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A CAG repeat-targeting artificial miRNA lowers the mutant huntingtin level in the YAC128 model of Huntington's disease.

ABSTRACT: Among the many proposed therapeutic strategies for Huntington's disease (HD), allele-selective therapies are the most desirable but also the most challenging. RNA interference (RNAi) tools that target CAG repeats selectively reduce the mutant huntingtin level in cellular models of HD. The purpose of this study was to test the efficacy, selectivity, and safety of two vector-based RNAi triggers in an animal model of HD. CAG repeat-targeting short hairpin RNA (shRNA) and artificial miRNA (amiRNA) were delivered to the brains of YAC128 mice via intrastriatal injection of AAV5 vectors. Molecular tests demonstrated that both the shRNA and amiRNA reduced the mutant huntingtin level by 50% without influencing endogenous mouse huntingtin. In addition, a concentration-dependent reduction in HTT aggregates in the striatum was observed. In contrast to the shRNA, the amiRNA was well tolerated and did not show signs of toxicity during the course of the experiment up to 20 weeks post injection. Interestingly, amiRNA treatment reduced the spleen weight to values characteristic of healthy (WT) mice and improved motor performance on the static rod test. These preclinical data demonstrate that the CAG-targeting strategy and amiRNA could make an original and valuable contribution to currently used therapeutic approaches for HD.

SUBMITTER: Kotowska-Zimmer A

PROVIDER: S-EPMC9126840 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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A CAG repeat-targeting artificial miRNA lowers the mutant huntingtin level in the YAC128 model of Huntington's disease.

Kotowska-Zimmer Anna A Przybyl Lukasz L Pewinska Marianna M Suszynska-Zajczyk Joanna J Wronka Dorota D Figiel Maciej M Olejniczak Marta M

Molecular therapy. Nucleic acids 20220505

Among the many proposed therapeutic strategies for Huntington's disease (HD), allele-selective therapies are the most desirable but also the most challenging. RNA interference (RNAi) tools that target CAG repeats selectively reduce the mutant huntingtin level in cellular models of HD. The purpose of this study was to test the efficacy, selectivity, and safety of two vector-based RNAi triggers in an animal model of HD. CAG repeat-targeting short hairpin RNA (shRNA) and artificial miRNA (amiRNA) w ...[more]

PMID: 35664700

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Project description:Huntington's disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion that encodes a polyglutamine tract in the huntingtin (HTT) protein. The mutant CAG repeat is unstable and expands in specific brain cells and peripheral tissues throughout life. Genes involved in the DNA mismatch repair pathways, known to act on expansion, have been identified as genetic modifiers; therefore, it is the rate of somatic CAG repeat expansion that drives the age of onset and rate of disease progression. In the context of an expanded CAG repeat, the HTT pre-mRNA can be alternatively processed to generate the HTT1a transcript that encodes the aggregation prone and highly pathogenic HTT1a protein. This may be a mechanism through which somatic CAG repeat expansion exerts its pathogenic effects, as the longer the CAG repeat, the more HTT1a and HTT1a is produced. The allelic series of knock-in mouse models, HdhQ20, HdhQ50, HdhQ80, HdhQ111, CAG140 and zQ175 with polyglutamine expansions of 20, 50, 80, 111, 140 and ∼190, can be used to model the molecular and cellular consequences of CAG repeat expansion within a single neuron. By western blot of cortical lysates, we found that mutant HTT levels decreased with increasing CAG repeat length; mutant HTT was only 23 and 10% of wild-type levels in CAG140 and zQ175 cortices, respectively. To identify the optimal bioassays for detecting the full-length HTT and HTT1a isoforms, we interrogated the pairwise combinations of seven well-characterized antibodies on both the 'homogeneous time-resolved fluorescence' and 'Meso Scale Discovery' platforms. We tested 32 assays on each platform to detect 'full-length mutant HTT', HTT1a, 'total mutant HTT' (full-length HTT and HTT1a) and 'total full-length HTT' (mutant and wild type). None of these assays recapitulated the full-length mutant HTT levels as measured by western blot. We recommend using isoform- and species-specific assays that detect full-length mutant HTT, HTT1a or wild-type HTT as opposed to those that detect more than one isoform simultaneously. Our finding that as the CAG repeat expands, full-length mutant HTT levels decrease, while HTT1a and HTT1a levels increase has implications for therapeutic strategies. If mutant HTT levels in cells containing (CAG)200 are only 10% of wild-type, HTT-lowering strategies targeting full-length HTT at sequences 3' to Intron 1 HTT will predominantly lower wild-type HTT, as mutant HTT levels in these cells are already depleted. These data support a therapeutic strategy that lowers HTT1a and depletes levels of the HTT1a protein.

Dataset Information

A CAG repeat-targeting artificial miRNA lowers the mutant huntingtin level in the YAC128 model of Huntington's disease.

Publications

A CAG repeat-targeting artificial miRNA lowers the mutant huntingtin level in the YAC128 model of Huntington's disease.

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