Title
Does the Mutant CAG Expansion in Huntingtin mRNA Interfere with Exonucleolytic Cleavage of its First Exon
UMMS Affiliation
RNA Therapeutics Institute; Department of Medicine, Division of Endocrinology and Metabolism; Gene Therapy Center; Department of Pediatrics, Division of Pulmonary and Allerg
Publication Date
2016-3
Document Type
Article
Disciplines
Genetics and Genomics | Nervous System Diseases
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.
OBJECTIVES: 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.
Keywords
CAG repeats, Huntington’s disease, RNAi, mRNA degradation
DOI of Published Version
10.3233/JHD-150183
Source
J Huntingtons Dis. 2016;5(1):33-8. doi: 10.3233/JHD-150183. Link to article on publisher's site
Journal/Book/Conference Title
Journal of Huntington's disease
Related Resources
PubMed ID
27003665
Repository Citation
Liu W, Pfister EL, Kennington LA, Chase KO, Mueller C, DiFiglia M, Aronin N. (2016). Does the Mutant CAG Expansion in Huntingtin mRNA Interfere with Exonucleolytic Cleavage of its First Exon. Open Access Publications by UMass Chan Authors. https://doi.org/10.3233/JHD-150183. Retrieved from https://escholarship.umassmed.edu/oapubs/2880