Affiliations: [a] RNA Therapeutics Institute and Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA | [b] Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, USA | [c] Mass General Institute for Neurodegenerative Disease, Massachusetts General Hospital, Charlestown, MA, USA
Correspondence:
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Correspondence to: Neil Aronin, RNA Therapeutics Institute and Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA. Tel.: +1 508 856 6559; Fax: +1 508 856-6696; E-mail: [email protected].
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.
