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, 116 (12), 3204-10

Lentivector-mediated RNAi Efficiently Suppresses Prion Protein and Prolongs Survival of Scrapie-Infected Mice


Lentivector-mediated RNAi Efficiently Suppresses Prion Protein and Prolongs Survival of Scrapie-Infected Mice

Alexander Pfeifer et al. J Clin Invest.


Prion diseases are fatal neurodegenerative diseases characterized by the accumulation of PrP(Sc), the infectious and protease-resistant form of the cellular prion protein (PrP(C)). We generated lentivectors expressing PrP(C)-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 PrP(Sc) accumulation. After intracranial injection, lentiviral shRNA reduced PrP(C) 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 PrP(C). 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.


Figure 1
Figure 1. Targeting of PrPC by lentivector-mediated RNAi.
(A) The HIV-based lentivector (LVshPrPC, top) used carries an H1 promoter–driven shRNA cassette and a PGK-EGFP expression cassette. Ψ, packaging signal; ppt, polypurine tract; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; triangle, self-inactivating mutation; arrows, direction of transcription. The lentivector-encoded anti-PrPC shRNA (shPrPC, bottom) consists of 19- or 21-bp stems separated by a loop. (B) Representative bright-field (left) and fluorescence microscope (right) images of N2a cells 72 hours after transduction with LVshPrPC. (C) LVshPrPC-induced knock down of PrPC in N2a cells. Control, uninfected cells. (D) PrPC expression in N2a cells infected with a low (×1) and high (×10) concentration of LVsh512 and LVshscr. (E) Analysis of PrPC expression in granule cells 72 hours after infection with LVsh512 or a control vector carrying only the EGFP expression cassette (LVEGFP). Western blots (CE) were probed with the indicated antibodies. (F) Effect of LVsh512 on the accumulation of protease K–resistant (PK-resistant) PrPSc in ScN2a cells. Western blot analysis 72 hours after transduction with the indicated lentivectors. (G and H) Intracranial injection of LVshscr (G) and LVsh512 (H) in tga20 mice overexpressing PrPC. Analysis of EGFP (left) and PrPC (right) expression 3 weeks after injection. Arrows indicate the lentivector-transduced area.
Figure 2
Figure 2. Silencing of PrPC in chimeric mice derived from LVsh512-infected ES cells.
(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. (CJ) 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 CF and 50 μm in GJ. (K and L) Western blot analysis of PrPC expression in the cerebrum and cerebellum of chimeric animals.
Figure 3
Figure 3. Survival times of LVsh512-chimeric and control mice after Rocky Mountain Laboratory Inc.
(RML) scrapie infection. Chimeric mice derived from LVsh512-derived ES cells survived prion disease up to 231 days. All control animals (GFP.3, green; C57×129sv, black; 129sv, blue) died within 174 dpi with prions. Dashed lines, controls; Red line, LVsh512-chimeric mice.
Figure 4
Figure 4. Analysis of PrPSc accumulation and neuropathological changes in prion-inoculated chimeric siblings.
(A) Histological analysis of a scrapie-infected chimeric mouse (20%) that died at 172 dpi and a 70% chimera that was sacrificed at the same time. Analysis of spongiform changes (top) and gliosis (bottom). Coronal sections of the posterior cerebrum at the hippocampal level of the 20% (left) and 70% (right) chimeric siblings are shown. Top: H&E staining; bottom: glial fibrillary acidic protein (GFAP) staining (brown). (B) Western blots of samples from the same chimeric mice as shown in A and a wild-type control. Levels of PrPSc (+PK) and total PrP (PrPtotal, –PK) in 2 different brain regions: cerebrum including hippocampus (left) and cerebellum (right).

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