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# Translational research for Parkinson's disease: The value of pre-clinical primate models

Romina Aron Badin, Marta Vadori, Emanuele Cozzi, Philippe Hantraye

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Romina Aron Badin, Marta Vadori, Emanuele Cozzi, Philippe Hantraye. Translational research for Parkinson's disease: The value of pre-clinical primate models. European Journal of Pharmacology, 2015, 759, pp.118 - 126. 10.1016/j.ejphar.2015.03.038. hal-04453956

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# Translational research for Parkinson's disease: The value of pre-clinical primate models

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Romina Aron Badin<sup>a,*</sup>, Marta Vadori<sup>b</sup>, Emanuele Cozzi<sup>b,c</sup>, Philippe Hantraye<sup>a</sup>

## ARTICLE INFO

**Article history:**

Received 16 January 2015

Received in revised form

10 March 2015

Accepted 12 March 2015

Available online 2 April 2015

**Keywords:**

NHP

Viral vectors

Cell transplantation

Functional assessment

Translational neuroscience

## ABSTRACT

Animal models have been highly questioned for their ability to predict the efficacy of different therapeutic strategies for neurodegenerative diseases. The increasing number of phase I/II clinical trials that fail to proceed to further stages of drug development has discredited the pertinence of such investigations. However, critical analysis of the data has often revealed errors and partially explained the lack of efficacy, opening the way to a refinement in designing pre-clinical studies.

In parallel, many promising methods of drug delivery to the brain such as gene therapy or cell therapy have considerably advanced thanks to the clinical failures in the past 10 years. As methodological advances appear and knowledge becomes available, scientists will be faced with the choice of how to test new strategies or re-test old ones. With the hardening of social views and legislation regarding animal experimentation, there is increasing pressure to find alternative methods of assessment that predict efficacy (such as computational based models), or to perform efficacy trials directly in patients and only safety assays in animals.

In this review we will focus on Parkinson's disease and on the impact of a body of data issued from NHP studies. We will attempt to critically examine the advantages and limitations of various approaches from the perspective of the animal model used to address specific questions.

© 2015 Elsevier B.V. All rights reserved.

## 1. Introduction

Translational research in neurodegenerative diseases is still struggling to make significant advances in prevention, diagnosis and treatments despite the wealth of information provided by preclinical research. Although many experimental models have given invaluable insight on the pathological processes underpinning neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease

(PD), our understanding of the triggering events, and the biological correlates of the rates of progression and severity of a given disease still remain unclear. However, many advances have been made and have helped to develop new therapeutic strategies against protein unfolding for example or to characterize the contribution of specific genes to the neuropathological process. These strategies have been mainly tested in rodent models of disease, some have been tested in NHP models and a small percentage is currently being tested in the clinic. The key to their success will be to have enough insight into the mechanisms leading to the disease, in order to identify the molecular/cellular dysfunctions driving the pathological cascade and target them with either neuroprotection or disease modification strategies. This remains one of the main reasons for developing animal models of diseases (Fig. 1).

The clinical description of a constellation of symptoms triggers a working hypothesis of a particular molecular/cellular aspect of the human disease of interest. NHPs can be used to mimic the phenotypic, genetic, histopathological aspects of the disorder (bed-to-bench approach). As fundamental knowledge of the disease arises from observations made in the models, these can be confronted to novel clinical findings. Such progressive iterative loops allow both the refinement of the models and a better understanding of the

**Abbreviations:** PD, Parkinson's disease; SNpc, substantia nigra pars compacta; L-DOPA, Levodopa, 3,4-dihydroxyphenylalanine; 6OHDA, 6-hydroxydopamine; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; AADC, L-aromatic amino acid decarboxylase; TH, tyrosine hydroxylase; GCH1, GTP cyclohydrolase 1; BH4, 5,6,7,8-tetrahydro-L-biopterin; AAV, adeno-associated viruses; EIAV, equine infectious anemia virus; UPDRS, unified Parkinson's disease rating scale; PET, positon emission tomography; [¹⁸F]-FMT, fluoro-meta-tyrosine; GABA, gamma-amino butyric acid; GAD, glutamic acid decarboxylase; GDNF, glial-cell-derived neurotrophic factor; NRTN, neurturin; MRI, magnetic resonance imaging; GP, Globus pallidum (i, internal, e, external); GI, gastro-intestinal; VMAT2, vescicular monoamine transporter 2; NA, noradrenaline; 5HT, Serotonin; AchE, acetylcholine; HLA-DR, human leukocyte antigen complex; Iba1, Ionized calcium-binding adapter molecule 1

* Corresponding author. Tel.: +33 146548529; fax: +33 146548451.

<footnote><sup>a</sup> MIRCen, CEA UMR 9199, 18 Route du Panorama, Fontenay-aux-Roses 92265, France</footnote>

<footnote><sup>b</sup> CORIT (Consortium for Research in Organ Transplantation), Padua, Italy</footnote>

<footnote><sup>c</sup> Transplant Immunology Unit, Department of Transfusion Medicine, Padua University Hospital, Padua, Italy</footnote>
<figure><img src="image_3.png" /><figcaption>Fig. 1. General principles in translational research: step 1.</figcaption></figure>

human disorder. In this process, multiple models can therefore be generated at any given time to describe different aspects of the disease thus enriching their face and construct validity.

Ideally, a model of neurodegenerative disease should be triggered by the same (or at least similar) molecular/cellular alterations as the human disease itself. It should also replicate at best the diversity of cell loss that has been identified in patients as well as their known rate of progression. Finally, the model should also mimic the diversity of symptoms, which will depend heavily on the behavioral repertoire of the animal species selected to develop the model.

In the case of PD, the rationale adopted by the community to develop a model was first to postulate that a dopamine deficiency was the primary consequence of this human pathology. Then, the next step relied on the elaboration of an experimental paradigm capable of mimicking/testing this hypothesis in an animal species. To this purpose, well-controlled methods capable of reproducing this primary event leading to PD had to be developed and validated. Finally, it remained to expect that in the animal species selected for this purpose, a progressive dopaminergic dysfunction would trigger the symptoms, disease evolution and cell degeneration similar to those observed in the human condition.

## 2. Parkinson's disease

Parkinson's disease is a progressive neurodegenerative disorder clinically characterized by motor symptoms like tremor, rigidity, akinesia and postural imbalance. Although it is well established that it is the decrease in dopamine availability in the striatum that gives rise to these symptoms, the causes are still unknown. Dopaminergic deafferentation of the caudate and putamen in particular results from the loss of axonal projections coming from dopaminergic neurons located in the substantia nigra pars compacta (SNpc) and 50-70% of these neurons are lost before the appearance of clinical symptoms (Lee et al., 1994; Bonnet et al., 2012).

The focus over the last decades has been to palliate the motor component of the disease but PD is a multisymptomatic neurological disease that includes cognitive and psychiatric non-motor symptoms. In fact, manifestations such as depression, hypomania and visual hallucinations are not uncommon, alongside with executive symptoms and dementia. Depression, often associated with anxiety, classically precedes the first motor symptoms in 50% of cases. Other common symptoms are reduced appetite, sleep disturbances, loss of libido, fatigue, and inability to work (Wirdefeldt et al., 2011; Bonnet et al., 2012).

Currently, there is not curative treatment to prevent or slow the progression of disease in PD patients. Therapies available aim at controlling symptoms and include both pharmacological replacement and surgical approaches but display severe adverse events. Among these, the most effective and the most commonly prescribed symptomatic pharmacotherapy remains the precursor of dopamine, L-3,4-dihydroxyphenylalanine (L-DOPA). However, chronic administration

leads to an increase in motor fluctuations and abnormal movements termed dyskinesias in 80% of patients between 5 and 8 years of use, that are thought to arise with the increasing loss of dopaminergic neurons over time and the intermittent availability of L-DOPA (Lee et al., 1994; Wirdefeldt et al., 2011; Bonnet et al., 2012).

The pathological hallmark of the disease are Lewy bodies, inclusions present in the brain that result from the accumulation of α-synuclein and other proteins that are thought to begin years before the appearance of motor symptoms. Only a minority of PD patients carries genetic mutations and over 90% are sporadic cases where other genetic predispositions, age and environmental factors such as toxin exposure are thought to be determinant risk factors (Wirdefeldt et al., 2011).

<figure><img src="image_4.png" /><figcaption>Fig. 2. General principles in translational research: step 2.</figcaption></figure>

## 3. Modeling Parkinson's disease in animals: a translational bed-to-bench approach

After the initial description of the disease by James Parkinson which was essentially describing a syndrome with no indication of any mechanism of cell death or even a description of a particular cell degeneration associated with this condition, the development of a pertinent animal model of PD remained difficult (Parkinson, 1817). It is only after a specific method was developed to identify the dopamine-producing cells in the brain (Fuxe et al., 1970) that the severe degeneration of the nigrostriatal dopamine pathway was identified in Parkinson's disease. Moreover, it was only after Hornykiewicz et al. discovered that striatal tissue levels of dopamine and its metabolites were profoundly depleted in parkinsonian patients that some experimental models could be proposed and evaluated and even an effective palliative treatment (L-DOPA therapy) tested (Ehringer and Hornykiewicz, 1960).

Although PD is now recognized as a more complex disorder, in those early days, the pathology was principally recognized as a 'pure' dopaminergic deficiency. It is therefore understandable that the first attempts made to develop an animal model were initially targeting the nucleus at the origin of the dopaminergic nigrostriatal path i.e. the ventral mesencephalon. At that stage, the only experimental method available to induce a lesion of a given brain nucleus was the in situ delivery of an electric current delivered through stereotactic placement of an electrode (Agid et al., 1974), so-called electrolytic lesion. Despite the fact that lesioned animals presented some symptoms reminiscent of akinesia, one of the cardinal features of PD, it appeared subsequently that this type of model was suboptimal – as it severed passing fibers in addition to the cell bodies of the 'treated' region.

Again, it was only after the discovery of 6-hydroxydopamine, a neurotoxic analog of dopamine, that an almost pure severing of the nigral dopaminergic neurons could be achieved in experimental animals, giving raise to the well-known 6-OHDA lesion models,
which became the gold standard replicates for PD (Viallet et al., 1984). However, it was quickly recognized that the acute nature of the cell loss induced in these models could not reproduce the typical progressive degeneration observed in the human condition. This was a major limitation in view of the potential use of this model in preclinical trials to assess neuroprotective strategies.

Serendipitously, another dopamine selective toxin, MPTP, was discovered in the mid 80s (Langston et al., 1983) inducing chronic parkinsonism in healthy humans following systemic repeated injections. For many years, the MPTP lesion model, in particular when developed in NHP, has been and still is considered as the most predictive animal model of PD. However, whereas the pathological and biochemical changes produced by this neurotoxin are quite similar to the well-established changes observed in patients with parkinsonism, the model is not able to reproduce some of the histopathological features typical of PD, the Lewy bodies (Halliday et al., 2009; Fornai et al., 2005). The current research efforts in this field focus now on the development of models reproducing the α-synucleinopathy of the nigrostriatal pathway (Maries et al., 2003). This remains highly dependent on our ability to either overexpress locally in the substantia nigra various α-synuclein protein species or create pertinent transgenic animal models (Niu et al., 2014).

All this explains why modeling PD in animals has always been heavily dependent on a bed-to-bench and bench-to-bed translational approach allowing a true cross-fertilization between clinical and preclinical findings for a better understanding of the physiopathology of this disorder (Fig. 2).

For a given model of the disease that has been fully characterized and that best reproduces one or more specific aspects of the human condition, the translational therapeutic intervention paradigm allows to test the functional efficacy of a wide variety of treatment strategies. An iterative process may then allow the direct comparison of different treatments (i.e. drug-, cell-, gene-based therapy) as well as the progressive refinement of a given approach (i.e. different viral vectors, cell types or drug formulations for the same target).

In addition, the development of the model itself deeply relied on the timely availability of specific technical know-how, enabling preclinical researchers to reproduce those clinical observations in experimental animals. This also shows that over the years, modeling PD in animals has been a very active line of research (Bezard and Przedborski, 2011; Porras et al., 2012). However, despite these long-standing efforts, a robust model of PD, bearing both motor and non-motor complications, still remains to be developed and validated. A summary of the different features described in the currently available NHP models of PD is reported in the table below.

<table><thead><tr><th rowspan="3">PD features</th><th rowspan="3"></th><th colspan="4">NHP MODELS</th></tr><tr><th>MPTP chronic</th><th>6-OHDA</th><th>α-synuclein</th><th>Tg α-synuclein</th></tr><tr><th>Systemic neurotoxin injections</th><th>Systemic and local neurotoxin injections</th><th>Local unilateral viral vector injection</th><th>Lentivirus</th></tr></thead><tbody><tr><td rowspan="4">Motor</td><td>Akinesia/ bradykinesia</td><td>Severe bradykinesia and akinesia. Stable parkinsonian syndrome; partial behavioral recovery possible (Burns et al., 1983; Hantraye et al., 1993; Bezard et al., 2001)</td><td>Drug-induced rotations and reduced spontaneous motor activity. Hemineglect on sensorimotor tests (Annett et al., 1992). Hypokinesia and deficits in operant tasks (Apicella et al., 1990). Akinesia and climbing deficits. Drug-induced rotations. Fine motor skill deficits. Spontaneous recovery (Apicella et al., 1990; Annett et al., 1992; Eslamboli et al., 2003a). No change (Joers et al., 2014a)</td><td>Motor coordination deficits (Eslamboli et al., 2007), no robust amphetamine rotation deficits (Kirik et al., 2003)</td><td>None (Niu et al., 2014)</td></tr><tr><td>Tremor</td><td>Resting and action tremor present (Burns et al., 1983; Hantraye et al., 1993)</td><td>No tremor (Annett et al., 1992; Eslamboli et al., 2003a; Joers et al., 2014a)</td><td>No tremor (Kirik et al., 2003; Eslamboli et al., 2007)</td><td>None (Niu et al., 2014)</td></tr><tr><td>Posture/ rigidity</td><td>Postural changes present (Burns et al., 1983; Hantraye et al., 1993)</td><td>Head posture change (Annett et al., 1992; Eslamboli et al., 2003a)</td><td>Head posture change (Kirik et al., 2003; Eslamboli et al., 2007)</td><td>None</td></tr><tr><td>Cognitive</td><td>Executive function, memory and attentional deficits present (Schneider, 1990)</td><td>Postural change and falling (Apicella et al., 1990)</td><td>Not assessed</td><td>(Niu et al., 2014)</td></tr><tr><td colspan="2">Non-motor</td><td></td><td>Not assessed</td><td>Not assessed</td><td>Learning and memory deficits</td></tr></tbody></table>
<table><tr><td></td><td>Decamp and Schneider, 2004)</td><td></td><td></td><td>(Niu et al., 2014)</td></tr><tr><td rowspan="2">Non-cognitive</td><td>Sleep disturbances (McDowell and Chesselet, 2012; Hyacinthe et al., 2014).</td><td>Cardiac sympathetic neurodegeneration and disautonomia (Joers et al., 2014b)</td><td>Not assessed</td><td>Only sleep disturbance assessed and absent (Niu et al., 2014)</td></tr><tr><td>Circadian rythm changes (Fifel et al., 2014).</td><td>GI, urinary, sexual, mood/apathy: not addressed</td><td>Not assessed</td><td>Not assessed</td></tr><tr><td>Pathology</td><td>Electrophysiology</td><td>Changes in GPe, GPi and STN neuronal firing rates (Filion et al., 1991; Nambu and Tachibana, 2014)</td><td>Not assessed</td><td>Not assessed</td></tr><tr><td></td><td>Neurotransmission</td><td>Severe DA loss in striatum. Severe decrease in striatal 5TH, NA and ACEh levels (Pifl et al., 1991)</td><td>DA decrease in striatum and frontal ctx; NA decrease in frontal and parietal ctx, hypothalamus; 5HT decrease in striatum (Annett et al., 1992). DA depletion in striatum and GP (Apicella et al., 1990)</td><td>Not assessed</td></tr><tr><td rowspan="3">Histology</td><td>Severe TH depletion in SN and striatum (Burns et al., 1983; Verastet et al., 1994; Bezard et al., 2001; Jan et al., 2003)</td><td>Severe TH depletion in SN and striatum (Annett et al., 1992). Decreased 5HT in GP. Decreased cell counts in SN (Apicella et al., 1990). Mild TH decrease in SN and striatum (Eslamboli et al., 2003a).</td><td>DA degeneration in nigro-striatal neurons, decrease in TH cell counts and VMAT2; α-synuclein positive inclusions in SN; Iba1 positive microglia in SN (Barkholt et al., 2012)</td><td>Not completed (Niu et al., 2014)</td></tr><tr><td>α-synuclein aggregates (Kowall et al., 2000; Purisai et al., 2005; Fornai et al., 2005)</td><td>HLA-DR increased staining throughout brain vasculature; no robust change in TH; no change in α-synuclein (Joers et al., 2014a).</td><td>No Lewy bodies</td><td></td></tr><tr><td>No Lewy bodies</td><td>No Lewy bodies</td><td></td><td></td></tr></table>

### 4. The MPTP model of Parkinson's disease: strengths and pitfalls

The use of the NHP model of PD has not been always successful in predicting the efficacy of a therapeutic strategy in the clinic. Nonetheless, the MPTP model has been used to test effects unilaterally by creating a hemi-parkinsonism, or bilaterally, by systemic injection of the toxin. In the latter case, many different intoxication regimes exist by varying the doses, the frequency and duration of the intoxication period (Bezard et al., 2014). So even if the MPTP model has good face validity by recapitulating the motor symptoms of the disease, it bears limited validity for the increasingly important non-motor symptoms that remain less characterized than in the clinic. Although the predictive validity to test pharmacotherapies has been explored in this model for decades, clinical trials have shown that the degree of functional recovery in PD patients tends to be less significant and often confounded by a big placebo effect.

The construct validity is hard to achieve in a model of acute or semi-chronic intoxication compared to a progressive disease mechanism that takes decades to manifest symptoms. Some of

the main limitations are the possibility of a spontaneous functional recovery and compensation, the lack of the protein aggregates that characterize the pathology in humans and the lack of environmental factors that could be determinant in the manifestation and severity of symptoms. However, MPTP primates show a characteristic histopathological gradient of tyrosine hydroxylase loss in the caudate and putamen and in the SN as well as other non-striatal structures like the brainstem and cortex that is also observed in patients (Bezard et al., 2014). Moreover, α-synuclein protein levels are altered in MPTP primates even if Lewy bodies have never been reported to date (Hantraye et al., 1993; Fornai et al., 2005). Finally, environmental stresses related to infection, toxin exposure, nutritional habits or social interaction cannot be adequately assessed in this model nor can genetic predispositions and gene variants that increase the risk of developing sporadic PD. Some researchers have however developed the model in aged primates in order to better mimic the metabolic, cellular and immune instability that overcomes with age (Zhang et al., 2000). So although construct validity is not fully met, the model can address specific questions of the disease and help elucidate certain mechanisms triggered by the intoxication that resemble the pathological outcome seen in
patients. Causative links for biological phenomena must be explored in different model systems including, for example, patient-derived induced pluripotent stem cells, and then tested in NHP as a validation of a potential therapeutic target. Other hypotheses, however, can benefit from the use of primate models to test the mechanism of disease progression. In fact, the role of α-synuclein deposition has been debated for decades as causative or a consequence of the disease. Researchers are currently testing the hypothesis that the accumulation of α-synuclein is causative and precedes the loss of dopaminergic neurons in the SN by injecting fibrils or Lewy body extracts into the brain but also into the gastrointestinal tract (Maries et al., 2003; Chu and Kordower, 2007). A new model could then be developed and characterized, ideally yielding a slower progressive neurodegeneration accompanied by protein aggregation and chronic neuroinflammation. Moreover, the unique opportunity that NHP offer is to test circuitry effects thanks to the similarity in neuronal populations to humans. In fact, it is well established that several neurotransmitter systems are affected in PD and the balance and compensatory increase or decrease in such neurotransmitters gives rise to motor and non-motor symptoms such as depression, compulsivity, apathy and executive dysfunction (Alexander, 2004). These symptoms do not normally respond to dopaminergic drugs and a better understanding and management of the disease could be achieved by reproducing and characterizing these symptoms biochemically and phenotypically in primate models.

**5. Primate models in the evaluation of novel therapeutic strategies**

Because the dopaminergic neurons in the substantia nigra pars compacta have the striatum as their main target, alternative strategies to pharmacotherapy include dopamine replacement therapy using viral vectors or cell therapy.

**5.1. Gene therapy**

Dopamine synthesis in the brain requires the activity of many enzymes but the two rate limiting ones are tyrosine hydroxylase (TH) and the L-aromatic amino acid decarboxylase (AADC). For TH to function correctly, the GTP cyclohydrolase 1 (GCH1) together with two other enzymes are needed to produce its cofactor, 5,6,7,8-tetrahydro-L-biopterin (BH4). On the basis of this well known enzymatic pathway, gene therapy strategies have been developed using viral vectors such as lentiviruses of adeno-associated viruses (AAV). In fact, several clinical trials for PD using such approaches are currently ongoing (Gene Therapy Clinical Trials Worldwide, 2013). These trials involve single vectors or co-injections of several vectors into the striatum that express TH-AADC-GCH1, TH-GCH1 or only AADC (Muramatsu et al., 2002; Shen et al., 2000; Azzouz et al., 2002; Jarraya et al., 2009). These strategies have been tested in 6OHDA rats and macaques monkeys, both rhesus and cynomolgus. The two most advanced strategies in the clinic are the use of the equine EIAV lentiviral vector overexpressing TH-AADC-GCH1 and the use of an AAV vector overexpressing AADC in putaminal cells. Both therapeutic strategies have reached their primary safety end-points in phase I/II. Both secondary efficacy end-points show a modest 30% improvement of motor symptoms in OFF medication UPDRS scores, the most commonly used clinical scale to follow progression of PD (Palfi et al., 2014; Christine et al., 2009; Eberling et al., 2008; Muramatsu et al., 2010; Novel Partnership, 2013). Both have been tested in macaques and the therapeutic efficacy of the vectors assessed by clinical scoring adapted from UPDRS scales, dopamine production measured by microdialysis or positron emission tomography (PET) imaging with fluoro-meta-tyrosine ([¹⁸F]-FMT) suggest much higher effects. The

model used was always the MPTP model, but the lentiviral vector in the ProSavin® study was injected into the post-commissural putamen bilaterally whereas the AAV-hAADC was delivered unilaterally.

A different strategy looked at increasing the production of the inhibitory neurotransmitter GABA by overexpressing the gamma-aminobutyric acid gene in glutamatergic neurons of the subthalamic nucleus. Since the hyperactivity of the sub-thalamic nucleus has been identified in PD patients and successfully treated by deep brain stimulation, this strategy provides an alternative to a risky surgical procedure to implant electrodes in an atrophied brain and prevents the presence of electric currents that affect the targeted but also unwanted neighboring cell populations.

This strategy was shown to be efficacious in pre-clinical models of PD (Emborg et al., 2007; Lee et al., 2005; Luo et al., 2002), and safe in an open label phase I clinical trial where patients received unilateral sub-thalamic nucleus mixed injections of AAV-GAD65 and AAV-GAD67 viral vectors (Kaplitt et al., 2007). The OFF medication UPDRS scores were improved at 6 months post-injection and no further information on this clinical trial is currently available (LeWitt et al., 2011).

An alternative way of improving dopaminergic function indirectly is by boosting neuroprotection using trophic factors. Two compounds have been heavily characterized, the glial-cell-derived neurotrophic factor (GDNF) and neurturin (NRTN). Direct intraventricular or intracerebral delivery of the GDNF protein showed efficacy in neurotoxic models of PD (Beck et al., 1995; Gash et al., 1996; Kirik et al., 2000; Miyoshi et al., 1997; Tomac et al., 1995) while results were only significant when GDNF was delivered intraputamenally in PD patients (Gill et al., 2003; Lang et al., 2006; Amgen, 2004a, 2004b). However, the phase II trial did not meet its endpoint since no improvement in UPDRS scores was observed at 6 months and 10% of advanced patients were found to carry antibodies against the GDNF protein (41–43). In order to avoid immunization against the therapeutic protein, a lentivirus over-expressing GDNF was unilaterally delivered into the striatum and SN of healthy aged (Kordower et al., 1999) and young parkinsonian (Kordower et al., 2000) NHP. Both experiments showed an increase in fluorodopa uptake in the treated hemisphere compared to baseline [¹⁸F]-F-DOPA PET scans and a concordant increase in TH immunostaining in the striatum. Aged healthy primates were injected and assessed three months following viral delivery whereas young primates were injected 1 week before inducing parkinsonism via a unilateral intracarotid injection of the MPTP toxin. The neuroprotective effects of GDNF were then further confirmed using an AAV2-GDNF viral vector in unilaterally 6OHDA-lesioned marmosets where the viral vector was delivered into the SN 4 weeks before lesioning (Eslamboli et al., 2003b; Eslamboli et al., 2005). More data was accumulated in healthy aged and unilaterally lesioned MPTP monkeys and results showed an improvement in clinical score and in [¹⁸F]-FMT uptake in the injected putamen in PET scans (Eberling et al., 2009; Johnston et al., 2009; Kells et al., 2010). Although these results were robust and replicated in different laboratories, the predictive value of such an improvement is conditioned to the models used to tests it: intact aged monkeys or monkeys bearing unilateral lesions in order to use the contralateral hemisphere as an internal control are not representative of the clinical situation. Moreover, the neuroprotective effect of this therapy will not be helpful in the clinical setting unless it is administered early and possibly at a pre-symptomatic stage of the disease and tested as a disease modifying agent that prevents further degeneration of niagral dopaminergic neurons. Nevertheless, an open label phase I clinical trial using AAV2-GDNF is currently ongoing (uniQure, 2012; uniQure, 2013; AMT, 2008).

Similarly to GDNF, direct protein infusion or intraparenchymal delivery of engineered cells expressing another trophic factor, NTRN, was shown to promote dopaminergic neuronal survival
in vitro and in vivo in the 6-OHDA rat model of PD. The AAV2-NTRN viral vector was shown to be efficacious and safe in several rodent and primate models of PD where functional recovery and dopaminergic neuron preservation were observed in the absence of adverse effects or virally-induced immunogenicity. The clinical product, CERE-120 showed efficacy in unilaterally-injected primates 8 months following viral delivery as shown by ¹⁸F-Fluorodopa PET scans and TH staining in the striatum (Fjord-Larsen et al., 2005; Herzog et al., 2007; Horger et al., 1999; Kordower et al., 2006; Kotzbauer et al., 1996). The clinical trials initiated thereafter showed safety (Marks et al., 2008) but not efficacy at 12 month post-injection in 38 PD patients (Marks et al., 2010). The availability of brain tissue from 2 of these patients stained for NTRN showed poor viral transduction in the targeted putamen and SN neurons compared to previous primate studies. Since an improvement in clinical scores was observed only at 15 and 18 months post-viral delivery, the authors suggested that unlike primate models, advanced PD patients could have differences in retrograde transport of the therapeutic protein that delayed the protection of nigral neurons (Bartus et al., 2011a). In the light of these findings, the next clinical trial targeted viral delivery to both SN and putamen and the primary end-point was assessed at 15 and 24 months after treatment (Bartus et al., 2011b; Su et al., 2009). The lack of efficacy on UPDRS scores of this clinical trial, however, resulted in the discontinuation of this therapeutic strategy altogether (Baumann et al., 2012; Ceregene, 2013a, 2013b).

## 5.2. Cell therapy

A different approach to replenishing the striatum with dopamine is grafting dopaminergic cells that continuously supply neurotransmitters to the neighboring tissue. For decades now researchers have tested and showed the feasibility and efficacy of different types of grafts into the striatum including adrenal medullary dopaminergic cells, human fetal ventral mesencephalic cells, porcine fetal ventral mesencephalic tissue and retinal pigment epithelial cells (reviewed in Freeman and Widner (1998); Backlund et al. (1985); Arjona et al. (2003); Schumacher et al. (2000)). The long-term survival of allogenic (Redmond et al., 2008) and human xenografts (up to 24 months) (Lee et al., 2014) can be achieved in the primate brain even in the complete absence of immunosuppression. Only 12 week survival was reported for porcine neural xenografts in immunosuppressed primates (Cicchetti et al., 2003), although up to 6 months survival was obtained with encapsulated cells (Luo et al., 2013). In human recipients, long-term survival of allogeneic fetal ventral mesencephalic cell transplants has been demonstrated and sustained clinical benefits have been reported up to 18 years following transplantation (Kefalopoulou et al., 2014; Ma et al., 2010). In contrast, much poorer outcomes have been reported when porcine fetal neural precursors have been transplanted in PD patients. A significant clinical improvement was observed only in 3 out of 12 immunosuppressed patients followed for up to 12 months (Schumacher et al., 2000) even if survival of the pig graft containing pig dopaminergic neurons could be shown at autopsy in one of the patients 7.5 months following transplantation (Deacon et al., 1997). Altogether, the clinical trials initiated on the basis of such robust pre-clinical data failed to show unequivocal efficacy in PD patients (Kefalopoulou et al., 2014; Bjorklund et al., 2003; Barker et al., 2013). The controversial nature of the use of human embryonic tissue from an ethical perspective (Turner and Kearney, 1993), the limited sources of tissue required, the immunogenicity of the grafted tissue and the variability introduced by dissection and surgical implantation techniques (Barker and Widner, 2004; Drouin-Ouellet and Barker, 2013; Piquet et al., 2012) favor the use of embryonic stem cells and inducible

pluripotent stem cells. Survival, safety and efficacy of these transplanted cells that differentiate into dopaminergic neurons and restore motor function in small animal models of PD has been shown (Doi et al., 2014; Kirkeby et al., 2012; Kriks et al., 2011). Now that appropriate differentiation protocols have been developed (Studer, 2012; Grealish et al., 2014) and large international consortiums like TransEuro (http://www.transeuro.org.uk) or NYS-TEM (http://stemcell.ny.gov) join forces to assess the validity of this strategy in PD patients, it becomes of vital importance to reconsider scaling-up issues.

## 6. Scaling up

A challenging aspect in scaling up from pre-clinical disease models is achieving sufficient brain penetration and suitable availability of therapeutic product. The role of the blood-brain-barrier and the immune system in inactivating (via antibodies) or diminishing the effect of such therapies can also be explored in primate models. Moreover, the fact that primates have larger brain size than rodents and a significantly similar anatomical organization makes them more suitable to test disease hypotheses and new therapeutic strategies. For gene and cell therapy, for example, transduction efficiency and volume of biological product needed can be estimated more closely in primates than in rodents. More importantly, because of a similar anatomical organization, the migration or transport of therapeutic products to connected regions can also be evaluated. The caveat remains that for regulatory safety reasons, the highest dose of biological product tested in humans is normally the highest dose tested in NHP which does not allow appropriate scaling-up in terms of brain size.

It is important to understand not only the disease but also the limitations of the models used in translational neuroscience. Comparing the effect of treatment in a primate brain that has received acute or partial lesions to the dopaminergic system with a patients suffering from a chronic pathology does not have the same impact in terms of metabolism and connectivity or, more importantly, compensatory mechanisms and plasticity that underlie any neurodegenerative process. The stage of the disease in PD patients at the time of therapeutic intervention needs to be considered when comparing efficacy across species. So far, PD patients receiving experimental treatments have been selected at an advanced stage of the disease where it is less likely to count on the integrity of the endogenous neuronal network to produce dopamine or halt degeneration. This selection criteria is already changing as it is frequently advised to initiate clinical trials at early stages of the disease.

Finally, methodological considerations favor the use of NHP models in that the same follow-up techniques can be used. Clinical scoring scales adapted from the clinic have been used for decades, complemented by the quantification of locomotor activity using telemetric devices or video-based analysis. More interestingly, tactile screens have been used to test executive dysfunction, memory and perseverative behaviour, and could potentially be useful for the assessment of compulsivity. Moreover, imaging tools and in particular PET imaging are invaluable in the in vivo assessment of the metabolic state and availability of receptors and transporters of neurotransmitters in the human and NHP brain. This is an excellent means to evaluate the effect of a therapeutic strategy not only on the specific neuronal population of interest but also any potentially desirable or undesirable circuitry effects. Magnetic resonance imaging (MRI) techniques can help monitor changes in the anatomy such as atrophy or monitoring of cell transplants, whereas functional MRI can link increasing demands in blood flow with activity readouts in specific territories of the brain.
Unlike rodents, primates also offer the possibility to screen and test biomarkers of disease progression in peripheral fluids like blood or cerebrospinal fluid longitudinally which, once appropri- ate markers are defined, have high translational value for PD patients. All of the abovementioned tools can be readily used in primates and, combined with thorough post-mortem analysis of the brains, can provide insight towards pathological mechanisms and the consequences of therapeutic interventions.

## 7. Conclusion

In summary, the variable outcomes from both pre-clinical and clinical data highlight the need for a reproducible, measurable and clinically translatable methodology to fully and objectively evalu- ate the translational potential of novel therapies for PD patients. Scaling-up issues regarding the species, the models and the technical and practical aspects of the therapeutic products designed can all influence the predictability of the outcome of clinical studies and should be taken into consideration when interpreting pre-clinical data.

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