Targeting CAG repeat RNAs reduces Huntington’s disease phenotype independently of huntingtin levels
Laura Rué, … , Xavier Estivill, Eulàlia Martí
Laura Rué, … , Xavier Estivill, Eulàlia Martí
Published October 10, 2016
Citation Information: J Clin Invest. 2016;126(11):4319-4330. https://doi.org/10.1172/JCI83185.
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Concise Communication Neuroscience

Targeting CAG repeat RNAs reduces Huntington’s disease phenotype independently of huntingtin levels

Abstract

Huntington’s disease (HD) is a polyglutamine disorder caused by a CAG expansion in the Huntingtin (HTT) gene exon 1. This expansion encodes a mutant protein whose abnormal function is traditionally associated with HD pathogenesis; however, recent evidence has also linked HD pathogenesis to RNA stable hairpins formed by the mutant HTT expansion. Here, we have shown that a locked nucleic acid–modified antisense oligonucleotide complementary to the CAG repeat (LNA-CTG) preferentially binds to mutant HTT without affecting HTT mRNA or protein levels. LNA-CTGs produced rapid and sustained improvement of motor deficits in an R6/2 mouse HD model that was paralleled by persistent binding of LNA-CTG to the expanded HTT exon 1 transgene. Motor improvement was accompanied by a pronounced recovery in the levels of several striatal neuronal markers severely impaired in R6/2 mice. Furthermore, in R6/2 mice, LNA-CTG blocked several pathogenic mechanisms caused by expanded CAG RNA, including small RNA toxicity and decreased Rn45s expression levels. These results suggest that LNA-CTGs promote neuroprotection by blocking the detrimental activity of CAG repeats within HTT mRNA. The present data emphasize the relevance of expanded CAG RNA to HD pathogenesis, indicate that inhibition of HTT expression is not required to reverse motor deficits, and further suggest a therapeutic potential for LNA-CTG in polyglutamine disorders.

Authors

Laura Rué, Mónica Bañez-Coronel, Jordi Creus-Muncunill, Albert Giralt, Rafael Alcalá-Vida, Gartze Mentxaka, Birgit Kagerbauer, M. Teresa Zomeño-Abellán, Zeus Aranda, Veronica Venturi, Esther Pérez-Navarro, Xavier Estivill, Eulàlia Martí

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Figure 1

LNA-CTG preferentially binds to expanded HTT mRNA in HD fibroblasts, without inhibiting HTT protein levels.

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LNA-CTG preferentially binds to expanded HTT mRNA in HD fibroblasts, wit...
(A) Scheme showing the binding sites of the primers used for PCR amplification in HTT exon 1 (HTT_e1* and HTT_e1 sets of primers) and HTT exons 29-30 (HTT_e29-30 set of primers). The black line represents introns. (B) HTT expression in HD fibroblasts (44_CAG repeats) transfected with different concentrations of LNA-CTG or LNA-SCB, 48 hours after transfection. Graph shows the RQ of HTT using the primer set HTT-e1* detecting WT (HTT-e1*-WT) or mutant (HTT-e1*-Mut) HTT-exon 1 and the primer set HTT_e29-30 detecting HTT-e29-30. Densitometric determinations were normalized to the β-actin PCR product and referred to the mock-transfected condition with a value of 1. Results are expressed as the mean ± SEM (n = 6). Box plot shows quartile values of the RQ of WT versus mutant HTT-e1* mRNA levels normalized to β-actin levels in HD fibroblasts transfected with LNA-CTG or LNA-SCB. A representative gel electrophoresis of the PCR products is shown. *P < 0.05, by Mann-Whitney U test with Bonferroni’s correction for multiple comparisons (n = 6 independent transfections). (C) HTT expression at different time points after transfection with 15 nM LNA-ASOs in HD fibroblasts (68_CAG repeats). The mean determinations ± SEM of HTT-e1*-WT and HTT-e1*-Mut and HTT-e29-30 relative to β-actin PCR products and a representative PCR gel electrophoresis are shown (n = 3). (D) Dot plots show Western blot densitometric analysis of WT HTT (WT-HTT) versus mutant HTT (mHTT) protein levels and total HTT (WT and mutant alleles) protein levels normalized to β-actin and expressed relative to an LNA-SCB–transfected sample with a value of 1. A representative immunoblot is shown (n = 3).