rAAV-mediated shRNA ameliorated neuropathology in Huntington disease model mouse

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Abstract

Huntington disease (HD) is a fatal progressive neurodegenerative disorder associated with expansion of a CAG repeat in the first exon of the gene coding the protein huntingtin (htt). Although the feasibility of RNA interference (RNAi)-mediated reduction of htt expression to attenuate HD-associated symptoms is suggested, the effects of post-symptomatic RNAi treatment in the HD model mice have not yet been certified. Here we show the effects of recombinant adeno-associated virus (rAAV)-mediated delivery of RNAi into the HD model mouse striatum after the onset of disease. Neuropathological abnormalities associated with HD, such as insoluble protein accumulation and down-regulation of DARPP-32 expression, were successfully ameliorated by the RNAi transduction. Importantly, neuronal aggregates in the striatum were reduced after RNAi transduction in the animals comparing to those at the time point of RNAi transduction. These results suggest that the direct inhibition of mutant gene expression by rAVV would be promising for post-symptomatic HD therapy.

Section snippets

Materials and methods

HD model mouse. The HD190QG transgenic mouse was used as a HD model in this study. The HD190QG transgenic mouse harbors mutant truncated N-terminal htt containing 190 CAG repeats fused with EGFP in its genome. This animal shows progressive motor abnormality, and neuropathology such as formation of aggregates in brain, and shorter viability [21]. All the experiments with mice were approved by the Animal Experiment Committee of the RIKEN Brain Science Institute.

Construction and production of rAAV.

Gene silencing by rAAV-shRNA

In vitro screening was used to identify the efficiency of mRNA ablation of shRNAs directed to EGFP and EGFP-fused truncated htt-polyQ. EGFP-fused truncated htt-190Q is identical to the pathogenic transgene present in the HD190QG mouse [21]. The gene silencing function of 10 candidate shRNA sequences targeting EGFP was evaluated by the EGFP expression of co-transfected Neuro2A cells with shRNA and EGFP (data not shown). An shRNA targeting EGFP sequence 5′-GCAAGCTGACCCTGAAGTTCAT-3′ (shEGFP)

Discussion

In this study, we demonstrated that neuropathological abnormalities associated with HD, such as insoluble protein accumulation and down-regulation of DARPP-32 expression, were successfully ameliorated by RNAi transduction. Following shRNA transduction, the number of neuronal aggregates in the striatum detected by ubiquitin antibody was reduced to 34.1% of that in the sham-treated striatum. Importantly, the number of aggregates in the shEGFP-transduced striatum was less than that in the striatum

Acknowledgments

The authors thank Dr. John A. Chiorini for providing pAAV5-RNL and pAAV5-RepCap (identical to 5RepCapB) and Avigen, Inc. (Alameda, CA) for providing pAAV-LacZ, pHLP19, and pAdeno. We also thank Drs. Nobuhisa Iwata, Takaomi Saido, Mayumi Okada, and Ms. Miyoko Mitsu for their technical supports and Drs. Joanna Doumanis and Hong-Kit Wong for their critical readings. This work was supported in part by grants from Grants-in-Aid for Scientific Research on Priority Areas 17025044 from The Ministry of

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