Introduction

Dopamine is normally synthesized and stored in the terminals of neurons that originate in the substantia nigra and project to the striatum (caudate and putamen). Once dopamine is released into the extracellular space, it binds to postsynaptic striatal receptors and starts a cascade of signaling events that control and coordinate movement through direct and indirect pathways. In Parkinson disease (PD), atrophy of these nigral projections causes a dopamine deficiency in the striatum, which leads in turn to the central neuropathology of the disorder ^1,2,7. The mesolimbic pathway is also involved in idiopathic PD ¹⁷, but degeneration occurs at a much slower rate. Imbalances in dopaminergic activity within the nigrostriatal pathway and between the mesolimbic and the nigrostriatal systems become pronounced as the disease progresses.

The biosynthetic pathway for dopamine requires both tyrosine hydroxylase (TH), which converts tyrosine to L-3, 4-hydroxyphenylalanine (L-Dopa), and aromatic L-amino acid decarboxylase (AADC), which decarboxylates L-Dopa to generate dopamine. Because dopamine cannot cross the blood–brain barrier, L-Dopa has become the standard pharmacological therapy for PD. L-Dopa is given orally several times daily and is highly effective in early stages of the disease, presumably because there is still enough AADC left in the diminishing nigral terminals to exceed a rate-limiting level ^22,23. With disease progression, however, AADC levels decline, and both increased and more frequent amounts of substrate (L-Dopa) are required for clinical response ²⁶. We hypothesize that increased L-Dopa doses (and dopamine end-product) result in excessive stimulation of the relatively intact mesolimbic system, which may engender some of the side effects such as hallucinations, although it should be noted that there are a number of competing hypotheses, notably the pulsatile dopamine hypothesis of Chase and colleagues ⁸. Nonuniform degeneration within the nigrostriatal system may be responsible for some complications of treatment such as involuntary movements termed L-Dopa-induced dyskinesias (LID) ^4,23. Thus, dose escalations of L-Dopa are invariably associated with the development of LID and other deleterious side effects, the management of which represents one of the greatest challenges in the long-term treatment of PD ^25,27. We have developed a strategy to overcome this progressive loss of efficiency in conversion of L-Dopa to dopamine. In our experience, overexpression of AADC in the striatum of MPTP-lesioned monkeys leads to substantial restoration of response to L-Dopa ⁵. Infusion of a recombinant adeno-associated virus (AAV) encoding human AADC (AAV-hAADC) into the lesioned striatum produced marked improvements in L-Dopa responsiveness. A limitation in translation of this approach to humans was whether such improvement in the short term of only a few months would persist over a longer time frame. In this study, therefore, we show that restoration of L-Dopa sensitivity in MPTP-lesioned monkeys is pronounced, is stable, and persists for more than 2 years after administration of therapy. These studies support the current clinical study of this vector in patients suffering from dose-limiting L-Dopa toxicity in advanced PD.

Results

We made eight male nonhuman primates (Macaca mulatta) hemiparkinsonian as described briefly under Materials and Methods and as detailed previously ^3,6,11,13. Two to four months later, we infused the animals stereotactically by convection-enhanced delivery (CED) unilaterally into the lesioned striatum with AAV-hAADC (n = 4) or a nontherapeutic gene control (AAV-LacZ; n = 4) ⁵. Availability of resources impacted the study design, as follows. We euthanized control animals at 24 months (n = 4). We euthanized two test animals at 36 months and continue to evaluate the other two clinically and with PET. The most recent testing was performed at 72 months.

PET imaging of gene expression

A critical issue with gene therapy targeted to brain has been the difficulty of measuring transgene expression in vivo. We employed 6-[¹⁸F]fluoro-meta-tyrosine (FMT) PET to monitor AADC activity both before and at several times after gene transfer, as described previously ⁵. Logistical issues prevent simultaneous measures of FMT signal. Thus, individual scans vary somewhat as to timing of scans over the entire time course. However, they were all routinely scanned within 2 weeks of one another. Fig. 1A shows a low level of AADC activity after MPTP-lesioning in animals from both treatment groups. In the AAV-hAADC-treated animals only, [¹⁸F]FMT signals were restored to near-normal levels within the first few months and remained stable over time (over 2 years), indicating sustained expression of AADC. The remaining two animals appeared to maintain expression beyond 2 years, although the small number of animals precludes any statistically meaningful analysis. The PET signal on the AAV-hAADC-infused lesioned side of the brain remained comparable to endogenous signal measured on the contralateral (nonlesioned, nontreated) side throughout the 2-year (fully powered) phase of the study, suggesting a return to balanced dopaminergic activity within the nigrostriatal system. Fig. 1B shows representative PET images from an AAV-hAADC-treated animal prior to, and at 10 and 24 months after, gene transfer.

Figure 1.

PET imaging of hAADC activity in hemilesioned monkey striatum. We measured AADC activity, prior to and after AAV transfer, by PET with [¹⁸F]FMT, a specific ligand for AADC. (A) The K_i for the side ipsilateral to MPTP infusion and AADC gene transfer is shown over time. One animal (solid triangle) was scanned (see baseline data point) prior to MPTP administration to determine the normal levels of [¹⁸F]FMT. After MPTP administration, [¹⁸F]FMT was significantly reduced in all animals ipsilateral to the lesion. In AAV-hAADC-treated monkeys (solid triangles), [¹⁸F]FMT values increased and appeared stable over time, demonstrating sustained expression of AADC. Little, if any, increase was observed in animals that received an otherwise identical infusion of a control (AAV-LacZ) vector (open circles). (B) PET images from an AAV-hAADC-treated animal were taken 80–90 min after administration of the radioactive tracer. Shown are raw coronal [¹⁸F]FMT PET images at baseline (1) and at 10 and 60 months after unilateral AAV-hAADC administration to the right side of the brain.

Full figure and legend (113K)

Behavioral response

Behavioral assessments (clinical rating scale or CRS) were performed by a blinded clinical rater throughout the course of the experiment, either in the absence of L-Dopa dosing or 45 and 90 min after a single intramuscular (im) injection of L-Dopa. Prior to gene transfer, animals were clinically evaluated and scored as moderately affected with parkinsonian signs, as expected, predominantly on the side contralateral to the intracarotid MPTP infusion. Intramuscular administration of 15 mg/kg L-Dopa induced clinical recovery in all animals both before and after gene transfer. However, improvement was associated with adverse side effects, such as considerable hyperactivity, apparent hallucinations, stereotypical behavior, and distractibility. Acute challenge with a lower dose of L-Dopa (3 mg/kg, im) prior to gene transfer did not result in clinical improvement in control animals. However, improvement was seen with this dose in all four AAV-hAADC-treated animals beyond about 6 months after gene transfer. This improvement occurred with minimal side effects, if any, and continued throughout the course of the study (Fig. 2). Also, CRS scores for the AAV-hAADC treatment group at 90 min after L-Dopa treatment, as with control animals, remained virtually unchanged compared to scores at 45 min (data not shown), suggesting that AAV-hAADC does not compromise the duration of the L-Dopa response. Because destruction of nigral projections into the striatum via MPTP eliminates vesicular dopamine storage capacity, behavioral response to L-Dopa is driven by tonic generation of extracellular dopamine and not by phasic, vesicular release ¹⁶. Thus, there is no absolute requirement for storage of dopamine in AADC gene therapy, although there is some in vitro evidence that combined expression of VMAT2 and AADC in striatal tissues might confer added benefit beyond that of AADC alone ²¹.

Figure 2.

Clinical responses to low-dose L-Dopa in parkinsonian monkeys treated with AAV-hAADC or AAV-LacZ. The data show behavioral response (mean CRS SD) to acute challenges with L-Dopa (3 mg/kg im) in unilaterally lesioned monkeys 1 month before (pre) and 3, 6, 9, 12, 24, 36, 42, 54, and 60 months after infusion with either a control vector (AAV-LacZ; n = 4 until 24 months) or AAV-hAADC (n = 4 until 24 months). Control group testing ceased at 24 months, and testing for two of the AAV-hAADC-treated monkeys ceased at 36 months. Progressive improvement was seen in AAV-hAADC-treated animals at 6 months, reached a plateau at approximately 12 months, and was sustained for the duration of the experiment. Slight improvement was seen in the control group, which may be attributed to one outlier animal that appeared to recover from MPTP lesioning. Statistical significance is indicated by asterisks: **P < 0.001 by t test that compares the data to prevector CRS (pre).

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Immunohistochemistry

Fig. 3A shows a representative FMT-PET image of hAADC transgene expression on the right side of the brain at three coronal levels through striatum. The left hemisphere shows normal endogenous AADC activity because this side was not lesioned with MPTP. Corresponding TH-immunoreactive staining at three levels, anterior to posterior, in Figs. 3B and 3C from two representative animals reveals the persistent loss of nigral projections to the right striatum, but those on the left side appear normal. In contrast, however, AADC staining is robust on both the lesioned and the nonlesioned side, confirming the almost complete restoration of AADC activity in lesioned striatum induced by infusion of AAV-hAADC.

Figure 3.

Immunohistochemistry of AADC in hemilesioned monkeys before and after AAV-hAADC treatment. (A) Three [¹⁸F]FMT PET coronal images of an AAV-hAADC-treated monkey striatum at 18 months show the strongest AADC transgene expression in targeted area (midstriatum) and correlate with AADC immunostaining. (B–D) In (B) and (C), color-coded immunostaining against TH and AADC in progressive anterior to posterior coronal slices in two representative AAV-hAADC monkeys is shown at 3 years after AAV delivery. (D) shows TH and AADC staining in a representative AAV-LacZ monkey at 2 years after AAV delivery. The pattern of TH and AADC immunoreactivity, normally colocalized in intact dopaminergic pathways, is very different in these MPTP-lesioned animals. Note the dramatic reduction of anti-TH staining in the right striatum of all animals compared to the intact left side, confirming profound loss of dopaminergic fibers and terminals on the lesioned side (B–D). Anti-AADC staining shows endogenous AADC in the nonlesioned, left striatum and almost complete restoration of the enzyme within the right striatum of AAV-hAADC animals, demonstrating widespread distribution of AADC transgene expression (B and C). No such AADC staining or [¹⁸F]FMT PET signal is seen on right side of control animals (D). The box in the PET image in (D) indicates the region of infusion of AAV-LacZ.

Full figure and legend (343K)

Fig. 3D, in comparison, confirms the marked reduction of nigrostriatal fibers in the striatum of a representative AAV-LacZ control animal on the MPTP-lesioned side and no AADC activity when the right striatum was infused with the AAV-LacZ control. This observation is consistent with the lack of PET signal on the infused side of the striatum indicated in Fig. 3D on the right. Immunoperoxidase staining indicates further the even distribution of AADC activity in AADC-transduced tissue (Figs. 3B and 3C). We observed little AADC staining in control (AAV-LacZ) tissue (Fig. 3D).

Confocal microscopy revealed that AAV-transduced cells have a typical medium spiny neuron morphology (Fig. 4A). AADC immunostaining colocalized with the neuronal marker NeuN, suggesting that the majority of expressing cells were neurons. The broad distribution of AADC activity revealed by immunoperoxidase staining (Fig. 4B) compares well with parallel staining of a representative adjacent section with NeuN antibody (Fig. 4C). No adverse histopathology was present in these tissue sections or in any other sections examined by H&E staining (data not shown). The density of neurons in regions containing AADC-expression did not appear to be reduced as determined by counting of NeuN-positive cells in brains transduced with control or AADC vectors (data not shown).

Figure 4.

Confocal microscopy of hAADC staining in striatum of representative AAV-hAADC-treated monkey. (A) Confocal immunofluorescent staining ⁵ for both AADC and NeuN immunoreactivity of putaminal sections revealed that AADC and NeuN colocalized well, and the indicated neuron has a typical medium spiny morphology. The image is a confocal example of many such images from AAV-hAADC-treated monkeys. (B and C) Immunoperoxidase staining with anti-hAADC and anti-NeuN antibodies of striatal tissue showed that many neurons, indicated by NeuN staining (C), also stained positive for AADC (B). Data are from a representative hAADC-treated monkey.

Full figure and legend (318K)

Discussion

Individuals with idiopathic PD progress inexorably toward dependence on L-Dopa (Sinemet) therapy. The dilemma faced universally by such patients is that L-Dopa therapy, although very effective initially, begins to fail within a few years. There are few alternatives to the drug, and patients and clinicians share in the frustration of severely limited therapeutic options, such as dopamine agonists, MAO-B, and COMT inhibitors ^2,28,30. L-Dopa exerts its therapeutic effect through its conversion to dopamine in the brain. Progressive elimination of nigral neurons, the major source of AADC in the striatum, leads to a serious loss of efficiency of conversion of L-Dopa to dopamine. It should be noted, however, that human striatum already contains AADC⁺ neurons that do not express tyrosine hydroxylase ¹⁹. Presumably these neurons provide a residual level of L-Dopa responsiveness in human striatum, even when nigral innervation is completely eliminated. Nevertheless, progression of PD is associated with a steady increase in L-Dopa requirement, and there is evidence that L-Dopa has a pharmacological activity independent of its role as a dopamine precursor ^10,14. It seems reasonable to assume that at least part of the problem of L-Dopa-dependent side effects is due to the need for ever-increasing doses of the drug as PD advances and that improvement in conversion efficiency may alleviate at least some L-Dopa-dependent symptoms. A potential therapeutic strategy, therefore, is to use gene therapy to restore adequate levels of AADC in the striatum and thereby reestablish the satisfactory L-Dopa responsiveness initially enjoyed by PD patients.

As previously reported ⁵, expression of AADC in hemiparkinsonian monkeys induced a robust recovery in L-Dopa-dependent dopamine synthesis. In this study, we studied the functional consequences of such treatment over a very long period of time. In fact, to our knowledge, this is the longest reported monitoring of AAV-mediated transgene expression in primates. By means of the AADC-specific PET reagent, FMT, we were able to follow AADC expression in a single cohort of four monkeys for over 2 years. Two of these monkeys are still under observation and were scanned most recently at the 6-year time point. A significant increase in FMT PET signal was observed only in animals treated with AAV-hAADC, and this increase was seen only on the treated (ipsilateral) hemisphere; the nonlesioned, nontreated (contralateral) side showed no consistent changes in signal. In all AADC-treated animals, the PET signal in the treated striatum remained very stable over a long period of time. Another remarkable feature of the data (Fig. 1) is the apparent reliability of our delivery method. All four AADC-treated monkeys displayed robust restoration of striatal AADC activity and behavioral response to L-Dopa. To achieve this reliability, a technique known as CED along with a unique, reflux-resistant cannula has been developed ³¹. CED uses a slow infusion under pressure to take advantage of the pulsation of the perivascular tissue that permeates the brain down to the level of capillaries to drive very efficient distribution of viral particles ¹⁸.

We measured behavioral improvement in both control and AADC-treated animals with acute L-Dopa challenge. AADC-treated animals displayed a sharp improvement in acute response to low-dose L-Dopa (3 mg/kg) that was not apparent until more than 3 months after vector infusion. This slow improvement in clinical response is clearly at odds with the PET imaging, which reaches a plateau after 1–2 months. The reason for this discrepancy is unclear. We are still investigating this issue ⁹; but it is worth noting that AAV2, the serotype of the vector, has been reported to resist uncoating ³². Thus, the time course of transgene expression from AAV2 vectors may be more complicated than previously suspected. Expression of AADC was localized to medium spiny neurons (Fig. 4), the major neuronal population in the striatum. These GABAergic neurons express dopamine D2 receptors and mediate motor output signals from the cortex to pallidal pathways. Production of dopamine from exogenously supplied L-Dopa in these neurons would be expected to act in an autocrine fashion, primarily acting to restore dopamine D2 signaling in these cells. In this respect, one might expect the density of AADC-expressing neurons in the striatum, in addition to the overall level of enzyme per cell, to be an important driver of efficacy in this therapy. It is for this reason that we have placed great emphasis on the development of convection-enhanced delivery of AAV to the striatum to maximize the distribution of AADC expression in the primate brain.

After about 12 months, AADC-treated animals achieved a stable, much improved sensitivity to acute L-Dopa. The question of whether improved responsiveness reflects a widened therapeutic window is a difficult one to answer definitively in a small population of hemiparkinsonian animals. Ultimately, the question of whether AAV-hAADC therapy really does improve the L-Dopa therapeutic window may come from its application to human disease. We note that clinical investigation of this therapy is now under way.

Materials and methods

Induction of parkinsonism in rhesus monkeys

Parkinsonism in nonhuman primates (M. mulatta) was induced by administration of 2–4 mg MPTP into the right internal carotid artery that was followed by intravenous dosing with MPTP (0.25–0.40 mg/kg) until a baseline CRS indicating moderate to severe parkinsonism was obtained. This model shows almost complete lesioning in the ipsilateral hemisphere ¹³ and virtually none in the contralateral hemisphere (Fig. 3D).

Clinical rating scale

The modified Parkinson CRS employed here was developed by our laboratory and closely approximates those reported in the literature ²⁰. The scale evaluates 14 parkinsonian features, each of which receives a score from 0 to 3 in order of increasing severity. Individual scores are summed to arrive at a final score. Features evaluated include tremor (right and left sides), locomotion, "freezing," fine motor skills (right and left sides), bradykinesia (right and left sides), hypokinesia, balance, posture, startle response, and gross motor skills (right and left sides). Normal animals score in the range 0–4, and severely parkinsonian monkeys score in the range 30–42. In these studies, we obtained animals with a CRS from 15 to 25. An experienced rater, blind to whether animals were test or control, performed all measurements.

AAV vector

The human AADC cDNA was cloned into an AAV2 shuttle plasmid, and a recombinant AAV2 containing hAADC under the control of the cytomegalovirus promoter was generated by a triple-transfection technique ^24,33. AAV-hAADC was purified from cell extracts by CsCl centrifugation and was routinely concentrated to approximately 2–4 10¹² vector genomes (vg)/ml as determined by quantitative PCR. More recently, we have introduced a unit measurement of AAV in which 1 10⁹ vg of AAV is defined as 1.0 Unit ¹⁵. This innovation, in our opinion, facilitates dose comparisons and simplifies presentation of data.

Convection enhanced delivery

Animals were sedated, readied for surgery, and placed in an MRI-compatible stereotactic frame. Vital signs were monitored, and a bone flap was made in the skull with a dental drill to expose areas of the dura over target sites. Fused silica cannulae, prepared as previously described ⁵, were stereotactically guided into the brain with coordinates generated by MRI. Approximately 10 min after infusion, the cannulae were raised at a rate of 1 mm/min until clear of both the striatum and the overlying cortex. Animals were monitored for full recovery and clinically observed twice per day over the next 7 days.

The infusion parameters used were essentially the same as described previously ⁵. Briefly, AAV-hAADC at a concentration of 2000 Units/ml, or control AAV-LacZ at a concentration of 800 Units/ml, was infused into right hemispheres at six sites at the following rates: 0.1 l/min (60 min), 0.2 l/min (60 min), and 0.4 l/min (30 min). Total infusion time was approximately 150 min; volume per site was 30 l, and total volume per brain (lesioned side only) was 180 l. Total dose of AAV vector was 360 or 144 Units per monkey for AAV-hAADC and AAV-LacZ animals, respectively.

Positron emission tomography

PET imaging was performed essentially as described previously ^5,12. Briefly, prior to PET imaging, the monkeys were sedated with an intramuscular injection of ketamine (10 mg/kg), intubated, and maintained in a sedated state with 1–3% isoflurane ⁵. The animals were placed in a stereotactic frame identical to that used for MRI such that coronal images could be acquired. All animals were pretreated with an intravenous injection of benserazide (2.5 mg/kg), a peripheral AADC inhibitor, 30 min before injection of 3–5 mCi [¹⁸F]FMT tracer. A transmission scan (20 min) was obtained prior to the emission scan to correct for photon attenuation by means of a rotating ⁶⁸Ge source consisting of three rods of about 2 mCi/rod. Emission data for these studies were acquired immediately after the transmission scan and were collected for 1.5 to 2 h, beginning at the time of injection of the tracer. Regions of interest (ROIs) were drawn for the striatum in both hemispheres and for the cerebellum. Tissue time–activity data were obtained from each ROI and striatal uptake constants (K_i) were obtained by multiple-time graphical analysis with the cerebellum used as reference tissue ²⁹. The cerebellum was selected as a reference region because FMT uptake should be negligible and should not change between baseline and posttreatment studies. After transmission scans, animals were injected with FMT and emission data were collected for 30 min, beginning 45 min after tracer injection. Radioactivity counts were determined for each ROI and radioactivity count ratios were created with the cerebellum used as a reference tissue.

Immunohistochemistry

Sectioning and immunostaining of brain tissue were performed as described ⁵. Briefly, animals were lethally overdosed with intravenous sodium pentobarbital (25 mg/kg) and then perfused sequentially with PBS and PBS/4% formaldehyde. Brains were placed in a graded series of sucrose in PBS as a cryoprotective measure. The tissue was then cut coronally into 11 large blocks. Blocks of interest were then further sectioned into 40-m free-floating sections that were subjected to standard immunofluorescent and immunoperoxidase staining as described ⁵. Rabbit polyclonal antibodies against tyrosine hydroxylase, AADC, and NeuN were obtained from Chemicon (Temecula, CA, USA) and were used at a dilution of 1:1000, 1:2000, and 1:500, respectively.

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Acknowledgements

We thank Avigen, Inc., for the AAV-hAADC vector and are indebted to the staff of the NIH PET facility and Dr. M. Daadi for technical support. We also thank Dr. Irwin Kopin and Dr. Michael Aminoff for helpful discussions. This work was supported by the NINDS Intramural Research Program (Bethesda, MD, USA) and by Avigen, Inc. (Alameda, CA, USA).