Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits

DiFiglia et al. 10.1073/pnas.0708285104.

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SI Figure 7
SI Table 1
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Fig. 7. Cellular immune response after siRNA administration. Shown are sections from the striata of mice that had been injected with 3 ml of PBS alone (A and C) or cc-siRNA-Luc (B and D). Immunoperoxidase labeling is shown for GFAP (A and B) and CD11B (C and D). The extent of GFAP and CD11B immunoreactive cells in striatum is similar whether mice were injected with cc-siRNA-Luc or cc-siRNA-Htt (not shown) or PBS. The arrows demarcate the regions of the analysis.





Fig. 8. Neuronal delivery of Cy3-cc-siRNA in primary striatal neurons in culture. We incubated Cy3-siRNA (50 mM final concentration) in a primary culture of murine striatal neurons (gestational day 15.5), to test whether adeno-associated virus was needed to deliver cc-siRNA into neurons. Cy3 labeling was detected in neurons (detail in Inset; N, nucleus).





Fig. 9. cc-siRNA silences endogenous huntingtin in a transgenic murine model of HD. To determine whether cc-siRNA-Htt required adeno-associated virus (AAV) coinjection, we studied Htt silencing by direct injection of cc-siRNA (same dose used in AAV-Htt experiments) into mouse striatum of YAC128 transgenic mice [Slow EJ, van Raamsdonk J, Rogers D, Coleman SH, Graham RK, Deng Y, Oh R, Bissada N, Hossain SM, Yang YZ, et al. (2003) Hum Mol Genet 12:1555-1567; gift of M. Hayden, University of British Columbia, Vancouver, BC, Canada]. Injections were made on days 1 and 3, with striatal tissue extracted on day 4. Endogenous huntingtin after cc-siRNA-Htt administration was decreased on Western blot compared with cc-siRNA-Luc injection (n = 6 in each group; cc-siRNA-Luc versus cc-siRNA-Htt, 9.22 ± 2.71 versus 6.62 ± 1.86; P £ 0.04, Student's t test, one-tailed.) No difference was found in the mutant human huntingtin, which is overexpressed in this transgenic model. Htt, cc-siRNA-Htt; Luc, denotes cc-siRNA-Luc.





Table 1. Animal temperature and weight changes after siRNA administration

 

Average temperature, ºC

Average weight, g

AAV Htt100Q siRNA-Luc (n = 14)

37.3 ± 0.47

24.1 ± 2.1

AAV Htt100Q siRNA-Htt (n = 14)

37.3 ± 0.52

24.0 ± 1.6

AAV Htt18Q siRNA-Luc (n = 9)

37.4 ± 0.43

25.3 ± 2.3

AAV Htt18Q siRNA-Htt (n = 9)

37.5 ± 0.42

24.5 ± 1.9

Animal temperatures or weights did not differ based on administration of siRNA-Luc or siRNA-Htt. Means ± SD are shown.

This Article

  1. Published online before print October 16, 2007, doi: 10.1073/pnas.0708285104 PNAS October 23, 2007 vol. 104 no. 43 17204-17209
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