Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice
Alexander Pfeifer, Sabina Eigenbrod, Saba Al-Khadra, Andreas Hofmann, Gerda Mitteregger, Markus Moser, Uwe Bertsch, Hans Kretzschmar
Alexander Pfeifer, Sabina Eigenbrod, Saba Al-Khadra, Andreas Hofmann, Gerda Mitteregger, Markus Moser, Uwe Bertsch, Hans Kretzschmar
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Research Article Neuroscience

Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice

Abstract

Prion diseases are fatal neurodegenerative diseases characterized by the accumulation of PrPSc, the infectious and protease-resistant form of the cellular prion protein (PrPC). We generated lentivectors expressing PrPC-specific short hairpin RNAs (shRNAs) that efficiently silenced expression of the prion protein gene (Prnp) in primary neuronal cells. Treatment of scrapie-infected neuronal cells with these lentivectors resulted in an efficient and stable suppression of PrPSc accumulation. After intracranial injection, lentiviral shRNA reduced PrPC expression in transgenic mice carrying multiple copies of Prnp. To test the therapeutic potential of lentiviral shRNA, we used what we believe to be a novel approach in which the clinical situation was mimicked. We generated chimeric mice derived from lentivector-transduced embryonic stem cells. Depending on the degree of chimerism, these animals carried the lentiviral shRNAs in a certain percentage of brain cells and expressed reduced levels of PrPC. Importantly, in highly chimeric mice, survival after scrapie infection was significantly extended. Taken together, these data suggest that lentivector-mediated RNA interference could be an approach for the treatment of prion disease.

Authors

Alexander Pfeifer, Sabina Eigenbrod, Saba Al-Khadra, Andreas Hofmann, Gerda Mitteregger, Markus Moser, Uwe Bertsch, Hans Kretzschmar

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Figure 2

Silencing of PrPC in chimeric mice derived from LVsh512-infected ES cells.

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Silencing of PrPC in chimeric mice derived from LVsh512-infected ES cell...
(A) Western blot analysis of ES cell clones infected with LVsh512 or LVEGFP. The number of integrants (Integr.) and levels of PrPC expression (PrPC) are given above the blot. (B) Fluorescence imaging of freshly isolated brains from a control mouse (WT, left) and a transgenic animal (no. 1917, right). Shown are the fluorescence (top) and the bright-field (bottom) images. (C–J) Immunohistochemical analysis of PrPC and EGFP expression in sections of hippocampus from a transgenic (no. 1917) and an age-matched WT animal. (C and G) Analysis of PrPC expression in WT hippocampus. (E and I) Staining for PrPC revealed reduced expression of PrPC in the chimeric hippocampus as compared with the WT. (D and H) Expression of EGFP in the WT mouse. (F and J) Staining for EGFP in the chimeric hippocampus, indicating the presence of the LVsh512 provirus. Higher magnifications are shown in G and H for the WT and I and J for the chimeric mouse. Scale bars: 200 μm in C–F and 50 μm in G–J. (K and L) Western blot analysis of PrPC expression in the cerebrum and cerebellum of chimeric animals.