Dose Optimization for Long-term rAAV-mediated RNA Interference in the Nigrostriatal Projection Neurons

• Published in: Molecular therapy Vol. 17; no. 9; pp. 1574 - 1584
• Main Authors: Ulusoy, Ayse, Sahin, Gurdal, Björklund, Tomas, Aebischer, Patrick, Kirik, Deniz
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2009; Elsevier Limited; Nature Publishing Group
• Subjects: Adenoviridae - genetics; Animals; Basic Medicine; Brain; Cell death; Cell Line; Chromatography, High Pressure Liquid; Dopamine; Dopamine - metabolism; Female; Gene expression; Gene Silencing - physiology; Genetic Vectors - genetics; Humans; Levodopa; Medical and Health Sciences; Medical Genetics; Medicin och hälsovetenskap; Medicinsk genetik; Medicinska och farmaceutiska grundvetenskaper; MicroRNAs; Nervous system; Neurons - metabolism; Optimization; Original; Proteins; Rats; Rats, Sprague-Dawley; RNA Interference - physiology; Substantia Nigra - cytology; Toxicity; Tyrosine 3-Monooxygenase - genetics; Tyrosine 3-Monooxygenase - metabolism; Tyrosine 3-Monooxygenase - physiology; Variance analysis; Sweden
• ISSN: 1525-0016; 1525-0024; 1525-0024
• DOI: 10.1038/mt.2009.142

Short-hairpin RNA (shRNA)–mediated gene knockdown is a powerful tool for targeted gene silencing and an emerging novel therapeutic strategy. Recent publications, however, reported unexpected toxicity after utilizing viral-mediated shRNA knockdown in vivo. Thus, it is currently unclear whether shRNA-mediated knockdown strategy can be used as a safe and efficient tool for gene silencing. In this study, we have generated rAAV vectors expressing shRNAs targeting the rat tyrosine hydroxylase (TH) mRNA (shTH) for testing the efficacy of in vivo TH knockdown in the nigral dopaminergic neurons. At high titers, not only the shTH vectors but also the scrambled and green fluorescence protein (GFP)–only controls caused cell death. In a dose–response study, we identified a dose window leading to >60% decrease in TH+ neurons without any change in vesicular monoamine transporter-2 (VMAT2) expression. Moreover, using the safe and efficient dose, we showed that dopamine (DA) synthesis rate was significantly reduced and this lead to emergence of motor deficits in the shTH-expressing rats. Interestingly, these animals showed very robust and long-lasting recovery after a single systemic L-3,4-dihydroxyphenylalanine (L-DOPA) administration beyond what can be achieved in 6-hydroxydopamine (6-OHDA)–lesioned rats. Our results have implications for both mechanistic and therapeutic studies utilizing long-term shRNA-mediated gene silencing in the nigrostriatal projection system.