dopamine gene therapy for parkinson's disease in a nonhuman primate without associated...

DOI: 10.1126/scitranslmed.3000130, 2ra4 (2009);1 Sci Transl Med

, et al.Béchir JarrayaPrimate Without Associated DyskinesiaDopamine Gene Therapy for Parkinson's Disease in a Nonhuman

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R E S EARCH ART I C L E

GENE THERAPY

Dopamine Gene Therapy for Parkinson’s Disease in aNonhuman Primate Without Associated DyskinesiaBéchir Jarraya,1,2,3,4* Sabrina Boulet,1,2† G. Scott Ralph,5† Caroline Jan,1,2 Gilles Bonvento,1,2

Mimoun Azzouz,6 James E. Miskin,5 Masahiro Shin,1,2 Thierry Delzescaux,1,2 Xavier Drouot,3,7

Anne-Sophie Hérard,1,2 Denise M. Day,5 Emmanuel Brouillet,1,2 Susan M. Kingsman,5

Philippe Hantraye,1,2 Kyriacos A. Mitrophanous,5* Nicholas D. Mazarakis,8 Stéphane Palfi1,2,3,4*
(Published 14 October 2009; Volume 1 Issue 2 2ra4)
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In Parkinson’s disease, degeneration of specific neurons in the midbrain can cause severe motor deficits, in-cluding tremors and the inability to initiate movement. The standard treatment is administration of pharma-cological agents that transiently increase concentrations of brain dopamine and thereby discontinuouslymodulate neuronal activity in the striatum, the primary target of dopaminergic neurons. The resulting inter-mittent dopamine alleviates parkinsonian symptoms but is also thought to cause abnormal involuntary move-ments, called dyskinesias. To investigate gene therapy for Parkinson’s disease, we simulated the disease inmacaque monkeys by treating them with the complex I mitochondrial inhibitor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which induces selective degeneration of dopamine-producing neurons. In this model, wedemonstrated that injection of a tricistronic lentiviral vector encoding the critical genes for dopamine synthesis(tyrosine hydroxylase, aromatic L-amino acid decarboxylase, and guanosine 5′-triphosphate cyclohydrolase 1)into the striatum safely restored extracellular concentrations of dopamine and corrected the motor deficitsfor 12 months without associated dyskinesias. Gene therapy–mediated dopamine replacement may be ableto correct Parkinsonism in patients without the complications of dyskinesias.

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INTRODUCTION

The striatum (putamen and caudate nucleus) is the primary target nu-cleus of afferent input to the basal ganglia, a network of intercon-nected subcortical brain nuclei [striatum, globus pallidus, substantianigra, and subthalamic nucleus (STN)] that serve motivation, motorplanning, and procedural learning functions (fig. S1) (1). The neuro-transmitter dopamine is released by midbrain substantia nigra neu-rons into the striatum where it modulates neuronal firing leading toa fine tuning of basal ganglia activity (1). In Parkinson’s disease (PD),these midbrain dopaminergic neurons degenerate, which leads to do-pamine loss within the striatum and causes severe motor impairmentthat includes akinesia (the inability to initiate movement), rigidity, andtremor (2). Increasing dopamine concentrations in the striatum is themost effective therapeutic strategy for PD. This is usually achieved byoral administration of the dopamine precursor 3,4-dihydroxy-L-phenylalanine (L-dopa), which is converted to dopamine upon crossingthe blood-brain barrier into the brain (3). Initially, patients with PD ex-perience excellent benefits from L-dopa. After long-term L-dopa intake,however, most PD patients develop disabling complications that includemotor fluctuations and dyskinesias. Patients can alternate between

1CEA, DSV, I²BM, Molecular Imaging Research Center (MIRCen), F-92265 Fontenay-aux-Roses, France. 2CEA, CNRS URA 2210, F-92265 Fontenay-aux-Roses, France. 3UniversiteParis 12, Faculte de Medecine, F-94010 Creteil, France. 4AP-HP, Groupe Henri-MondorAlbert-Chenevier, UF Neurochirurgie Fonctionnelle, F-94010 Creteil, France. 5OxfordBioMedica Ltd., Medawar Centre, Oxford Science Park, Oxford OX4 4GA, UK. 6NeurologyUnit, Medical School, Sheffield University, Sheffield S10 2RX, UK. 7AP-HP, Groupe Henri-Mondor Albert-Chenevier, Service de Neurophysiologie, F-94010 Creteil, France.8Department of Gene Therapy, Division of Medicine, Imperial College London, St Mary’sCampus, London W2 1PG, UK.*To whom correspondence should be addressed. E-mail: [email protected](S.P.); [email protected] (B.J.); [email protected] (K.A.M.)†These authors contributed equally to this work.

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“ON” states, which can be complicated by disabling dyskinesias, and“OFF” states, in which patients are akinetic. Dyskinesias are involuntaryabnormalmovements that include chorea (brief, irregularmuscle contrac-tions that are not repetitive or rhythmic) and dystonia (sustained musclecontractions causing twisting repetitivemovements or abnormal postures)(2). Increasing evidence suggests that dyskinesias and motor fluctuationsare at least partially caused by the intermittent oral intake of L-dopa, result-ing in fluctuating dopamine concentrations and subsequent pulsatile stim-ulation of striatal dopamine receptors (2, 4–6). Continuous delivery ofdopamine may prevent dyskinesias by restoring sustained “background,”or tonic (7), concentrations of dopamine in the striatum (5).

Another complication of long-term oral L-dopa treatment is cog-nitive impairment. PD is characterized primarily by dopamine deple-tion in the dorsal striatum (“motor” striatum) particularly in thepostcommissural area (caudal to the anterior commissure level). Do-pamine function in the ventral striatum (“cognitive” striatum) and theprefrontal cortex is relatively intact or even increased (8). Because oralL-dopa stimulates all of the dopaminergic systems in the brain, theintact ventral striatum receives excessive dopamine stimulation, andcognitive function is impaired (8). This off-target effect of oral L-dopatherapy may also account for other side effects, such as psychoticsymptoms (9) or pathological gambling (10). Thus, improved treat-ment of PD would be achieved by the selective restoration of sustainedtonic dopamine concentrations to the motor striatum only (5).

Gene therapy allows the continuous production of proteins in thecentral nervous system in a target-specific manner (11). For dopamineto be synthesized in nondopaminergic neurons of the striatum, theseneurons must express tyrosine hydroxylase (TH), aromatic L-aminoacid decarboxylase (AADC), and guanosine 5′-triphosphate cyclohy-drolase 1 (CH1) (fig. S2) (12, 13). Gene transfer of the AADC genealone, for example, with adeno-associated virus (AAV) vectors will

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only produce functional recovery in animal models of advanced PD ifL-dopa is also given to the animal (13, 14). The use of separate AAVvectors to transfer two or three of the critical dopamine biosynthesisgenes together has resulted in some behavioral benefit in rat and non-human primate models of PD (13, 15). However, transfer of all threegenes to striatal cells with a single AAV vector has not been achieveddue to the packaging constraints of these vectors (13, 15–18). Al-though it is theoretically possible to deliver three separate vectors tothe human brain, this is impractical and does not ensure that all threegenes get into each cell. A more feasible strategy is to deliver all threegenes to target cells with a delivery system, such as a lentiviral-basedvector, that can carry all three genes in a single vector. Thus, we havegenerated a tricistronic lentiviral vector derived from the equine in-fectious anemia virus (EIAV) encoding TH, AADC, and CH1 in asingle vector (Lenti-TH-AADC-CH1). Delivery of an earlier genera-tion of this vector into the striatum resulted in local dopamine syn-thesis and significant behavioral improvement in a rat model of PD(19). Here, we provide evidence of biochemical and functional efficacyof Lenti-TH-AADC-CH1 in nonhuman primate models of PD.

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RESULTS

Lentiviral vector technologyTo improve vector-mediated dopamine production, we alteredpONY8.1TSIN, the previously reported EIAV vector genome thatexpressed the tricistronic cassette (19), to generate a new vector,pONY8.9.4TY (Lenti-TH-AADC-CH1), that showed improveddopamine production by approximately two orders of magnitude,as assessed in vitro after transduction of human embryonic kidney(HEK) 293T cells with the two vectors (fig. S3).

Local dopamine depletion in a primate modelTo simulate advanced PD in nonhuman primates, we systemicallyadministered the selective neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to adult macaques until they reacheda severe and stable bilateral parkinsonian syndrome, including akine-sia, flexed posture, balance impairment, and tremor (20) (videos S1and S2). Before MPTP treatment, all primates scored 0 on a clinicalrating scale (CRS) of parkinsonian severity. After MPTP treatment,but before lentiviral injection, macaques displayed a significant in-crease in the CRS to 10.4 (maximum = 14) compared to the controlpre-MPTP state (normal; Fig. 1A) (n = 18). We further quantified theseverity of motor impairment by MPTP using quantitative video move-ment analysis (VMA) and found that, compared to the normal state,MPTP-treated macaques displayed marked akinesia (8% of the dis-tance traveled by normal animals) (Fig. 1B) (n = 18) and posturalimpairment as assessed by the frequency of rearing motions (7%of normal state) (fig. S4) (n = 18). The severity of MPTP-inducedparkinsonism was stable for the 12-month duration of the experi-ment, as assessed in the control MPTP-treated animals (MPTP–longterm group) (Fig. 1 and fig. S2). Analysis of extracellular concen-trations of dopamine in the putamen with microdialysis, performed10 months after MPTP cessation, showed a decreased dopamineconcentration to 27% of normal concentrations in MPTP-treated ani-mals, indicating a severe dopamine depletion in these animals (posthoc Mann-Whitney test, P < 0.001) (see below). Postmortem neuropath-ological analysis performed 12 weeks after MPTP cessation demon-

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strated that MPTP treatment caused pronounced cell loss and decreasedmetabolic function in the substantia nigra pars compacta (SNpc) (fig.S5) and a dramatic decrease of TH- and AADC-immunoreactive fibersin the striatum (Fig. 2). Striatal denervation, as assessed by loss of TH(Fig. 2), resulted in a heterogeneous pattern of degeneration resemblingthat observed in patients with PD: the putamen was more affected thanthe caudate nucleus and the dorsolateral part of the putamen (motorputamen) (Fig. 2B, white arrowhead) was more affected than its ventralpart (cognitive putamen) (Fig. 2B, black arrowhead).

Long-term motor behavioral restorationTo investigate the ability of gene transfer of TH, AADC, and CH1 tocorrect parkinsonism, we assigned 18 MPTP-treated macaques tothree behaviorally equivalent groups after the cessation of MPTPtreatment. The first group (MPTP-Lenti-TH-AADC-CH1) (n = 6) re-ceived bilateral injections of Lenti-TH-AADC-CH1 into the commis-sural and postcommissural motor putamen (five stereotactic injectionsof 10 ml; that is, 50 ml per striatum and a total of 100 ml per animal).The second group (MPTP-Lenti-lacZ) (n = 6) received bilateral injec-

Fig. 1. Injection of Lenti-TH-AADC-CH1 vector to MPTP-treated macaquesimproves parkinsonian syndrome.Macaques treatedwithMPTP (black) (n=
18) were significantly impaired relative to their control pre-MPTP state, dis-playing severe parkinsonismas assessedbyCRS (A) and VMA (B) of distancetraveled. Twoweeks after lentiviral injection, the animals that received Lenti-TH-AADC-CH1 (blue) (n = 6 until W8, then n = 3 until M12) demonstratedsignificant improvement in akinesia (B) compared to theMPTP animals thatreceived Lenti-lacZ (red) (n = 6 until W8, then n = 3 until M12) or no viralinjection (gray) (n = 6 until W8, then n = 3 until M12). W, week after genetransfer; M, month after gene transfer. *P < 0.05 relative to MPTP-Lenti-lacZandMPTP–long term animals; **P < 0.01 relative to normal pre-MPTP lesionstate. All data are expressed as the mean ± SEM.



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tions of a control EIAV vector encoding the LacZ reporter gene intothe motor putamen (five stereotactic injections of 10 ml; that is, 50 mlper striatum and a total of 100 ml per animal). The third group(MPTP–long term) (n = 6) did not receive any surgical interventionbut was included as an additional control to evaluate the stability ofthe MPTP model. All of these animals were maintained throughoutthe study without L-dopa or any other dopaminergic drug.

MPTP animals treated with Lenti-TH-AADC-CH1 demonstratedsignificant improvements in akinesia and posture starting 2 weeks aftervector injection compared with controls (Friedman test, P < 0.001; posthoc Mann-Whitney test: MPTP–long term, P < 0.05; MPTP-Lenti-lacZ,P < 0.05) (Fig. 1B and fig. S4). Clinical observations also showed a sus-tained and significant improvement (decrease) in the global CRS of theMPTP-Lenti-TH-AADC-CH1 group compared with controls 6 weeksafter vector injection (Friedman test, P < 0.001; post hocMann-Whitneytest:MPTP–long term,P<0.05;MPTP-LacZ,P<0.05) (Fig. 1AandvideosS3 and S4). MPTP-treated animals that were injected with Lenti-TH-AADC-CH1 continued to recover from akinesia and postural impair-ment, without any additional dopamine-altering agents, reaching 77%of normal for total distance moved (Fig. 1B) and 85% of normal posture(fig. S4). Behavioral recovery was sustained for the entire 12-month ob-servation period. One of the Lenti-TH-AADC-CH1–treated animals wasleft on study and exhibited a sustainedmotor improvement for 44monthsafter lentiviral injection, at which time point it was euthanized for histo-logical analysis. The control MPTP–long term and MPTP-Lenti-lacZanimals remained severely disabled at all time points (Fig. 1 and fig. S4).

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Dyskinesia studiesBecause standard oral dopamine replacement corrects parkinsonismbut also induces uncontrolled, disabling drug-induced dyskinesias,we performed a series of experiments to investigate the incidence ofdyskinesias after vector injection with and without L-dopa intake. Toinvestigate whether the vector induced abnormal dyskinetic move-ments in the MPTP-treated (dopamine-depleted) animals, we quanti-fied dyskinesias using video dyskinesia analysis (VDA). This methodassesses the entire range of dyskinetic movements during continuousobservation of a video of a macaque using a standardized protocol (fig.S7). We assessed both ON drug dyskinesia (after the administration ofL-dopa or the dopaminergic drug apomorphine) and OFF drug dys-kinesia (generally dystonia, which was observed in MPTP-treated pri-mates without any drug administration).

The administration of Lenti-TH-AADC-CH1 (Fig. 1) or oral L-dopa(20 mg/kg) (fig. S8A) produced a similar degree of correction of par-kinsonian symptoms inMPTP-treated primates as measured by VMA.Nevertheless, repeated discontinuous daily oral L-dopa administration tocontrol MPTP-treated animals induced sustained and severe dyskinesias[L-dopa–induced dyskinesias (LID)] (LID-MPTP animals; n = 5) (Fig. 3and video S5), whereas Lenti-TH-AADC-CH1 injection did not induceany dyskinetic movements during the observation period (Fig. 3 andvideo S4). Furthermore, MPTP-treated primates that received Lenti-TH-AADC-CH1 displayed significantly less OFF drug dyskinesia (dysto-nia)when compared toMPTP–long termandMPTP-Lenti-lacZprimates(Mann-Whitney test, P < 0.05; n = 6) (Fig. 4, A and B).

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Fig. 2. Transgenes for TH, AADC,and CH1 are expressed after stri-atal delivery of lentiviral vectors.Immunoreactivity for TH, AADC,and CH1 was reduced by MPTPtreatment, especially in the dorsalaspect of the striatum. Comparecoronal brain sections fromnormal[left column, (A), (E), and (I)] toMPTP-Lenti-lacZ [second column,(B), (F), and (J)]macaques. Injectionof the Lenti-TH-AADC-CH1 vectorto MPTP-treated animals resultedin significant increases (third col-umn) in TH (C), AADC (G), andCH1 (K) in thevicinity of theneedletrack in the commissural and post-commissural putamen. Higher-magnification photomicrographsof Lenti-TH-AADC-CH1–infusedareas (far right column) show im-munoreactive fibers throughoutthe putamen neuropil and neu-rons positive for TH (D), AADC(H), and CH1 (L). Arrows, needletracts; white arrowhead, dorsal pu-tamen; black arrowhead, ventralputamen; dotted line, delineationof the striatum; P, putamen; Cd,caudate nucleus. Scale bar in (A),500 mm, applies to all panels ex-
cept (D), (H), and (L); scale bar in (D), 20 mm, applies to (D), (H), and (L).



To mimic the clinical condition of a patient with PD who would beinjected with Lenti-TH-AADC-CH1 and also receive oral dopaminer-gic agents, we challenged MPTP-treated, drug-naïve animals with

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acute systemic administration of dopaminergic drugs (L-dopa and aprodyskinetic short-acting D1-D2 dopaminergic agonist, apomor-phine) on different days with a suitable washout time between drugs(21). Oral L-dopa intake and apomorphine injection improved thelocomotor activity of both MPTP–long term (n = 3) and MPTP-Lenti-lacZ (n = 3) primates to concentrations similar to those of normal pri-mates (n = 3) (Mann-Whitney test, P < 0.05; fig. S8, A and B). However,neither drug treatment altered the locomotor activity of drug-naïvenormal (n = 3) or MPTP-Lenti-TH-AADC-CH1 primates (n = 3)(fig. S8, A and B). Furthermore, normal and MPTP-Lenti-TH-AADC-CH1 animals showed no signs of significant dyskinesias after an acutestandard dose of either L-dopa or apomorphine, whereas MPTP–longterm and MPTP-Lenti-lacZ animals displayed debilitating dyskinetic(choreiform and dystonic) movements with both drugs (Mann-Whitneytest, P < 0.05) (Fig. 4, A and C).

Having demonstrated the capability of our gene therapy approachto correct parkinsonism without inducing dyskinesia, we subsequentlyinvestigated the potential of Lenti-TH-AADC-CH1 to reduce dyski-netic movement in a primate model of LID (LID-MPTP primatemodel). This model mimics the clinical condition of a patient whohas received long-term L-dopa therapy and has developed LID. Agroup of six MPTP-treated primates was treated with repeated, dailyoral L-dopa, from 20 to 100 mg/kg per day, adjusted for each individ-ual, until animals developed sustained and severe LID (LID-MPTP

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Fig. 4. Lenti-TH-AADC-CH1gene therapyplus L-dopaoral treatment results inless dyskinesia than L-dopa alone in parkinsonian macaques. (A) Dyskinesia

dystonia) in MPTP–long term and MPTP-Lenti-lacZ animals (Mann-Whitneytest, ***P < 0.05) compared to normal (Mann-Whitney test, **P < 0.05) and

events as rated by VDA before and after L-dopa administration. Without anyL-dopa,MPTP–long term (n= 3) andMPTP-Lenti-lacZ (n= 3) animals displayedspontaneous dyskinetic movements (OFF) compared to the normal (n = 3)(Mann-Whitney test, *P < 0.05) and MPTP-Lenti-TH-AADC-CH1 (n = 3)(Mann-Whitney test, **P < 0.05) animals. Acute oral L-dopa intake (20 mg/kg)significantly amplified the number of dyskinetic events (both chorea and

MPTP-Lenti-TH-AADC-CH1 (Mann-Whitney test, *P < 0.05) groups. (B and C)Schematic illustration of all dyskinetic events in a representative animal ofeach group during 120 min of observation before (B) and after (C) L-dopaadministration, as rated with VDA. Each chart depicts all dystonic (upper part)and choreiform (lower part) movements in face, trunk, neck, and upper andrear limbs. Each bar represents a rated dyskinetic movement (see fig. S7).

Fig. 3. Daily oral L-dopa, but not Lenti-TH-AADC-CH1, induces dyskinesia inMPTP-treated animals. P < 0.01, Friedman test; *P < 0.05, post hoc Mann-
Whitney test. W, week after initiation of treatment. Data are expressed asthe mean ± SEM (n = 5).



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animals) (n = 6). The animals then received bilateral injections intothe motor striatum (as performed in the MPTP-treated, drug-naïveprimates) of either Lenti-TH-AADC-CH1 (n = 3) or Lenti-lacZ(n = 3).

To closely mimic the clinical trial situation in LID PD patients, wethen adjusted the daily dose of L-dopa treatment for each individualanimal (in the MPTP-LacZ and MPTP-Lenti-TH-AADC-CH1group) to maintain the daily locomotor activity at its pre-MPTP–lesioning value. Thus, changes in the dose of the daily L-dopa treat-ment were based only on the OFF drug motor state (as evaluatedwith VMA). This approach is now used to find optimal L-dopa dosesin PD patients implanted with deep brain stimulation electrodes (22).This treatment management protocol led to a progressive decrease ofaverage L-dopa intake in the LID-MPTP-Lenti-TH-AADC-CH1animals from 70 to 30 mg/kg per day 6 months after vector injection,whereas daily L-dopa treatment was stable at 67 mg/kg per day inLID-MPTP-Lenti-lacZ. This experimental design led to different dai-ly L-dopa intake in the two groups but maintained their locomotoractivity at the same value.

When assessed in the OFF L-dopa state, LID-MPTP-Lenti-TH-AADC-CH1–treated macaques demonstrated improvement in mo-tor function from 8 weeks after vector injection (Fig. 5A) (Friedmantest, P < 0.05). However, motor recovery in LID-MPTP-Lenti-TH-AADC-CH1 animals (n = 3) was delayed and did not reach the samelevel of behavioral benefit as in L-dopa–naïve MPTP-Lenti-TH-AADC-CH1 animals (n = 3; 46 and 76%, respectively, of pre-MPTPstate; P < 0.05).

Dyskinesia assessment was performed by administering an acutedose of L-dopa (20 mg/kg) that was previously demonstrated to me-diate dyskinesias in all the study animals before vector administra-tion. In the ON L-dopa state (challenge with L-dopa at 20 mg/kg),LID-MPTP-Lenti-TH-AADC-CH1 animals (n = 3) displayed a de-crease in dyskinesias by up to 60% of the baseline dyskinesia score asearly as 1 month after vector injection (Fig. 5B and video S6). Con-versely, LID-MPTP-Lenti-lacZ control primates (n = 3) continued toshow severe dyskinesias in response to L-dopa challenge throughoutthe study (Fig. 5B and video S5). These results indicate that the com-bination of continuous release of dopamine in the striatum obtainedwith Lenti-TH-AADC-CH1 and the reduction of L-dopa intake de-creased the incidence of LID in MPTP-Lenti-TH-AADC-CH1.

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Local and continuous dopamine productionTo investigate the effect of lentiviral gene transfer on dopamineproduction in vivo, we performed histological analysis of transgeneexpression and measured local dopamine concentrations in the stri-atum of our study animals. Animals treated with Lenti-lacZ had anaverage of 54,947 transduced cells (b-galactosidase–immunoreactivecells) per injected putamen, which were mostly neurons (NeuN-immunoreactive cells >90%) (fig. S9). TH-, AADC-, and CH1-positiveneurons were evident in the vicinity of the injection site in MPTP-Lenti-TH-AADC-CH1–treated animals (representing ~15% of thetotal putamen volume, see Supplementary Material) but not inMPTP-Lenti-lacZ controls (Fig. 2).

To quantify lentiviral-mediated dopamine production in the puta-men, we performed two types of analysis: (i) whole tissue dopamineconcentrations ([DA]wt), measured by postmortem analysis of freshstriatal punches, which quantifies both intracellular dopamine (pre-

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synaptic substantia nigra dopaminergic terminals and striatal neuronsthat were transduced by Lenti-TH-AADC-CH1) and extracellular do-pamine, and (ii) extracellular dopamine concentrations ([DA]ec),measured by in vivo microdialysis, which specifically quantifies the do-pamine release within the extracellular space of the striatum.

Lenti-TH-AADC-CH1 injection of MPTP-treated macaques sig-nificantly increased [DA]wt in the dorsal putamen (fig. S10A) com-pared to Lenti-lacZ injection. Measurement of [DA]wt in putamenpunches from normal unlesioned macaques (n = 2) revealed thatthe amount of dopamine in the dorsal putamen was increased bya factor of 3, from 0.4% of normal concentrations in MPTP-Lenti-lacZ animals to 1.2% in MPTP-Lenti-TH-AADC-CH1 animals. Theseconcentrations of dopamine are low relative to normal concentrations,reflecting the nigral neurodegeneration that dramatically decreasespresynaptic dopamine pools. These presynaptic intracellular reservescontain most of the total dopamine in the striatum, which highlights

Fig. 5. Striatal injection of Lenti-TH-AADC-CH1 reduces L-dopa–induceddyskinesias in dyskinetic parkinsonian macaques. (A) Without L-dopa treat-
ment (OFF L-dopa), animals treated with MPTP (black) (n = 6) displayed asignificant -reduction in movement compared to their control pre-MPTPstate. After lentiviral injections, MPTP-LID animals that received Lenti-TH-AADC-CH1 (blue) (n = 3) significantly increased their movement comparedto MPTP-LID animals that received Lenti-lacZ (red) (n = 3) (Friedman test, *P <0.05 relative to MPTP-LID-Lenti-lacZ). All data are expressed as the mean ±SEM. (B) After L-dopa treatment (ON L-dopa) (acute challenge, 20 mg/kgby mouth), MPTP-LID-Lenti-TH-AADC-CH1 (blue bars; n = 3) animalsdisplayed a significant reduction of the total number of dyskinesias com-pared to MPTP-LID-Lenti-lacZ (red bars; n = 3) (Mann-Whitney test, *P <0.05 relative to MPTP-LID-Lenti-lacZ). All data are expressed as the mean ±SEM. W, week after gene transfer; M, month after gene transfer.



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the importance of analyzing [DA]ec. The effects of Lenti-TH-AADC-CH1 were specific to the putamen region; no unregulated release ofdopamine was observed in other brain regions, such as cortex, globuspallidus, and caudate nucleus (fig. S10B).

To gain a more precise understanding of the dopamine availableto neurons within the striatum in our animals, we next measured[DA]ec concentrations in individual brain hemispheres from normal(n = 6), MPTP–long term (n = 6), MPTP-Lenti-lacZ (n = 5), andMPTP-Lenti-TH-AADC-CH1 (n = 6) primates. Microdialysis probeswere placed in the motor putamen for each animal (see Materialsand Methods) (fig. S11). Significant [DA]ec differences were demon-strated between the groups (Kruskal-Wallis test, P < 0.001) (Fig. 6A).In the control groups (MPTP–long term and MPTP-Lenti-lacZanimals), baseline [DA]ec was reduced to 27 and 26% of normal do-pamine concentrations, respectively, indicating a severe dopaminedepletion in these animals (post hoc Mann-Whitney test, P < 0.001)(Fig. 6A). Lenti-TH-AADC-CH1 significantly increased baseline[DA]ec compared to both MPTP–long term and MPTP-Lenti-lacZgroups, which reached 47%of normal concentrations in themotor pu-tamen (post hoc Mann-Whitney test, P < 0.05) (Fig. 6A). We alsomeasured striatal [DA]ec and [L-dopa]ec after intramuscular adminis-tration of L-dopa (40 mg/kg). Analysis of [DA]ec 2 hours after L-dopatreatment showed that [DA]ec [area under the curve (AUC)] wasincreased by a factor of 6 in the MPTP-Lenti-TH-AADC-CH1 condi-tion, compared with a factor of 2.7 increase in the MPTP–long termcondition and a factor of 3.5 increase in the MPTP-Lenti-lacZ condi-tion (Fig. 6B). This analysis indicates that Lenti-TH-AADC-CH1mediated a higher conversion rate of L-dopa to dopamine through itsAADC enzyme activity. After L-dopa injection, L-dopa in the striatumwas increased only in the MPTP–long term and MPTP-Lenti-lacZgroups, suggesting that a larger proportion of L-dopa is not metabolizedto dopamine, most likely because the L-dopa–converting enzymeAADC was depleted by MPTP treatment (Figs. 2 and 6C). In contrast,

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most of the injected L-dopawas converted into dopamine in normal andin MPTP-Lenti-TH-AADC-CH1 animals where there is presumably suf-ficient AADC (Figs. 2 and 6C).

The improvement in the parkinsonian deficits observed in MPTP-Lenti-TH-AADC-CH1 animals was not due to the presence of a lesssevere nigrostriatal lesion in these animals. Stereological counts of do-paminergic neurons in the SNpc showed a decrease in the number ofTH-positive neurons of both MPTP-Lenti-TH-AADC-CH1 andMPTP-Lenti-lacZ groups compared to normal animals (Kruskal-Wallis test, P < 0.001; post hoc Mann-Whitney test, P < 0.001) (fig.S12). Furthermore, no difference was observed between the number ofTH-immunoreactive neurons in the two lesioned MPTP groups (posthoc Mann-Whitney test, P = 0.51) (fig. S12).

Dopamine restoration of parkinsonian neural circuitryTo determine the mechanism by which Lenti-TH-AADC-CH1corrected motor dysfunction, we investigated neuronal activity withinthe basal ganglia system of MPTP-treated animals injected with thevector. The current model of basal ganglia dysfunction in PD suggeststhat abnormal overactivity of output nuclei such as the internal globuspallidus (GPi) accounts for the motor symptoms in this disorder (23).To determine whether Lenti-TH-AADC-CH1 normalized neuronalelectrical activity in basal ganglia output nuclei, we performed unitaryrecordings in the GPi of normal and MPTP-treated macaques. Inagreement with previous reports (21, 23), we found a significant in-crease [52%; 62.5 ± 3.6 Hz (n = 8 neurons) (MPTP) versus 41.0 ± 3.8Hz (n = 8 neurons) (normal); Mann-Whitney test, P < 0.01] in themean firing rate of GPi neurons in a drug-naïve untreated MPTP ma-caque compared to a normal macaque (fig. S13, A and B). StriatalLenti-TH-AADC-CH1 administration restored the firing rate of GPineurons to normal [39.8 ± 3.9 Hz (n = 10 neurons) (MPTP-Lenti-TH-AADC-CH1) versus 62.5 ± 3.6 Hz (n = 8 neurons) (MPTP); Mann-Whitney test, P < 0.01] (fig. S13B).

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Fig. 6. Lenti-TH-AADC-CH1 restores striatal [DA]ec. (A) Baseline [DA]ec innormal (unlesioned, no gene transfer; white bar) and in MPTP-treated pri-

but not Lenti-lacZ, significantly increased striatal [DA]ec. (B) [DA]ec in the pu-tamenat thebaseline (–) andafter L-dopa challenge (+) in thedifferent groups

mates that received Lenti-TH-AADC-CH1 (blue bar), Lenti-lacZ (red bar), orno treatment (gray bar). Microdialysis probes were placed in the post-commissural putamen for each animal as demonstrated by in vivo T2* MRIimaging after the microdialysis procedure (see fig. S11). Lenti-TH-AADC-CH1,

(data are represented asAUC). (C) [L-dopa]ec in theputamenat thebaseline (–)and after L-dopa challenge (+) in the different groups (data are represented asAUC). P< 0.05,Mann-Whitney test. *P< 0.05 relative toMPTP-Lenti-TH-AADC-CH1 animals; **P < 0.05, ***P < 0.01 relative to normal animals.




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Because the pattern of neuronal firing in theGPi is also important inthe pathophysiology of PD (21), we analyzed the burst activity of re-cordedGPi neurons. Theproportionof spikes per burst and thenumberof burst events significantly increased inMPTPprimates (15.9% and 9.7events per cell per minute, respectively) compared to normal primates(3.8% and 1.7 events per cell per minute, respectively; Mann-Whitneytest, P < 0.05). Treatment with Lenti-TH-AADC-CH1 significantly de-creased the proportion of spikes per burst and the number of burstevents in GPi neurons to concentrations similar to those in normal un-lesioned animals (5.3% and 1.6 events per cell per minute, respectively;Mann-Whitney test: P < 0.05 compared to MPTP primates, not signif-icant compared to normal primates) (fig. S13C).

Neuronal hyperactivity in the STN is also a key pathophysiologicalfeature of PD, and its electrical modulation by deep brain stimulationhas been therapeutically successful in both MPTP macaques and PDpatients (24, 25). Metabolic studies of basal ganglia in both PD pa-tients and primate models of nigrostriatal degeneration have demon-strated alterations in the neural circuitry that interconnect cortex andbasal ganglia (26–28). With [14C]2-deoxyglucose (2-DG) (150-mmspatial resolution) (fig. S14, A and B) functional imaging, the STNin an MPTP control macaque showed an increase in metabolic ac-tivity compared to that of a normal macaque (fig. S14C). At 36 weeksafter treatment with Lenti-TH-AADC-CH1, the metabolic activity ofboth the right and the left STN resembled those of a normal animal(fig. S14D).

Toxicology studiesWe investigated the potential of Lenti-TH-AADC-CH1 to induce ad-verse events that could prevent translation of this work into the clinic.Having ruled out any behavioral adverse event (specifically no OFFdrug dyskinesia and no increase in LID up to 44 months after Lenti-TH-AADC-CH1 injection), we investigated the potential of Lenti-TH-AADC-CH1 to induce pathological structural changes withinthe putamen, which might lead to striatal dysfunction (29). We firstchecked that the needle tracks were all located in the putamen (fig.S11). Markers for striatal neurons (NeuN) exhibited no tissue altera-tions in all brain structures of Lenti-TH-AADC-CH1–injected animal.Glial fibrillary acidic protein (GFAP) immunoreactivity (an astrocytemarker) and ionized calcium binding adapter molecule 1 (Iba1) im-munoreactivity (a microglia marker) were all limited to the vicinity ofthe injection area in the putamen 2 months after vector injection (fig.S15). At 44 months after Lenti-TH-AADC-CH1 injection, one animalshowed an even more restricted area of both GFAP and Iba1 immu-noreactivity (fig. S15). We performed a neuroimaging study usingT2*-weighted magnetic resonance imaging (MRI) sequence, which isvery sensitive to a wide range of brain pathologies. The putamen andthe remaining brain areas were free from any abnormal T2* signal.

DISCUSSION

The dyskinesias that are seen in patients with PD who have been treatedwith L-dopa for long periods are thought to result from the intermittentdopaminergic stimulation of postsynaptic receptors in the striatumcombined with extensive degeneration of the presynaptic dopaminergicneurons of the nigrostriatal tract (2, 4, 5). Here, we demonstrate thatcontinuous delivery of dopamine to the striatum, with a lentiviral vec-tor, provided long-term correction of motor deficits (up to 44 months)

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in a primate model of PD and, in contrast to the usual PD treatmentof repeated oral L-dopa treatment (30), did not initiate dyskinesias inL-dopa–naïve parkinsonian animals.

One explanation for our results is that the gene therapy restoresdopamine sufficiently to maintain sustained concentrations (dopa-minergic tone) in the striatum and restores normal functioning ofthe basal ganglia networks, as demonstrated by our data showing nor-malization of neuronal activity in the GPi and of STN metabolism.We found no other neurological or anatomical changes that couldsupport an alternative hypothesis. Our data support the notion thatit is not the extent of substantia nigra degeneration per se that causesdyskinesia but rather the absence of constant dopaminergic tone inthe striatum.

We observed that the LID-MPTP animals recovered from motordeficits to a lesser extent than the L-dopa–naïveMPTP primates despitereceiving the same vector in their putamen. One possible explanationfor this observation could be attributed to the length of elapsed timebetween the cessation of MPTP treatment and vector injection inLID-MPTP animals (average of 19 weeks) compared to L-dopa–naïveMPTP animals (average of 4 to 5 weeks). Alternatively, differences inphysiology between MPTP and LID-MPTP animals, which are poorlyunderstood, might also account for this variation in response time (6).Therefore, to closely reflect the clinical scenario of patients that havereceived long-term treatment with L-dopa and develop dyskinesias,we decided to adapt the daily dopaminergic treatment for each animalafter vector administration (including controls) according to the behav-ioral recovery observed, that is, to correct the parkinsonismwithout un-derdosing or overdosing the animal with dopamine in our LID-MPTPdyskinesia study. This experiment was designed to anticipate treatmentalternatives in our LID PD patients and provide a basis for the clinicalmanagement of potential adverse events during the clinical trial ratherthan to demonstrate LID reversal. Accordingly, we assessed the inci-dence of dyskinesia after a fixed dose of L-dopa (20 mg/kg) duringthe acute challenge across all the dyskinesia studies. This dose isconsistent with the preclinical literature in nonhuman primate modelsof PD (31, 32) [see (33) for a review] andwith the clinical practice in PDpatients. Clinical evidence showing that the severity of dyskinesia is notstrictly dose related in advanced PD was previously confirmed by de-monstrating in severely MPTP-lesioned primates the absence of alinear dose relation between L-dopa and the severity of dyskinesia,where the severity of dyskinesia reached a maximum level at an L-dopadose far below the dose used in our LID nonhuman primate study(33–35).

The therapeutic effects that we describe have been achieved withmodest and localized gene transfer, which generates subnormal con-centrations of dopamine. Assuming that the amount of dopaminethat we measured by microdialysis corresponds to the active dopa-mine concentration in the extracellular space, our gene therapyprocedure restores dopamine to ~50% of normal concentrations. Dra-matic behavioral correction is consistent with the observation in hu-man PD that motor symptoms are observed when >60% of thedopaminergic neurons degenerate. Therefore, a modest replacementof dopamine in the striatum would be expected to provide therapeuticbenefit. In addition, the behavioral improvement observed in thisstudy may also reflect a combination of the subnormal lentiviral do-pamine production and the reduced concentrations of the dopaminetransporter (DAT) in the parkinsonian striatum due to the decrease inthe number of presynaptic nigral dopaminergic terminals. DAT is




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known to control the spatial and temporal activity of released dopa-mine, and lower concentrations of DAT may increase the activityand/or distribution of dopamine released from the Lenti-TH-AADC-CH1–transduced striatal neurons (36).The current accepted surgicaltreatment for advanced PD involves electrical stimulation of theSTN or GPi, and this prevents L-dopa–induced motor complicationsby restoring normal electrical activity within these nuclei (37, 38)through selective stimulation of afferent projecting axons (26, 39). Itis possible that gene therapy may provide these therapeutic benefitsbut without the neuropsychological side effects observed by unwantedelectrical stimulation of nonmotor regions of the STN region (40, 41).

A unique feature of our gene therapy vector is its ability to conferlocal dopamine production using a single vector encoding all three ofthe critical biosynthesis enzymes (TH, AADC, and CH1 expression).This provides an advantage over AAV vectors, for example, which aresize constrained and currently unable to package such a large geneticcargo. Furthermore, delivery of the AADC gene allows enhanced con-version of standard L-dopa therapy into dopamine, which allows theadjustment of the L-dopa dose for each individual at different stages oftheir PD progression.

How can our dopamine gene therapy approach be translated into aclinical reality? Our study was designed and conducted as a “primateclinical trial.” Accordingly, after we demonstrated the efficacy of Lenti-TH-AADC-CH1 in the drug-naïve, MPTP-treated macaques (primatemodel of drug-naïve PD) and investigated its mechanism, we extendedour study to the LID-MPTP–treated macaques (primate model of phar-macologically treated PD with concurrent drug-induced dyskinesias).Because a previous biotherapy approach for PD (fetal grafting) could ini-tiate OFF drug dyskinesias in clinical trials (42, 43), we evaluated thepotential for this serious adverse event in our primate models. Becauseneither naïve nor LID parkinsonian primates displayed any Lenti-TH-AADC-CH1–induced dyskinesia, we suggest that a clinical trial couldbe safely designed for PD patients who remain responsive to L-dopawhile experiencing motor complications related to intermittent oralL-dopa. A phase 1/2 clinical trial in PD patients that investigates thesafety and efficacy of this gene therapy is therefore ongoing (44).

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MATERIALS AND METHODS

AnimalsAll animal studies were conducted in accordance with the Europeanconvention for animal care (86-406) and the National Institutes ofHealth’s Guide for the Care and Use of Laboratory Animals. A total of26 adult male Macaca fascicularis, weighing 5 to 7 kg, were housedindividually with a 12:12-hour light-dark cycle. After lentiviral vec-tor injections, experiments were performed using level III Biosafetyprocedures.

Viral vector construction, generation, andin vivo administrationTo improve vector-mediated dopamine production, a number ofchanges weremade to the original EIAV vector genome that expressedthe tricistronic cassette called pONY8.1TSIN (fig. S3). The updatedconstruct (pONY8.9.4TY) contains the catalytic isoforms of thefollowing three genes: human aromatic AADC (E.C. 4.1.1.28), humanTH (catalytic domain only) (E.C. 1.14.16.2), and human CH1 (E.C.3.5.4.16) (GenBank accession number GQ872121). These changes

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led to at least an approximately two–order of magnitude increase indopamine production per integrated genome as assessed in vitro aftertransduction of human HEK293T cells (fig. S3). A LacZ encoding ver-sion of this vector (pONY8.9NCZ, Lenti-lacZ) was used as a control.This construct encodes the LacZ reporter gene in place of the tricistro-nic cassette. The method for generating EIAV-based vectors has beendescribed (45–49). Viral vector was injected bilaterally to the motorpostcommissural putamen using MRI-guided stereotaxy (see Supple-mentary Material).

BehaviorAll animals were assessed daily for their general clinical condition,with special attention paid to their nutritional requirements. In addi-tion, the general neurological state of each macaque was assessedbefore and after lentiviral injection. To objectively quantify our neu-rological observations, animals were videotaped for 30 min before andafter MPTP administration and at regular intervals after lentiviralinjection. The videos were then analyzed off-line by an examinerblinded to the experimental conditions. A CRS was adapted from thePapa andChaseMPTP primate parkinsonian scale (posture, 0 to 2; gait,0 to 2; tremor, 0 to 2; general mobility, 0 to 4; hand movements, 0 to 2;climbing, 0 to 2; a score of 0 corresponds to a normal monkey) (50).VMA was performed with a motion tracking software (Ethovision 3,Noldus) that allowed an objectivemeasurement of total distancemoved(traveled distance, centimeters), maximum velocity (maximal velocity,centimeters per second), and rearing behavior (rearing, number ofevents) during the video-recording period (26). The quantitative anal-ysis of dyskinesia was performed with VDA, video event–relatedsoftware (TheObserver 7.0, Noldus) that counts predefinedmovements(upper and lower limb, trunk, face and neck chorea, and dystonia fordyskinetic movements) during the video-recording period.

MPTP lesionAll primates received a daily intramuscular dose of MPTP (Sigma-Aldrich) at 0.2 mg/kg until they reached a severe stable bilateral parkin-sonian syndrome. MPTP administration was halted when animalsreached a CRS of ≥10 and a decrease of traveled distance of ≤500cm/30 min and rearing of ≤5/30 min. The long-term stability of par-kinsonian syndrome was confirmed with the same behavioral test.

Immunohistochemistry and double-labelingimmunofluorescence procedureSee Supplementary Material for immunohistochemistry and double-labeling immunofluorescence procedure.

Stereological analysisStereological analysis was used for cell counts, as described (51–53).See Supplementary Material for details.

L-Dopa administrationFor the study of dyskinesia induction by Lenti-TH-AADC-CH1 treat-ment compared with chronic L-dopa treatment, the animals in theL-dopa groupwere treated chronically with an average daily oral dose ofL-dopa (20mg/kg) and benserazide (at a 4:1 ratio, Madopar dispersible,Roche; termed L-dopa thereafter). For the acute L-dopa challenge ofMPTP–long term, MPTP-Lenti-lacZ, and MPTP-Lenti-TH-AADC-CH1 nonprimed primates, animals received a single oral dose of L-dopa(20 mg/kg). To test whether Lenti-TH-AADC-CH1 reduced LID in




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primed dyskinetic MPTP animals, oral daily doses of L-dopa wereadministered to obtain a stable dyskinetic state over a period of 2 to4 months. After lentiviral vector injection, oral daily discontinuousL-dopa treatment was maintained at a “customized” dose for each LIDanimal, as performed in patients with PD who are treated by deepbrain stimulation (22). This dose was adjusted according to the motorstatus observed (based on OFF drug VMA using specifically thetraveled distance parameter). For example, a 50% improvement inOFF traveled distance would result in a 50% reduction in L-dopa dose.However, the dose of L-dopa used for the dyskinesia assessments wasmaintained constant (20 mg/kg) as described above.

Short-acting D1-D2 agonist administrationApomorphine (Aguettant), a short-acting nonselectiveD1-D2 agonist,was administered systemically at a dose (0.1 mg/kg intramuscularly)known to induce dyskinesia in MPTP-treated macaques (54).

Microdialysis procedurePrimates were anesthetized with ketamine and xylazine (15 mg/kg +1.5 mg/kg, every hour) and placed in a stereotactic frame. Body tem-perature was stabilized at 37°C throughout the experiment with a ther-mostatic blanket. The microdialysis probes (CMA/12; membranelength, 5 mm; cutoff, 20 kD; CMAMicrodialysis) were implanted bilat-erally in the striatum. Microdialysis probes were placed into the post-commissural putamen of four normal unlesioned animals, four MPTPanimals, three MPTP-Lenti-lacZ animals, and four MPTP-Lenti-TH-AADC-CH1 animals. Probes were perfused with artificial cerebrospinalfluid (147mMNaCl, 2.7mMKCl, 1.2mMCaCl2, and 0.85mMMgCl2)(CMA Microdialysis) at a rate of 2 ml/min. Microdialysates werecollected every 15 min into a refrigerated fraction collector and frozenat –80°C until analysis. After implantation of each probe into the pri-mate brain,microdialysis sampleswere taken over a 2-hour stabilizationperiod, allowing recovery from any transient increase in neurotransmitterrelease due to procedural trauma. Baseline sampleswere then taken overthe next hour. After collection of baseline samples, animals were sub-jected to an acute L-dopa challenge (L-dopa methyl ester, 40 mg/kgintramuscularly) and additional microdialysis samples were taken con-tinuously over a 2-hour period. By the end of the microdialysis session,additional control samples were generated by performing microdialysisfor 30min in a dopamine solution of known concentration (1 mM) afterremoval of the probe from the putamen. After each experiment, the lo-cation of probes was confirmed by T2*-weighted MRI. In cases wherethe probe was not placed in the putamen, these samples were omittedfrom dopamine measurements.

Postmortem measurement of dopamine: [DA]wt

High-performance liquid chromatography (HPLC) with electrochemicaldetectionwas used tomeasure striatal concentrations of catecholaminesin brain punches as previously described (19) (see SupplementaryMaterial).

Measurement of dopamine in vivo: [DA]ecSee Supplementary Material for measurement of dopamine in vivo([DA]ec).

Microdialysis data analysesThe mean baseline value of dopamine ([DA]ec) and L-dopa ([L-dopa]ec)was calculated by averaging the concentration of the four basal dialysate

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samples. The AUC values were calculated at baseline and immediatelyafter L-dopa pharmacological challenge.

ElectrophysiologyData collection and analysis were performed as described (26). Burst-ing discharge was quantified with the Poisson “surprise” method ofburst detection (55) with a Poisson surprise value of >10 (see Supple-mentary Material).

Local cerebral glucose utilization using 2-DGLocal cerebral glucose utilization was measured as described (56) (seeSupplementary Material). Optical densities determined in the STN onthe autoradiograms (10 to 13 sections per STN) were converted toradioactivity and then to glucose consumption values using the 14Cstandards and the modified operational equation of Sokoloff (57).

Statistical analysisValues are the mean ± SEM. Data were analyzed with Kruskal-Wallisor Friedman test (the nonparametric equivalent of the repeated mea-sures analysis of variance) and then withMann-Whitney post hoc testat the individual time points, corrected for multiple comparisons bythe method of Bonferroni, using the SPSS (Statistical Package for theSocial Sciences) software. Both asymptotic and exact P (one-tailed)values were calculated.

SUPPLEMENTARY MATERIAL

www.sciencetranslationalmedicine.org/cgi/content/full/1/2/2ra4/DC1Experimental DesignMaterials and MethodsFig. S1. The anatomy of the basal ganglia.Fig. S2. Dopamine biochemical synthesis pathway.Fig. S3. Dopamine production by lentiviral vectors in HEK293T cells.Fig. S4. Effect of Lenti-TH-AADC-CH1 on postural impairment in MPTP-treated macaques.Fig. S5. Neuronal loss in substantia nigra pars compacta (SNpc) following systemic adminis-tration of neurotoxin MPTP.Fig. S6. Dopamine transporter (DAT) immunoreactivity after striatal delivery of lentiviral vectors.Fig. S7. An example of quantification of dyskinesia with video dyskinesia analysis (VDA) (TheObserver 7.0 software).Fig. S8. Effects of dopaminergic agents on akinesia.Fig. S9. Neurotropism of EIAV lentiviral vector.Fig. S10. Postmortem [DA]wt.Fig. S11. In vivo localization of microdialysis probes and needle tracks.Fig. S12. Stereological count of SNpc neurons after MPTP intoxication.Fig. S13. Restoration of normal firing rate of basal ganglia output (globus pallidus internalis,GPi) neurons by Lenti-TH-AADC-CH1.Fig. S14. Normalization of metabolic activity within the subthalamic nucleus (STN) afterinjection of Lenti-TH-AADC-CH1 into the motor striatum of a MPTP-treated primate.Fig. S15. Astrocyte and microglia activation after striatal delivery of lentiviral vectors.Videos S1–S6. Videos are available upon request and may be obtained by contacting thecorresponding author directly.References

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58. Acknowledgments: We thank the individuals at Oxford BioMedica who contributed tothe ProSavin project; C. Jouy, H. Juin, J. Busson, P. Flament, J. C. Mascaro, J. Mitja, P.Pochard, D. Renault, and J.-C. Wilk for excellent care of the primate colony; F. Wilkes, L.Walmsley, R. Barber, J. B. Rohll, O. Bekaert, F. Condé, J. M. Hermel, L. Uhrig, M. Guillermier,and F. Petit for technical assistance; M. Dhenain and V. Lebon for help during the MRI studies;E. Werner for supplying antibody against CH1; and R. LeGrand, E. Bouchoux, and C. Joubertfor logistic help.

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Funding: This study was supported by Oxford BioMedica, Commissariat à l’Energie Atomique(CEA), and Association pour la Recherche sur la Stimulation Cérébrale (ARSC). B.J. receivedfellowships from CEA, ARSC, Société Française de Neurochirurgie, and Assistance Publique-Hôpitaux de Paris.Author contributions: B.J., S.B., S.M.K., P.H., K.A.M., N.D.M., and S.P. designed and organized theexperiments. G.S.R., M.A., J.E.M, K.A.M., S.M.K., and N.D.M. designed, generated, and producedthe lentiviral vectors. B.J., S.B., M.S., and S.P. performed the animal models. B.J., S.B., C.J., M.S.,T.D., P.H., and S.P. performed the MRI studies. B.J., S.B., M.S., X.D., and S.P. performed thebehavioral studies. B.J., S.B., and S.P. performed the pharmacological studies. B.J., S.B., C.J.,M.S., and S.P. performed the stereotactic surgeries. B.J., C.J., and S.P. performed the histo-logical studies. B.J. and E.B. extracted the brain samples for the biochemical studies. G.S.R.performed the HPLC measurements of monoamines. B.J., M.S., X.D., and S.P. performed theelectrophysiology study. B.J., G.B., C.J., T.D., A.-S.H., and E.B. performed the 2-DG metabolicstudy. B.J., S.B., G.S.R., C.J., G.B., M.S., and S.P. performed the microdialysis experiments. B.J., S.B.,G.S.R., S.M.K., K.A.M., N.D.M., and S.P. wrote the manuscript. S.P. taught and supervised B.J.Competing interests: Authors affiliated with Oxford BioMedica hold shares or share options inOxford BioMedica. Authors not affiliated with Oxford BioMedica declare no competing finan-cial interests.GenBank accession number: GQ872121.

Submitted 7 May 2009Accepted 25 September 2009Published 14 October 200910.1126/scitranslmed.3000130

Citation: B. Jarraya, S. Boulet, G. Scott Ralph, C. Jan, G. Bonvento, M. Azzouz, J. E. Miskin,M. Shin, T. Delzescaux, X. Drouot, A.-S. Hérard, D. M. Day, E. Brouillet, S. M. Kingsman,P. Hantraye, K. A. Mitrophanous, N. D. Mazarakis, S. Palfi, Dopamine gene therapy forParkinson’s disease in a nonhuman primate without associated dyskinesia. Sci. Transl. Med.1, 2ra4 (2009).

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