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

Abstract

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.

Studies of Efficacy and Liver Toxicity Related to Adeno-Associated Virus–Mediated RNA Interference

“…Subsequently, several other groups reported that shRNA overexpression from AAV vectors, most using the strong U6 promoter, causes cytotoxicity in different cell types and organs (McBride et al, 2008;Boudreau et al, 2009;Bish et al, 2011;Martin et al, 2011). Lowering the AAV dose reduced the adverse effects (Grimm et al, 2006;Ulusoy et al, 2009;Ehlert et al, 2010), suggesting that the toxic effect is dependent upon shRNA levels, a result supported by our findings. Analysis of cellular microRNAs was carried out in several of these studies, and the results showed that some microRNAs were decreased to various levels, but others were not changed (Grimm et al, 2006(Grimm et al, , 2010Witting et al, 2008;Ahn et al, 2011;Bish et al, 2011;Martin et al, 2011;Pan et al, 2011).…”

Prostaglandin-dependent modulation of dopaminergic neurotransmission elicits inflammation-induced aversion in mice

“…The GFP vector contained a FLEX cassette (66) resulting in Cre-dependent expression. The AAV vectors were produced using a double-transfection method with the appropriate transfer plasmid and the helper plasmid containing the essential adenoviral packaging genes, as described previously (67). Vectors were purified by iodixanol step gradients and Q Sepharose column chromatography (Sigma-Aldrich).…”

Comparison of AAV Serotypes for Gene Delivery to Dopaminergic Neurons in the Substantia Nigra

“…In this study not only TH was diminished, but also the expression of VMAT2. In subsequent experiments with low titer AAV5-GFP injections this effect was not seen anymore (Ulusoy et al, 2009). We speculate that in our case, the high tropism of AAV7 for DAergic neurons induces more GFP expression, and as a consequence causes DAergic neurotoxicity.…”

“…In the mouse SN, AAV vectors with the SYN promoter were superior to the AAV vectors containing the CMV promoter. In contrast, in the rat, a range of promoters have been used very successfully to direct the transgene expression in the DAergic neurons of the SN via an AAV vector including the CMV (McFarland et al, 2009), CBA (Burger et al, 2004;Klein et al, 2006;Paterna et al, 2004;Ulusoy et al, 2009), CMV/CBA hybrid (Klein et al, 2008) and PDGV (Paterna et al, 2000)) promoters. This suggests that for experiments in the rat the choice of the promoter is not as critical as it appears to be for the mouse.…”

“…The skull level was controlled by measurement of bregma and lambda. 2µl of each AAV virus was injected at the following coordinates with VD being measured from dura: AP -5.2, L -2.0, and VD 7.2 (Ulusoy, Sahin, Bjorklund, Aebischer, & Kirik, 2009). All of the animals recovered on a heating pad at 37°C and were allowed to survive for 4 weeks post surgery after which they were sacrificed by an IP overdose with Pentobarbital (50mg/µl) and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (PFA, Sigma-Aldrich Co., St. Louis, MO, USA) in PBS pH 7.4.…”