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dc.contributor.authorLiu, Wanzhao
dc.contributor.authorPfister, Edith L.
dc.contributor.authorKennington, Lori A.
dc.contributor.authorChase, Kathryn O.
dc.contributor.authorMueller, Christian
dc.contributor.authorDiFiglia, Marian
dc.contributor.authorAronin, Neil
dc.date2022-08-11T08:09:45.000
dc.date.accessioned2022-08-23T16:42:21Z
dc.date.available2022-08-23T16:42:21Z
dc.date.issued2016-03-01
dc.date.submitted2016-11-30
dc.identifier.citationJ Huntingtons Dis. 2016;5(1):33-8. doi: 10.3233/JHD-150183. <a href="http://dx.doi.org/10.3233/JHD-150183">Link to article on publisher's site</a>
dc.identifier.issn1879-6397 (Linking)
dc.identifier.doi10.3233/JHD-150183
dc.identifier.pmid27003665
dc.identifier.urihttp://hdl.handle.net/20.500.14038/40076
dc.description.abstractBACKGROUND: 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.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=27003665&dopt=Abstract">Link to Article in PubMed</a>
dc.relation.urlhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4816656/
dc.subjectCAG repeats
dc.subjectHuntington’s disease
dc.subjectRNAi
dc.subjectmRNA degradation
dc.subjectGenetics and Genomics
dc.subjectNervous System Diseases
dc.titleDoes the Mutant CAG Expansion in Huntingtin mRNA Interfere with Exonucleolytic Cleavage of its First Exon
dc.typeJournal Article
dc.source.journaltitleJournal of Huntington's disease
dc.source.volume5
dc.source.issue1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/oapubs/2880
dc.identifier.contextkey9425517
html.description.abstract<p>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.</p> <p>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.</p> <p>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.</p> <p>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.</p> <p>CONCLUSIONS: Our results demonstrate that degradation is equally distributed across the human mutant huntingtin mRNA following RNAi-induced cleavage.</p>
dc.identifier.submissionpathoapubs/2880
dc.contributor.departmentDepartment of Pediatrics, Division of Pulmonary and Allerg
dc.contributor.departmentGene Therapy Center
dc.contributor.departmentDepartment of Medicine, Division of Endocrinology and Metabolism
dc.contributor.departmentRNA Therapeutics Institute
dc.source.pages33-8


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