1051. A Fail-Safe System for Gene Therapy of Parkinson's Disease; Application of Removable Expression Cassette To Prevent Overproduction of Dopamine in a Rat Model

Top of pageAbstract Parkinson's disease (PD) is a progressive movement disorder characterized by selective degeneration of dopaminergic neurons of the substantia nigra and a severe decrease in the dopamine (DA) content of the striatum. We demonstrated previously that adeno-associated viral (AAV) vector-mediated gene transfer of three DA-synthesizing enzymes, namely tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), and GTP cyclohydrolase I (GCH), resulted in behavioral recovery in animal models of PD with efficient and long-term transduction of striatal neurons. However, before the introduction of this strategy to the clinical arena, regulation of gene expression is necessary to avoid overproduction of DA. Inducible transgenesis based on bacteriophage P1 Cre recombinase has been developed to shut down gene expression when creating conditional knock out mice. To investigate the potential application of this technique to gene therapy for PD, we generated a recombinant AAV vector that expresses an improved variant of the tamoxifen-inducible Cre recombinase (CreERT2; provided by Pierre Chambon and Daniel Metzger, IGBMC), where the recombinase is fused to the mutated ligand-binding domain of the human estrogen receptor. The AAV vector that expresses TH was modified by adding flanking loxP sites to the TH coding sequence (AAV-floxed TH). Hemiparkinsonian rats were obtained by injecting a selective neurotoxin, 6-OHDA, into the left medial forebrain bundle. The animals received a mixture of AAV-CreERT2, AAV-floxed TH, AAV-AADC, and AAV-GCH. Half of them were further treated with 4-OH tamoxifen (1mg/kg, i.p., five days) in the course of the experiments. Control rats were injected with AAV-LacZ alone, or AAV-Cre that expresses Cre recombinase instead of AAV-CreERT2 with AAV-floxed TH/-AADC/-GCH. Behavioral recovery was observed in the rats that received AAV vectors expressing DA-synthesizing enzymes except for those that received AAV-Cre simultaneously. After tamoxifen treatment, the AAV-CreERT2 injected rats showed behavioral impairment again. The DA content in the lesioned striatum was significantly lower and there were less TH-immunoreactive cells in the tamoxifen-treated AAV-CreERT2 group compared with the tamoxifen-untreated group. The AADC activity did not change after the tamoxifen treatment, suggesting selective inactivation of TH. With this method, orally administrated L-DOPA, a substrate for AADC, could be converted to DA in the striatum and retain therapeutic effects even after the patients were treated with tamoxifen in case of DA overproduction. Inducible reduction of TH expression using AAV-CreERT2 should be a molecular switch for increasing the safety of AAV vector-mediated gene therapy for PD.