Phase I study wasnot confirmed in a randomized controlled trial (Lang et al.,2006). Recently, a clinical trial for gene therapy for Parkinson'sdisease using an unregulated AAV vector expressing neurturin(a GDNF analog) has been launched (http://www.clinicaltrials.gov/ct/show/NCT00252850).

The undesirable effects of GDNF could be avoided by usingtightly controlled inducible gene transfer vectors that wouldallow dose-dependent induction and rapid extinction uponaddition/removal of the inducer as well as low basal level oftransgene expression.

The tetracycline-inducible system is based upon the interac-tion between the tetracycline reverse transactivator (rtTA)(Gossen et al., 1995) and the tetracycline-responsive promoter(Gossen and Bujard, 1992). In this system, the rtTA trans-activator binds to tetracycline operator sites after a conforma-tional change induced by antibiotics of the tetracycline familysuch as doxycycline (Gossen et al., 1995) or minocycline(Chtarto et al., 2003b; Matis et al., 2001). However, the rtTA – achimerical transcription factor which consists of a mutated formof the bacterial tetracycline repressor (tetR) fused to theactivation domain (AD) of the herpes simplex virus VP16protein – suffers from several limitations for its use in eucaryoticcells. Indeed, the VP16 AD is known to elicit cellular toxicitydue to the sequestration of cellular factors of the transcriptionmachinery (Matis et al., 2001). The toxicity of rtTA has beendrastically reduced by replacing VP16 AD by 3 repeats of a 12amino acids minimal activation domain (Baron et al., 1997).Furthermore, the mutated tetR initially isolated by Gossen et al.

(1995) had a relatively low affinity for the tetracycline operatorsites in the induced state and a residual binding in the non-induced state. Interestingly, new mutants with a higher affinityand specificity for tetO have been described (Urlinger et al.,2000). Particularly, the M2 mutant of rtTA described byUrlinger and coll. (2000) has been shown to be induced bydoxycycline at doses 10-fold lower than the initial rtTA (Gossenet al., 1995).

Tight regulation of the expression of various transgenesmediated by viral vectors has been obtained in various organs(Stieger et al., 2006; Xiong et al., 2006; Yao et al., 2006).However, the high stability of GDNF protein in the brain(Georgievska et al., 2004b) has precluded the development oftightly regulated viral vectors for GDNF gene delivery.Recently, two different studies have reported efficientlyregulated GDNF gene expression from lentiviral vectors(Georgievska et al., 2004b, Szulc et al., 2006).

We have previously developed a single tetracycline-in-ducible autoregulated AAV vector (AAV-pTetbidiON) (Chtartoet al., 2003a). In the present study, it is reported that animproved version of AAV-pTetbidiON vector harboring the M2mutant rtTA transactivator (Urlinger et al., 2000), allows todeliver GDNF in the striatum at biological active concentrationsleading to downregulation of tyrosine hydroxylase in doxycy-cline-treated rats but not in uninduced controls.

Materials and methods

Plasmids

The pAC1 plasmid comprising AAV ITRs bracketing thebidirectional tetracycline-responsive cassette expressing bothrtTA and EGFP has been described previously (Chtarto et al.,2003a; Fig. 1A). The human GDNF cDNA (a kind gift from Dr.Nicole Deglon, Lausanne, Switzerland) (Bensadoun et al.,2000) has been introduced in this autoregulatory tetracycline-inducible vector by using the multiple cloning site upstream toEGFP and the NotI site at the end of the EGFP coding sequence.To obtain pAC1-M2, the rtTA transactivator has been replacedby rtTAM2 (a kind gift from Prof. W. Hillen, Erlangen,Germany) (Urlinger et al., 2000), using XbaI and BamHI sites(Fig. 1B). To obtain pAC1-M2-WPRE, a BamHI-BglII frag-ment from PCR2.1-WHV (a kind gift from Dr. Nicole Deglon,Orsay, France) comprising the Woodchuck hepatitis virusposttranscriptional regulatory element (Donello et al., 1998;WPRE) has been introduced in pAC1 immediately downstreamto the rtTA sequence into a BamHI site, then the rtTA has beenreplaced by rtTAM2 as described above (Fig. 1C).

Cell line

The U87-MG human astrocytoma cell line was obtainedfrom American Type Culture Collection (Bethesda, MD, USA).Cells were cultured in Dulbecco's modified Eagle's medium(DMEM; Life Technologies) supplemented with 10% FCS(Sigma).

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Recombinant AAV (rAAV) viral preparations

Recombinant AAV2 viruses conditionally expressing EGFPor hGDNF were produced by co-transfection of the vectorplasmids together with the pDG plasmid expressing the AAV2rep and cap genes as well and the adenoviral gene required forthe AAV helper effect (Grimm et al., 1998) as previouslydescribed (Chtarto et al., 2003a).

Recombinant viruses generated from pAC1, pAC1-M2 andpAC1-M2-WPRE are designated hereafter as rAAV2-rtTA,rAAV2-rtTAM2, rAAV2-rtTAM2-WPRE, respectively. Viralstocks were purified using the iodixanol/heparin method(Zolotukhin et al., 1999) as previously described (Chtarto etal., 2003a). The infectious titers of the viral stocks used for invitro experiments were evaluated by in situ focus hybridiza-tion assay on HeLa cells using 32P-labeled probes for EGFPor hGDNF and expressed in infectious units (I.U.) permilliliter as previously described (Chtarto et al., 2003a). Theinfectious titers were: rAAV2-rtTA-EGFP, 8.1×107 I.U/ml;rAAV2-rtTAM2-EGFP, 1.6×108 I.U./ml; rAAV2-rtTAM2-WPRE-EGFP 3.4×108 I.U./ml; rAAV2-rtTAM2-GDNF,2.6×108 I.U./ml; rAAV2-rtTAM2-WPRE-GDNF, 2.8×108

I.U./ml. High titers recombinant AAV1 viral stocks for invivo experiments were produced at the Gene Vector Produc-tion Network (Laboratoire de Thérapie Génique, Nantes,France) as described (Salvetti et al., 1998). Titers expressed asviral genomes per milliliter were: rAAV1-rtTAM2-WPRE-EGFP, 1.7×1011; rAAV1-rtTAM2-GDNF, 7.0×1011; rAAV1-rtTAM2-WPRE-GDNF, 8.8×1011.

Infections

Twenty five thousand cells seeded in 48-well plates wereincubated in the presence of rAAV at the indicated multiplicityof infection in serum-free medium containing 30 μM etoposide

(Sigma, Belgium). After 2 h, the cells were rinsed twice withserum-supplemented medium and further cultured for theindicated time without passaging. The culture medium contain-ing or not doxycycline at the indicated concentration wasreplaced every 2 days.

Fluorescence-activated cell sorter (FACS) analysis

Ten thousand infected cells were analyzed in a FACStaranalyzer/sorter (Becton Dickinson, Erembodegem-Aalst, Bel-gium) using the CellQuest Software (Becton Dickinson).

Stereotaxic injections

Adult female Sprague-Dawley rats of 250 g (Harlan, TheNetherlands) were used for unilateral intracerebral injections aspreviously described (Tenenbaum et al., 2000). The animalswere anesthetized using a mixture of ketamine (Imalgene 1000,Merial; 100 mg kg−1 ip) and xylazine (Rompun, Bayer; 10 mgkg−1 ip). Injections were made according to coordinates definedby Paxinos and Watson (1997) using a Kopf stereotaxicapparatus (David Kopf, Tujunga, California). The injectioncoordinates were 0.5 mm anterior, 2.5 mm lateral to bregma and5.5 mm below the dural surface. Viral particles diluted in 2 μl D-PBS (BioWittaker) were infused using a motor-driven Hamiltonsyringe (0.2 μl/min) with a 30-G needle. After injection, theneedle was left in place for 5 min in order to allow diffusion ofthe viral suspension in the parenchyma. The needle was thenslowly lifted 1 mm up and left in place 5 min, then slowlyremoved. When indicated, the animals were given watercontaining 3% sucrose and doxycycline (600 μg/ml) continu-ously from the day of injection until sacrifice.

Animals were killed at the indicated time after surgery by anoverdose of anesthetic (200 mg kg−1 ketamine and 20 mg kg−1

xylazine). For immunohistological analysis, animals were

Fig. 1. Schematic diagram of tetracycline-regulatable AAV vectors used in this study. ITR, inverted terminal repeat; SV40 PA, SV40 fragment containing the early andlate polyadenylation signals; intron, SV40 small intron; rtTA, reverse tetracycline transactivator; rtTAM2, mutated reverse tetracycline transactivator (Baron et al.,1997); Ptet-bidi, seven tetracycline operator sites flanked by two minimal CMV promoters; MCS, multiple cloning site; WPRE,Woodchuck hepatitis posttranscriptionalregulatory element (Donello et al., 1998); EGFP, enhanced green fluorescent protein gene; hGDNF, human glial cell line-derived neurotrophic factor cDNA.

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perfused through the ascending aorta first with 150–200 ml ofsaline (NaCl 0.9%), then with 300 ml 4% paraformaldehyde in0.1 M phosphate buffer (PF4). After overnight post-fixation inPF4 at 4 °C, brains were transferred to phosphate-bufferedsaline (PBS) and stored at 4 °C.

GDNF ELISA assay

For infected U87-MG cells, the culture medium washarvested 2 h (transfected cells) or 24 h (infected cells) afterchanging the medium and used to measure secreted GDNFconcentrations using a commercial ELISA assay (HumanGDNF CytoSets, catalog #CHC2423, BioSource, Nivelles,Belgium).

For determination of GDNF brain tissue levels, animals weredecapitated and the brains were removed, gradually frozen inisopentane/dry ice (−10 °C for 10 s then −20 °C for 20 s) andstored at −80 °C. Two-hundred-micrometer coronal slices werecut using a cryostat and striata and midbrains were dissected out,weighed and processed for ELISA assay. Tissue was sonicated(Branson Sonifier 250, output 2, 30% duty cycle, 10 s) in M-Perbuffer (Pierce) supplemented with protease inhibitors cocktail(Boehringer). Samples were centrifuged at 10,000×g andsupernatants were harvested and their protein concentrationanalysed using the BCA Assay kit (Pierce). GDNF tissue levelswere measured according to the manufacturer's protocol(BioSource, Nivelles, Belgium) and expressed in pg permilligram proteins. Recombinant human GDNF (R&D System)was used to establish the standard curve. A cross-reaction of100% was demonstrated with recombinant rat GDNF (Onco-gene Research Products).

Detection of native fluorescence

Coronal sections (50 μm) from paraformaldehyde-fixedbrains, obtained using a vibratome (Leica) were mountedusing FluorSave mounting fluid (Calbiochem) and photo-graphed using a Zeiss Axiophot 2 microscope equipped withan FITC filter and an AxioCam digital camera (Carl Zeiss,Gottingen, Germany). Images were acquired as jpeg filesusing the KS300 software (Zeiss) with a constant exposuretime (1000 ms, gain 2).

Immunohistochemistry

For GDNF staining, coronal sections (50 μm) obtained usinga vibratome (Leica) were sequentially incubated in: (i) THST(50 mM Tris, 0.5 M NaCl, 0.5% Triton X100 (Merck) pH7.6)for 30 min; (ii) polyclonal rabbit anti-GDNF (R&D Systems,cat. no. AF-212-NA) diluted in THST overnight at 4 °C; (iii)donkey anti-rabbit–biotin (Amersham) diluted 1:100 in TBS(Tris 10 mM, NaCl 0.9%, pH7.6), 1 h at room temperature.Streptavidin-peroxidase staining was performed using the ABCElite vectastain kit and diaminobenzidine (Vector, NTLLaboratories, Brussels, Belgium), according to the manufac-turer's protocol. Sections were mounted on gelatin-coated

slides, dehydrated and mounted using Entellan mounting fluid(Merck, Darmstadt, Germany). Sections were photographedusing a Zeiss Axiophot 2 microscope. Images were acquired asjpeg files using the KS300 software (Zeiss).

For TH staining, the sections (50 μm) were sequentiallyincubated (i) THST (50 mM Tris, 0.5 M NaCl, 0.5% TritonX100 (Merck) pH7.6) for 30 min; (ii) monoclonal anti-TH(Chemicon) diluted in THST overnight at 4 °C; (iii) anti-mouse–biotin (Amersham) diluted 1:100 in TBS (Tris 10 mM,NaCl 0.9%, pH7.6), 1 h at room temperature; (iv) streptavidin-peroxidase staining using the ABC Elite vectastain kit anddiaminobenzidine (Vector, NTL Laboratories, Brussels, Bel-gium), according to the manufacturer's protocol. Sections weremounted on gelatin-coated slides, dehydrated and mountedusing Entellan mounting fluid (Merck, Darmstadt, Germany).Sections were photographed using a Zeiss Axiophot 2microscope. The intensity of the TH staining was evaluated as

Fig. 2. Induction of EGFP gene expression driven by rAAV2-tetON vectorsafter infection of U87-MG cells at two different multiplicities. U87-MG cellswere infected with rAAV2-rtTA-EGFP, rAAV2-rtTAM2-EGFP or rAAV2-rtTAM2-WPRE-EGFP at a multiplicity of 1 (A) or 10 (B) I.U./cell in thepresence of 30 μM etoposide and further cultured in the absence (blackboxes) or in the presence (gray boxes) of doxycycline at a concentration of1 μg/ml. At 5 days postinfection, cells were harvested and analyzed byFACS. The values, representing the percentage of green fluorescent cellsmultiplied by the mean fluorescence intensity of the induced cell population,are means of 5 independent infections from 1 representative out of 2experiments. The induction factor representing the ratio between the valuesobtained in the induced (Dox) versus uninduced (No Dox) states is shown.Bars represent standard deviations. The differences between the basal level ofEGFP expression in rAAV2-rtTA versus rAAV2-rtTAM2 (*) and rAAV2-rtTAversus rAAV2-rtTAM2-WPRE (°) infected cells are significant at the twoMOIs (**, p<0.01; ***, p<0.001). Except for rAAV2-rtTA at MOI 1, thedifferences between EGFP expression in doxycycline-treated and untreatedcultures (#) are significant (##, p<0.01; ###, p<0.001; one-way ANOVANewman–Keuls post hoc test).

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follows. Images were acquired using the KS300 software(Zeiss, Göttingen, Germany) with a constant exposure time. Thearea harboring GDNF staining was delimited and the intensityof the TH staining measured in the same area on subsequentsections. Grey levels (GL) were measured after subtracting thebackground using the Image J software (NIH, Bethesda). Thevalues were converted into optical densities (OD) using thefollowing formula: OD=log (GL/255).

Statistical analyses

Means±standard deviations are shown.Data were analyzed using the Graph Pad Software Prism 3.0.

Results

Construction of improved tetracycline-inducible autoregulatoryAAV vectors expressing EGFP or human GDNF cDNA

The previously described autoregulatory tetracycline-indu-cible pAC1 plasmid comprises AAV ITRs bracketing thebidirectional tetracycline-responsive cassette expressing bothrtTA tetracycline transactivator (Gossen et al., 1995) and EGFP(Chtarto et al., 2003a; Fig. 1A). When recombinant AAVprepared from this construct was used to infect cells in culture aslow inducibility upon doxycycline addition was observed(approximately 10 days; see Fig. 5 in Chtarto and coll., 2003a).In contrast, when a high copy number of the construct wasintroduced into cultured cells by calcium phosphate-mediatedtransfection, a high level of transgene expression was readilyobserved (see for example, EGFP expression 48 h posttransfec-tion in Chtarto and coll. (2003a; Fig. 2)). We speculated thatupon doxycycline addition, a threshold concentration of rtTAhas to be present to efficiently bind the tetracycline operatorsites present in Ptet-bidi promoter and to induce the self-accelerating transcription of the rtTA transactivator. Newconstructs were therefore derived in order to improve theinducibility of the pAC1 vector.

In order to obtain a vector which can be induced at lowertetracycline concentration, while harboring a lower basaltransgene expression, the M2 mutant which has been shownto interact more specifically with the tetO sites (Urlinger et al.,2000), has been substituted for the rtTA sequence (Fig. 1B).When placed downstream to a coding sequence, the WPREsequence has been shown to stabilize its mRNA and to augmentits translation (Donello et al., 1998). In order to obtain a higherintracellular level of rtTAM2 in the uninduced state, we haveintroduced the WPRE sequence downstream to the rtTAM2coding sequence (Fig. 1C). Finally, the human GDNF cDNAhas been substituted for the EGFP coding sequence.

Tetracycline-inducible EGFP expression inrAAV-pTetbidiON-infected U87-MG cells

Synthesis of a DNA strand complementary to the AAV viralsingle-stranded DNA is a limiting step affecting the kinetics of

transgene expression in several cell lines (McCarty et al., 2003;Alexander et al., 1994). In order to optimize the efficiency oftransduction mediated by rAAV-tetON vectors, U87-MG cellswere treated with etoposide, a topoisomerase inhibitor which isknown to stimulate second-strand synthesis (Wang et al., 2003).

Comparison of the different vectors in etoposide-treated U87-MG cells (Fig. 2) showed that at both multiplicities of infection(MOI) of 1 (Fig. 2A) and 10 (Fig. 2B) I.U./cell, the M2 mutationsignificantly reduced the uninduced expression level (rAAV2-rtTA versus rAAV2-rtTAM2, p<0.001) but had no significanteffect on the induced expression level (p>0.05). The WPREsequence significantly increased the induced level of transgeneexpression (rAAV2-rtTAM2 versus rAAV2-rtTAM2-WPRE,p<0.001). However, in the absence of doxycycline, the WPREsequence also significantly increased the level of transgeneexpression at MOI 10 (p<0.05; Fig. 2B; compare M2 and M2-WPRE viruses) but not at MOI 1 (p>0.05; Fig. 2A).

In conclusion, in comparison to the initial rAAV2-rtTAvector, the rAAV2-rtTAM2 vector provided a similarly inducedbut a lower uninduced level of transgene expression, resulting ina respectively 40-fold (Fig. 2A) and 23-fold (Fig. 2B) inductionfactor at a multiplicity of infection of 1 and 10 I.U./cell. Incontrast, the rAAV2-rtTAM2-WPRE provided a higher inducedand a lower uninduced level of transgene expression than theinitial rAAV2-rtTA vector, resulting in a more than 100-foldinduction factor at lowMOI (1 I.U./cell). However, as comparedto the rAAV2-rtTAM2-WPRE vector, the basal level oftransgene expression driven by the rAAV2-rtTAM2 vector was30-fold (MOI=1; see Fig. 2A) and 5-fold (MOI=10; see Fig.2B) lower. In the case of potent neurotrophic factors which areactive at low doses, a vector harboring a lower leakiness willhopefully constitute a more valuable tool to regulate thebiological effects of transgene expression (see below).

Rapid induction/extinction of GDNF transgene expression byrAAV-pTetbidiON in vitro in human glioma cells

U87-MG human astrocytoma cells, treated with etoposidewere infected with rAAV2 viruses conditionally expressinghGDNF at a multiplicity of 10 I.U./cell. The next day, cells weretreated or not with doxycycline (Figs. 3A and B) and theamounts of hGDNF present in the medium measured by ELISAevery day post-induction.

The amount of GDNF secreted by the rAAV2-rtTAM2-WPRE-infected cells started to be significantly different fromthe non-induced control after 2 days (Fig. 3A). In contrast, whenusing rAAV2-rtTAM2 virus, the amount of GDNF producedstarted to be significantly different from the non-induced controlonly 5 days after induction (Fig. 3A).

In the absence of doxycycline, the level of GDNF expressedby the rAAV2-rtTAM2 vector remained under the detectionthreshold (less than 7 pg/ml) whereas it started to be significantat 6 days postinfection by rAAV2-rtTAM2-WPRE (Fig. 3B).

In conclusion, the WPRE element significantly increasedboth the basal and the induced levels of hGDNF transgeneexpression. These data are in accordance with the data obtainedwith the EGFP transgene (see above).

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For both vectors, after reaching a maximum, transgeneexpression levels gradually declined, presumably reflectingdilution of the non-integrated vector sequences in dividingastrocytoma cells.

The extinction of transgene expression after removal ofdoxycycline from cultures containing induced vectors, wascomplete at 5 days after doxycycline removal with the M2and M2-WPRE constructs (Fig. 3C). However, at respectively2 and 3 days after doxycycline removal, less than 10% of themaximal level of hGDNF was present in the medium. Theamount of GDNF present inside the cells, evaluated aftersonication of cell pellets was less than 10% of the amount

secreted in the medium (data not shown) and was not takeninto account.

Controlled amounts of hGDNF secreted by U87-MG cellsinfected by rAAV2-pTetbidiON

We further analyzed if it was possible to modulate the amountof hGDNF secreted by the rAAV-infected cells by adjusting thedoxycycline concentration in the culture medium. Fig. 3D showsthat variations of the doxycycline concentration from 3 to1000 ng/ml resulted in a sigmoid-shaped increase in hGDNFlevels. Curve fitting evidenced a maximal amount of hGDNF at

Fig. 3. Time and dose-dependent modulation of human GDNF gene expression driven by rAAV2-pTetbidiON vectors after infection of U87-MG cells. (A) Kinetics ofdoxycycline-mediated induction of hGDNF gene expression driven by rAAV2-rtTAM2-hGDNF and rAAV2-rtTAM2-WPRE-hGDNF in infected U87-MG cells. U87-MG cells were infected with rAAV2-rtTAM2-hGDNF (circles) or rAAV2-rtTAM2-WPRE-hGDNF (triangles) at a multiplicity of 10 I.U./cell in the presence of 30 μMetoposide and further cultured in the presence (closed symbols) or in the absence (open symbols) of doxycycline at a concentration of 1 μg/ml. At various time points,medium was harvested and analyzed by ELISA. The values, representing the amounts of hGDNF per milliliter medium per 24 h, are means of 4 samples in a typicalexperiment out of 3 experiments. Amounts of GDNFwere significantly different from the basal level (in the absence of doxycycline) from 2 days post-induction for therAAV2-rtTAM2-WPRE-hGDNF vector (***, p<0.001) and from 5 days post-induction for the rAAV2-rtTAM2-hGDNF vector (°°°, p<0.001). At 4 days post-induction, there was a tendency for significant induction of the M2 vector (p=0.07) (one-way ANOVA Newman–Keuls post hoc test). Bars represent standarddeviations. (B) Basal level of hGDNF gene expression driven by rAAV2-rtTAM2-hGDNF and rAAV2-rtTAM2-WPRE-hGDNF in infected U87-MG cells. U87-MGcells were infected with rAAV2-rtTAM2-hGDNF (circles) or rAAV2-rtTAM2-WPRE-hGDNF (triangles) at a multiplicity of 10 I.U./cell in the presence of 30 μMetoposide and further cultured in the absence of doxycycline. The scale of the y axis was enlarged to visualize differences between the basal levels of transgeneexpression. Values were significant from 6 days postinfection *, p<0.05; **, p<0.01; ***, p<0.001 (one-way ANOVA Newman–Keuls post hoc test). (C) Kineticsof extinction of hGDNF gene expression driven by rAAV2-rtTAM2-hGDNF and rAAV2-rtTAM2-WPRE-hGDNF in infected U87-MG cells after removal ofdoxycycline. U87-MG cells infected with rAAV2-rtTAM2-hGDNF (filled circles) or rAAV2-rtTAM2-WPRE-hGDNF (empty circles) at a multiplicity of 10 I.U./cell inthe presence of 30 μM etoposide were cultured in the presence of doxycycline at a concentration of 1 μg/ml. Five days after infection, doxycycline was removed fromthe medium and cells further cultured in the absence of antibiotics. At various time points, medium was harvested and analyzed by ELISA. The values, representing theamounts of hGDNF per milliliter medium per 24 h are means of 4 samples (one-way ANOVA Newman–Keuls post hoc test). Bars represent standard deviations. (D)Induction of rAAV2-rtTAM2-WPRE-hGDNF-mediated transgene expression in function of the doxycycline dose. U87-MG cells were infected with rAAV2-rtTAM2-WPRE-hGDNF at a multiplicity of 10 I.U./cell in the presence of 30 μM etoposide were cultured in the presence of doxycycline at a concentrations of 1–1000 ng/ml.Six days after infection, medium replaced 24 h before, was harvested and analyzed by ELISA. The values, representing the amounts of hGDNF per milliliter mediumper 24 h, are means of 4 samples. Bars represent standard deviations. Curve fitting and regression analysis were performed using GraphPad PRISM Software.

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a plateau level of 5.2 ng/ml/24 h with EC50 of 31.4 ng/mldoxycycline. These data indicate that hGDNF expression levelscan be modulated over a 2-log dose range of doxycycline.

Modulation of human GDNF transgene expression in vivo inthe rat brain

Regardless of the promoter used for transgene expression,AAV serotype 1 vectors were shown to transduce the striatum

more efficiently than equivalent AAV2 vectors (Wang et al.,2003; Passini et al., 2003; Burger et al., 2004). In order tooptimize the transduction efficiency of the rAAV-pTetbidiONvector in the striatum, the pAC1-M2 and pAC1-M2-WPREvector were transencapsidated into serotype 1 capsids generatingrAAV1-rtTAM2-hGDNF and rAAV1-rtTAM2-WPRE-hGDNF.

The performances of rAAV1-rtTAM2-hGDNF and rAAV1-rtTAM2-WPRE-hGDNF were compared after injection in thestriatum (Fig. 4). Eight weeks post-surgery, the animals were

Fig. 4. Distribution of GDNF in the striatum of doxycycline-induced rats after rAAV-pTetbidiON injection. Eight weeks after injection of 3.5×108 rAAV1-rtTAM2-hGDNF (A–C) or rAAV1-rtTAM2-WPRE-hGDNF (D–F) viral particles, animals continuously fed with doxycycline (A, B, D, E) or not (C, F) were sacrificed andbrains processed into 4 sets of equivalent 50 μM vibratome sections. One fourth of the sections was used for immunohistochemistry using anti-GDNF antibodiesevidenced by peroxidase staining. (B and E) Higher magnification pictures representing transduced striatal neurons in the area inside the dashed line in rAAV2-rtTAM2-hGDNF (A)- or rAAV1-rtTAM2-WPRE-hGDNF (D)-injected animals. Images were acquired using a 2.5-fold (A, C, D, F) or a 20-fold objective (B, E). Scalebars correspond to 800 μm (A, C, D, F) and 100 μm (B, E).

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divided into 2 groups: in the first group, animals were perfusedand brain sections were processed for GDNF immunohisto-chemistry; in the second group, brains were frozen and proteinswere extracted as described (see Materials and methods).

Immunohistochemistry data show that the spread of GDNFimmunostaining was similar for rAAV1-rtTAM2-hGDNF (Fig.4A) and rAAV1-rtTAM2-WPRE-hGDNF (Fig. 4D) doxycy-cline-induced animals (approximately 1.5 mm in the antero-posterior, ventral and lateral axis). Higher magnificationpictures (Figs. 4B and E) suggested that the density ofimmunolabeled cell bodies was similar for both viruses butthat extracellular GDNF concentration was higher with theWPRE construct. The spread of GDNF staining was also similarin non-induced rAAV1-rtTAM2-hGDNF (Fig. 4C) and rAAV1-rtTAM2-WPRE-hGDNF-injected animals (Fig. 4F) (approxi-mately 1 mm in all directions) but similar to induced animals,the intensity of the staining was stronger for the WPREconstruct (compare Figs. 4C and F).

The amounts of GDNF protein present in the striatum as well asin the midbrain of animals injected with rAAV1-rtTAM2-hGDNF,rAAV1-rtTAM2-WPRE-hGDNF, control rAAV1-rtTAM2-WPRE-EGFP or D-PBS (sham) were then determined byELISA assay (Table 1). Using the rtTAM2 constructs, adminis-tration of doxycycline in drinking water, resulted in a 15-foldenhancement of the GDNF protein levels present in the striatum(365.2 pg/mg proteins corresponding to approximately 60 pg/mgtissue). In addition, in the absence of doxycycline, the GDNFconcentration (24.2 pg/mg proteins corresponding to approxi-mately 4.5 pg/mg tissue) was not significantly higher than in thestriatum of sham-injected animals (25.4 pg/mg proteins) oranimals injected with an equivalent construct expressing EGFP(24.4 pg/mg proteins; see Table 1). In contrast, when using thertTAM2-WPRE construct, the induced level of GDNF was similarto that expressed by the rtTAM2 construct but the basal level ofGDNF was 65.1 pg/mg proteins, i.e., approximately 2.5-foldhigher than in the negative controls and therefore the inductionfactor was only approximately 5 (see Table 1). In the substantianigra, detectable levels of GDNF, statistically different from theendogenous level, were obtained in doxycycline-treated but not inuntreated animals with both vectors (see Table 1).

Transduction at distance from the injection site

With both viruses, increased density of GDNF immunostain-ing was observed at a distance from the injection site in the areabordering the external capsule (see Figs. 5A and B). SinceGDNF is secreted and was suggested to accumulate into cellsexpressing the GFRα receptor (Kirik et al., 2000), thisobservation could reflect either a selective tropism of theAAV1-tetON virus or a higher density of GFRα receptor in thisarea. To discriminate between these 2 hypotheses, we injectedrAAV1-rtTAM2-WPRE-EGFP into the striatum and nativefluorescence was observed 5 weeks post-injection. A highdensity of gfp-positive cells was present in the same area asobserved for GDNF immunostaining (Figs. 5C to F). These datasuggest that GDNF-immunopositive cells are cells expressingthe transgene rather than cells accumulating GDNF protein.

In addition, these data suggest that rAAV1 does not infect allneurons with a similar affinity. This uneven tropism should betaken into account when designing clinical trials in order toavoid undesirable effects of GDNF in non-targeted area.

Modulation of GDNF-mediated downregulation of tyrosinehydroxylase in vivo in the rat striatum

GDNF was shown to downregulate the expression oftyrosine hydroxylase in both nigral dopaminergic cell bodiesand striatal dopaminergic terminals (Rosenblad et al., 2003;Georgievska et al., 2004a). In order to determine whetherfunctional effects of GDNF delivered via rAAV-pTetbidiON-hGDNF in healthy brain could be modulated by doxycycline,animals injected by rAAV1-rtTAM2-hGDNF, rAAV1-rtTAM2-WPRE-hGDNF, or D-PBS were treated or not with doxycyclineduring 8 weeks (Fig. 6). The expression and localization ofGDNF were evidenced by immunohistochemistry (Fig. 6A).Tyrosine hydroxylase-positive dopaminergic fibers were evi-denced by immunohistochemistry on both injected (Fig. 6C)and contralateral striata (Fig. 6B). A reduced tyrosine hydro-xylase staining was evidenced in the doxycycline-treatedanimals in the area of the striatum in which the GDNF labelingwas positive (Fig. 6C, area inside the dashed line) as compared

Table 1Doxycycline induction of rAAV1-tetON-mediated hGDNF transgene expression in the adult rat striatum

Vector Doxycycline treatment GDNF in the striatum (pg/mg proteins) GDNF in the substantia nigra (pg/mg proteins)

rAAV1-M2-GDNF (n=4) − 24.19±2.35 27.65±10.33rAAV1-M2-GDNF (n=4) + 365.24±141.62 89.54±58.48rAAV1-M2-WPRE-GDNF (n=5) − 65.09±26.65 29.93±10.42rAAV1-M2-WPRE-GDNF (n=5) + 346.07±84.40 75.67±24.97Sham (n=3) − 25.41±6.39 21.11±1.65rAAV1-M2-WPRE-EGFP (n=4) + 24.35±2.93 32.46±17.11

Eight weeks after injection of 3.5×108 viral particles rAAV1-rtTAM2-hGDNF,rAAV1-rtTAM2-WPRE-hGDNF, rAAV1-rtTAM2-WPRE-EGFP or D-PBS (shamgroup), extracts from the right (injected) striatum of animals fed or not with doxycycline (600 μg/ml) in the drinking water were processed for ELISA assay. Theamounts of GDNF protein per milligram of total proteins in the striatum and in the substantia nigra are shown. Means±SD are shown.The number of rats per group is indicated. Values are significantly different from control groups (sham, rAAV1-M2-WPRE-EGFP) for both doxycycline-treated groups(p<0.001, one-way ANOVA Newman–Keuls post hoc test) and for untreated rAAV1-rtTAM2-WPRE-hGDNF (p<0.01) but not for untreated rAAV1-rtTAM2-hGDNF (p>0.05).Differences between rAAV1-M2-GDNF versus rAAV1-M2-WPRE-GDNF are not significant (p>0.05) for doxycycline-treated groups but are significant for untreatedgroups (p<0.05).

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to the equivalent region in the contralateral striatum (Fig. 6B).Tyrosine hydroxylase staining was quantified by measuring theoptical densities of the deliminated area corresponding toGDNF immunopositivity on representative sections fromdoxycycline-treated or non-treated animals as compared to thecontralateral sides (Fig. 6D). Statistically significant decreasesof the optical density (∼27% for the rtTAM2 vector and ∼46%for the rtTAM2-WPRE vector; p<0.05 and p<0.01, respec-tively) were obtained in doxycycline-treated animals (Fig. 6D).In contrast, uninduced animals were not statistically differentfrom the sham operated animals for both constructs (Fig. 6D).

Discussion

In the present study, we describe tetracycline-inducible AAVvectors (AAV-pTetbidiON) in which transcription of both an

optimized version of the reverse tetracycline transactivator(rtTAM2; Urlinger et al., 2000) and a transgene (EGFP orhuman GDNF cDNA) is initiated from a bidirectionaltetracycline-responsive promoter and terminated at bidirectionalSV40 polyadenylation sites flanking both ITRs.

In vitro, in accordance with Urlinger and collaborators, ascompared to the first generation of rAAV-pTetbidiON (Chtarto etal., 2003a) harboring the rtTA transactivator (Gossen et al.,1995), the rtTAM2 mutant was induced at lower doxycyclinedoses and resulted in a lower level of transgene expression inthe uninduced state (data not shown). It is shown, in addition,that the introduction of the WPRE element (Donello et al.,1998) downstream to the rtTAM2 coding sequence allowed afaster induction of the tet system. However, the rAAV-rtTAM2-WPRE vector harbored a higher basal level of transgeneexpression than the rAAV-rtTAM2 vector.

Fig. 5. Transduction at distance outside the striatum after injection of rAAV1-tetON vectors. (A and B) Ten weeks after injection of 1.8×109 rAAV1-rtTAM2-hGDNF(A) or rAAV1-rtTAM2-WPRE-hGDNF (B), animals fed with doxycycline (600 μg/ml) were sacrificed and brains processed into 50 μMvibratome sections and stainedwith anti-GDNF antibodies. (C–F) Five weeks after injection of 4.0×108 rAAV1-rtTAM2-WPRE-EGFP, animals, fed with doxycycline (600 μg/ml) were sacrificedand 50 μM vibratome sections were examined under visible (C) or U.V. (D–F) light to detect native gfp fluorescence. (C and D) Same region. (E) Enlargement ofstriatal area designated by dashed arrow. (F) Enlargement of external capsule designated by continuous arrow. Scale bars correspond to 800 μm (A–D) and 100 μm(E and F).

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In vivo, injections of the rAAV-rtTAM2-(WPRE) vectortransencapsidated into AAV1 (Rabinowitz et al., 2002) in the ratstriatum, resulted in widespread transduction as shown by usingan EGFP-expressing vector.

Stereotaxic injections of rAAV1-rtTAM2 vector harboringthe human GDNF cDNA resulted in a 15-fold increase ofGDNF concentration in doxycycline-treated but no detectablelevels of GDNF above the endogenous striatal level inuntreated rats as measured by ELISA assay on striatalextracts. However, GDNF immunohistochemistry revealed aweak staining in uninduced rats, suggesting that locally GDNFwas present at a concentration higher than the endogenouslevel. The low basal level of transgene expression in theabsence of inducer that was observed using the rAAV1-rtTAM2 vector might originate from 3 sources: (i) transcrip-

tion initiated in the ITRs (Flotte et al., 1993; Haberman et al.,2000) incompletely arrested at bidirectional SV40 polyAsequences; (ii) basal transcription of the transgene from theminimal CMV promoter (Fender et al., 2002); (iii) unspecificbinding of rtTAM2 to tetracycline operator sites (Urlinger etal., 2000). Nevertheless, it should be stressed out that the basallevel of GDNF expressed by the rAAV1-rtTAM2-hGDNFvector in vivo, was below the endogenous GDNF tissueconcentration and was not sufficient to induce tyrosinehydroxylase downregulation.

In contrast, stereotaxic injections of the rAAV1-rtTAM2-WPRE vector produced a 2.5-fold increased concentration ofGDNF in the striatum in the absence of inducer which wasdetectable by the ELISA assay and GDNF immunostainingwas stronger. These data, in accordance with the in vitro

Fig. 6. Doxycycline-dependent downregulation of tyrosine hydroxylase mediated by rAAV1-pTetbidiON-hGDNF transgene expression in the adult rat striatum. (A–C)Eight weeks after injection of 3.8×108 rAAV1-rtTAM2-hGDNF or rAAV1-rtTAM2-WPRE-hGDNF, animals, fed with doxycycline (600 μg/ml) were sacrificed andbrains processed into 4 sets of equivalent 50 μM vibratome sections. Two sets of the sections were used for anti-GDNF (A) and anti-tyrosine hydroxylase (B, C)immunohistochemistry, respectively. Immunoreactivity was evidenced by peroxidase/DAB staining. (A, C) Injected side; (B) contralateral side of the same section aspanel C. Images were acquired using a 2.5-fold objective. Scale bar corresponds to 1 mm. (D) Optical density of tyrosine hydroxylase-labeled sections from animalsinjected with D-PBS (sham), with rAAV1-rtTAM2-hGDNF (rtTAM2) or with rAAV1-rtTAM2-WPRE-hGDNF (rtTAM2-WPRE) fed (+dox) or not (−dox) withdoxycycline. Means of the ratio between the optical density of the injected and contralateral sides are shown (sham, n=3; rAAV1-rtTAM2-hGDNF−dox, n=5;rAAV1-rtTAM2-hGDNF+dox, n=6; rAAV1-rtTAM2-WPRE-hGDNF−dox, n=4; rAAV1-rtTAM2-WPRE-hGDNF+dox, n=4). The differences between virus-and PBS-injected animals were significant for doxycycline-treated (p<0.05 and p<0.01, respectively for rAAV1-rtTAM2-hGDNF and rAAV1-rtTAM2-WPRE-hGDNF) but not for untreated animals. The statistical analysis was performed using one-way ANOVA with a Dunnett's post hoc test. Bars represent standarddeviations.

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experiments, suggest that (i) the addition of the WPREsequence, and hence, increasing the rtTAM2 intracellularconcentration, results in increased non-specific binding to thetetracycline operator sites or that (ii) enhancer sequencespresent in the WPRE sequence (Flajolet et al., 1998)transactivate the tetracycline-responsive promoter at distance,independently of doxycycline-mediated activation.

In the presence of doxycycline, the concentrations of GDNFin striatal extracts obtained in both rAAV1-rtTAM2- andrAAV1-rtTAM2-WPRE-injected rats, were found to be 15-fold higher than the endogenous level, i.e., approximately 60 pgGDNF/mg of tissue from the entire striatum obtained after asingle vector injection. However, immunohistochemistryrevealed that GDNF did not spread in the entire striatum butwas localized mainly in the medio-lateral dorsal part of thestriatum. This suggests that a concentration of GDNF higherthan 60 pg/mg tissue was achieved locally. Consistently,downregulation of tyrosine hydroxylase expression wasobserved mainly in the area of GDNF immunopositivity.Multiple injection sites will probably be necessary to achieveprotection of dopaminergic terminals throughout the striatum inlesioned animals. In a previous study (Kirik et al., 2000), 9delivery sites of rAAV2 vector constitutively expressing GDNFwere necessary to obtain a neuroprotective effect in a ratParkinson's disease model. In this study, a concentration ofapproximately 200 pg GDNF/mg tissue was obtained.

It has been shown that long-term functional recovery andregeneration of the lesioned nigro-striatal projections can beobtained using an AAV vector constitutively delivering GDNFin the striatum (Kirik et al., 2000). However, GDNF over-expression in intact striatum resulted in increased locomotoractivity and spontaneous as well as amphetamine-inducedcontralateral rotational bias (Kirik et al., 2000; Georgievska etal., 2004a,b). Furthermore, long-term unregulated expression ofGDNF in the striatum by a lentiviral vector resulted intranscriptional downregulation of tyrosine hydroxylase (Geor-gievska et al., 2004a). In addition, serious side effects have beenobserved in a Phase I clinical trial in which GDNF recombinantprotein was delivered intraventricularly (Nutt et al., 2003).Therefore, it will be of particular importance to use regulatableviral vectors allowing to modulate GDNF levels in function ofthe therapeutical requirements as well as of the appearance ofundesirable effects. However, the threshold level of GDNFconcentration inducing biological activities is currently notdetermined.

In the present report, it is shown that downregulation oftyrosine hydroxylase mediated by rAAV-tetONbidi-expressedGDNF could be induced by doxycycline. In the absence ofdoxycycline, the rAAV1-rtTAM2 vector did not express adetectable level of GDNF and did not cause any reduction intyrosine hydroxylase staining. In contrast, the rAAV1-rtTAM2-WPRE vector expressed a basal level of GDNF 2.5-fold higher(∼12 pg/mg tissue) than the endogenous level and induced aslight (approximately 20%) although not significant reductionin tyrosine hydroxylase. Similarly to our single AAV-rtTAM2-WPRE vector, in a previous study using a two-vector lentiviraltetracycline-inducible system in an optimized ratio to regulate

GDNF gene expression (Georgievska et al., 2004b), a basallevel of 11 pg/mg tissue, significantly higher than the endo-genous level, was shown to induce a ∼20% (non-significant)decrease in tyrosine hydroxylase enzymatic activity.

In conclusion, the herein described rAAV1-rtTAM2 vectorallowed to induce a doxycycline-dependent biological effect ofGDNF, i.e., downregulation of tyrosine hydroxylase. Thesuccessful modulation of a biological activity of GDNF invivo demonstrated in the present study using a single AAVconstruct, will constitute a valuable tool to determine theminimal concentration of GDNF at which activities can beobserved at the electrophysiological, metabolic and behaviorallevels and hopefully for controlled AAV-mediated GDNF genetherapy.

Acknowledgments

We thank the vector core of the University Hospital ofNantes supported by the Association Française contre lesMyopathies (AFM) for preparing AAV vectors used for in vivoexperiments.

We thank Drs. Jean-Denis Fransen (EuroScreen, Belgium)and Francis Dupont (Henogen, Belgium) for their helpfuldiscussions and technical advice as well as Michel Arcq(BioSource, Belgium) for the excellent technical assistance. Weare grateful to Dr. W. Hillen for the gift of the M2 plasmid, Dr. J.Kleinschmidt for the gift of the pDG plasmid and Dr. N. Deglonfor the gifts of hGDNF cDNA and the WPRE sequence.Plasmid pAC1: US patent no. 6,780,639.

A.C. and E.L. were recipients of a predoctoral fellowshipfrom the Belgian national research foundation (FNRS-Télévie).O.V. was recipient of a predoctoral fellowship from the Belgian“FRIA” (Fonds pour la Recherche dans l'Industrie et l'Agri-culture). D.B. was a “chargé de recherches” of the FNRS.

This work was also supported by a FIRST objectif I grantfrom Wallonnia and Europe (Fonds Social Européen) and bygrants from the «Région Bruxelles-Capitale», the Belgian«Société Générale» and the Hereditary Disease foundation.

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