D CNS Disease

Parkinson's disease (PD) is a common progressive neurodegenerative movement disorder that affects 5% of the population over 65 years of age. In this disease there is loss of dopa-minergic neurons, mainly in the substantia nigra, which leads to deficiency of the neurotransmitter dopamine (DA) in the striatum. The clinical symptoms typically appear after extensive loss of 60% to 80% of the dopaminergic neurons has occurred, which is correlated with the DA deficiency. DA is synthesized from tyrosine, first through conversion to L-dihydroxyphenylalanine (L-DOPA) by tyrosine hydroxylase (TH), and then to DA by aromatic amino acid decarboxylase (AADC). L-DOPA therapy is very efficacious, but response declines as the disease progresses and is complicated by adverse side effects. Thus, the different enzymes involved in DA synthesis have been targeted for gene transfer replacement therapy in order to restore dopaminergic stimulation of the striatum.

Dopamine-deficient mice (DAW~), lacking TH in dopa-minergic neurons, become hypoactive and aphagic and die by 4 weeks of age but can be rescued by daily treatment with L-DOPA. Bilateral coinjection of AAV vectors expressing human TH or GTP cyclohydrolase 1 (GTPCH1) into the stria-tum of these mice restored feeding behavior for several months. However, locomotor activity and coordination were only partially improved. A virus expressing only TH was less effective, and one expressing GTPCH1 alone was ineffective. TH immunoreactivity and DA were detected in the ventral striatum and adjacent posterior regions of rescued mice, suggesting that these regions mediate a critical DA-dependent aspect of feeding behavior (236).

Sustained and long-term transduction of striatal neurons and glial cells accompanied by significant behavioral recovery following administration of an AAV vector encoding the human TH cDNA has been demonstrated in the 6-hydroxydo-pamine (6-OHDA) rat model of PD (53). Also, using the 6-OHDA-lesioned rat model of PD, a single intrastriatal infusion of an AAV2-AADC vector led to an enhanced conversion of L-DOPA to restore DA to 50% of normal levels 12 weeks after vector administration (237). The vector-mediated increase in striatal decarboxylase activity remained undiminished over a 6-month period and persistence of transgene expression was demonstrated for at least 1 year (238). Coexpression of TH and AADC, using 2 separate AAV vectors in this Parkinsonian rat model, resulted in more effective DA production and more remarkable behavioral recovery compared with the expression of TH alone (239).

Convection-enhanced delivery of AAV2-AADC into 6 sites in the striatum of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys restored AADC activity to levels that exceeded the normal range (240). Injection of a bicistronic AAV vector expressing genes for human TH and AADC to asymptomatic but dopamine-depleted monkeys that had been treated with MPTP demonstrated successful transduction of the primate neurons in the striatum over a period up to 2.5 months with suggestive evidence of biochemical phenotypic effects and without significant toxicity (241). Finally, triple transduction with AAV-TH, AAV-AADC, and AAV-GTPCH1 to the striatum of either 6-OHDA-lesioned rats or MPTP-treated monkeys has resulted in restoration of DA synthesis and motor function (242,243).

Another strategy for gene therapy of PD involves the use of neuronal-specific growth factors to regenerate or halt ongoing degeneration of dopaminergic neurons of the substantia nigra. Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic molecule for nigral dopaminergic neurons both in vitro (244) and in vivo (245). Using the 6-OHDA-lesioned rat model of PD, perinigral injection of an AAV vector encoding the rat GDNF 3 weeks prior to a striatal 6-OHDA treatment resulted in stable transgene expression for 10 weeks and sig nificant protection of neurons from degeneration in the substantia nigra. However, there was no recovery of striatal TH-containing fibers and no recovery of motor function, suggesting that striatal dopaminergic recovery is necessary for functional improvement (246). In another study, both nigral and striatal long-term transduction of up to 6 months provided significant protection of nigral DA neurons against 6-OHDA-induced degeneration. However, only the rats receiving AAV-GDNF in the striatum displayed behavioral recovery, accompanied by significant reinnervation of the lesioned striatum. (247). In both studies (246,247), AAV-GDNF was administered before or shortly after the injection of the neurotoxin 6-OHDA. To better model the progressive DA depletion and significant neuron degeneration, which typically occur before symptoms appear, additional studies were carried out to examine the effect of AAV-mediated GDNF gene delivery into the striatum 4 weeks after injection of 6-OHDA. These studies showed that there was retrograde transport of GDNF to the substantia nigra that halted the ongoing degeneration of nigro-striatal DA neurons, with functional recovery, even after substantial numbers of DA cells had been lost (243,248,249).

Several clinical trials of AAV vectors in the CNS may begin soon. First, a novel approach to treatment of PD was proposed (250). In this clinical study, patients suffering from PD will be administered 2 different AAV vectors, each expressing, respectively, the gene for the 2 isoforms of the enzyme glutamic acid decarboxlysase (GAD-65 and GAD-67) by coinjection into the subthalamic nucleus (STN) region of the brain. The STN, which has a central role in the region of the brain that is responsible for regulation of movement, becomes disinhibited in PD. Preclinical evidence suggests that the gene delivery can inhibit the STN.

A clinical trial to treat Canavan's disease, a childhood leukodystrophy, has been proposed (251). This disease results from an autosomal recessive mutation in the gene for aspartoa-cyclase, which causes a toxic accumulation of the metabolite N-acetyl-aspartate. In this trial, up to 21 patients ages 3 months to 6 years will be administered in up to 6 sites in the frontal, varietal, and occipital regions of the brain.

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