 Dominant neurodegenerative disease  Polyglutamine repeat expansions (CAG, codon, Q) in exon 1 of huntingtin gene (htt). Usually >35 CAG repeats.  Toxic gain of function mutation causing gradually damage to certain areas in brain

 Behavioral and cognitive disturbances  Involuntary movements (chorea)  Neuronal inclusions  Striatal and cortical neurodegeneration

 Expressed in all mammalian cells, highest in brain and testes  Function not entirely clear in humans  Interacts with proteins involved in transcription, cell signaling, and intracellular transporting

 Glutamine is polar and causes interactions with other proteins when overproduced in htt  Then, htt forms H-bonds with each other, resulting in a protein aggregate instead of normal folding protein.  Aggregates over time result in neuronal inclusions.

 By directly inhibiting the expression of mutant htt, HD associated symptoms may be reduced or prevented.  This study tested if RNAi induced by short hairpin RNAs (shRNAs) could improve HD- associated abnormalities by reducing expression of mutant htt in a transgenic HD mouse model.

 HD-N171-82Q gene expressed from the pCMV-HD-N171-82G plasmid.  HD-N171-82Q is a truncated htt fragment  shRNAs and U6 promoter were amplified with PCR to target human htt (shHD2.1), eGFP (shGFP) or E. coli β-galactosidase (shLacZ).

 PCR products were cloned, sequenced, and inserted into adenoassociated virus (AAV) plasmid pAAV.CMV.hrGRP with AAV serotype 2 inverted terminal repeats, CMV-humanized Renilla GFP (hrGFP)-simian virus 40 poly(A) reporter cassette.

 HEK293 cells were transfected with pCMV-HD-N171-82G and plasmid expressing shHD2.1, shGFP, or shLacZ.  RNA was isolated 48 hours after transfection and Northern blot analysis performed with human htt probes or human GAPDH probes as a control.

 HEK293 transfected cells were lysed to recover total protein and Western blot analysis was performed with actin as a control.

 Mice were injected with AAV plasmids containing U6-driven shHD2.1 or shLacZ at four-weeks old and analyzed at four- months.  After injection into mouse striatum, shHD2.1 expression was analyzed by isolating total RNA from grGFP-positive striata using Northern blot analysis.

 To test the effect of RNAi on neuronal inclusions associated with HD, tissues were harvested from mice at about 5.5 months old and RNA was isolated.  In striata from mice injected with AAV.shHD2.1, htt-reactive inclusions were absent and mutant htt expression was reduced.

 Coronal sections were isolated from mice and stained with mEM48 antibody followed by goat anti-mouse secondary antibody  Images were captured using fluorescent microscopy

 Stride length measurements were taken by injected mice walking across a paper-lined chamber and into an enclosed box and measuring footprint tracings.  There was a noticable weight difference between HD-N171-82Q and wild type mice that was not normalized by RNAi directly to the striatum.

 Mice were injected at 4 weeks of age and tested at 10 and 18 weeks old.  Amount of time it took mice to fall was measured.

 Motor and neuropathological abnormalities in a HD mouse model are significantly improved using AAV delivered shRNA to reduce striatal expression of pathogenic htt allele.  Suggests feasibility of treating HD with direct reduction of mutant htt gene expression using RNAi.

 Harper, Q. S. et al. (2005) RNA interference improves motor and neuropathological abnormalities in a Huntington’s disease mouse model. PNAS, 102:  /introduction-animals.aspx