Adeno-associated virus-mediated gene transfer of human aromatic L-amino acid decarboxylase protects mixed striatal primary cultures from L-DOPA toxicity Mohammad M. Doroudchi, Jason Liauw, Krista Heaton, Zhu Zhen and John R. Forsayeth 1 Avigen Inc., Alameda, California, USA Abstract Although L-DOPA is the drug of choice for Parkinson’s dis- ease, prolonged L-DOPA therapy results in decreased drug effectiveness and the appearance of motor complications. This may be due in part to the progressive loss of the enzyme, aromatic L-amino acid decarboxylase (AADC). We have developed an adeno-associated virus vector (AAV–hAADC) that contains human AADC cDNA under the control of the cytomegalovirus promoter. Infusion of this vector into the striatum of parkinsonian rats and monkeys improves L-DOPA responsiveness by improving AADC-mediated conversion of L-DOPA to dopamine. This is now the basis of a proposed therapy for advanced Parkinson’s disease. A key concern has been that over-production of dopamine in striatal neurons could cause dopamine toxicity. To investigate this possibility in a controlled system, mixed striatal primary rat neuronal cultures were prepared. Exposure of cultures to high con- centrations of L-DOPA induced the following changes: cell death in nigral and striatal neurons, aggregation of neurofila- ments and focal axonal swellings, abnormal expression of DARPP-32, and activation of astroglia and microglial cells. Transduction of cultures with AAV–hAADC resulted in efficient and sustained neuronal expression of the AADC protein and prevented all the L-DOPA-induced toxicities. The protective effects were due primarily to AADC-dependent conversion of L-DOPA to dopamine and an increase in induction of vesicular monoamine transporter resulting in dopamine storage in cul- tured cells. These results suggest a neuroprotective role for AADC gene transfer against L-DOPA toxicity. Keywords: adeno-associated virus, gene therapy, L-DOPA, neuroprotection, neurotoxicity, Parkinson’s disease. J. Neurochem. (2005) 93, 634–640. Parkinson’s disease (PD) is a neurodegenerative disorder that is caused primarily by degeneration of dopaminergic neurons projecting into the striatum from the substantia nigra (Forno 1982; Marsden 1986). At present, the cause of the degen- eration and death of neurons in idiopathic PD is unknown. Although L-DOPA, the metabolic precursor of dopamine, is the drug of choice in the management of PD (Lloyd et al. 1975; Land and Lozano 1998), prolonged L-DOPA therapy is associated with decreased drug effectiveness and the appear- ance of side effects such as dyskinesia and on–off phenom- ena (Lesser et al. 1979; Obeso et al. 2000). This may be due in part to progressive loss of the enzyme, aromatic L-amino acid decarboxylase (AADC), which is required for efficient conversion of L-DOPA to dopamine (Poewe and Wenning 2002). Recent evidence shows that L-DOPA is itself phar- macologically active in a manner independent of PD pathology (Pearce et al. 2001; Smith et al. 2003). Gene transfer technology offers the possibility of achiev- ing prolonged delivery of AADC protein to the striatum. Recombinant adeno-associated virus (AAV)-based vectors show promise in gene therapy due to their relative safety and long-term gene expression (Xiao et al. 1996; Bennett 1999; High 2001; Pfeifer and Verma 2001). An AAV vector (AAV– hAADC) has been developed that contains human AADC cDNA under the control of the cytomegalovirus promoter. Infusion of this vector into the striatum of parkinsonian rats Received November 16, 2004; revised manuscript received December 14, 2004; accepted December 15, 2004. Address correspondence and reprint requests to Dr Mehdi Doroudchi, Avigen Inc., 1301 Harbor Bay Pkwy, Alameda, CA 94502, USA. E-mail: mdoroudchi@avigen.com 1 The present address of John R. Forsayeth is Department of Neurological Surgery, University of California San Francisco, San Francisco, CA 94103–0555, USA. Abbreviations used: AADC, aromatic L-amino acid decarboxylase; AAV, adeno-associated virus; GFAP, glial fibrillary acidic protein; PD, Parkinson’s disease; TH, tyrosine hydroxylase; VMAT2, vesicular monoamine transporter. Journal of Neurochemistry , 2005, 93, 634–640 doi:10.1111/j.1471-4159.2005.03048.x 634 Ó 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 634–640