Gene Therapy For Parkinson's Disease Research Articles

Safety and tolerability of putaminal AADC gene therapy for Parkinson disease

In Parkinson disease (PD), the benefit of levodopa therapy becomes less marked over time, perhaps because degeneration of nigrostrial neurons causes progressive loss of aromatic l-amino acid decarboxylase (AADC), the enzyme that converts levodopa into dopamine. In a primate model of PD, intrastriatal infusion of an adeno-associated viral type 2 vector containing the human AADC gene (AAV-hAADC) results in robust response to low-dose levodopa without the side effects associated with higher doses. These data prompted a clinical trial. Patients with moderately advanced PD received bilateral intraputaminal infusion of AAV-hAADC vector. Low-dose and high-dose cohorts (5 patients in each) were studied using standardized clinical rating scales at baseline and 6 months. PET scans using the AADC tracer [(18)F]fluoro-L-m-tyrosine (FMT) were performed as a measure of gene expression. The gene therapy was well tolerated, but 1 symptomatic and 2 asymptomatic intracranial hemorrhages followed the operative procedure. Total and motor rating scales improved in both cohorts. Motor diaries also showed increased on-time and reduced off-time without increased "on" time dyskinesia. At 6 months, FMT PET showed a 30% increase of putaminal uptake in the low-dose cohort and a 75% increase in the high-dose cohort. This study provides class IV evidence that bilateral intrastriatal infusion of adeno-associated viral type 2 vector containing the human AADC gene improves mean scores on the Unified Parkinson's Disease Rating Scale by approximately 30% in the on and off states, but the surgical procedure may be associated with an increased risk of intracranial hemorrhage and self-limited headache.

Doxorubicin augments rAAV-2 transduction in rat neuronal cells

Doxorubicin, an approved drug for cancer therapy, was recently found to be a potent agent to augment adeno-associated virus (AAV)-mediated transgene expression, especially in airway cells. Recombinant AAV type 2 (rAAV-2) has been shown to preferentially transduce neural tissues and is considered as the primary viral vector for the treatments of various neurodegenerative diseases including Parkinson's disease (PD). The goal of this study is to investigate whether doxorubicin can be applied to increase the efficacy of rAAV-2 transduction in the central nervous system. We co-administrated doxorubicin with AV2.luc/EGFP into the rat striatum, a preferred target site for PD gene therapy, and found that doxorubicin augmented rAAV-2 transduction dramatically without significant cytotoxicity and alteration of rAAV-2 tropism. By evaluating the effects of doxorubicin on rAAV-2 transduction in PC12 and MN9D neuronal cells, we found that doxorubicin appeared to promote the nuclear accumulation of rAAV-2, but did not affect viral binding or uptake. Our data suggested that doxorubicin might play an important role in modulating rAAV-2 intracellular trafficking in neuron-like cells. Our study also provided the initial in vivo evidence to facilitate AAV-mediated gene expression in the midbrain with the treatment of doxorubicin.

Results from a phase I safety trial of hAADC gene therapy for Parkinson disease

In a primate model of Parkinson disease (PD), intrastriatal infusion of an adeno-associated viral (AAV) vector containing the human aromatic l-amino acid decarboxylase (hAADC) gene results in robust gene expression. After gene transfer, low doses of systemically administered l-dopa are converted to dopamine in the transduced striatal neurons, resulting in behavioral improvement without the side effects typically associated with higher doses of l-dopa. These studies led to the initiation of a phase I safety trial. Here we report the findings for the first cohort of five patients. Patients with moderate to advanced PD received bilateral infusion of a low dose of the AAV-hAADC vector into the putamen. PET scans using the AADC tracer, 6-[18F]fluoro-l-m-tyrosine (FMT), were performed at baseline and at 1 and 6 months after infusion as an in vivo measure of gene expression. PET results showed an average 30% increase in FMT uptake (K(i)(c)) in the putamen after gene transfer. Preliminary analysis of clinical data indicates a modest improvement, but absence of a control and the nonblinded analyses make interpretation difficult. Thus far, this gene therapy approach has been well tolerated and shows PET evidence of sustained gene expression. These initial findings demonstrate the safety of the therapy; higher doses of adeno-associated viral vector containing the human aromatic l-amino acid decarboxylase gene in the next cohort of patients may further increase dopamine production in the putamen and provide more profound clinical benefit.

Design of clinical trials of gene therapy in Parkinson disease

No current therapy for Parkinson disease has been shown to slow or reverse the progressive course of the disease. As a departure from traditional treatments, gene therapy approaches provide a new hope for realizing this long-sought goal; but before they can be widely employed for use in patients, they must first be submitted to the rigorous safety and efficacy standards of the clinical trial. Some of the challenges of gene therapy clinical trial design are similar to those in studies of conventional pharmacological agents and include addressing the heterogeneity of the disease, the need for clinical and surrogate endpoints, and the issue of distinguishing “symptomatic” from “neuroprotective” effects. Gene therapy trials also raise the issues of the risks of viral therapy, issues of dose–response, the need for sham surgery, and the long duration of risks and benefits. We conclude that the most feasible designs are for those treatments that are expected to produce a rapid improvement in directly observable symptoms. Trials of agents which are expected to produce only a slowing of progression and not a reversal of the disease course are likely to take much longer and will require the development of methods to assess quality of life and other non-motor aspects of the disease.

DJ-1 and Parkin Modulate Dopamine-dependent Behavior and Inhibit MPTP-induced Nigral Dopamine Neuron Loss in Mice

Parkin-deficient animals exhibit mitochondrial degeneration and increased oxidative stress vulnerability, and both mice and flies lacking DJ-1 are hypersensitive to environmental toxins associated with Parkinson's disease (PD). We used recombinant adeno-associated virus (AAV) gene transfer to study the influence of DJ-1 and Parkin on the dopaminergic system of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, a model for sporadic PD. After MPTP lesioning, significantly more dopamine neurons survived in the virus-injected substantia nigra of the AAV-DJ-1 and AAV-Parkin mice when compared with AAV-enhanced green fluorescent protein injected controls. Protection at the neuronal level was supported by increased amphetamine-induced contralateral turning behavior. Normal mice expressing DJ-1 showed apomorphine-induced ipsilateral turning, suggesting a hyporesponsiveness of striatal dopamine D1 receptors in the DJ-1-expressing hemisphere. MPTP drastically reduced dopamine to 19% of normal levels and neither DJ-1 nor Parkin protected against MPTP-induced catecholamine loss under these conditions. Our results show that Parkin and DJ-1 inhibit dopamine neuron death and enhance amphetamine-induced dopaminergic function in a mouse model of idiopathic PD. However, DJ-1 overexpression also reduced postsynaptic dopamine receptor responses in normal mice. These results warrant further exploration of DJ-1 and Parkin gene therapy for PD, although a better understanding of their effects on behavior and dopamine neurotransmission is required before these proteins can be safely used.

Gene therapy using AAV

AAV (adeno-associated virus) vectors are considered to be promising gene-delivery vehicles for gene therapy, because they are derived from non-pathogenic virus, efficiently transduce non-dividing cells, and cause long-term gene expression. Appropriate AAV serotypes are utilized depending on the type of target cells. Among various neurological disorders, Parkinson's disease (PD) is one of the most promising candidates of gene therapy. PD is a progressive neurodegenerative disorder that predominantly affects dopaminergic neurons in the substantia nigra. One of the major approaches to gene therapy of PD is the intrastriatal expression of dopamine (DA)-synthesizing enzyme genes. As for the initial step of clinical application, AAV vector-mediated AADC (aromatic L-amino acid decarboxylase; the enzyme converting L-DOPA to DA) gene transfer in combination with oral administration of L-DOPA would be appropriate, since DA production can be regulated by adjusting the dose of L-DOPA. Second, intramuscular injection of AAV vectors is appropriate to protein-supplement gene therapy. Monogenic diseases such as hemophilia and Fabry disease are suitable candidates. Regarding cancer gene therapy, AAV vectors may be utilized to inhibit tumor angiogenesis, metastasis, and invasion. When long-term transgene expression in stem cells is needed, a therapeutic gene should be introduced with a minimal risk of insertional mutagenesis. To this end, site-specific integration into the AAVS1 locus on the chromosome 19 (19q13.4) by using the integration machinery of AAV would be particularly valuable.

244. New Gene Therapy for Parkinson's Disease by Down Regulation of α-Synuclein

Parkinson's disease (PD) is characterized by selective degeneration of substantia nigra dopamine neurons and abnormal cytoplasmic aggregates of |[alpha]|-synuclein. There is substantial evidence from molecular genetics, transgenic animal studies, and aggregation/toxicity studies with recombinant |[alpha]|-synuclein to suggest that conversion of this protein from soluble monomers to aggregated insoluble forms in the brain is a key event in the pathogenesis of PD. In the present study, adeno-associated virus (AAV) vector delivery of |[alpha]|-synuclein ribozyme was tested for its silencing effect on degenerating nigrostriatal neurons in MPP+ model of PD. We designed |[alpha]|-synuclein ribozyme against human |[alpha]|-synuclein gene expression and constructed |[alpha]|-synuclein ribozyme carried rAAV vector (designated rAAV-SynRz). Co-transfection of rAAV-SynRz and rAAV-|[alpha]|- synuclein into HEK293 cells resulted down regulating |[alpha]|-synuclein protein expression in vitro. Then, rAAV-SynRz was injected into SN of the MPP+-treated rats. Cell counts of TH-positive neurons in the substantia nigra revealed that rAAV-SynRz protected a significant number of TH positive cells when compared with cell counts of rAAV-EGFP or no rAAV injected rats. In close agreement, 56.2% of TH-positive cells remained in the nigral rAAV-SynRz group vs only 30.9% of in the EGFP group or 31.8% of no-rAAV treatment. These data indicated that using of rAAV-SynRz could survive more TH-positive neuron in SN in MPP+ model. Down regulation of |[alpha]|-synuclein expression might be a possible therapeutic tool for gene therapy of PD.

The assays of activities and function of TH, AADC, and GCH1 and their potential use in ex vivo gene therapy of PD

In the past decades, there have been numerous studies in the gene therapy for Parkinson's disease (PD), especially in delivering genes of enzymes for dopamine (DA) synthesis. Gene therapy in PD appears to be at the brink of the clinical study phase. However, there are many questions that need to be solved before this approach can be contemplated clinically, especially the question about the control of DA production because too much DA could cause toxicity. Until recently, few studies have investigated the relation between DA production and PD improvement and respective expressed human tyrosine hydroxylase (hTH), human GTP-cyclohydrolase 1 (hGCH1), and human aromatic acid decarboxylase (hAADC) in ex vivo gene therapy for PD. Now, we have developed a simple, fast, and reliable method to assay the activities of TH and AADC and have provided the possibility of ex vivo gene therapy for PD by genetically modifying cells with separate hTH, hGCH1, and hAADC genes. Using the method, we found though hTH, hGCH1, and hAADC genes were expressed, respectively, they could fulfil the function of DA synthesis by incubating together in vitro, and more DA was synthesized in vitro when hTH, hGCH1, and hAADC genes were expressed together rather than hTH and hAADC genes expressed or hTH expressed. The result suggests that we could easily control DA production in ex vivo gene therapy before transplantation. By combining this method and microdialysis, we also could further investigate the DA production in vitro and in vivo and then decide the optimal number and ratio of different transduced cells to improve the therapy of PD. Thus, the method has potential use in ex vivo gene therapy of PD.

Brain Transplantation of Neural Stem Cells Cotransduced with Tyrosine Hydroxylase and GTP Cyclohydrolase 1 in Parkinsonian Rats

Neural stem cells (NSCs) of the central nervous system (CNS) recently have attracted a great deal of interest not only because of their importance in basic research on neural development, but also in terms of their therapeutic potential in neurological diseases, such as Parkinson's disease (PD). To examine if genetically modified NSCs are a suitable source for the cell and gene therapy of PD, an immortalized mouse NSC line, C17.2, was transduced with tyrosine hydroxylase (TH) gene and with GTP cyclohydrolase 1 (GTPCH1) gene, which are important enzymes in dopamine biosynthesis. The expression of TH in transduced C17.2-THGC cells was confirmed by RT-PCR, Western blot analysis, and immunocytochemistry, and expression of GTPCH1 by RT-PCR. The level of L-DOPA released by C17.2-THGC cells, as determined by HPLC assay, was 3793 pmol/10(6) cells, which is 760-fold higher than that produced by C17.2-TH cells, indicating that GTPCH1 expression is important for L-DOPA production by transduced C17.2 cells. Following the implantation of C17.2-THGcC NSCs into the striata of parkinsonian rats, a marked improvement in amphetamine-induced turning behavior was observed in parkinsonian rats grafted with C17.2-THGC cells but not in the control rats grafted with C17.2 cells. These results indicate that genetically modified NSCs grafted into the brain of the parkinsonian rats are capable of survival, migration, and neuronal differentiation. Collectively, these results suggest that NSCs have great potential as a source of cells for cell therapy and an effective vehicle for therapeutic gene transfer in Parkinson's disease.

Tyrosine Hydroxylase Replacement in Experimental Parkinson's Disease with Transvascular Gene Therapy

Transvascular gene therapy of Parkinson's disease (PD) is a new approach to the gene therapy of PD and involves the global distribution of a therapeutic gene to brain after an intravenous administration and transport across the blood-brain barrier (BBB). This is enabled with the development of a nonviral gene transfer technology that encapsulates plasmid DNA inside pegylated immunoliposomes or PILs. An 85- to 100-nm liposome carries the DNA inside the nanocontainer, and the liposome surface is conjugated with several thousand strands of 2000-Da polyethyleneglycol (PEG). This PEGylation of the liposome stabilizes the structure in the blood stream. The liposome is targeted across the BBB via attachment to the tips of 1-2% of the PEG strands of a receptor-specific monoclonal antibody (mAb) directed at a BBB receptor, such as the insulin receptor or transferrin receptor (TfR). Owing to the expression of the insulin receptor or the TfR on both the BBB and the neuronal plasma membrane, the PIL is able to reach the neuronal nuclear compartment from the circulation. Brain-specific expression is possible with the combined use of the PIL gene transfer technology and brain-specific gene promoters. In the 6-hydroxydopamine rat model of experimental PD, striatal tyrosine hydroxylase (TH) activity is completely normalized after an intravenous administration of TfRmAb-targeted PILs carrying a TH expression plasmid. A treatment for PD may be possible with dual gene therapy that seeks both to replace striatal TH gene expression with TH gene therapy, and to halt or reverse neurodegeneration of the nigro-striatal tract with neurotrophin gene therapy.

518. Long-Term Lentiviral Vector-Mediated Transgene Expression in Neural Progenitor Cells Following Implantation into the Injured Rat Spinal Cord

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons (DAN) in the substantia nigra (SNi). The ultimate treatment of PD is to prevent DAN from the cell death. We previously showed that hepatocyte growth factor (HGF) resulted in effective prevention of DAN from cell death and remarkable behavioral recovery in 6-hydroxy-dopamine-lesioned parkinsonian rats. In this study, we have extend the preclinical exploration to primate model of PD. Seven days after stereotaxic transfection of human HGF plasmid or pVAX1 control plasmid into the unilateral striatum, infusion of MPTP into the right internal carotid artery of animals produces toxin-induced injury to the right nigro-striatal pathway with sparing of other dopaminergic neurons on the infused side and with negligible or little injury to the opposite, untreated side. After 2 to 3 weeks period, animals exhibited stable moderate parkinsonian features. But animals transfecterd with human HGF plasmid showed normal states. Interestingly, animals transfected with pVAX1 plasmid demonstrated the apomorphine and amphetamine-induced rotational asymmetry. However, the transfection of human HGF plasmid resulted in a significant inhibition of abnormal rotation over 3 months. In immunohistochemistry, more than 90% doperminergic neurons of PD model animals transfecred with pVAX1 plasmid were lost, while more than 70% those of animals transfected with human HGF plasmid were survived. Microdialysis demonstrated that concentrations of dopamine in striatum and SNi in animals transfected with human HGF plasmid were higher compared with PD model. Overall, the present study demonstrated that over-expression of human HGF prevented neuronal death in primate PD model, providing a potential of novel gene therapy for PD using human HGF plasmid.

50. AAV-Mediated Expression and Secretion of ADNF-9 Protects Substantia Nigra Neurons from 6-OHDA Damage

Parkinson's disease (PD) is a progressive movement disorder marked by selective degeneration of dopaminergic neurons of the substantia nigra and severe decrease in the dopamine (DA) content of the striatum. Replacement of DA is important to alleviate the motor symptoms of the disease. The DA precursor, L-3,4-dihydroxyphenylalanine (L-dopa), which is converted to DA by aromatic L-amino acid decarboxylase (AADC), provides clinical benefit to virtually all patients and has been mainstay in pharmacotherapy for PD. However, as the disease progresses, severe loss of dopaminergic nerve terminals is associated with an 80–95% depletion of striatal AADC activity resulting in loss of L-dopa efficacy. We demonstrated previously that AAV vector-mediated gene transfer of three DA-synthesizing enzymes, namely tyrosine hydroxylase, AADC, and GTP cyclohydrolase I, resulted in behavioral recovery in animal models of PD with efficient and long-term transduction of striatal neurons. In the present experiments, we investigated the therapeutic potential of the combination therapy, gene transfer of AADC with oral administration of L-dopa, in a primate bilateral model of PD. Cynomolgus macaques (Macaca fascicularis) were chronically treated with 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to make bilateral striatal lesions. Animals became behaviorally stable and motor symptoms did not ameliorate by the administration of L-dopa with peripheral AADC inhibitor. Then, adeno-associated virus (AAV) vector expressing AADC was injected into the unilateral putamen stereotaxically. Expression of AADC in the unilateral putamen resulted in remarkable improvement in manual dexterity on the contralateral to the AAV-AADC-injected side after the oral administration of L-dopa with peripheral AADC inhibitor. Fine motor task consists of capturing a piece of an apple improved on the contralateral hand with L-dopa. Amelioration of motor symptoms persisted about four hours each time after the oral intake of L-dopa. This duration of efficacy corresponds to the effective blood concentrations of the drug. Positron emission tomography showed remarkable and persistent increase of [11C]-L-dopa uptake in the AAV-AADC injected putamen. Gene transfer of AADC alone in combination with oral administration of L-dopa could be a shortcut to starting clinical trials of gene therapy for PD. With this method, patients still need to take L-dopa for the control of PD symptoms, but DA production can be regulated by the dose of L-dopa.

889. Optimization of Lentiviral EIAV Tricistronic Vectors for Parkinson's Disease Gene Therapy

889. Optimization of Lentiviral EIAV Tricistronic Vectors for Parkinson's Disease Gene Therapy

Parkinson's disease gene therapy: GAD Zooks! Excitement to inhibition in one easy step?

Parkinson's disease gene therapy: GAD Zooks! Excitement to inhibition in one easy step?

Gene therapy for Parkinson's disease.

We review recent progress in gene therapy utilizing experimental parkinsonian models including our data. Investigation of ex vivo gene therapy for Parkinson's disease (PD) is to provide L-dopa by transplantation of genetically modified cells into the striatum. Recently, neuronal progenitor cells (NPC) are recognized as the most appropriate target population for such genetic and cellular therapy of PD. We have developed modified pseudo-typed retrovirus production system. Using this gene transfer system, it is easy and efficient to introduce the gene into NPC because high titer virus vector is easily obtained. For the in vivo gene therapy, adeno-associated virus (AAV) vector is best virus vector because it is easy to introduce gene into neurons without inflammatory reaction. We established in vivo models of the inhibition of the caspase-cascade by overexpression of apoptotic protease activating factor-1-dominant negative inhibitor (Apaf-1-DN) using AAV vector. We showed that Apaf-1-DN delivery using an AAV vector system could prevent nigrostriatal degeneration in MPTP mice, suggesting that it might be an anti-mitochondrial apoptotic gene therapy for PD.

Parkinson's disease: changes in apoptosis-related factors suggesting possible gene therapy.

Specific degeneration of the nigrostriatal dopamine (DA) neurons of the substantia nigra pars compacta and the resulting loss of nerve terminals accompanied by DA deficiency in the striatum are responsible for most of the movement disturbances called parkinsonism, i.e., muscle rigidity, akinesia, and resting tremor, observed in Parkinson's disease (PD). We and other workers have found changes in the levels of cytokines, neurotrophins, and other apoptosis-related factors in the nigro-striatal region of postmortem brain and/or in the cerebrospinal fluid (CSF) from PD patients, or from animal models of PD such as 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP)-induced PD in mice or 6-hydroxydopamine (6-OHDA)-induced PD in rats. The most remarkable changes observed specifically in the nigrostriatal region were decreased levels of neurotrophins supporting DA neurons. These results indicate that the process of cell death in the nigrostriatal DA neurons in PD may be the so-called programmed cell death, i.e., apoptosis. Thus gene therapy for PD should aim both at supplementing the decreased striatal DA level by introducing the genes of DA-synthesizing enzymes into non-DA cells in the striatum and at supporting or restoring DA neurons by preventing apoptosis by introducing genes that block the process of apoptosis.

Triple transduction with adeno-associated virus vectors expressing tyrosine hydroxylase, aromatic-L-amino-acid decarboxylase, and GTP cyclohydrolase I for gene therapy of Parkinson's disease.

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.

Gene therapy of Parkinson's disease using adeno-associated virus (AAV) vectors.

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.

Behavioral Recovery in 6-Hydroxydopamine-Lesioned Rats by Cotransduction of Striatum with Tyrosine Hydroxylase and Aromatic L-Amino Acid Decarboxylase Genes Using Two Separate Adeno-Associated Virus Vectors

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 enzymes in the biosynthetic pathway for dopamine. Tyrosine hydroxylase (TH) catalyzes the synthesis of L-dopa, which must be converted to dopamine by aromatic L-amino acid decarboxylase (AADC). Since the endogenous AADC activity in the striatum is considered to be low, coexpression of both TH and AADC in the same striatal cells would increase the dopamine production and thereby augment the therapeutic effects. In the present study, the TH gene and also the AADC gene were simultaneously transduced into rat striatal cells, using two separate adeno-associated virus (AAV) vectors, AAV-TH and AAV-AADC. Immunostaining showed that TH and AADC were coexpressed efficiently in the same striatal cells in vitro and in vivo. Moreover, cotransduction with these two AAV vectors resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine (6-OHDA)-lesioned rats, compared with rats receiving AAV-TH alone (p < 0.01). These findings suggest an alternative strategy for gene therapy of PD and indicate that the simultaneous transduction with two AAV vectors can extend their utility for potential gene therapy applications.

Behavioral recovery in 6-hydroxydopamine-lesioned rats by cotransduction of striatum with tyrosine hydroxylase and aromatic L-amino acid decarboxylase genes using two separate adeno-associated virus vectors.

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 enzymes in the biosynthetic pathway for dopamine. Tyrosine hydroxylase (TH) catalyzes the synthesis of L-dopa, which must be converted to dopamine by aromatic L-amino acid decarboxylase (AADC). Since the endogenous AADC activity in the striatum is considered to be low, coexpression of both TH and AADC in the same striatal cells would increase the dopamine production and thereby augment the therapeutic effects. In the present study, the TH gene and also the AADC gene were simultaneously transduced into rat striatal cells, using two separate adeno-associated virus (AAV) vectors, AAV-TH and AAV-AADC. Immunostaining showed that TH and AADC were coexpressed efficiently in the same striatal cells in vitro and in vivo. Moreover, cotransduction with these two AAV vectors resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine (6-OHDA)-lesioned rats, compared with rats receiving AAV-TH alone (p < 0.01). These findings suggest an alternative strategy for gene therapy of PD and indicate that the simultaneous transduction with two AAV vectors can extend their utility for potential gene therapy applications.