Abstract
RNA interference (RNAi) is a powerful strategy for unraveling gene function and for drug target validation, but exogenous expression of short hairpin RNAs (shRNAs) has been associated with severe side effects. These may be caused by saturation of the microRNA pathway. This study shows degenerative changes in cell morphology and intrusion of blood vessels after transduction of the ventromedial hypothalamus (VMH) of rats with a shRNA expressing adeno-associated viral (AAV) vector. To investigate whether saturation of the microRNA pathway has a role in the observed side effects, expression of neuronal microRNA miR-124 was used as a marker. Neurons transduced with the AAV vector carrying the shRNA displayed a decrease in miR-124 expression. The decreased expression was unrelated to shRNA sequence or target and observed as early as 1 week after injection. In conclusion, this study shows that the tissue response after AAV-directed expression of a shRNA to the VMH is likely to be caused by shRNA-induced saturation of the microRNA pathway. We recommend controlling for miR-124 expression when using RNAi as a tool for studying (loss of) gene function in the brain as phenotypic effects caused by saturation of the RNAi pathway might mask true effects of specific downregulation of the shRNA target.
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Acknowledgements
This work was supported by TIPharma, project T5-210-1, and by a VIDI grant from the Netherlands Organization for Health Research and Development and a grant from Neuroscience and Cognition Utrecht (to RJP). We acknowledge Eljo van Battum, Rudolf Magnus Institute of Neuroscience, for her technical assistance and Dr Joost Verhaagen, Netherlands Institute for Neuroscience, for critically reading the manuscript.
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van Gestel, M., van Erp, S., Sanders, L. et al. shRNA-induced saturation of the microRNA pathway in the rat brain. Gene Ther 21, 205–211 (2014). https://doi.org/10.1038/gt.2013.76
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DOI: https://doi.org/10.1038/gt.2013.76
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