title: Does the Mutant CAG Expansion in Huntingtin mRNA Interfere with Exonucleolytic Cleavage of its First Exon?
abstract: BACKGROUND
  Silencing mutant huntingtin mRNA by RNA interference (RNAi) is a therapeutic strategy for Huntington's disease. RNAi induces specific endonucleolytic cleavage of the target HTT mRNA, followed by exonucleolytic processing of the cleaved mRNA fragments.
  
  
  OBJECTIVE
  We investigated the clearance of huntingtin mRNA cleavage products following RNAi, to find if particular huntingtin mRNA sequences persist. We especially wanted to find out if the expanded CAG increased production of a toxic mRNA species by impeding degradation of human mutant huntingtin exon 1 mRNA.
  
  
  METHODS
  Mice expressing the human mutant HTT transgene with 128 CAG repeats (YAC128 mice) were injected in the striatum with self-complementary AAV9 vectors carrying a miRNA targeting exon 48 of huntingtin mRNA (scAAV-U6-miRNA-HTT-GFP). Transgenic huntingtin mRNA levels were measured in striatal lysates after two weeks. For qPCR, we used species specific primer-probe combinations that together spanned 6 positions along the open reading frame and untranslated regions of the human huntingtin mRNA. Knockdown was also measured in the liver following tail vein injection.
  
  
  RESULTS
  Two weeks after intrastriatal administration of scAAV9-U6-miRNA-HTT-GFP, we measured transgenic mutant huntingtin in striatum using probes targeting six different sites along the huntingtin mRNA. Real time PCR showed a reduction of 29% to 36% in human HTT. There was no significant difference in knockdown measured at any of the six sites, including exon 1. In liver, we observed a more pronounced HTT mRNA knockdown of 70% to 76% relative to the untreated mice, and there were also no significant differences among sites.
  
  
  CONCLUSIONS
  Our results demonstrate that degradation is equally distributed across the human mutant huntingtin mRNA following RNAi-induced cleavage.
authors.affiliation: RNA Therapeutics Institute and Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Wanzhao
authors.initials: W
authors.lastname: Liu
authors.affiliation: RNA Therapeutics Institute and Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Edith L
authors.initials: EL
authors.lastname: Pfister
authors.affiliation: RNA Therapeutics Institute and Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Lori A
authors.initials: LA
authors.lastname: Kennington
authors.affiliation: RNA Therapeutics Institute and Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Kathryn O
authors.initials: KO
authors.lastname: Chase
authors.affiliation: Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Mueller
authors.affiliation: Mass General Institute for Neurodegenerative Disease, Massachusetts General Hospital, Charlestown, MA, USA.
authors.forename: Marian
authors.initials: M
authors.lastname: DiFiglia
authors.affiliation: RNA Therapeutics Institute and Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Neil
authors.initials: N
authors.lastname: Aronin
mesh_terms: D000818:Animals; D003342:Corpus Striatum / Q000378:metabolism; D004195:Disease Models, Animal; D005091:Exons / Q000235:genetics; D055785:Gene Knockdown Techniques; D000071058:Huntingtin Protein / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D006816:Huntington Disease / Q000235:genetics*; D008099:Liver / Q000378:metabolism; D051379:Mice; D009154:Mutation / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D018911:Trinucleotide Repeats / Q000235:genetics*
chemical_list: C086055:HTT protein, human; D000071058:Huntingtin Protein; D012333:RNA, Messenger
keywords: CAG repeats; Huntington’s disease; RNAi; mRNA degradation