revue neurologique 170 (2014) 763–769
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Biotherapies in neurological diseases
Biotherapies for Parkinson disease Biothe´rapies dans la maladie de Parkinson P. Remy a,b,c,* a
Centre expert Parkinson, neurologie, universite´ Paris-Est, CHU Henri-Mondor, AP–HP, 51, avenue Mare´chal-deLattre-de-Tassigny, 94010 Cre´teil, France b ´ Equipe 2, MIRCEN, CEA/DSV/I2BM, 18, route du Panorama, baˆtiment 61, 92265 Fontenay-aux-Roses cedex, France c NEURATRIS, MIRCen, 18, route du Panorama I, baˆtiment 61, pie`ce 121, BP 6 I, 92265 Fontenay-aux-Roses cedex, France
info article
abstract
Article history:
The clinical use of biotherapies in Parkinson disease already has 30 years history. The
Received 7 October 2014
transplantation of dopamine fetal cells in the striatum of advanced patients has proved to be
Accepted 8 October 2014
relevant in some patients but randomized efficacy trials in the US have provided disap-
Available online 20 November 2014
pointing results. However, cell therapies might come back on stage with the use of stem cells in the future. Gene therapy is a more recent strategy relying on viral vectors able to
Keywords:
transduce genes coding either for the enzymes that can increase neurotransmitters pro-
Graft
duction or genes for trophic factors. Several approaches have been developed in PD and have
Transplantation
been experimented in patients. Although, some of the studies have evidenced insufficient
Gene therapy
clinical benefit, other programs, such as those using dopamine replacement techniques are
Viral vector
promising. We find fresh hope in this field that might be the future of PD treatment. It
Review
remains however that advanced PD might not be the ideal condition to properly benefit from biotherapies and there is a need of studies at earlier stages of the disease, a time where major change in the disease course might be expected. # 2014 Elsevier Masson SAS. All rights reserved.
r e´ s u m e´ Mots cle´s :
Les premiers essais de biothe´rapie dans la maladie de Parkinson datent maintenant d’il y a
Greffe
30 ans. Les greffes de cellules fœtales dans le striatum de patients a` un stade avance´ de la
La transplantation
maladie ont montre´ qu’elles pouvaient ame´liorer certains patients. Ne´anmoins, les essais
La the´rapie ge´nique
controˆle´s re´alise´s aux E´tats-Unis ont e´te´ de´cevants. La the´rapie cellulaire pourrait toutefois
Vecteurs viraux
revenir au premier plan avec l’e´mergence prochaine des cellules souches. La the´rapie
Avis
ge´nique est plus re´cente et repose sur l’utilisation de vecteurs viraux capables de transmettre des ge`nes codant soit pour des enzymes permettant la fabrication de neurotransmetteurs, soit pour des facteurs trophiques. Plusieurs approches diffe´rentes ont de´ja` e´te´ expe´rimente´es chez les parkinsoniens. Si certaines de ces strate´gies ont apporte´ des be´ne´fices insuffisants, il semble que les techniques visant a` faire produire de la dopamine
* Correspondance. Centre expert Parkinson, CHU Henri-Mondor, 51, avenue Mare´chal-de-Lattre-de-Tassigny, 94010 Cre´teil, France. E-mail address: [email protected]. http://dx.doi.org/10.1016/j.neurol.2014.10.002 0035-3787/# 2014 Elsevier Masson SAS. All rights reserved.
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soient prometteuses. Elles pourraient re´volutionner le champ the´rapeutique de la maladie. Il n’en demeure pas moins que les formes avance´es de maladie de Parkinson ne sont peuteˆtre pas le stade ide´al pour be´ne´ficier de ces biothe´rapies. Nous avons besoin d’essais a` des stades plus pre´coces qui pourraient s’ave´rer en particulier be´ne´fiques sur l’e´volution de la maladie. # 2014 Elsevier Masson SAS. Tous droits re´serve´s.
Parkinson disease (PD) has been the main disease for which biotherapies, i.e., cell or gene therapy, have been experimented in the last 30 years. Although considering that PD is only related to dopamine (DA) cell loss in the substantia nigra is an oversimplification, it has allowed the development of different strategies to replace DA cells or DA production in the striatum of advanced patients. This review aims to draw a schematic view of the main results and drawbacks of these strategies when applied to PD patients.
1.
Cell therapies
The idea of replacing DA neurons in PD has emerged in the 1970s. The rationale was to use DA-producing cells able to overcome the neurochemical deficiency in the striatum of patients with PD and to reduce motor fluctuations and dyskinesias observed at the late stage of the disease. This strategy has been extensively experimented in rodent models of PD until the demonstration of the survival of human cells obtained from the ventral mesencephalon of abortion fetuses and the recovery observed in rats following striatal implantation of these cells [1]. These pivotal results opened the road for a human safety trial. It is important to remember that these strategies were designed and experimented long before the emergence of deep-brain stimulation (DBS). From 1988 to 2003, several trials have been performed most of them being open-labelled and two were blinded and randomized. It is important to add that there was a general agreement across the different teams involved in transplantation trials to use common evaluation methods relying both on clinical protocols (CAPIT and CAPSIT [2,3]) and imaging of dopamine production using positron emission tomography (PET) and 18F-DOPA. Reviewing all these studies in detail is not in the scope of this paper, therefore, we selected the main results to illustrate the benefits and limits of cell therapy in PD. First of all, the proof of concept has been validated. Indeed, it is possible to graft DA fetal cells in an ectopic location, i.e., the striatum, instead of the natural site, which is the substantia nigra, and to obtain both survival and function of the grafted DA cells. Survival has been evidenced by postmortem observations occurring more than 10 years after transplantation, revealing nerve fibres growing from grafted cells into the host striatum. The function of the transplanted cells has been demonstrated by PET studies, showing both the increase of 18F-DOPA uptake induced in the striatum by the grafted tissue [4,5], and that DA produced by grafted cells reaches the D2 receptors of the host striatal neurons [6]. Moreover, the increase of DA production obtained with
transplantation is able to improve the function of corticostriatal loops implicated in motor behavior [7]. Clinically, grafts can be associated with sustained motor improvement in some but not all transplanted PD patients. Some patients still have a graft-benefit more than 15 years after tissue implantation. Although open-label trials might be biased by marked placebo effects [8,9], correlations observed between 18 F-DOPA uptake improvement and clinical changes, even in small cohorts [5] suggest that the clinical changes are likely to reflect the real pharmacological action of transplants. The marked variability of the clinical changes induced by the grafts have been attributed to discrepancies in grafting procedures, or immunosuppressive regimens or age of fetal donors, but this would not explain intra-centre variability observed in all openlabelled studies. However, it seems that the number of donors used for each transplantation plays a major role in the clinical result [10], probably because the majority of grafted neurons do not survive in the host striatum. Altogether, recent reviews agree on the fact that open-labelled studies besides heterogeneous results have not allowed the emergence of a validated procedure that could be applied in efficacy, randomized trials. Nevertheless, two such trials have been funded by the NIH at the end of the 1990s. Their originality is the use of ‘‘sham surgery’’: the non-grafted patients (‘‘placebo’’ arm) are anaesthetized and have a similar surgical procedure except that no needle pass the dura and enters the brain. Patients randomized in the placebo arm can be grafted if they wish at the end of the trial. Sham surgery has raised many ethical issues in European countries but is likely to be the only strategy able to overcome the major placebo effect associated with surgical techniques in PD patients [11]. Both NIH-funded trials failed to demonstrate a clinical benefit of the grafts when looking to the primary endpoint. Post-hoc analysis of the Colorado study suggested that younger patients (aged less than 60) had a significant improvement (34% reduction of motor UPDRS) compared to complete failure in patients aged more than 60 [12]. Unfortunately, 5 of the 33 patients who ultimately were grafted (20 + 13 from placebo group, all aged less than 60 at study entry) developed severe dyskinesias that were still present after complete L-DOPA withdrawal. The second randomized trial aimed to evidence a ‘‘dosing effect’’ for transplants by comparing sham surgery, to 1 vs. 4 fetal donors transplanted bilaterally in the post-commissural putamen [13]. The study failed to demonstrate motor improvement on UPDRS-motor scale 2 years after surgery in both active arms of the trial, despite the fact that 18F-DOPA uptake increased in the putamen of transplanted patients. It seems however that some of the patients selected for this trial did not have a satisfying levodopa response before surgery. It
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has also been suggested that early withdrawal of immunosuppressive treatment, as soon as 6 months after surgery, might be in part responsible for the failure of transplantation. Moreover, 57% of grafted patients (n = 13) exhibited graftinduced dyskinesias that were severe enough in three cases to necessitate DBS. The pathophysiology of these graft-induced dyskinesias has been debated. Briefly, some authors have suggested misplacement of the grafts in the ventral striatum [14], however, this is inconsistent and therefore unlikely to be the main mechanism. Alternatively, it has been suggested that dysregulation of dopamine production was induced by the presence of serotonine neurons in the transplants [15,16]. Interestingly, it has also been shown that Lewy bodies (LB) could be found in neurons transplanted in the striatum of PD patients. Although, the proportion of LB+ neurons was low (< 5%) and its’ role uncertain, this raises the question on the pathogeny of striatal environment for future graft programs [17]. Eventually, a complete review of the results of grafts across open-label and randomized trial has been performed by Roger Barker et al. [18,19]. This analysis suggests that on average, grafts induced a motor improvement, but that older patients are less likely to benefit from the grafts. It seems that the number of donors might play a role in the clinical response. The need for immunosuppression could not be assessed considering the heterogeneity of procedures across the studies.
1.1.
Other sources of dopamine cells?
As mentioned above, a major issue of the benefit of grafts is the number of cells that survive and produce dopamine in the host striatum. Although some groups have used as much as 6 fetal donors to graft a single striatum, obviously it is ethically and technically impossible to envisage grafts as a curing procedure for PD with a ratio of 12 fetuses for one treated patient. Since dopamine neurons cannot be maintained in culture, alternative sources of DA-producing cells have been explored with little or no success. The first attempt to use DA-producing cells was the graft of adrenal chromaffin cells. Although pre-clinical studies have been initially encouraging, first trials in PD patients in Sweden were disappointing [20,21]. Unfortunately, a Mexican neurosurgeon reported dramatic improvement in 2 patients that received pieces of adrenal medulla placed in a premade cavity in the head of the caudate nucleus on one side using an open surgery [22]. However, these preliminary results, already in contradiction with Swedish cases, were not reproduced subsequently by other teams. A multicenter North American trial obtained disappointing results and conclude ‘‘that the widespread use of this procedure outside of research centres is premature’’ [23]. However, the technique was used over 5 years by several centres, although most of cases were never reported. This procedure relied on insufficient pre-clinical data and inadequate clinical studies. Another possible source, theoretically unlimited, of dopamine neurons is the pig mesencephalon. Ole Isacson group experienced graft of porcine ventral mesencephalon in 12 PD
765
patients. However, the motor benefit was limited and there was no improvement of 18F-levodopa PET [24], despite graft survival revealed by one case studied post-mortem 7 months after transplantation [25]. Eventually, levodopa can be released by human retinal pigment epithelial (RPE) cells as an intermediate product in the melanin biosynthetic pathway. A Californian company used a process to bind human RPE cells to a microcarrier support matrix (Spheramine, Titan, San Francisco). After pre-clinical studies in monkey considered as encouraging, a phase I trial was performed in six patients in whom an averaged of approximately 325,000 RPE cells were implanted in the most affected post-commissural putamen. No safety issue was raised and motor UPDRS in off was improved by 42% at 9 months, which is remarkable [26]. Based on this open-label study, the Spheramine was used in a randomized double-blind trial using sham surgery for the placebo arm in 36 patients and spheramine administration bilaterally in 35 cases. However, the trial was negative, since in the spheramine-treated group UPDRS-motor score in off was reduced by 21.5% at 12 months, which was not different from the sham surgery group (–20.7%) [27]. Moreover, several neurological or psychiatric serious adverse events, including one death were likely related to spheramine implantation. Ultimately, post-mortem results showed that only 0.036% of human RPE cells had survived 6 months after implantation [28].
1.2.
Is there a future for grafts in Parkinson disease?
The hope and disappointment that were associated with grafts in PD have stimulated research of new grafting procedures. European Union has granted a program, named TransEuro, to prepare the future transplantations in PD. A more thorough description of this program can be found elsewhere (http://www.transeuro.org.uk/), but huge efforts have been invested in technical procedure as well as patient selection and follow-up to be ready to launch a new graft trial. Although stem cells might be considered as the ideal cells to be transplanted, the development of safe dopamine neurons obtained from stem cells is not yet ready for clinical use. Moreover, in the near future, cell transplantation need to demonstrate a cost/benefit ratio that might compete both with DBS and gene therapy.
2.
Gene therapies
Gene therapy has recently emerged in Parkinson disease and relies on the use of viral vectors able to make brain cells to produce a neurotransmitter or a trophic factor. In this disease, all gene therapy approaches targeted the basal ganglia system, and were therefore administered using stereotactic surgery. Another common aspect of these strategies is the use of non-human primate models of PD to demonstrate the clinical improvement before applying such experimental treatments in humans.
2.1.
STN inhibition procedure
Among the first to reach the clinical phase was the use of adeno-asssociated virus tranducing the gene of glutamic acid
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decarboxylase (AAV-GAD) into the subthalamic nucleus (STN) of PD patients [29]. In this structure, the induction of this tranduced GAD activity would increase the GABA concentrations therefore reducing the activity of the STN. Twelve PD patients, aged 50–67 years were treated using an escalation dose program (3 doses, 4 patients per dose). All had a UPDRSmotor score 30, but their dose of anti-parkinsonian treatment varied from 250 (!) to 2300 mg of levodopa equivalents. They all had motor fluctuations for at least 3 months. Clinical improvement was observed as soon as 3 months after unilateral AAV-GAD deposition, and was maintained for one year. Surprisingly, the amplitude of UDPRS-3 decrease (meaning improvement) was similar in off and on conditions, respectively –23.8% and –27.2% at 12 months. This is different of observations in patients with STN-DBS in whom off motor scores decreases much more than on scores, probably because of the additional or confounding effect of treatment [30]. Among other results, no serious adverse event could be attributed to AAV-GAD gene therapy, and there was no evidence of a true dose effect although patients with the lowest does only exhibited minor improvement of motor scores. Considering the unilateral procedure in the safety trial, these results were encouraging and a randomized trial, with bilateral AAV-GAD infusion was performed in 45 patients, half of the subjects being sham operated and serving as controls [31]. However, the results were disappointing, since the motor improvement in AAV-GAD group although significantly higher than the one of the sham surgery group (–23.6% vs. –12.1%, P = 0.04) cannot compare to what is observed in STN-DBS patients. Moreover, this technique cannot allow any modulation and further improvement is unlikely to occur. Therefore, despite the demonstration of the proof of concept, we feel that this technique is not competitive enough in the field of biotherapies in PD.
2.2.
Gene therapy using trophic factors
Trophic factors might both delay disease progression and even restore dopaminergic function in PD patients by enhancing dopamine synaptic sprouting and function. Numerous studies in animal models have suggested that among trophic factors, glial-cell derived neurotrophic factor (GDNF) might be beneficial in PD [32]. First administration in man was performed in the lateral ventricle using a pump [33], but there was no clinical benefit and many adverse events ranging from nausea to severe weight loss and depression. However, it is unlikely that GDNF reached its’ target, i.e., the striatum, and the only post-mortem case reported confirmed this assumption [34]. Direct infusion of GDNF in the striatum of advanced PD patients has provided clinical benefit in two open-labelled studies [35–37], with motor improvements by 39% and 34%, respectively on UPDRS-off motor scores. No significant adverse events were reported in these studies. Amgen Inc. (Thousand Oaks, CA, USA) who owns the GDNF license sponsored a randomized trial with bilateral administration of GDNF vs. placebo in 34 patients, which failed to demonstrate a significant benefit (press release, unpublished data). Alternative routes of administration of GDNF were therefore investigated. Ex-vivo gene therapy, such as encapsulated
mice fibroblasts transduced to produce GDNF, were envisaged but did not reach the clinical phase. Eventually, in vivo gene therapy using either AAV or lentiviral vectors has been investigated in animal models even in non-human primates with encouraging results (review in [32]), but none of these vectors has reached the clinical stage, possibly because of matters of industrial properties. An alternative product, neurturin has been developed for clinical use. Neurturin has a 40% homology with GDNF and has similar mechanism of action. Neurturin gene was integrated in an AAV2 vector, the product being referred as CERE-120 (Ceregene Inc. San Diego CA, USA). After, successful preclinical investigations reviewed elsewhere [32], a phase I trial including 12 PD patients with two dose levels was performed [38]. A marked improvement was observed with a 36% reduction of UPDRS-off motor score without any significant adverse event [39]. There was no difference between the two doses. However, the clinical benefit was not associated with increase of putamenal 18F-DOPA uptake (+1.2% on average), raising the question of the mechanism by which neurturin was able to improve motor condition in these patients. This study was followed by a multicentre randomized trial including 58 patients with a 2:1 allocation to treatment and sham surgery, respectively. AAV2-neurturin was injected bilaterally in the putamen. The UPDRS-motor score in off was similar in the two groups at baseline (39 and 38.7) and improved by 18.5% and 17.8% in the treated and sham group, respectively (P = 0.91). Among other results, treated patients had a small but significant benefit on quality-of-life scales, compared to controls. Finally, more frequent serious adverse events were seen in AAV2-neurturin group, and specifically, five patients (3 treated, 2 sham) developed tumors, a glioblastoma occurring in a treated patient. Two patients died in this group but for reasons likely unrelated to treatment, and post-mortem analysis revealed that neurturin was expressed over 15% of the putamen. However, conversely to monkey studies, no TH induction was found in the substantia nigra compacta, suggesting strong differences in the function of nigrostriatal neurons between PD and animal models of PD. This result was the rationale of a following phase 1 study aiming to transduce neurturin gene both in the putamen and directly in the substantia nigra to obtain a better neuroprotective action. This was performed bilaterally in 6 patients with two dose levels, without any serious adverse event. In this open-label study, the UPDRS-motor in off improved by 13% and 14% at 12 and 24 months, respectively [40]. However, if one considers that neurturin should slow down disease progression rather than improve symptoms, this result is encouraging. The main limitation would be to find centres ready to perform such high-risk surgery for an efficacy trial.
2.3.
Gene therapy of the dopamine synthesis
This approach might be the most promising since it consists of making brain cells producing the main neurotransmitter lacking in PD, with a more specific objective to get a smooth dopamine production less dependent on ‘‘pulsatile’’ stimulation induced by drug intake. To understand the different strategies explored for gene therapy of dopamine synthesis, it is necessary to briefly remind the dopamine biosynthesis. The
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first step is the conversion of tyrosine into L-DOPA by tyrosinehydroxylase. This enzyme is present in dopaminergic, noradrenergic, and adrenergic neurons and requires the cofactor 5,6,7,8-tetrahydro-L-biopterin (BH4). BH4 is generated through a three-step enzymatic reaction, where GTP-cyclohydrolase 1 (GCH1), is rate limiting in the biosynthesis. L-DOPA is transformed into dopamine with the aromatic L-amino acid decarboxylase (AADC) that is expressed in different brain areas. In the striatum, it seems that the large majority of AADC enzyme is found in dopaminergic and serotonergic terminals [41]. Under physiological conditions, all synthesized DOPA can be readily converted to dopamine, which will be mainly maintained in the presynaptic vesicles. Gene therapy for dopamine replacement might be divided in three different strategies depending on the number of enzymes that are tranduced by the viral vector. The ultimate goal is to make striatal cells able to produce dopamine and therefore reconstituting the dopaminergic tone in the striatum. However, the number of genes that can be integrated in a vector is obviously related to the size of the virus. Whereas the Herpes-Simplex virus type 1 (HSV-1) or Equine infectious anemia virus (EIAV) have a loading capacity large enough to encompass several genes, the use of AAV has required a mix of separate vectors each coding for a different gene, although the latter technique has been found to be efficient in animal studies (review in [41]). Accordingly, three main strategies have been developed to date. The ‘‘continuous dopamine delivery’’ approach makes transduced striatal cells completely able to produce dopamine by delivery of the TH, AADC, and GCH1 genes. A second approach referred as ‘‘pro-drug’’ tranduces solely the AADC gene to increase the transformation of peripheral L-DOPA, provided by drug intake, into dopamine. Finally, the ‘‘continuous DOPA delivery’’ approach aims to increase L-DOPA synthesis by transducing only the TH and GCH1 genes. This approach relies on residual AADC activity in remaining dopaminergic or serotonergic terminals to convert newly produced L-DOPA into dopamine. The numerous pre-clinical studies performed in this field are not in the scope of this paper and should be read elsewhere (see reviews in [41,42]). We focus here on the vectors that have been experimented in humans. Therefore, since it is not yet the case for the continuous DOPA delivery technique, it will not be discussed further. The San Francisco group has developed a pro-drug approach using AAV-AADC gene product. A theoretical advantage of this treatment is that it relies on the exogenous supply of L-DOPA, which can be reduced if side effects occur. A phase I clinical trial performed in 5 advanced PD patients was published in 2008 [43]. Surprisingly, the reduction of UPDRSmotor score was significant in the off state (–34%, P = 0.005) but not under levodopa medication (–27.8%, P = 0.077), and daily doses of levodopa were not reduced after surgery. Meanwhile, PET measurement of 18F-fluoro-methyl-tyrosine, that evaluates macroscopically AADC activity, found an increase by 25% of tracer binding in the putamen of the patients 6 months after surgery. No significant adverse event could be related to the gene therapy itself. The same group published an extension of this study with five additional patients treated with a higher concentration of viral vectors [44]. Again, in the high dose cohort, a similar profile was obtained with a significant 37%
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reduction of UPDRS-motor score in off and a lower and nonsignificant reduction on medication. These clinical results were discordant with PET data since, the highest dose induced a putamenal increase of 18F-fluoro-methyl-tyrosine that was three-fold higher than the lowest dose [44]. Finally, a four-year follow-up of the same cohort confirmed the significantly increased PET value compared with baseline values. However, the improvement of UPRDS-motor score in off condition observed in the first 12 months displayed a slow deterioration in subsequent years [45]. The continuous dopamine delivery technique was made possible thanks to a vector construct using a lentiviral vector derived from the EIAV which is large enough to transport the genes for TH, AADC and GCH1. In a MPTP monkey model of PD, this vector, named ProSavin, was deposited in the posterior putamen safely restored extracellular concentrations of dopamine and improved motor behavior without inducing dyskinesias [46]. The phase I trial, recently reported by the Creteil group [47], included 15 patients distributed in three dose levels. The UPDRS-motor score in off was improved on average by 31% at 12 months after surgery with a trend toward better improvement in the group treated with the highest dose (34%, n = 6) compared to the two other groups (28%, n = 9). The UPDRS score in on was not significantly modified (7% reduction, n = 15). Doses of levodopa equivalents were supposed to be kept constant during the study, but 11 of 15 patients needed a reduction of medication because of dyskinesias, which were more frequent in the group receiving the highest dose of ProSavin. Interestingly, the evaluation with PET of synaptic dopamine production, indirectly measured by D2 receptors occupancy, showed a significant dose effect (P = 0.02): the highest dose group had a more important reduction of D2 availability, which means that D2 receptors were occupied by more dopamine than in the lower doses groups [47]. These promising results should be now confirmed by additional studies.
3.
Immunotherapies
In PD, the target of immunotherapy is obviously a-synuclein, and two strategies can be proposed [48]:
passive immunization using antibodies against this protein, if such antibodies cross the blood-brain barrier; or active immunotherapy which relies on vaccine. Studies in mice have demonstrated that antibodies against the C-terminus of a-synuclein can pass the blood-brain barrier and reduce pathology in a transgenic mouse model, mimicking the striato-nigral and motor deficits of PD [49]. Motor behavior of the rodents was also improved. However, further pre-clinical studies are necessary to envisage such treatments in humans.
4.
Conclusions
Biotherapies in PD have pioneered the field and both cell and gene therapies have already been experimented in humans.
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Cell therapies have raised hopes but were disappointing considering the complexity of the procedure and scarcity of fetal tissue. However, this strategy might be revived if stem cells become available for clinical use. Gene therapies have failed to demonstrate a benefit when the genes of trophic factors are employed. However, using these genes earlier in the course of the disease with a real neuroprotective goal and evaluation might put this technique back in the field. To date, gene therapy of the dopamine synthesis remains the most promising technique considering the safety of the procedure and the consistency of the results obtained, especially when the dose, i.e., number of viral vector units deposited, is sufficient. Efficacy studies are impatiently expected. Eventually, a common question for all these biotherapies is the stage of the disease at which they should be used. Indeed, all clinical studies have been performed in advanced PD patients considering that the available drugs at earlier stages would make unethical to rely on such experimental treatments. However, several of the techniques discussed above might be more efficient in younger, less advanced patients. Applying cell therapy or gene therapy when the basal ganglia system is not completely ‘‘exhausted’’ might really amplify the benefit not only in terms of immediate relief but also on disease progression itself. Although clinical trials in more early stages of the disease might be considered risky, recent demonstration of the benefit of early DBS [50] legitimates this question in the biotherapies field.
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
Disclosure of interest [19]
The author declares that he has no conflicts of interest concerning this article.
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[28] Farag ES, Vinters HV, Bronstein J. Pathologic findings in retinal pigment epithelial cell implantation for Parkinson disease. Neurology 2009;73:1095–102. [29] Kaplitt MG, Feigin A, Tang C, et al. Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson’s disease: an open label, phase I trial. Lancet 2007;369:2097–105. [30] Krack P, Batir A, Van Blercom N, et al. Five-year follow-up of bilateral stimulation of the subthalamic nucleus in advanced Parkinson’s disease. N Engl J Med 2003;349: 1925–34. [31] LeWitt PA, Rezai AR, Leehey MA, et al. AAV2-GAD gene therapy for advanced Parkinson’s disease: a double-blind, sham-surgery controlled, randomised trial. Lancet Neurol 2011;10:309–19. [32] Kordower JH, Bjorklund A. Trophic factor gene therapy for Parkinson’s disease. Mov Disord 2013;28:96–109. [33] Nutt JG, Burchiel KJ, Comella CL, et al. Randomized, doubleblind trial of glial cell line-derived neurotrophic factor (GDNF) in PD. Neurology 2003;60:69–73. [34] Kordower JH, Palfi S, Chen EY, et al. Clinicopathological findings following intraventricular glial-derived neurotrophic factor treatment in a patient with Parkinson’s disease. Ann Neurol 1999;46:419–24. [35] Gill SS, Patel NK, Hotton GR, et al. Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson disease. Nat Med 2003;9:589–95. [36] Slevin JT, Gash DM, Smith CD, et al. Unilateral intraputamenal glial cell line-derived neurotrophic factor in patients with Parkinson disease: response to 1 year of treatment and 1 year of withdrawal. J Neurosurg 2007;106:614–20. [37] Slevin JT, Gerhardt GA, Smith CD, et al. Improvement of bilateral motor functions in patients with Parkinson disease through the unilateral intraputaminal infusion of glial cell line-derived neurotrophic factor. J Neurosurg 2005;102:216–22. [38] Bartus RT, Herzog CD, Bishop K, et al. Issues regarding gene therapy products for Parkinson’s disease: the development of CERE-120 (AAV-NTN) as one reference point. Parkinsonism Relat Disord 2007;13(Suppl. 3):S469–77.
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[39] Marks Jr WJ, Ostrem JL, Verhagen L, et al. Safety and tolerability of intraputaminal delivery of CERE-120 (adenoassociated virus serotype 2-neurturin) to patients with idiopathic Parkinson’s disease: an open-label, phase I trial. Lancet Neurol 2008;7:400–8. [40] Bartus RT, Baumann TL, Siffert J, et al. Safety/feasibility of targeting the substantia nigra with AAV2-neurturin in Parkinson patients. Neurology 2013;80:1698–701. [41] Bjorklund T, Kirik D. Scientific rationale for the development of gene therapy strategies for Parkinson’s disease. Biochim Biophys Acta 2009;1792:703–13. [42] Varenika V, Kells A, Valles F, et al. Controlled dissemination of AAV vectors in the primate brain. Prog Brain Res 2009;175:163–72. [43] Eberling JL, Jagust WJ, Christine CW, et al. Results from a phase I safety trial of hAADC gene therapy for Parkinson disease. Neurology 2008;70:1980–3. [44] Christine CW, Starr PA, Larson PS, et al. Safety and tolerability of putaminal AADC gene therapy for Parkinson disease. Neurology 2009;73:1662–9. [45] Mittermeyer G, Christine CW, Rosenbluth KH, et al. Longterm evaluation of a phase 1 study of AADC gene therapy for Parkinson’s disease. Hum Gene Ther 2012;23:377–81. [46] Jarraya B, Boulet S, Ralph GS, et al. Dopamine gene therapy for Parkinson’s disease in a nonhuman primate without associated dyskinesia. Sci Transl Med 2009;1:2ra4. [47] Palfi S, Gurruchaga JM, Ralph GS, et al. Long-term safety and tolerability of ProSavin, a lentiviral vector-based gene therapy for Parkinson’s disease: a dose escalation, openlabel, phase 1/2 trial. Lancet 2014;383:1138–46. [48] Lindstrom V, Fagerqvist T, Nordstrom E, et al. Immunotherapy targeting alpha-synuclein protofibrils reduced pathology in (Thy-1)-h[A30P] alpha-synuclein mice. Neurobiol Dis 2014;69:134–43. [49] Games D, Valera E, Spencer B, et al. Reducing C-terminaltruncated alpha-synuclein by immunotherapy attenuates neurodegeneration and propagation in Parkinson’s disease-like models. J Neurosci 2014;34:9441–54. [50] Schuepbach WM, Rau J, Knudsen K, et al. Neurostimulation for Parkinson’s disease with early motor complications. N Engl J Med 2013;368:610–22.
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Biotherapies in neurological diseases
Biotherapies for Parkinson disease Biothe´rapies dans la maladie de Parkinson P. Remy a,b,c,* a
Centre expert Parkinson, neurologie, universite´ Paris-Est, CHU Henri-Mondor, AP–HP, 51, avenue Mare´chal-deLattre-de-Tassigny, 94010 Cre´teil, France b ´ Equipe 2, MIRCEN, CEA/DSV/I2BM, 18, route du Panorama, baˆtiment 61, 92265 Fontenay-aux-Roses cedex, France c NEURATRIS, MIRCen, 18, route du Panorama I, baˆtiment 61, pie`ce 121, BP 6 I, 92265 Fontenay-aux-Roses cedex, France
info article
abstract
Article history:
The clinical use of biotherapies in Parkinson disease already has 30 years history. The
Received 7 October 2014
transplantation of dopamine fetal cells in the striatum of advanced patients has proved to be
Accepted 8 October 2014
relevant in some patients but randomized efficacy trials in the US have provided disap-
Available online 20 November 2014
pointing results. However, cell therapies might come back on stage with the use of stem cells in the future. Gene therapy is a more recent strategy relying on viral vectors able to
Keywords:
transduce genes coding either for the enzymes that can increase neurotransmitters pro-
Graft
duction or genes for trophic factors. Several approaches have been developed in PD and have
Transplantation
been experimented in patients. Although, some of the studies have evidenced insufficient
Gene therapy
clinical benefit, other programs, such as those using dopamine replacement techniques are
Viral vector
promising. We find fresh hope in this field that might be the future of PD treatment. It
Review
remains however that advanced PD might not be the ideal condition to properly benefit from biotherapies and there is a need of studies at earlier stages of the disease, a time where major change in the disease course might be expected. # 2014 Elsevier Masson SAS. All rights reserved.
r e´ s u m e´ Mots cle´s :
Les premiers essais de biothe´rapie dans la maladie de Parkinson datent maintenant d’il y a
Greffe
30 ans. Les greffes de cellules fœtales dans le striatum de patients a` un stade avance´ de la
La transplantation
maladie ont montre´ qu’elles pouvaient ame´liorer certains patients. Ne´anmoins, les essais
La the´rapie ge´nique
controˆle´s re´alise´s aux E´tats-Unis ont e´te´ de´cevants. La the´rapie cellulaire pourrait toutefois
Vecteurs viraux
revenir au premier plan avec l’e´mergence prochaine des cellules souches. La the´rapie
Avis
ge´nique est plus re´cente et repose sur l’utilisation de vecteurs viraux capables de transmettre des ge`nes codant soit pour des enzymes permettant la fabrication de neurotransmetteurs, soit pour des facteurs trophiques. Plusieurs approches diffe´rentes ont de´ja` e´te´ expe´rimente´es chez les parkinsoniens. Si certaines de ces strate´gies ont apporte´ des be´ne´fices insuffisants, il semble que les techniques visant a` faire produire de la dopamine
* Correspondance. Centre expert Parkinson, CHU Henri-Mondor, 51, avenue Mare´chal-de-Lattre-de-Tassigny, 94010 Cre´teil, France. E-mail address: [email protected]. http://dx.doi.org/10.1016/j.neurol.2014.10.002 0035-3787/# 2014 Elsevier Masson SAS. All rights reserved.
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soient prometteuses. Elles pourraient re´volutionner le champ the´rapeutique de la maladie. Il n’en demeure pas moins que les formes avance´es de maladie de Parkinson ne sont peuteˆtre pas le stade ide´al pour be´ne´ficier de ces biothe´rapies. Nous avons besoin d’essais a` des stades plus pre´coces qui pourraient s’ave´rer en particulier be´ne´fiques sur l’e´volution de la maladie. # 2014 Elsevier Masson SAS. Tous droits re´serve´s.
Parkinson disease (PD) has been the main disease for which biotherapies, i.e., cell or gene therapy, have been experimented in the last 30 years. Although considering that PD is only related to dopamine (DA) cell loss in the substantia nigra is an oversimplification, it has allowed the development of different strategies to replace DA cells or DA production in the striatum of advanced patients. This review aims to draw a schematic view of the main results and drawbacks of these strategies when applied to PD patients.
1.
Cell therapies
The idea of replacing DA neurons in PD has emerged in the 1970s. The rationale was to use DA-producing cells able to overcome the neurochemical deficiency in the striatum of patients with PD and to reduce motor fluctuations and dyskinesias observed at the late stage of the disease. This strategy has been extensively experimented in rodent models of PD until the demonstration of the survival of human cells obtained from the ventral mesencephalon of abortion fetuses and the recovery observed in rats following striatal implantation of these cells [1]. These pivotal results opened the road for a human safety trial. It is important to remember that these strategies were designed and experimented long before the emergence of deep-brain stimulation (DBS). From 1988 to 2003, several trials have been performed most of them being open-labelled and two were blinded and randomized. It is important to add that there was a general agreement across the different teams involved in transplantation trials to use common evaluation methods relying both on clinical protocols (CAPIT and CAPSIT [2,3]) and imaging of dopamine production using positron emission tomography (PET) and 18F-DOPA. Reviewing all these studies in detail is not in the scope of this paper, therefore, we selected the main results to illustrate the benefits and limits of cell therapy in PD. First of all, the proof of concept has been validated. Indeed, it is possible to graft DA fetal cells in an ectopic location, i.e., the striatum, instead of the natural site, which is the substantia nigra, and to obtain both survival and function of the grafted DA cells. Survival has been evidenced by postmortem observations occurring more than 10 years after transplantation, revealing nerve fibres growing from grafted cells into the host striatum. The function of the transplanted cells has been demonstrated by PET studies, showing both the increase of 18F-DOPA uptake induced in the striatum by the grafted tissue [4,5], and that DA produced by grafted cells reaches the D2 receptors of the host striatal neurons [6]. Moreover, the increase of DA production obtained with
transplantation is able to improve the function of corticostriatal loops implicated in motor behavior [7]. Clinically, grafts can be associated with sustained motor improvement in some but not all transplanted PD patients. Some patients still have a graft-benefit more than 15 years after tissue implantation. Although open-label trials might be biased by marked placebo effects [8,9], correlations observed between 18 F-DOPA uptake improvement and clinical changes, even in small cohorts [5] suggest that the clinical changes are likely to reflect the real pharmacological action of transplants. The marked variability of the clinical changes induced by the grafts have been attributed to discrepancies in grafting procedures, or immunosuppressive regimens or age of fetal donors, but this would not explain intra-centre variability observed in all openlabelled studies. However, it seems that the number of donors used for each transplantation plays a major role in the clinical result [10], probably because the majority of grafted neurons do not survive in the host striatum. Altogether, recent reviews agree on the fact that open-labelled studies besides heterogeneous results have not allowed the emergence of a validated procedure that could be applied in efficacy, randomized trials. Nevertheless, two such trials have been funded by the NIH at the end of the 1990s. Their originality is the use of ‘‘sham surgery’’: the non-grafted patients (‘‘placebo’’ arm) are anaesthetized and have a similar surgical procedure except that no needle pass the dura and enters the brain. Patients randomized in the placebo arm can be grafted if they wish at the end of the trial. Sham surgery has raised many ethical issues in European countries but is likely to be the only strategy able to overcome the major placebo effect associated with surgical techniques in PD patients [11]. Both NIH-funded trials failed to demonstrate a clinical benefit of the grafts when looking to the primary endpoint. Post-hoc analysis of the Colorado study suggested that younger patients (aged less than 60) had a significant improvement (34% reduction of motor UPDRS) compared to complete failure in patients aged more than 60 [12]. Unfortunately, 5 of the 33 patients who ultimately were grafted (20 + 13 from placebo group, all aged less than 60 at study entry) developed severe dyskinesias that were still present after complete L-DOPA withdrawal. The second randomized trial aimed to evidence a ‘‘dosing effect’’ for transplants by comparing sham surgery, to 1 vs. 4 fetal donors transplanted bilaterally in the post-commissural putamen [13]. The study failed to demonstrate motor improvement on UPDRS-motor scale 2 years after surgery in both active arms of the trial, despite the fact that 18F-DOPA uptake increased in the putamen of transplanted patients. It seems however that some of the patients selected for this trial did not have a satisfying levodopa response before surgery. It
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has also been suggested that early withdrawal of immunosuppressive treatment, as soon as 6 months after surgery, might be in part responsible for the failure of transplantation. Moreover, 57% of grafted patients (n = 13) exhibited graftinduced dyskinesias that were severe enough in three cases to necessitate DBS. The pathophysiology of these graft-induced dyskinesias has been debated. Briefly, some authors have suggested misplacement of the grafts in the ventral striatum [14], however, this is inconsistent and therefore unlikely to be the main mechanism. Alternatively, it has been suggested that dysregulation of dopamine production was induced by the presence of serotonine neurons in the transplants [15,16]. Interestingly, it has also been shown that Lewy bodies (LB) could be found in neurons transplanted in the striatum of PD patients. Although, the proportion of LB+ neurons was low (< 5%) and its’ role uncertain, this raises the question on the pathogeny of striatal environment for future graft programs [17]. Eventually, a complete review of the results of grafts across open-label and randomized trial has been performed by Roger Barker et al. [18,19]. This analysis suggests that on average, grafts induced a motor improvement, but that older patients are less likely to benefit from the grafts. It seems that the number of donors might play a role in the clinical response. The need for immunosuppression could not be assessed considering the heterogeneity of procedures across the studies.
1.1.
Other sources of dopamine cells?
As mentioned above, a major issue of the benefit of grafts is the number of cells that survive and produce dopamine in the host striatum. Although some groups have used as much as 6 fetal donors to graft a single striatum, obviously it is ethically and technically impossible to envisage grafts as a curing procedure for PD with a ratio of 12 fetuses for one treated patient. Since dopamine neurons cannot be maintained in culture, alternative sources of DA-producing cells have been explored with little or no success. The first attempt to use DA-producing cells was the graft of adrenal chromaffin cells. Although pre-clinical studies have been initially encouraging, first trials in PD patients in Sweden were disappointing [20,21]. Unfortunately, a Mexican neurosurgeon reported dramatic improvement in 2 patients that received pieces of adrenal medulla placed in a premade cavity in the head of the caudate nucleus on one side using an open surgery [22]. However, these preliminary results, already in contradiction with Swedish cases, were not reproduced subsequently by other teams. A multicenter North American trial obtained disappointing results and conclude ‘‘that the widespread use of this procedure outside of research centres is premature’’ [23]. However, the technique was used over 5 years by several centres, although most of cases were never reported. This procedure relied on insufficient pre-clinical data and inadequate clinical studies. Another possible source, theoretically unlimited, of dopamine neurons is the pig mesencephalon. Ole Isacson group experienced graft of porcine ventral mesencephalon in 12 PD
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patients. However, the motor benefit was limited and there was no improvement of 18F-levodopa PET [24], despite graft survival revealed by one case studied post-mortem 7 months after transplantation [25]. Eventually, levodopa can be released by human retinal pigment epithelial (RPE) cells as an intermediate product in the melanin biosynthetic pathway. A Californian company used a process to bind human RPE cells to a microcarrier support matrix (Spheramine, Titan, San Francisco). After pre-clinical studies in monkey considered as encouraging, a phase I trial was performed in six patients in whom an averaged of approximately 325,000 RPE cells were implanted in the most affected post-commissural putamen. No safety issue was raised and motor UPDRS in off was improved by 42% at 9 months, which is remarkable [26]. Based on this open-label study, the Spheramine was used in a randomized double-blind trial using sham surgery for the placebo arm in 36 patients and spheramine administration bilaterally in 35 cases. However, the trial was negative, since in the spheramine-treated group UPDRS-motor score in off was reduced by 21.5% at 12 months, which was not different from the sham surgery group (–20.7%) [27]. Moreover, several neurological or psychiatric serious adverse events, including one death were likely related to spheramine implantation. Ultimately, post-mortem results showed that only 0.036% of human RPE cells had survived 6 months after implantation [28].
1.2.
Is there a future for grafts in Parkinson disease?
The hope and disappointment that were associated with grafts in PD have stimulated research of new grafting procedures. European Union has granted a program, named TransEuro, to prepare the future transplantations in PD. A more thorough description of this program can be found elsewhere (http://www.transeuro.org.uk/), but huge efforts have been invested in technical procedure as well as patient selection and follow-up to be ready to launch a new graft trial. Although stem cells might be considered as the ideal cells to be transplanted, the development of safe dopamine neurons obtained from stem cells is not yet ready for clinical use. Moreover, in the near future, cell transplantation need to demonstrate a cost/benefit ratio that might compete both with DBS and gene therapy.
2.
Gene therapies
Gene therapy has recently emerged in Parkinson disease and relies on the use of viral vectors able to make brain cells to produce a neurotransmitter or a trophic factor. In this disease, all gene therapy approaches targeted the basal ganglia system, and were therefore administered using stereotactic surgery. Another common aspect of these strategies is the use of non-human primate models of PD to demonstrate the clinical improvement before applying such experimental treatments in humans.
2.1.
STN inhibition procedure
Among the first to reach the clinical phase was the use of adeno-asssociated virus tranducing the gene of glutamic acid
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decarboxylase (AAV-GAD) into the subthalamic nucleus (STN) of PD patients [29]. In this structure, the induction of this tranduced GAD activity would increase the GABA concentrations therefore reducing the activity of the STN. Twelve PD patients, aged 50–67 years were treated using an escalation dose program (3 doses, 4 patients per dose). All had a UPDRSmotor score 30, but their dose of anti-parkinsonian treatment varied from 250 (!) to 2300 mg of levodopa equivalents. They all had motor fluctuations for at least 3 months. Clinical improvement was observed as soon as 3 months after unilateral AAV-GAD deposition, and was maintained for one year. Surprisingly, the amplitude of UDPRS-3 decrease (meaning improvement) was similar in off and on conditions, respectively –23.8% and –27.2% at 12 months. This is different of observations in patients with STN-DBS in whom off motor scores decreases much more than on scores, probably because of the additional or confounding effect of treatment [30]. Among other results, no serious adverse event could be attributed to AAV-GAD gene therapy, and there was no evidence of a true dose effect although patients with the lowest does only exhibited minor improvement of motor scores. Considering the unilateral procedure in the safety trial, these results were encouraging and a randomized trial, with bilateral AAV-GAD infusion was performed in 45 patients, half of the subjects being sham operated and serving as controls [31]. However, the results were disappointing, since the motor improvement in AAV-GAD group although significantly higher than the one of the sham surgery group (–23.6% vs. –12.1%, P = 0.04) cannot compare to what is observed in STN-DBS patients. Moreover, this technique cannot allow any modulation and further improvement is unlikely to occur. Therefore, despite the demonstration of the proof of concept, we feel that this technique is not competitive enough in the field of biotherapies in PD.
2.2.
Gene therapy using trophic factors
Trophic factors might both delay disease progression and even restore dopaminergic function in PD patients by enhancing dopamine synaptic sprouting and function. Numerous studies in animal models have suggested that among trophic factors, glial-cell derived neurotrophic factor (GDNF) might be beneficial in PD [32]. First administration in man was performed in the lateral ventricle using a pump [33], but there was no clinical benefit and many adverse events ranging from nausea to severe weight loss and depression. However, it is unlikely that GDNF reached its’ target, i.e., the striatum, and the only post-mortem case reported confirmed this assumption [34]. Direct infusion of GDNF in the striatum of advanced PD patients has provided clinical benefit in two open-labelled studies [35–37], with motor improvements by 39% and 34%, respectively on UPDRS-off motor scores. No significant adverse events were reported in these studies. Amgen Inc. (Thousand Oaks, CA, USA) who owns the GDNF license sponsored a randomized trial with bilateral administration of GDNF vs. placebo in 34 patients, which failed to demonstrate a significant benefit (press release, unpublished data). Alternative routes of administration of GDNF were therefore investigated. Ex-vivo gene therapy, such as encapsulated
mice fibroblasts transduced to produce GDNF, were envisaged but did not reach the clinical phase. Eventually, in vivo gene therapy using either AAV or lentiviral vectors has been investigated in animal models even in non-human primates with encouraging results (review in [32]), but none of these vectors has reached the clinical stage, possibly because of matters of industrial properties. An alternative product, neurturin has been developed for clinical use. Neurturin has a 40% homology with GDNF and has similar mechanism of action. Neurturin gene was integrated in an AAV2 vector, the product being referred as CERE-120 (Ceregene Inc. San Diego CA, USA). After, successful preclinical investigations reviewed elsewhere [32], a phase I trial including 12 PD patients with two dose levels was performed [38]. A marked improvement was observed with a 36% reduction of UPDRS-off motor score without any significant adverse event [39]. There was no difference between the two doses. However, the clinical benefit was not associated with increase of putamenal 18F-DOPA uptake (+1.2% on average), raising the question of the mechanism by which neurturin was able to improve motor condition in these patients. This study was followed by a multicentre randomized trial including 58 patients with a 2:1 allocation to treatment and sham surgery, respectively. AAV2-neurturin was injected bilaterally in the putamen. The UPDRS-motor score in off was similar in the two groups at baseline (39 and 38.7) and improved by 18.5% and 17.8% in the treated and sham group, respectively (P = 0.91). Among other results, treated patients had a small but significant benefit on quality-of-life scales, compared to controls. Finally, more frequent serious adverse events were seen in AAV2-neurturin group, and specifically, five patients (3 treated, 2 sham) developed tumors, a glioblastoma occurring in a treated patient. Two patients died in this group but for reasons likely unrelated to treatment, and post-mortem analysis revealed that neurturin was expressed over 15% of the putamen. However, conversely to monkey studies, no TH induction was found in the substantia nigra compacta, suggesting strong differences in the function of nigrostriatal neurons between PD and animal models of PD. This result was the rationale of a following phase 1 study aiming to transduce neurturin gene both in the putamen and directly in the substantia nigra to obtain a better neuroprotective action. This was performed bilaterally in 6 patients with two dose levels, without any serious adverse event. In this open-label study, the UPDRS-motor in off improved by 13% and 14% at 12 and 24 months, respectively [40]. However, if one considers that neurturin should slow down disease progression rather than improve symptoms, this result is encouraging. The main limitation would be to find centres ready to perform such high-risk surgery for an efficacy trial.
2.3.
Gene therapy of the dopamine synthesis
This approach might be the most promising since it consists of making brain cells producing the main neurotransmitter lacking in PD, with a more specific objective to get a smooth dopamine production less dependent on ‘‘pulsatile’’ stimulation induced by drug intake. To understand the different strategies explored for gene therapy of dopamine synthesis, it is necessary to briefly remind the dopamine biosynthesis. The
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first step is the conversion of tyrosine into L-DOPA by tyrosinehydroxylase. This enzyme is present in dopaminergic, noradrenergic, and adrenergic neurons and requires the cofactor 5,6,7,8-tetrahydro-L-biopterin (BH4). BH4 is generated through a three-step enzymatic reaction, where GTP-cyclohydrolase 1 (GCH1), is rate limiting in the biosynthesis. L-DOPA is transformed into dopamine with the aromatic L-amino acid decarboxylase (AADC) that is expressed in different brain areas. In the striatum, it seems that the large majority of AADC enzyme is found in dopaminergic and serotonergic terminals [41]. Under physiological conditions, all synthesized DOPA can be readily converted to dopamine, which will be mainly maintained in the presynaptic vesicles. Gene therapy for dopamine replacement might be divided in three different strategies depending on the number of enzymes that are tranduced by the viral vector. The ultimate goal is to make striatal cells able to produce dopamine and therefore reconstituting the dopaminergic tone in the striatum. However, the number of genes that can be integrated in a vector is obviously related to the size of the virus. Whereas the Herpes-Simplex virus type 1 (HSV-1) or Equine infectious anemia virus (EIAV) have a loading capacity large enough to encompass several genes, the use of AAV has required a mix of separate vectors each coding for a different gene, although the latter technique has been found to be efficient in animal studies (review in [41]). Accordingly, three main strategies have been developed to date. The ‘‘continuous dopamine delivery’’ approach makes transduced striatal cells completely able to produce dopamine by delivery of the TH, AADC, and GCH1 genes. A second approach referred as ‘‘pro-drug’’ tranduces solely the AADC gene to increase the transformation of peripheral L-DOPA, provided by drug intake, into dopamine. Finally, the ‘‘continuous DOPA delivery’’ approach aims to increase L-DOPA synthesis by transducing only the TH and GCH1 genes. This approach relies on residual AADC activity in remaining dopaminergic or serotonergic terminals to convert newly produced L-DOPA into dopamine. The numerous pre-clinical studies performed in this field are not in the scope of this paper and should be read elsewhere (see reviews in [41,42]). We focus here on the vectors that have been experimented in humans. Therefore, since it is not yet the case for the continuous DOPA delivery technique, it will not be discussed further. The San Francisco group has developed a pro-drug approach using AAV-AADC gene product. A theoretical advantage of this treatment is that it relies on the exogenous supply of L-DOPA, which can be reduced if side effects occur. A phase I clinical trial performed in 5 advanced PD patients was published in 2008 [43]. Surprisingly, the reduction of UPDRSmotor score was significant in the off state (–34%, P = 0.005) but not under levodopa medication (–27.8%, P = 0.077), and daily doses of levodopa were not reduced after surgery. Meanwhile, PET measurement of 18F-fluoro-methyl-tyrosine, that evaluates macroscopically AADC activity, found an increase by 25% of tracer binding in the putamen of the patients 6 months after surgery. No significant adverse event could be related to the gene therapy itself. The same group published an extension of this study with five additional patients treated with a higher concentration of viral vectors [44]. Again, in the high dose cohort, a similar profile was obtained with a significant 37%
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reduction of UPDRS-motor score in off and a lower and nonsignificant reduction on medication. These clinical results were discordant with PET data since, the highest dose induced a putamenal increase of 18F-fluoro-methyl-tyrosine that was three-fold higher than the lowest dose [44]. Finally, a four-year follow-up of the same cohort confirmed the significantly increased PET value compared with baseline values. However, the improvement of UPRDS-motor score in off condition observed in the first 12 months displayed a slow deterioration in subsequent years [45]. The continuous dopamine delivery technique was made possible thanks to a vector construct using a lentiviral vector derived from the EIAV which is large enough to transport the genes for TH, AADC and GCH1. In a MPTP monkey model of PD, this vector, named ProSavin, was deposited in the posterior putamen safely restored extracellular concentrations of dopamine and improved motor behavior without inducing dyskinesias [46]. The phase I trial, recently reported by the Creteil group [47], included 15 patients distributed in three dose levels. The UPDRS-motor score in off was improved on average by 31% at 12 months after surgery with a trend toward better improvement in the group treated with the highest dose (34%, n = 6) compared to the two other groups (28%, n = 9). The UPDRS score in on was not significantly modified (7% reduction, n = 15). Doses of levodopa equivalents were supposed to be kept constant during the study, but 11 of 15 patients needed a reduction of medication because of dyskinesias, which were more frequent in the group receiving the highest dose of ProSavin. Interestingly, the evaluation with PET of synaptic dopamine production, indirectly measured by D2 receptors occupancy, showed a significant dose effect (P = 0.02): the highest dose group had a more important reduction of D2 availability, which means that D2 receptors were occupied by more dopamine than in the lower doses groups [47]. These promising results should be now confirmed by additional studies.
3.
Immunotherapies
In PD, the target of immunotherapy is obviously a-synuclein, and two strategies can be proposed [48]:
passive immunization using antibodies against this protein, if such antibodies cross the blood-brain barrier; or active immunotherapy which relies on vaccine. Studies in mice have demonstrated that antibodies against the C-terminus of a-synuclein can pass the blood-brain barrier and reduce pathology in a transgenic mouse model, mimicking the striato-nigral and motor deficits of PD [49]. Motor behavior of the rodents was also improved. However, further pre-clinical studies are necessary to envisage such treatments in humans.
4.
Conclusions
Biotherapies in PD have pioneered the field and both cell and gene therapies have already been experimented in humans.
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Cell therapies have raised hopes but were disappointing considering the complexity of the procedure and scarcity of fetal tissue. However, this strategy might be revived if stem cells become available for clinical use. Gene therapies have failed to demonstrate a benefit when the genes of trophic factors are employed. However, using these genes earlier in the course of the disease with a real neuroprotective goal and evaluation might put this technique back in the field. To date, gene therapy of the dopamine synthesis remains the most promising technique considering the safety of the procedure and the consistency of the results obtained, especially when the dose, i.e., number of viral vector units deposited, is sufficient. Efficacy studies are impatiently expected. Eventually, a common question for all these biotherapies is the stage of the disease at which they should be used. Indeed, all clinical studies have been performed in advanced PD patients considering that the available drugs at earlier stages would make unethical to rely on such experimental treatments. However, several of the techniques discussed above might be more efficient in younger, less advanced patients. Applying cell therapy or gene therapy when the basal ganglia system is not completely ‘‘exhausted’’ might really amplify the benefit not only in terms of immediate relief but also on disease progression itself. Although clinical trials in more early stages of the disease might be considered risky, recent demonstration of the benefit of early DBS [50] legitimates this question in the biotherapies field.
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Disclosure of interest [19]
The author declares that he has no conflicts of interest concerning this article.
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