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.

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Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive lethal disease that involves selective annihilation of motoneurons. Glial cell line-derived neurotrophic factor (GDNF) is proposed to be a promising therapeutic agent for ALS and other motor neuron diseases. Because adeno-associated virus (AAV) has been developed as an attractive gene delivery system with proven safety, we explored the therapeutic efficacy of intramuscular delivery of the GDNF gene mediated by an AAV vector (AAV-GDNF) in the G93A mouse model of ALS. We show here that AAV-GDNF leads to substantial and long-lasting expression of transgenic GDNF in a large number of myofibers with its accumulation at the sites of neuromuscular junctions. Detection of GDNF labeled with FLAG in the anterior horn neurons, but not beta-galactosidase expressed as a control, indicates that most of the transgenic GDNF observed there is retrogradely transported GDNF protein from the transduced muscles. This transgenic GDNF prevents motoneurons from their degeneration, preserves their axons innervating the muscle, and inhibits the treated-muscle atrophy. Furthermore, four-limb injection of AAV-GDNF postpones the disease onset, delays the progression of the motor dysfunction, and prolongs the life span in the treated ALS mice. Our finding thus indicates that AAV-mediated GDNF delivery to the muscle is a promising means of gene therapy for ALS.

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.

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 ␤-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

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.

Restoring dopamine production in the putamen through gene therapy is a straightforward strategy for ameliorating motor symptoms for Parkinson's disease (PD). In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity-based primate model of PD, we previously showed the safety and efficacy of adeno-associated viral (AAV) vector-mediated gene delivery to the putamen of three dopamine-synthesizing enzymes (tyrosine hydroxylase [TH], aromatic l-amino acid decarboxylase [AADC], and guanosine triphosphate cyclohydrolase I [GCH]) up to 10 months postprocedure. Although three of four monkeys in this study have previously undergone postmortem analysis, one monkey was kept alive for 15 years after gene therapy to evaluate long-term effects. Here, we report that this monkey showed behavioral recovery in the right-side limb that remained unchanged for 15 years, at which time euthanasia was carried out owing to onset of senility. Immunohistochemistry of the postmortem brain from this monkey revealed persistent expression of TH, AADC, and GCH genes in the lesioned putamen. Transduced neurons were broadly distributed, with the estimated transduction region occupying 91% of the left postcommissural putamen. No signs of cytotoxicity or Lewy body pathology were observed in the AAV vector-injected putamen. This study provides evidence of long-term safety and efficacy of the triple-transduction method as a gene therapy for PD.

"A propensity to bend the trunk forward" and "the chin is now almost immovably bent down upon the sternum" were described by James Parkinson in patients with Parkinson's disease (PD). The term "dropped head" was first reported in "Gerlier disease" in Switzerland and 'kubisagari' in Japan and since then also reported in myositis, myopathy, myasthenia gravis, amyotrophic lateral sclerosis, neuropathy, and hypothyroidism. Disproportionate antecollis occurs in about half cases of multiple system atrophy (MSA) and is considered dystonic in nature. Dropped head is considered rare in PD, both in advanced and early stages of PD. However, it is known to progress subacutely over a period of several days. In my experience, dropped head is relatively common in PD. The mechanism of dropped head in PD is either dystonia of flexor neck muscles or weakness of extensor neck muscles. The response of dropped head to various anti-parkinsonian medications is rather inconsistent. Levodopa is reported to induce amelioration in some patients while dopamine agonists can cause deterioration. Muscle afferent block with lidocaine and ethanol is reported to be effective, while the effect of botulinum toxin injection into the affected muscles is limited. The effect of stereotaxic neurosurgery on dropped head is controversial. Early diagnosis and prompt treatment is necessary to prevent muscle damage associated with longterm overstretch of extensor neck muscles.