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Clinical studies to date have failed to establish therapeutic benefit of glial cell-derived neurotrophic factor (GDNF) in Parkinson’s disease (PD). In contrast to previous non-clinical neuroprotective reports, this study shows clinically relevant and long-lasting regeneration of the dopaminergic system in rhesus macaques lesioned with 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 3–6 months prior to GDNF gene delivery (AAV2-GDNF). The observed progressive amelioration of functional deficits, recovery of dopamine and regrowth of fibers to the striatal neuropil, demonstrates that high GDNF expression in the putamen promotes restoration of the dopaminergic system in a primate model of advanced PD. Extensive distribution of GDNF within the putamen and transport to the severely lesioned substantia nigra, after convection-enhanced delivery (CED) of AAV2-GDNF into the putamen, indicates anterograde transport via striatonigral connections and is anticipated to occur in PD patients. Overall these data demonstrate non-clinical neurorestoration after putaminal infusion of AAV2-GDNF and suggest that clinical investigation in PD patients is warranted.
Because almost all Parkinson’s disease (PD) patients are clinically diagnosed only after substantial degeneration of the dopaminergic system there is a vital need to develop neurorestorative treatments. Current PD therapies provide only time-limited symptomatic relief of parkinsonian motor symptoms and do not prevent disease progression (Chen and Swope, 2007; Davie, 2008). Neurotrophic factor therapy has long been proposed as a way to mitigate neurodegenerative disease processes, and potentially stimulate regeneration of degenerated neurons (Dawbarn and Allen, 2003). Glial-derived neurotrophic factor (GDNF) is involved in prenatal development of mesencephalic dopaminergic neurons (Lin et al., 1993; Granholm et al., 2000) and is critical for postnatal survival of these neurons (Pascual et al., 2008). Therefore, GDNF and its homolog, Neurturin, have received considerable attention as therapeutic agents for the treatment of PD (Peterson and Nutt, 2008). Unfortunately, despite promising data generated in neurotoxicant models in rodents and nonhuman primates (NHP), clinical studies in PD patients so far have failed to meet primary efficacy endpoints. This suggests a deficiency in either the predictive ability of previously utilized nonclinical models or the ability to translate research protocols into effective clinical procedures.
The failure of previous attempts to efficaciously deliver neurotrophic factors to the human putamen of PD patients has been attributed to ineffective delivery (Patel and Gill, 2007). Clinical studies have revealed the significant technical challenges associated with intracranial delivery to targeted regions. Although less of an issue in small animal models, efficient distribution at physiologically relevant levels within the substantially larger adult human brain is critical for the efficacy of any neurotrophic factor therapy (Yin et al., 2009a). To facilitate translation from NHP to clinical studies, our group has invested considerable effort in the development of reflux-resistant cannulae and infusion protocols that enable delivery of clinically relevant volumes to precisely targeted structures within the brain (Varenika et al., 2009). The recent development of convection-enhanced delivery (CED) techniques, including clinically compatible real-time MRI-based targeting and visualization of vector infusions, will assist in translating this current study into an anticipated phase I AAV2-GDNF study for mid-stage PD (Fiandaca et al., 2008a).
The most universally accepted NHP model of PD pathology involves 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced lesioning of the dopaminergic system (Bankiewicz et al., 1986; Bankiewicz et al., 2001). We have previously demonstrated that the neurodegenerative process in this model occurs over a 4-week period after acute MPTP dosing (Eberling et al., 1997). Consequently, any therapeutic intervention prior to (Eslamboli et al., 2003; Li et al., 2003; Eslamboli et al., 2005) or during (Kordower et al., 2000; Kordower et al., 2006; Oiwa et al., 2006) this extended period of neurotoxin-induced lesioning should be viewed in the context of neuroprotection rather than restoration. This distinction is essential for future clinical study with PD patients who already have extensive nigrostriatal degeneration, and will likely require a neurorestorative treatment in order to gain meaningful clinical improvement. To our knowledge the present study is unique in that initiation of neurotrophic factor gene delivery was delayed until lesioning of the nigrostriatal pathway was complete to ensure a direct assessment of neuronal repair that is consistent with the expectation of the ongoing or future clinical trials testing neurorestorative potential of GDNF family of growth factors in PD.
This study was designed to assess the safety and efficacy of AAV2-GDNF in a cohort of adult Rhesus macaques rendered hemi-parkinsonian by MPTP administration. The animals were assessed in-life for up to 24 months after AAV2-GDNF or sham delivery. Three macaques were euthanized for early toxicity and distribution analysis at 1 month with the remaining animals euthanized in pairs at 6, 14, 20 or 24 months.
The protocol was approved by the Institutional Animal Care and Use Committee at the University of California San Francisco (San Francisco, CA) and the Animal Welfare and Research Committee at the Lawrence Berkeley National Laboratory (Berkeley, CA). Fourteen male rhesus monkeys (8–14 kg), and one female rhesus monkey (7 kg) were lesioned with MPTP as previously described (Bankiewicz et al., 1986; Eberling et al., 1998; Bankiewicz et al., 2000). Briefly, MPTP lesioning consisted of one or two right intracarotid artery infusions of 2.0–4.0 mg of MPTP-HCl followed by additional intravenous administrations of 0.2–0.5 mg/kg doses of MPTP-HCl. This method of lesioning produces nearly a complete dopaminergic lesion on the side of carotid artery infusion (right hemisphere) and a partial lesion in the contralateral (left) hemisphere of the brain creating a humane and stable model of PD with animals able to self maintain. In addition, this hemi-parkinsonian model allows investigation of a severe dopaminergic lesion reminiscent of advanced PD on the ipsilateral hemisphere, whereas the contralateral partially lesioned hemisphere models earlier stages of PD. Intravenous dosing with MPTP continued until each animal showed bilateral parkinsonian signs and a clinical rating scale (CRS) score between 21 and 26, as described below. Only macaques with CRS scores that were stable for at least 3 months prior to treatment were enrolled in the study. Macaques were randomly assigned to receive bilateral administration of either AAV2-GDNF (9.9 x 1011 vg; n=8) or PBS (n=7) into the putamen via CED that involved the ramped infusion of 75 μL into two stereotactic sites in each putamen (pre- and post-commissural). In previous studies, we have demonstrated that AAV2 vectors encoding control genes (i.e. LacZ) had no therapeutic effect on MPTP lesioned macaques (Bankiewicz et al., 2006; Forsayeth et al., 2006). To enable complete assessment of both the AAV2 vector and GDNF gene product the control macaques in this study received only a sham PBS infusion.
The AAV2-GDNF vector construct contained cDNA for human GDNF under the control of a CMV promoter with a 3′ human growth hormone polyadenylation sequence. The expression cassette was flanked by AAV2 inverted terminal repeats and packaged into recombinant AAV2 vector particles by Avigen Inc. by means of a standard helper free transfection method as previously described (Matsushita et al., 1998; Wright et al., 2003).
All ratings were performed by a single investigator blinded to the experimental conditions. The modified Parkinson’s CRS employed here was developed in our laboratory, and closely approximates those reported in the literature (Imbert et al., 2000). The scale evaluates 14 parkinsonian features, each of which receives a score from 0–3 in order of increasing severity (0 = normal, 1 = mild, 2 = moderate, 3 = severe). 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 hand), bradykinesia (right and left sides), hypokinesia, balance, posture, startle response, and gross motor skills (right and left hand). Normal animals score in the range 0–4, and severely parkinsonian monkeys score over 20. CRS assessments were performed after MPTP lesioning to determine a stable baseline prior to treatment. Animals were assessed both with and without L-dopa administration. Individual CRS ratings over 5–10 separate days were averaged for each time-point to provide unbiased CRS scores. To assess the L-dopa response the animals were treated with a twice-daily intramuscular injection of L-dopa at a therapeutic level determined pre-treatment and maintained throughout the study (3 – 10 mg/kg). CRS ratings were made 30 min after L-dopa administration.
Dopaminergic function was measured by 6-[18F]fluoro-l -m-tyrosine (FMT)-PET as previously described (Eberling et al., 2009). FMT-PET was performed 3 months before (baseline), and 6 months after, treatment for all animals. The long-term survival animals treated with AAV2-GDNF were rescanned 23 months after vector infusion. Parametric images of FMT influx (Ki) from both time points were generated and co-registered in order to identify functional changes after treatment. FMT influx in the putamen was thereby measured before and after treatment for each monkey.
Three macaques (2 AAV2-GDNF-treated and 1 PBS control) were euthanized 1-month after AAV2-GDNF or PBS delivery. Pairs of the remaining animals from each group were euthanized at the following time intervals after treatment, AAV2-GDNF: 6, 14 and 24 months; PBS: 6, 14 and 20 months. In brief, the unfixed brain was removed from the animal after perfusion with PBS only and sectioned into 3-mm coronal blocks. One putamen block and one block containing the substantia nigra were fresh-frozen for biochemical and protein analyses. All other blocks were fixed with 4% paraformaldehyde and processed for immunohistochemical staining.
Fresh tissue punches from the putamen were homogenized by sonication in 250 μL of 0.4 M perchloric acid, and then centrifuged for 15 min at 13,000 rpm. The supernatant solutions were filtered and the concentration of dopamine, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were determined by high-performance liquid chromatography (HPLC) as previously described (Johnston et al., 2009). Total protein was determined in the pellet fractions after re-suspension in 0.5M NaOH via a DC protein assay kit (Bio-Rad).
The tissue concentration of GDNF was determined from putamen and substantia nigra tissue punches with a commercially available GDNF ELISA kit (Promega). Fresh-frozen tissue punches were homogenized in 250 μL of lysis buffer (TropixR) supplemented with protease inhibitors (Complete mini; Roche Molecular Biochemicals). ELISA was performed as per the manufacturer’s guidelines except for color development and absorbance reading, which was replaced by a more sensitive chemiluminescent substrate kit (SuperSignalR ELISA Pico Chemiluminescent Substrate, Thermo Scientific). Total protein concentration was determined with a DC protein assay kit (Bio-Rad).
Coronal brain blocks fixed in 4% paraformaldehyde were either sectioned to 40 μm on a sliding microtome or paraffin-embedded and sectioned to 5 μm. Immunohistochemical staining was performed on sections from the caudate-putamen through to the mid-brain region with primary antibodies for GDNF (R&D Systems), TH (Chemicon), GFAP (Chemicon) and Iba1 (Biocare Medical). Polymer-based detection kits (Biocare Medical) and diaminobenzidine (DAB; Vector Laboratories) were used to complete the staining.
The extent of dopaminergic innervation of the putamen was assessed by densitometry analysis of 5 μm paraffin sections stained for TH. Images were taken of 4 different regions of each putamen in a single coronal section – dorsomedial, dorsolateral, central and ventral aspects – with a 20X objective lens. Three AAV2-GDNF-treated and 3 PBS-controls from the 14- and 24-month groups were included in this analysis. Images of the corpus callosum and cerebral cortex, devoid of TH staining, were also captured. Image J (NIH) software was used to analyze the images by thresholding the images to limit measurements to the TH-positive fibers and then obtaining an integrated density measurement by multiplying the area of TH-staining by the mean pixel intensity. To account for any staining variation between sections, the density measurements for the putaminal images were divided by the mean density of the corpus callosum and cerebral cortex from the same section. Microscope, camera and software settings remained consistent for all images.
MPTP-induced lesioning of the dopaminergic system in the adult NHP results in impaired motor control comparable to parkinsonian movement deficits and provides a reliable model for investigating restorative approaches for treating PD. The hemi-lesioned model used in this study produces a complete dopaminergic lesion in the right hemisphere, and a partial lesion in the left hemisphere. A modified parkinsonian clinical rating scale (CRS) was used to serially measure the extent of motor impairment after the initial intracarotid infusion of MPTP. All animals subsequently received systemic intravenous dosing of MPTP until they displayed bilateral motor impairment and achieved a stable CRS score of between 17–26 for at least 3 months prior to treatment. All AAV2-GDNF-treated macaques displayed considerable improvement in their CRS scores in the period between 6 and 24 months of the study, whereas PBS-treated control macaques (with the exception of one outlier) exhibited significantly less improvement in their CRS scores (Fig 1A). Although no reason for the substantial improvement of the outlier control animal was evident, it has previously been observed that infrequently an MPTP-lesioned NHP will show spontaneous behavioral recovery (Bankiewicz et al., 2006). Pairs of AAV2-GDNF-treated and PBS-control macaques were euthanized at 6, 14 and 20 (PBS controls) or 24 (AAV2-GDNF) months after treatment to provide a time-dependent immunohistochemical assessment of safety and toxicity. This protocol restricted statistical comparison of the CRS scores, although two-way ANOVA of CRS recovery revealed a very significant effect of treatment over the complete data set (p < 0.0001). When the spontaneously-recovered control macaque was excluded, separate two-way repeated measure ANOVA analyses showed significant treatment effects for animals remaining at each endpoint (6 months, p = 0.013; 12 months, p = 0.020; 18 months, p = 0.050). Improvements in CRS scores from pre-treatment baseline scores (ΔCRS) were evident three months after treatment (ΔCRS AAV2-GDNF -4.4 ± 1.0; PBS −0.5 ± 1.1; t-test p = 0.036), with progressive CRS improvement observed throughout the nine month period after AAV2-GDNF treatment with a mean 56 ± 4% reduction in CRS score (ΔCRS AAV2-GDNF −11.0 ± 0.8; PBS −2.7 ± 2.4; t-test p = 0.013).
To address the possibility that AAV2-GDNF treatment might be unsafe if combined with dopamine replacement therapy, we administered therapeutic doses of L-dopa to the macaques at various times throughout the study. All animals manifested measureable clinical responses to L-dopa with an improvement relative to their OFF L-dopa CRS scores (Fig 1B). The PBS-infused controls demonstrated a consistent L-dopa response throughout the study with a mean improvement in CRS score of 18% at both 6 and 12 months after treatment. This L-dopa response was less than the response pre-surgery (29% CRS improvement) and at 3 months (25%), probably related to the small improvement in OFF L-dopa CRS score exhibited by these controls after surgery. AAV2-GDNF-treated macaques exhibited a similar L-dopa response to the PBS-controls at 3 (25%) and 6 months (19%), but the CRS score was less affected at 12 months (11%) due to the already considerable CRS improvements due to GDNF activity. Macaques were maintained on a consistent therapeutic dose of L-dopa without any serious adverse effects. However, both AAV2-GDNF- and PBS-treated animals were more likely to become distracted from performing the tasks required to complete the CRS assessment when receiving L-dopa. One of the AAV2-GDNF treated macaques (NHP437) displayed L-dopa-induced dyskinetic movement in its left leg; however, this was not observed in any of the other macaques and may be related to the specific distribution of GDNF in this one animal (Fig S1).
PET scans were performed before and after treatment to assess in-life changes in dopaminergic terminal integrity. As detailed in our earlier report (Eberling et al., 2009), a bilateral increase in FMT uptake was observed in the AAV2-GDNF-treated macaques 6 months after vector infusion where Ki values were increased by 18 ± 5% in the moderately lesioned putamen and by 54 ± 19% in the severely lesioned putamen. PBS-control macaques did not show a significant change from baseline in FMT uptake in either hemisphere. The increased FMT uptake in AAV2-GDNF-treated macaques was directly correlated with improvements in CRS scores at 6 months. Additional PET scans performed 22 months after AAV2-GDNF treatment in the two long-term survival macaques showed further increases in FMT uptake compared to the baseline PET (Fig 1C; left putamen 37 ± 17% increase; right putamen 138 ± 63% increase). A larger increase in FMT uptake between 6 and 22 months was observed in the severely lesioned hemisphere (40–50% increase) than the moderately lesioned hemisphere (0–18% increase). The direct correlations between the increase in PET signal and improvement in CRS were maintained at 22 months for both the left (Pearson r = 0.88; p < 0.01) and right (Pearson r = 0.95, p < 0.001) hemispheres (Fig 1D,E).
To directly assess changes in dopamine metabolism after AAV2-GDNF treatment, putaminal levels of dopamine and dopamine metabolites (HVA and DOPAC) were quantified (Table 1). Fresh tissue punches were collected post mortem and analyzed by HPLC. We found no time-dependent changes in dopamine (Fig 2A,B), HVA or DOPAC levels within the AAV2-GDNF and PBS treatment groups euthanized at different time intervals (6–24 months) after treatment. As expected, after MPTP lesioning (Oiwa et al., 2003), control macaques showed reduced dopamine levels in both hemispheres with almost complete loss of dopamine in the severely lesioned right putamen (0.8 ± 0.3 ng/mg protein), and a ~70% loss in the moderately lesioned left putamen (68 ± 8 ng/mg protein) relative to dopamine levels in naïve macaques (Oiwa et al., 2003). Delivery of AAV2-GDNF into the putamen induced a three-fold increase in dopamine within the moderately lesioned left putamen (204 ± 33 ng/mg protein; unpaired t-test p < 0.01), almost completely restoring dopamine to non-lesioned levels (240 ± 19 ng/mg protein) (Oiwa et al., 2003). In contrast, dopamine levels in the severely lesioned right putamen were not substantially changed by AAV2-GDNF treatment (1.2 ± 0.5 ng/mg protein; unpaired t-test p = 0.4). Levels of dopamine metabolites were, however, substantially increased in both the severe and moderately lesioned hemispheres (Table 1). By calculating the ratio of dopamine metabolites (HVA and DOPAC) to dopamine, a relative rate of dopamine turnover can be determined. Naïve macaques typically have an HVA:dopamine ratio close to 1:1 and this ratio is substantially increased after MPTP lesioning. Consequently, both the AAV2-GDNF- and PBS-treated groups displayed bilaterally increased rates of dopamine turnover that were not significantly different between the three time points (Table 1; Fig 2C,D). Nevertheless, AAV2-GDNF delivery resulted in a further enhancement (unpaired t-test p < 0.01) of both the HVA:dopamine and DOPAC:dopamine ratios relative to the PBS controls within the severely lesioned putamen (Table 1). AAV2-GDNF treatment did not significantly alter the rate of dopamine turnover in the partially lesioned putamen, with equivalent increases of dopamine, HVA and DOPAC.
To further analyze the effect of AAV2-GDNF on dopamine in the putamen, we compared the level of dopamine and HVA with the corresponding rate of dopamine turnover in each hemisphere. In the severely lesioned hemisphere, there was a strong linear correlation between dopamine and HVA (Fig 2E; Pearson p < 0.0001) with AAV2-GDNF treatment largely resulting in an elevation of HVA levels but only a slight change in the actual amount of dopamine. This is reflected in the significantly enhanced rate of dopamine turnover (HVA:dopamine ratio) after AAV2-GDNF treatment that was highest in the AAV2-GDNF-treated macaques with the lowest dopamine levels (Fig 2G). In contrast, significantly increased HVA levels in the moderately lesioned hemisphere after AAV2-GDNF treatment were generally matched by increased dopamine levels (Fig 2F) with the result that the HVA:dopamine ratio for the majority of animals was equivalent to the PBS-treated controls (Fig 2H). Overall, this resulted in a strong correlation between dopamine levels and the HVA:dopamine ratio, where the turnover of dopamine was reduced to a relatively normal rate (~2:1) as the amount of dopamine increased to levels found in naïve macaques.
We investigated the extent of GDNF distribution by performing immunohistochemical staining on free-floating coronal sections from the caudate-putamen and mid-brain regions of the AAV2-GDNF-treated macaques (Fig 3). Extensive GDNF expression was present at the sites of CED infusion and covered a large proportion of the surrounding putamen in 12 of 16 infused hemispheres (Table 2). Without real-time imaging to monitor vector delivery (Fiandaca et al., 2008b; Su et al., 2010), it was not evident why GDNF expression was limited in the other 3 cases, although actual positioning of cannulae within the putamen largely determines the extent of vector distribution and, hence, vector transduction within the putamen (Yin et al., 2009b). Consistent with previous delivery of AAV2-GDNF vectors into the putamen, very few GDNF-positive cells in the putamen were identifiable, and most of the GDNF immunostaining did not co-localize with any cellular structures (Fig S2A). However, in our extensive experience with AAV2 vectors encoding other transgenes, including GFP (green fluorescent protein) and AADC (aromatic L-amino acid decarboxylase) (Bankiewicz et al., 2000; Hadaczek et al., 2006; Cunningham et al., 2008), we have demonstrated extensive transduction of striatal neurons that matched the pattern of GDNF distribution observed in the present animals. GDNF-positive neurons were identified in globus pallidus, subthalamic nucleus and substantia nigra, consistent with axonal transport of the AAV2-GDNF particles and/or GDNF protein (Fig 3 and Fig S2B-D). Substantial bilateral transport of GDNF to the substantia nigra was observed at all time-points indicating that transport to the midbrain is not dependent on the integrity of the dopaminergic nigrostriatal axonal projections, which were severely degenerated in the right hemisphere. Quantification of GDNF protein in the putamen and substantia nigra was obtained by ELISA of fresh tissue punches. GDNF levels in the putamen as high as 130 ng/mg total protein (mean = 24 ± 10 ng/mg total protein) were detected. No difference in GDNF expression was found between hemispheres. GDNF levels in the substantia nigra were similar for each hemisphere: Left (moderately-lesioned) 1.7 ± 0.8; Right (severely-lesioned) 1.5 ± 0.4 ng/mg protein. No GDNF protein expression was detected in any of the PBS-treated controls.
To investigate anatomical changes in the nigrostriatal pathway after AAV2-GDNF treatment, we performed immunohistochemical staining for tyrosine hydroxylase (TH) (Fig 3B and Fig 4). The severity of the MPTP-lesion was evident in the right caudate and putamen with almost complete loss of TH-positive fibers in the PBS controls. In contrast, AAV2-GDNF-treated macaques showed extensive TH-positive fiber networks within the lesioned putamen, partially along the ventral and medial regions. Increased TH-positive fiber staining was observed in the left moderately lesioned hemisphere of AAV2-GDNF-treated macaques, specifically in the putamen but also extending into the white matter tracts surrounding the globus pallidus. Integrated area-density measurements were used to quantify the enhanced TH-staining within the putamen (Fig 5). A significant increase in relative TH-staining was found in both moderately lesioned (staining intensity: AAV2-GDNF 520 ± 50; PBS 190 ± 20; unpaired t-test p < 0.01) and severely lesioned putamen (AAV2-GDNF 50 ± 10; PBS 5.5 ± 0.2; p < 0.05) of AAV2-GDNF-treated macaques compared with the PBS-controls. Enhanced TH-staining was observed across all regions of the moderately lesioned left hemisphere (2–3 fold increase compared to PBS-controls). However, in the severely lesioned right hemisphere, the ventral region of the putamen showed the greatest enhancement (~30-fold increase), with the medial and central areas of the putamen also displaying a significant 6-fold increase in TH staining.
In addition to the overall increase in TH-staining intensity in the caudate-putamen, we found large TH-positive structures primarily within the moderately lesioned hemisphere of AAV2-GDNF-treated animals at all time-points (Fig S3). We did not observe any significant change in the number, size or distribution of these structures between the 6-, 14- and 24-month animals. These TH-positive profiles were considerably less abundant in the severely lesioned hemisphere and were not observed after AAV2-GDNF delivery to intact aged macaques (Su et al., 2009). Further investigation of these TH-positive profiles revealed that they lacked nuclei (DAPI staining), did not co-localize with ubiquitin, and were not associated with activated microglia. Therefore, in agreement with our earlier assessment, we believe these profiles are caused by GDNF-induced enhancement of partially degenerated dopaminergic fibers. A potentially analogous enlargement of TH-positive fibers was observed in the putamen of a PD patient that received recombinant GDNF protein infusion (Love et al., 2005).
The extent of MPTP lesioning in the mid-brain was most apparent in the right hemisphere with almost complete loss of TH-positive neurons in the substantia nigra (Fig 4). Within the substantia nigra and ventral tegmental area (VTA) there was a considerable bilateral enhancement of TH expression in AAV2-GDNF-treated macaques relative to the PBS controls, and a clear increase in the density of TH-positive fibers. There was no evidence of any difference in the absolute number of TH-positive neurons between the AAV2-GDNF- and PBS-treated macaques. Unfortunately, quantification of the nigral neurons was not possible, since anatomically matched nigral sections were unavailable for all animals due to the collection of fresh tissue for GDNF and dopamine analysis. Disparity in the number of TH-positive neurons was also apparent within the control animals reflecting some inherent variability of the MPTP model within the substantia nigra. No change in TH immunoreactivity was evident in the left hemisphere of PBS controls when qualitatively compared with naïve macaques.
A consistent correlation was observed in the AAV2-GDNF-treated macaques between areas of GDNF immunoreactivity and enhanced TH expression within both the moderately lesioned left putamen and the mid-brain (Fig 3). The most evident correlation was observed in animals with a restricted region of GDNF expression, suggesting that GDNF has a strong local effect on TH-positive fibers within the putamen. The correlation between GDNF and TH was not as strong in the severely lesioned right putamen where the greatest increase in TH-positive fibers was found within the ventral and medial areas of the putamen, despite broad GDNF expression throughout the putamen and globus pallidus.
A detailed safety analysis was performed on all animals including a full body necropsy, blood analysis, anti-AAV2 capsid and anti-GDNF antibody titers, as well as a detailed neuropathology assessment. Consistent with the 6- and 14-month endpoints (Su et al., 2009), no long-term toxicity related to the AAV2-GDNF delivery into the putamen was observed during the 24-month study period. One of the AAV2-GDNF-treated animals in the 6-month group suffered a 20% decline in body weight. However, examination of the GDNF distribution in this animal showed high GDNF expression ventral to the anterior putamen on both sides that may have resulted in GDNF entering the CSF (Fig S1). It appeared that the anterior cannula was positioned too ventrally in this animal, thus highlighting the need for precise positioning of the cannula to ensure accurate delivery and to prevent AAV2-GDNF leakage into the CSF or other non-targeted regions. All of the other animals maintained stable food consumption and body weights throughout the study indicating that high GDNF expression within the basal ganglia after putaminal infusion does not influence appetite or body weight. No GDNF protein or antibodies against GDNF were detected in the CSF or serum. Neutralizing antibody titers against AAV capsid were measured in serum samples at up to 1:1280 post-surgery, consistent with previous reports and are now thought to be clinically unimportant (Cunningham et al., 2008; Herzog et al., 2009).
Both the AAV2-GDNF- and PBS-treated macaques exhibited enhanced GFAP expression along the cannula tracts in the putamen. Iba1-positive microglia were also found along the cannula tracts in most of the animals, including control subjects (Fig S4). None of the macaques showed any evidence of an immune response beyond the immediate site of infusion despite the extensive distribution of AAV2-GDNF and subsequent expression of GDNF in these animals.
Extensive amelioration of functional motor deficits demonstrates the clinically meaningful restorative potential of GDNF gene therapy in both moderately and severely lesioned dopaminergic systems reminiscent of early- and late-stage PD. Although protective properties of GDNF and Neurturin have repeatedly been demonstrated (Peterson and Nutt, 2008), Phase II clinical studies have failed to establish clinical efficacy (Lang et al., 2006; Ceregene Inc. press release 11/26/2008). One possible explanation for this discrepancy between nonclinical and clinical results is the integrity of the nigrostriatal neurons at the time of treatment. Previously reported gene delivery studies in NHP have, by design, primarily investigated protection of the dopaminergic system after GDNF (Kordower et al., 2000; Eslamboli et al., 2003; Eslamboli et al., 2005; Oiwa et al., 2006) or Neurturin (Li et al., 2003; Kordower et al., 2006) gene delivery in close temporal proximity to nigrostriatal lesioning. Eslamboli et al. (2005) demonstrated that GDNF expression as low as 0.04 ng/mg of tissue protected nigral dopaminergic neurons from neurotoxin-induced degeneration. It is possible that, although very low levels of neurotrophic factors are sufficient for protection, the much higher expression levels that we observed (24 ng/mg protein) combined with broad basal ganglia distribution may be necessary for restoration of the dopaminergic neurons. With an absence of early biomarkers for idiopathic PD, it is imperative that potential therapeutics are examined in animals with extensive degeneration of the dopaminergic system, with an emphasis on providing clinically relevant restoration. Development of PD models that reliably recapitulate the progressive neurodegeneration of PD will provide valuable opportunity to investigate therapeutic effects during ongoing cellular challenge, however no such model has yet been characterized in NHP. In the current study, macaques were rendered parkinsonian by MPTP intoxication and displayed stable motor impairments for at least 3 months preceding treatment. FMT PET prior to treatment confirmed the nearly complete loss of dopaminergic activity in the right putamen. This stable hemi-lesioned MPTP model was previously used in the pre-clinical development of AAV2-hAADC (Bankiewicz et al., 2006), with efficacy results that have proven to be highly predictive of clinical improvements observed in two independent Phase I human studies (Eberling et al., 2008; Christine et al., 2009; Muramatsu et al., 2009).
Recovery of motor function in AAV2-GDNF-treated NHP coincided with significant restoration of the dopaminergic system. The bilateral increase in FMT PET, an in-life measure of pre-synaptic dopaminergic activity, closely correlated with improvement in CRS scores 6 months (Eberling et al., 2009) and 24 months after AAV2-GDNF delivery. Post-mortem analysis confirmed increased dopaminergic activity and showed enhanced TH-positive fiber innervation of both the moderately and the severely lesioned putamen. An absence of change in actual dopamine levels between 6, 14 and 24 months suggest that the brain rapidly accommodates the enhanced GDNF levels, and that progressive improvements in functional behavior are associated with anatomical restoration of the lesioned dopaminergic system. It is important to note that additional functional recovery and FMT PET signal increases were observed beyond 6 months and that this recovery was fully maintained throughout the two-year study period. This relatively rapid but sustained recovery is supportive of the hypothesis that GDNF induces an up-regulation of TH in nigrostriatal neurons that survived MPTP lesioning, and that GDNF promotes the sprouting and branching of their terminal fiber networks within the putamen. GDNF has previously been shown to induce an asymptomatic down-regulation of TH in rats (Georgievska et al., 2002, 2004), but in concurrence with other NHP studies (Eslamboli et al., 2005), long-term GDNF expression in NHP results in a considerable enhancement of TH expression. Although we were unable to quantify neurons in the substantia nigra, no change in the number of TH-positive neurons was evident at any endpoint. In the severely lesioned hemisphere, we observed significant re-growth of TH-positive fibers, particularly in the medial and ventral aspects of the putamen where it is likely that some residual dopaminergic fibers survive lesioning and are able to respond rapidly to GDNF expression. In the moderately lesioned hemisphere, we observed enhanced TH expression, especially within the areas of GDNF expression, enlarged TH-positive fibers and the presence of large TH-positive profiles. Although we do not know what function, if any, these TH-positive profiles have, we found no associated pathology. Such profiles were not observed in unlesioned aged NHP that received higher doses of the same AAV2-GDNF vector (Su et al., 2009). The consistency of dopamine levels and the rate of dopamine turnover at the three different end-points is significant from a safety perspective, as this suggests that persistent high-level expression of GDNF in the basal ganglia after AAV2-GDNF delivery to the putamen is well tolerated.
In addition to significant efficacy data, AAV2-GDNF gene transfer in the NHP brain appeared to be well tolerated. The dose of AAV2-GDNF infused resulted in considerable spread of AAV2-GDNF into the surrounding white matter tracts, and both cortical and sub-cortical structures. This was designed to address safety issues from both high-level GDNF expression in the putamen and other associated areas under possible circumstances where AAV2-GDNF delivery might not be completely confined to the putamen. Therefore, in addition to the putamen, AAV2-GDNF-transduced neurons were observed within the globus pallidus, caudate nucleus, and cerebral cortex adjacent to the putamen or along the cannula tract. Possible adverse effects from GDNF expression were only observed in one macaque in which the cannula was too deeply positioned. All of the other macaques showed complete tolerance of the parenchymal GDNF expression. In contrast to AAV2-GDNF delivery to the mid-brain (Su et al., 2009), transport of GDNF to the substantia nigra after putaminal delivery did not induce weight loss. GDNF-induced weight loss has been reported after intra-ventricular protein delivery to humans (Kordower et al., 1999; Nutt et al., 2003) and also in rodents after gene delivery to the substantia nigra or hypothalamus (Tumer et al., 2006; Manfredsson et al., 2009). Overall, the lack of any apparent safety or toxicity after putaminal delivery, including immune responses, and continued responsiveness to L-dopa administration without serious adverse effect, supports initiation of a clinical AAV2-GDNF study in PD patients.
This study demonstrates that AAV2-GDNF infusion into the putamen by CED can provide broad GDNF distribution that is not limited to the putamen but includes both adjacent basal ganglia nuclei and distally located nuclei, such as subthalamic nucleus and substantia nigra. The presence of GDNF in the substantia nigra appeared to be largely dependent on achieving good distribution of GDNF in the putamen and was independent of the integrity of the dopaminergic nigrostriatal neurons, suggesting that this distribution is largely dependent on anterograde transport of AAV2-GDNF vector particles and/or GDNF protein. Therefore, it is anticipated that anterograde transport and distribution of GDNF will also occur in PD patients where the striatonigral projections to the substantia nigra pars reticulata are maintained, irrespective of degree of nigrostriatal degeneration. Although it is possible that some retrograde transportation of GDNF from putamen to substantia nigra may have occurred on the moderately lesioned side, we did not observe any retrograde transport to the cortex via corticostriatal projections, which is consistent with other studies where AAV2 vectors were delivered into the putamen (Hadaczek et al., 2006; Cunningham et al., 2008). In concordance with our previous observations of AAV2-GDNF transport from the substantia nigra to the striatum in aged NHP (Johnston et al., 2009) and along thalamocortical projections (Kells et al., 2009), anterograde transport is the predominant model of GDNF spread within the NHP brain after AAV2-GDNF delivery.
Negative results in previous neurotrophic factor Phase II human clinical studies for PD investigating either recombinant GDNF protein infusion (Nutt et al., 2003; Lang et al., 2006) or AAV2-Neurturin gene transfer have been attributed to a lack of protein distribution within the basal ganglia. Inconsistencies in the delivery system have each been identified as potential reasons for the disparity of results observed in the GDNF protein infusion trial (Patel and Gill, 2007). Although subjects in the open-label Phase I GDNF studies reported considerable clinical benefit after CED-based delivery (Gill et al., 2003; Patel et al., 2005), the multicenter Phase II study was performed with microinjection of GDNF and reported no significant clinical improvement compared with sham-treated subjects (Lang et al., 2006). In the recently reported Phase II AAV2-Neurturin study, distribution of Neurturin after microinjection into 8 sites per hemisphere was calculated in 2 subjects to have covered only 15% of the putamen (~1.5-mm radius of Neurturin coverage around infusion sites) with no transport to the substantia nigra (Ceregene Inc. NIH RAC meeting June 2009). The lack of clinical improvement in this study was attributed to absence of Neurturin in the substantia nigra and was suggested (in the absence of supporting data) to be due to deficient nigrostriatal retrograde transport rather than poor coverage of the putamen. Subsequently, a second Phase I/II clinical study has been initiated in which AAV2-Neurturin will be microinjected into both the putamen and substantia nigra (ClinicalTrials.gov NCT00985517). Although we agree that neurotrophic factor distribution within both the putamen and substantia nigra is key to the achievement of the full therapeutic potential of either GDNF or Neurturin, our current findings suggest that this can be safely and reliably accomplished by improving infusion to the putamen alone. Comparison of GDNF distribution in the macaque putamen to the dimensions of the human putamen (Fig S5) suggests that CED of AAV2-GDNF at the same volume (75 μL/site) delivered in this study would cover a significant proportion of the target region, although even larger volumes are anticipated in the clinical study. To ensure optimal delivery in clinical studies we have extensively evaluated image-guided positioning of reflux-resistant infusion cannula with real-time MR imaging of AAV2-GDNF distribution during CED (Fiandaca et al., 2008a; Su et al., 2010). Only by guaranteeing that the AAV2-GDNF vector is accurately delivered into the putamen, with extensive and reproducible coverage of the targeted region, can a fair assessment be made regarding the clinical safety and efficacy of AAV2-GDNF gene therapy as a treatment for PD.
This study was funded by an NIH-NINDS Cooperative Research Agreement U54 NS045309.