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Gene transfer of dopamine-synthesizing enzymes into the striatal neurons has led to behavioral recovery in animal models of Parkinson's disease (PD). We evaluated the safety, tolerability, and potential efficacy of adeno-associated virus (AAV) vector-mediated gene delivery of aromatic L-amino acid decarboxylase (AADC) into the putamen of PD patients. Six PD patients were evaluated at baseline and at 6 months, using multiple measures, including the Unified Parkinson's Disease Rating Scale (UPDRS), motor state diaries, and positron emission tomography (PET) with 6-[(18)F]fluoro-L-m-tyrosine (FMT), a tracer for AADC. The short-duration response to levodopa was measured in three patients. The procedure was well tolerated. Six months after surgery, motor functions in the OFF-medication state improved an average of 46% based on the UPDRS scores, without apparent changes in the short-duration response to levodopa. PET revealed a 56% increase in FMT activity, which persisted up to 96 weeks. Our findings provide class IV evidence regarding the safety and efficacy of AADC gene therapy and warrant further evaluation in a randomized, controlled, phase 2 setting.

Regulation of gene expression is necessary to avoid possible adverse effects of gene therapy due to excess synthesis of transgene products. To reduce transgene expression, we developed a viral vector-mediated somatic regulation system using inducible Cre recombinase. A recombinant adeno-associated virus (AAV) vector expressing Cre recombinase fused to a mutated ligand-binding domain of the estrogen receptor alpha (CreER(T2)) was delivered along with AAV vectors expressing dopamine-synthesizing enzymes to rats of a Parkinson disease model. Treatment with 4-hydroxytamoxifen, a synthetic estrogen receptor modulator, activated Cre recombinase within the transduced neurons and induced selective excision of the tyrosine hydroxylase (TH) coding sequence flanked by loxP sites, leading to a reduction in transgene-mediated dopamine synthesis. Using this strategy, aromatic L-amino acid decarboxylase (AADC) activity was retained so that l-3,4-dihydroxyphenylalanine (L-dopa), a substrate for AADC, could be converted to dopamine in the striatum and the therapeutic effects of L-dopa preserved, even after reduction of TH expression in the case of dopamine overproduction. Our data demonstrate that viral vector-mediated inducible Cre recombinase can serve as an in vivo molecular switch, allowing spatial and temporal control of transgene expression, thereby potentially increasing the safety of gene therapy.

Glial cell line-derived neurotrophic factor (GDNF) is a strong candidate agent in the neuroprotective treatment of Parkinson's disease (PD). We investigated whether adeno-associated viral (AAV) vector-mediated delivery of a GDNF gene in a delayed manner could prevent progressive degeneration of dopaminergic (DA) neurons, while preserving a functional nigrostriatal pathway. Four weeks after a unilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA), rats received injection of AAV vectors expressing GDNF tagged with FLAG peptide (AAV-GDNFflag) or beta-galactosidase (AAV-LacZ) into the lesioned striatum. Immunostaining for FLAG demonstrated retrograde transport of GDNFflag to the substantia nigra (SN). The density of tyrosine hydroxylase (TH)-positive DA fibers in the striatum and the number of TH-positive or cholera toxin subunit B (CTB, neuronal tracer)-labeled neurons in the SN were significantly greater in the AAV-GDNFflag group than in the AAV-LacZ group. Dopamine levels and those of its metabolites in the striatum were remarkably higher in the AAV-GDNFflag group compared with the control group. Consistent with anatomical and biochemical changes, significant behavioral recovery was observed from 4-20 weeks following AAV-GDNFflag injection. These data indicate that a delayed delivery of GDNF gene using AAV vector is efficacious even 4 weeks after the onset of progressive degeneration in a rat model of PD.

One potential strategy for gene therapy of Parkinson's disease (PD) is the local production of dopamine (DA) in the striatum induced by restoring DA-synthesizing enzymes. In addition to tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), GTP cyclohydrolase I (GCH) is necessary for efficient DA production. Using adeno-associated virus (AAV) vectors, we previously demonstrated that expression of these three enzymes in the striatum resulted in long-term behavioral recovery in rat models of PD. We here extend the preclinical exploration to primate models of PD. Mixtures of three separate AAV vectors expressing TH, AADC, and GCH, respectively, were stereotaxically injected into the unilateral putamen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys. Coexpression of the enzymes in the unilateral putamen resulted in remarkable improvement in manual dexterity on the contralateral to the AAV-TH/-AADC/-GCH-injected side. Behavioral recovery persisted during the observation period (four monkeys: 48 days, 65 days, 50 days, and >10 months, each). TH-immunoreactive (TH-IR), AADC-IR, and GCH-IR cells were present in a large region of the putamen. Microdialysis demonstrated that concentrations of DA in the AAV-TH/-AADC/-GCH-injected putamen were increased compared with the control side. Our results show that AAV vectors efficiently introduce DA-synthesizing enzyme genes into the striatum of primates with restoration of motor functions. This triple transduction method may offer a potential therapeutic strategy for PD.

Parkinson's disease (PD), a neurological disease suited to gene therapy, is biochemically characterized by a severe decrease in the dopamine content of the striatum. One current strategy for gene therapy of PD involves local production of dopamine in the striatum achieved by inducing the expression of enzymes involved in the biosynthetic pathway for dopamine. We previously showed that the coexpression of tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), using two separate adeno-associated virus (AAV) vectors, resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine-lesioned parkinsonian rats, compared with the expression of TH alone. Not only levels of TH and AADC but also levels of tetrahydrobiopterin (BH4), a cofactor of TH, and GTP cyclohydrolase I (GCH), a rate-limiting enzymes for BH4 biosynthesis, are reduced in parkinsonian striatum. In the present study, we investigated whether transduction with separate AAV vectors expressing TH, AADC, and GCH was effective for gene therapy of PD. In vitro experiments showed that triple transduction with AAV-TH, AAV-AADC, and AAV-GCH resulted in greater dopamine production than double transduction with AAV-TH and AAV-AADC in 293 cells. Furthermore, triple transduction enhanced BH4 and dopamine production in denervated striatum of parkinsonian rats and improved the rotational behavior of the rats more efficiently than did double transduction. Behavioral recovery persisted for at least 12 months after stereotaxic intrastriatal injection. These results suggest that GCH, in addition to TH and AADC, is important for effective gene therapy of PD.

Parkinson's disease (PD) is characterized by the progressive loss of the dopaminergic neurons in the substantia nigra and a severe decrease in dopamine in the striatum. A promising approach to the gene therapy of PD is intrastriatal expression of dopamine-synthesizing enzymes [tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC)]. The most appropriate gene-delivery vehicles for neurons are adeno-associated virus (AAV) vectors, which are derived from non-pathogenic virus. Therefore, TH and AADC genes were introduced into the striatum in the lesioned side using separate AAV vectors in parkinsonian rats, and the coexpression of TH and AADC resulted in better behavioral recovery compared with TH alone. Another strategy for gene therapy of PD is the protection of dopaminergic neurons in the substantia nigra using an AAV vector containing a glial cell line-derived neurotrophic factor (GDNF) gene. Combination of dopamine-supplement gene therapy and GDNF gene therapy would be a logical approach to the treatment of PD.

We evaluated the effects of mianserin, a relatively selective 5-HT2 receptor antagonist, on symptoms related to drug-induced psychosis in patients with Parkinson's disease (PD). A total of 12 patients with PD who had developed drug-induced psychosis showed delirium (DSM-III-R criteria; n = 10) and pure visual hallucinations (n = 2). The antiparkinsonian drugs involved in the drug-induced psychosis were L-DOPA/carbidopa, bromocriptine, trihexyphenidyl, and amantadine. They received mianserin (mean 36.7 mg, range 20-60 mg) given orally for 8 weeks. Complete relief or marked improvement in psychotic symptoms was noted in 8 patients, moderate improvement in 2 patients, and no effect in 2 patients. The parkinsonian disability also decreased slightly in 8 patients. These results suggest that serotonin antagonism at 5-HT2 receptors may not only play an important role in the treatment of drug-induced psychosis in PD, but may also ameliorate the symptoms of parkinsonism.