Cellular analysis of silencing the Huntington's disease gene using AAV9 mediated delivery of artificial micro RNA into the striatum of Q140/Q140 mice
AM Keeler, E Sapp, K Chase… - Journal of …, 2016 - content.iospress.com
Journal of Huntington's disease, 2016•content.iospress.com
Background: The genetic mutation in Huntington's disease (HD) is a CAG repeat expansion
in the coding region of the huntingtin (Htt) gene. RNAi strategies have proven effective in
substantially down-regulating Htt mRNA in the striatum through delivery of siRNAs or viral
vectors based on whole tissue assays, but the extent of htt mRNA lowering in individual
neurons is unknown. Objective: Here we characterize the effect of an AAV9-GFP-miRHtt
vector on Htt mRNA levels in striatal neurons of Q140/Q140 knock-in mice. Methods: HD …
in the coding region of the huntingtin (Htt) gene. RNAi strategies have proven effective in
substantially down-regulating Htt mRNA in the striatum through delivery of siRNAs or viral
vectors based on whole tissue assays, but the extent of htt mRNA lowering in individual
neurons is unknown. Objective: Here we characterize the effect of an AAV9-GFP-miRHtt
vector on Htt mRNA levels in striatal neurons of Q140/Q140 knock-in mice. Methods: HD …
Abstract
Background: The genetic mutation in Huntington’s disease (HD) is a CAG repeat expansion in the coding region of the huntingtin (Htt) gene. RNAi strategies have proven effective in substantially down-regulating Htt mRNA in the striatum through delivery of siRNAs or viral vectors based on whole tissue assays, but the extent of htt mRNA lowering in individual neurons is unknown.
Objective: Here we characterize the effect of an AAV9-GFP-miRHtt vector on Htt mRNA levels in striatal neurons of Q140/Q140 knock-in mice.
Methods: HD mice received bilateral striatal injections of AAV9-GFP-miRHtt or AAV9-GFP at 6 or 12 weeks and striata were evaluated at 6 months of age for levels of Htt mRNA and protein and for mRNA signal within striatal neurons using RNAscope multiplex fluorescence in situ hybridization.
Results: Compared to controls, the striatum of 6-month old mice treated at 6 or 12 weeks of age with AAV9-GFP-miRHtt showed a reduction of 40–50% in Htt mRNA and lowering of 25–40% in protein levels. The number of Htt mRNA foci in medium spiny neurons (MSNs) of untreated Q140/Q140 mice varied widely per cell (0 to 34 per cell), with∼ 10% of MSNs devoid of foci. AAV9-GFP-miRHtt treatment shifted the distribution toward lower numbers and the percentage of cells without foci increased to 14–20%. The average number of Htt mRNA foci per MSN was reduced by 43%.
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