Long-term in vivo and in vitro AAV-2-mediated RNA interference in rat retinal ganglion cells and cultured primary neurons Uwe Michel * ,1 , Ibrahim Malik 1 , Sandra Ebert, Mathias Ba ¨hr, Sebastian Ku ¨ gler Viral Vector Laboratory, Department of Neurology, University of Go ¨ ttingen, Waldweg 33, 37073 Go ¨ ttingen, Germany Received 18 October 2004 Available online 25 November 2004 Abstract Viral vector-based expression of small interfering RNAs is a promising tool for gene regulation, both in cultured cells and in animal models. In this study, we analysed the ability of adeno-associated virus-2 to function as an RNAi vector in cultured primary hippocampal neurons in vitro and in retinal ganglion cells in vivo. We demonstrate a long-lasting, highly efficient, and specific down- regulation of gene expression in vivo and in vitro by the use of bicistronic vectors. This is the first evidence of a cell type-specific long-term (more than three-month-long) RNAi in the eye. Furthermore, our results constitute the prerequisite for the use of this technique in models of neurodegeneration and neuroregeneration in vivo and in vitro. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Long-term RNAi; Retinal ganglion cells; Primary neuron culture; AAV-2 Neurodegeneration can be either due to neuronal loss, which occurs over a long period of time, like in AlzheimerÕs and ParkinsonÕs diseases, or to an acute neurological insult, as for example, in trauma or ischae- mia. Neurodegeneration is a major cause of mortality and morbidity, and causes tremendous costs in western societies. However, for most neurodegenerative disor- ders there are no effective therapies. One reason for the lack of therapeutic strategies is the inaccessibility of the brain, which is caused by the blood–brain barrier and the skull. In contrast to the brain, neurons of the eye and the optic nerve, which is made up of ganglion cell axons, are readily accessible and are therefore ideally suited to study neurodegeneration and neuroregenera- tion in the central nervous system (CNS) [1]. RNA interference (RNAi) is the sequence-specific down-regulation of mRNAs by double-stranded small interfering RNA (siRNA). This target gene-specific deg- radation of RNA can be used as an effective means of gene suppression and is therefore used to analyse gene function. A major challenge in applying the technique in vitro or in vivo is the introduction of the small inter- fering RNA (siRNA) into primary cell cultures or into target cells of higher living organisms. RNAi could be a useful technique for intervening in and targeting neu- rodegenerative or neuroprotective processes [2], although recent work has shown that neuronal cells can be difficult to transfect using synthetic siRNA or siRNA-expressing plasmids [3–5]. In the central nervous system, the serotype 2 of the adeno-associated virus (AAV-2) preferentially transdu- ces neurons [6]. We developed viral vectors based on hu- man AAV-2 that express a fluorescent protein directed from a polymerase II promoter as well as an siRNA di- rected from the human H1 promoter. Here, we show in vivo that in rat retinal ganglion cells an AAV-2, which expresses an siRNA directed against enhanced green fluorescent protein (EGFP), is capable of completely knocking down the human synapsin promoter-driven expression of EGFP from a co-transduced AAV-2. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.11.029 * Corresponding author. Fax: +49 551 3914476. E-mail address: umichel@gwdg.de (U. Michel). 1 Both authors contributed equally to the manuscript. www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 326 (2005) 307–312 BBRC