Characterization of Intrastriatal Recombinant Adeno-Associated Virus-Mediated Gene Transfer of Human Tyrosine Hydroxylase and Human GTP-Cyclohydrolase I in a Rat Model of Parkinson’s Disease

To achieve local, continuous l -DOPA delivery in the striatum by gene replacement as a model for a gene therapy for Parkinson’s disease, the present studies used high titer purified recombinant adeno-associated virus (rAAV) containing cDNAs encoding human tyrosine hydroxylase (hTH) or human GTP-cyclohydrolase I [GTPCHI, the rate-limiting enzyme for tetrahydrobiopterin (BH 4 ) synthesis] or both to infect the 6-OHDA denervated rat striatum. Striatal TH and GTPCHI staining was observed 3 weeks after rAAV transduction, with little detectable perturbation of the tissue. Six months after intrastriatal rAAV transduction, TH staining was present but apparently reduced compared with the 3 week survival time. In a separate group of animals, striatal TH staining was demonstrated 1 year after rAAV transduction. Double staining studies using the neuronal marker NeuN indicated that >90% of rAAV-transduced cells expressing hTH were neurons. Microdialysis experiments indicated that only those lesioned animals that received the mixture of MD–TH and MD–GTPCHI vector displayed BH 4 independent in vivo l -DOPA production (mean ∼4–7 ng/ml). Rats that received the hTH rAAV vector alone produced measurable l -DOPA (mean ∼1–4 ng/ml) only after receiving exogenous BH 4 . l -Aromatic amino acid decarboxylase blockade, but not 100 m m KCl-induced depolarization, enhanced l -DOPA overflow, and animals in the non-hTH groups (GTPCHI and alkaline phosphatase) yielded minimal l -DOPA. Although elevated l -DOPA was observed in animals that received mixed hTH and hGTPCHI rAAV vectors, there was no reduction of apomorphine-induced rotational behavior 3 weeks after intrastriatal vector injection. These data demonstrate that purified rAAV, a safe and nonpathogenic viral vector, mediates long-term striatal hTH transgene expression in neurons and can be used to successfully deliver l -DOPA to the striatum.