Gene Therapy (2003) 10, & 2003 Nature Publishing Group All rights reserved /03 $

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1 (2003) 10, & 2003 Nature Publishing Group All rights reserved /03 $ REVIEW Gene therapy progress and prospects: Parkinson s disease EA Burton 1, JC Glorioso 2 and DJ Fink 3,4 1 Department of Clinical Neurology, University of Oxford, Radcliffe Infirmary, Oxford, UK; 2 Department of Molecular Genetics and Biochemistry, University of Pittsburgh, USA; 3 Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA; and 4 Geriatric Research, Education and Clinical Center (GRECC), Pittsburgh VA Healthcare System, Pittsburgh, PA, USA (2003) 10, doi: /sj.gt In brief Progress Inhibition of apoptosis by gene delivery prevents development of the disease phenotype in animal models Transgene-mediated expression of glial cell linederived neurotrophic factor may prevent progression after an initial insult, and may even be restorative in animal models Combination of antiapoptotic and glial cell linederived neurotrophic factor (GDNF) gene therapy protects dopaminergic neurons against a toxic insult, more effectively than either intervention alone Transgene-mediated production of the inhibitory neurotransmitter g-amino butyric acid (GABA) in neurons of the subthalamic nucleus ameliorates the behavioral phenotype and may be neuroprotective, in an animal model Delivery of transgenes encoding enzymes involved in dopamine biosynthesis enhances dopamine production in the striatum Stem cells may be driven to differentiate into functioning dopaminergic cells by genetic modification Isolation of genes implicated in rare genetic forms of Parkinson s disease (PD) has allowed generation of new animal models and identification of new candidate targets for intervention One human gene therapy trial is about to commence in PD The optimal vector remains uncertain Prospects Development of presymptomatic diagnostic tests will facilitate neuroprotective studies Better understanding of the pathogenesis may lead to the development of improved animal models that more closely resemble the human disease Studies may broaden their scope to include the important nonmotor manifestations of PD Further characterization of ES and adult stem cell populations will establish whether ex vivo transduction can drive their differentiation into dopaminergic neurons in a therapeutically useful way Well-designed clinical trials for PD gene therapy may take their lead from cell transplantation trials Correspondence: Dr DJ Fink, Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA PD is an attractive target for central nervous system (CNS) gene therapy for several reasons. First, the pathology in early PD is, to a first approximation, limited to dopaminergic neurons projecting from the substantia nigra pars compacta (SNc) to the caudate aputamenl, so that localized gene delivery is a viable therapeutic strategy. Second, the neurochemical deficits and the functional consequences of dopaminergic cell loss on local basal ganglia circuitry are well characterized; gene transfer can be designed either to improve cell survival, or to modify functional activity in the damaged basal ganglia circuitry (summarized in Figures 1 and 2). Third, PD is common and disabling despite treatment; no current intervention is uniformly accepted as altering the natural history of disease progression; hence, development of novel therapeutics is desirable. A variety of therapeutic transgenes has been delivered in experimental models of PD, using a number of different vectors. In this article, we survey the literature from 2000 to 2003, and briefly review recent progress in the development of gene transfer strategies for treating PD. Inhibition of apoptosis by gene delivery prevents development of the disease phenotype in animal models The most frequently studied animal models of PD involve chemical induction of lesions to the SNc of rodents, using the toxins 6-hydroxydopamine (6-OHDA) or 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In each case, the toxic insult leads to a pathogenic cascade, resulting in apoptotic cell death of dopaminergic (DA) neurons. Local expression of apoptotic inhibitors, in the SNc of 6-OHDA- and MPTP-lesioned animals, prevents both the loss of DA neurons and the

2 1722 Figure 1 The human basal ganglia. A coronal section of a human brain is shown, illustrating the anatomical locations of the basal ganglia. (Photograph of human autopsy specimen kindly provided by Dr Olaf Ansorge, Department of Neuropathology, Radcliffe Infirmary, Oxford). Figure 2 Gene therapy strategies for PD. The putative events and functional consequences involved in loss of SNc neurons are depicted. The complex pathogenic and pathophysiological cascade provides several candidate targets for molecular intervention, which are labeled with black arrows and white text; some of these are supported by experimental evidence, which is discussed in the text. In addition, alternative strategies to gene delivery, involving functional neurosurgery, cell transplantation and neuropharmacology are shown for contextual comparison. Abbreviations: P, putamen; GPe, external segment of globus pallidus; GPi, internal segment of globus pallidus; STN, subthalamic nucleus; SNc, substantia nigra pars compacta; TH, tyrosine hydroxylase; GTPCH1, GTP-cyclohydrolase-1. development of a PD-like phenotype, following the chemical insult. Our previous work showed that expression of bcl-2 from a nonreplicating herpes simplex virus (HSV)-based vector within the SNc protected dopaminergic cells from apoptosis following administration of 6- OHDA, with the resulting preservation of motor func-

3 tion. These findings have now been extended to other experimental paradigms. The human neuronal apoptosis inhibitor protein (NAIP), delivered to the SN of rats by intrastriatal inoculation of a recombinant type 5 adenovirus (Ad) vector, 1 protected the animals against 6- OHDA toxicity administered 1 week after the vector, as measured by immunohistochemistry and motor phenotype studies up to 28 days later. Prevention of the apoptotic protease-activating factor-1 (apaf-1)-dependent activation of caspase 9, using a recombinant adenoassociated virus (AAV) vector expressing a dominantnegative apaf-1 derivative, 2 protected mice against the effects of intra-peritoneal injections of MPTP administered 2 weeks after vector inoculation. At 1 week following MPTP intoxication, neuronal survival was 75% on the transduced side of the brain compared with 25% on the contralateral control. These protein inhibitors of apoptosis must be expressed intracellularly in order to block the apoptotic cascade, so that gene transfer is uniquely suited to this approach. However, the pathogenic trigger for PD in humans is unknown, and the role of apoptosis in dopaminergic cell death in naturally occurring PD is controversial. To test the hypothesis that inhibition of apoptosis within the SNc of humans could arrest or slow the progression of PD, it would be necessary to identify and treat patients early in the course of their illness, or to generate better animal models that more directly model the pathogenesis of human PD. Finally, the effects of prolonged expression of antiapoptotic factors in the brain have not been fully explored; some of these proteins are proto-oncogene products, and there might be important issues regarding their safety. Transgene-mediated expression of glial cell line-derived neurotrophic factor may prevent progression after an initial insult, and may even be restorative in animal models GDNF was originally isolated by virtue of its trophic effects on dopaminergic cells in culture. It was subsequently demonstrated that GDNF could promote the survival of dopaminergic neurons in the face of a toxic insult in both rat and monkey models of PD, and a putative role for GDNF as a neuroprotective agent in PD was suggested. However, the delivery of potent biologically active peptides with short half-lives to the brain is difficult, and attempts at intraventricular infusion of recombinant GDNF were disappointing. The alternatives include continuous intraparenchymal infusion of recombinant GDNF, 3 transplantation of genetically modified cells as production sites for GDNF, 4 8 or vector-mediated transfer of the gene encoding GDNF into the CNS parenchyma. Several different vector systems have been successfully used to effect GDNF gene transfer in experimental models, including lentivirus, 9 11 adenovirus, adeno-associated virus and herpes simplex virus. 15 Various points emerge from these studies, which differ mainly in the details of the experimental paradigms used. First, robust GDNF expression can be seen after gene transfer into the striatum or substantia nigra, and anterograde transport of GDNF to nerve terminals after transduction of the neuronal soma seems to be a property of GDNF rather than the vector system used. Second, GDNF appears to provide trophic support, preventing degeneration of dopaminergic cells and loss of dopaminergic nerve terminals in both the 6-OHDA and MPTP models. This protection correlates both with some behavioral measures of nigrostriatal integrity and neurochemical assays examining dopamine production. Finally, in many circumstances, the application of GDNF is protective or restorative even after the toxic insult has taken place. As is the case with antiapoptotic gene therapy for PD, the applicability of the experimental studies to human patients is uncertain, because the etiology and pathogenesis of the human disease are likely to be different from the animal models. However, GDNF appears to provide generic trophic support to dopaminergic neurons in the face of a range of challenges, and a phase I study examining direct intraputaminal infusion of the recombinant protein in patients was recently reported. 16 Adverse events were limited to repositioning one infusion catheter and asymptomatic signal changes on MRI that resolved when the concentration of infused GDNF was reduced. Secondary end points in this nonblinded nonrandomized study implied possible clinical benefit and improvement in functional imaging surrogates of dopaminergic terminal integrity. Should the recombinant factor prove efficacious in phase II trials, it is possible that gene delivery will offer advantages for long-term focal treatment. A recent study has sounded a note of caution for GDNF therapy. 17 Careful study of 6-OHDA rats showed that, although pharmacologically induced circling behavior, a marker of dopaminergic neural function, was ameliorated in animals treated with lentivirus-expressing GDNF over periods of up to 9 months, the spontaneous motor behavior was abnormal. This correlated with abnormal axonal sprouting within the pallidum and other brain areas where GDNF expression occurred, and with loss of the tyrosine hydroxlase (TH)- positive phenotype in SNc neurons that were preserved by GDNF treatment. Further characterization of these models, in conjunction with the outcome of clinical trials, will determine whether these concerns relate appropriately to GDNF therapy for PD. Combination antiapoptotic and GDNF gene therapy protects dopaminergic neurons against a toxic insult, more effectively than either intervention alone Two recent studies have exploited simultaneous delivery of genes encoding an antiapoptotic factor and GDNF, to enhance the dopaminergic cell survival seen with the corresponding single interventions. Adenoviral delivery of X-linked inhibitor of apoptosis (XIAP), aimed at preventing apoptosis of SNc neurons in MPTP-treated animals, led to preservation of DA cells but did not prevent loss of striatal DA nerve terminals, resulting in failure of behavioral recovery. 18 Combination of adenovirus-expressing XIAP, with another adenovirus-expressing GDNF, however, produced a synergistic effect with functional recovery that was not seen in animals treated with the GDNF-encoding vector alone. 1723

4 1724 In another study, GDNF was combined with bcl-2 gene delivery using two HSV vectors encoding expression cassettes for each of the factors. 15 The 6-OHDA rat model was used, and either intervention (GDNF or Bcl-2) increased cell survival from 25% (control) to 55% (pretreated). However, coadministration of the two vectors increased cell survival to 75%, indicating that the effects of the different modalities were additive. Transgene-mediated production of the inhibitory neurotransmitter g-amino butyric acid (GABA) in neurons of the subthalamic nucleus ameliorates the behavioral phenotype and may be neuroprotective, in an animal model One functional disturbance found in the basal ganglia of PD patients is the overactivity of neurons within the subthalamic nucleus (STN) that project to the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNr). These excitatory neurons serve to increase the firing rate of GPi and SNr neurons that, in turn, inhibit brainstem and thalamic projections to downstream motor pathways, thereby inhibiting the initiation of voluntary movement. Inhibition of overactive STN neurons by stereotactic ablation or deep brain stimulation has been shown to ameliorate motor signs in late-stage PD. A gene transfer strategy based on this approach has recently been reported. 19 Transduction of STN neurons with glutamic acid decarboxylase (GAD), the rate-limiting enzyme for synthesis of the inhibitory neurotransmitter gamma-amino butyric acid (GABA), using an adeno-associated virus vector, resulted in synthesis and activity-dependent release of GABA from STN nerve terminals. Microelectrode studies in control animals showed that stimulation of the STN resulted in excitation of the majority of SNr neurons from which recordings were obtained, consistent with the known glutamatergic neurochemical phenotype of STN neurons. However, stimulation of GAD-transduced STN neurons produced a preponderance of inhibitory responses in the SNr neuron pool, suggesting that expression of GAD and consequent modification of the neurochemical phenotype had altered the physiological properties of the STN- SNr projection. Intriguingly, GAD transduction of the STN appeared to protect SNc dopaminergic neurons from a neurotoxic insult following administration of 6- OHDA. The protective effect seemed dependent on the induction of an inhibitory phenotype in the STN neurons, as destruction of the STN using ibotenic acid did not protect the SNc DA neurons from 6-OHDA. Combining neuroprotection with functional compensation is attractive; a phase I clinical trial has been approved to begin soon (see below). Delivery of transgenes encoding enzymes involved in dopamine biosynthesis enhances dopamine production in the striatum Pharmacologic therapy of PD involves correction of the neurochemical deficit by systemic delivery of the dopamine precursor, L-DOPA, or by use of agents that act directly on striatal dopamine receptors. In the first gene therapy study of PD, gene transfer was employed to deliver the rate-limiting enzyme for dopamine formation, tyrosine hydroxylase, to the striatum. This resulted in enhanced dopamine production and observable behavioral benefit in a rodent model. More recently, it has been demonstrated that simultaneous delivery of multiple genes encoding enzymes that drive DA synthesis, more effectively corrects the DAdeficient phenotype than single-enzyme replacement. Synthesis of DA from tyrosine depends on two reactions, catalyzed by the enzymes tyrosine hydroxylase (TH) and aromatic acid decarboxylase (AADC). The former step is rate limiting, and requires a cofactor that is synthesized by GTP-cyclohydrolase I (GCH1). Various vector systems have been used in recent preclinical studies to deliver different combinations of these enzymes. These include multicistronic lentiviruses simultaneously encoding GCH1, TH and AADC; 20 combinations of AAV vectors separately encoding GCH1 and TH 21 or GCH1, TH and AADC; 22,23 an HSV vector coexpressing AADC and TH. 24 In all cases, coexpression of the enzymes and functional recovery of the experimentally lesioned animals was observed. Long-term dopamine therapy in PD is associated with declining therapeutic efficacy and increasing adverse effects as the disease progresses. While transgenemediated dopamine expression effectively corrects the motor phenotype in lesioned rodent and primate models, it is unclear at present whether the nonphysiological sustained delivery of dopamine in the striatum by these kinds of approaches will alleviate or exacerbate the problem of adverse effects. Since the therapeutic and toxic doses of dopaminergic agents alter in individual patients over the course of the disease, control over the production of dopamine following gene transfer will be essential before the use of this approach can be contemplated clinically. This might be accomplished using either (i) vectors with inducible enzyme expression, (ii) enzymes with controllable activity, or (iii) a prodrug approach using AADC to activate L-DOPA. Stem cells may be driven to differentiate into functioning dopaminergic cells by genetic modification Restoration of the dopaminergic projection from the SNc to the striatum has been a major goal of cell transplantation strategies. One major hurdle for this approach to therapy has been the difficulty in obtaining a suitable source of donor tissue that is both accessible and acceptable. One potential means for achieving this might rely on the use of human stem cell populations, driven to differentiate into dopaminergic neurons by appropriate manipulations. Much effort has been invested in determining which extracellular cues to stem cells are important in directing their differentiation into the desired cell population. It is possible to direct the differentiation of ES cells, for example, into dopaminergic neurons by a series of tissue culture manipulations, with an efficiency of around 15%. 25 This can be enhanced to around 50% after transfection of the cells with a transgene encoding Nurr-1, 26 an orphan nuclear receptor of the retinoic acid receptor superfamily, which has been implicated in the later stages of dopaminergic neuronal

5 differentiation. It is possible that directed differentiation of dopaminergic neurons from a variety of stem cell sources might depend on ex vivo transduction of stem cell populations to effect genetic modifications that favor adoption of the desired cell fate. Dopaminergic cells formed from ES cells appear to have similar functional and neurochemical properties as native dopaminergic neurons, 26 and can rescue an animal model of PD. 26,27 Isolation of genes implicated in rare genetic forms of PD has allowed generation of new animal models and identification of new candidate targets for intervention Although the common form of PD is sporadic and of unknown etiology, rare genetic forms have allowed isolation of genes involved in their pathogenesis, and thus highlighted cellular pathways that may be vulnerable in dopaminergic neurons and form potential targets for molecular intervention in PD. Mutations have been described in the gene encoding a-synuclein, resulting in autosomal dominant PD. a-synuclein is abundantly expressed in the brain and normally localized at nerve terminals. Aggregates of a-synuclein comprise a major component of the Lewy body, which is the pathological hallmark of the common sporadic form of PD. A second form of familial PD is autosomal recessive, and results from mutations in the gene encoding Parkin, a ubiquitin ligase. Intriguingly, a-synuclein is a substrate of Parkin, 28 linking the two dissimilar proteins into a common functional pathway. Pathogenic mutations resulting in a PD phenotype have recently been described in two other genes: DJ-1, of unknown function, 29 and NR4A2, encoding Nurr-1, a nuclear receptor (see above). 30 Elucidation of the pathways involved in genetic forms of PD has provided new animal models of specific SN degeneration, which do not rely on toxicity caused by chemical insults. These may more closely resemble the pathogenesis of human disease. Thus, transgenic mice overexpressing either wild-type 31 or mutated forms of human a-synuclein develop neuronal inclusions and cell loss. In addition, rat 35 and monkey 36 models have been developed using virally mediated a-synuclein gene transfer. Finally, a transgenic Drosophila model of a- synucleinopathy has been described. 37 Work in Drosophila may accelerate understanding of the human disease by identifying candidate pathways for disease modification. 38 The genetic forms of PD are uncommon, but gene therapy targeting the a-synuclein, Parkin or other pathways may also turn out to be an appropriate intervention for idiopathic PD. Using a-synuclein transgenic mice, it was shown that overexpression of b-synuclein prevented aggregation of a-synuclein and the resulting abnormal phenotype. 39 In addition, it appears that Parkin is capable of blocking the toxic effects of mutant a- synuclein expression and proteasome inhibition in catecholaminergic neurons in culture. 40 If deposition of a-synuclein, formation of Lewy bodies, and proteasome dysfunction are pivotal events in the pathogenesis of PD, then b-synuclein or Parkin gene delivery might be effective measures to disrupt the pathogenic cascade causing neurodegeneration. One clinical gene therapy trial is about to commence in Parkinson s disease Despite the wealth of experimental data on preclinical studies of gene therapy for PD, only one gene therapy trial is poised to start recruiting patients. 41 The trial is based on GAD gene transfer to the subthalamic nucleus using an adeno-associated virus vector, as detailed in the section above. In all, 12 patients with asymmetric disease will be selected by standard criteria, to undergo unilateral STN stimulator implantation. 41 The trial is a dose-escalation safety study, and as approved by US Food and Drug Administration three cohorts of patients will receive between and particles of raav-gad at the time of STN stimulator implantation. The assessors will monitor the patients clinical state and PET scans. In the worst case, if GAD gene transfer has an unanticipated deleterious effect, then the STN can be either electrically silenced or ablated, both standard treatments for PD, using the stimulator leads without additional surgery. Although the molecular strategy used in this trial is highly specific to PD, the wider field will view this pioneering study of in vivo gene transfer to the brain to treat neurodegeneration with considerable interest. The optimal vector remains uncertain Is there an optimal gene transfer vector with special utility for the development of treatments for PD? Nonviral gene transfer (liposomes or naked plasmids) is in general ineffective for gene transfer to the brain parenchyma, but each of the major viral vectors have demonstrated utility in experimental models of PD. In both earlier and recent studies viral vectors based on Ad1, 42 lentivirus (LV), 11,20 AAV 13,19 and HSV 15,24 have all been used to transfer relevant genes to the substantia nigra or striatum of experimental animals. Owing to the differences between studies in the animal model of PD employed, the site and volume of vector inoculation, the transgene and promoter constructs tested, the vector dose and the outcome measures assessed, the published literature does not allow one to make a direct comparison between vectors. The immunogenicity of Ad is likely to exclude that vector from human trials for PD, but each of the remaining vector platforms are likely to come to trial in the next few years. Although AAV and LV result in high-level long-term expression in brain, the results of the human trial using retroviral gene transfer to treat X-linked SCID in which two treated children developed leukemia as a result of insertional mutagenesis, 43 coupled with the recent observation that AAV integrates more frequently into active genes than noncoding regions, 44 may favor the use of a non-integrating vector such as HSV for these applications. Prospects for the next 2 years The development of readily available presymptomatic diagnostic tests for PD will be necessary to enable the use of neuroprotective strategies to retard the progression of SNc cell loss. Better understanding of the pathogenesis 1725

6 1726 of the common idiopathic form of PD may lead to the development of improved animal models that more closely resemble the etiology and cellular pathophysiology of human PD. This advance might enable identification of further targets for molecular intervention, in addition to providing a necessary resource for studies aimed at tackling the nonmotor features of PD. Depression, cognitive and autonomic dysfunctions are important contributors to morbidity in PD; further research aimed at addressing these components of the illness would be welcome. Further characterization of ES and adult stem cell populations will establish whether ex vivo transduction can drive their differentiation into dopaminergic neurons in a therapeutically useful way. Finally, further clinical trials for PD gene therapy are likely to commence using a variety of strategies. The trial designs may take their lead from recent well-executed clinical trials of cell transplant therapy. 45,46 It will be important to measure disability, depression and quality of life in addition to motor outcome, in order to be certain about which aspects of the illness are favorably altered by gene transfer, and whether there is likely to be an overall beneficial effect from these interventions in patients. References 1 Crocker SJ et al. NAIP protects the nigrostriatal dopamine pathway in an intrastriatal 6-OHDA rat model of Parkinson s disease. Eur J Neurosci 2001; 14: Mochizuki H et al. An AAV-derived Apaf-1 dominant negative inhibitor prevents MPTP toxicity as antiapoptotic gene therapy for Parkinson s disease. Proc Natl Acad Sci USA 2001; 98: Grondin R et al. Chronic, controlled GDNF infusion promotes structural and functional recovery in advanced parkinsonian monkeys. Brain 2002; 125: Date I et al. 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