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RNA-Targeting CRISPR/Cas13d System Alleviates Disease-Related Phenotypes in Pre-clinical Models of Huntington’s Disease (Human).

ABSTRACT: Huntington's disease (HD) is a fatal, dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in exon 1 of the huntingtin (HTT) gene. Although the pathogenesis of HD remains complex, the CAG-expanded (CAGEX) HTT mRNA and protein ultimately causes disease through a toxic gain-of-function mechanism. As the reduction of pathogenic mutant HTT mRNA is beneficial as a treatment, we developed a mutant allele-sensitive CAGEX RNA-targeting CRISPR-Cas13d system (Cas13d/CAGEX) that eliminates toxic CAGEX RNA in HD patient-derived fibroblasts and iPSC-derived striatal neurons. We show that intrastriatal delivery of Cas13d/CAGEX via a single adeno-associated viral vector, serotype 9 (AAV9) mediates significant and selective reduction of mutant HTT mRNA and protein levels within the striatum of heterozygous zQ175 mice, an established mouse model of HD. Moreover, the reduction of mutant HTT mRNA renders a sustained partial reversal of HD phenotypes, including improved motor coordination, attenuated striatal atrophy, and reduction of mutant HTT protein aggregates. Importantly, phenotypic improvements were durable for at least 8 months without gross or behavioral adverse effects, and with minimal off-target interactions of Cas13d/CAGEX in the mouse transcriptome. Taken together, we demonstrate a proof-of-principle of an RNA-targeting CRISPR/Cas13d system as a therapeutic approach for HD, a strategy with broad implications for the treatment of other dominantly inherited neurodegenerative disorders.

ORGANISM(S): Homo sapiens

PROVIDER: GSE214108 | GEO | 2022/11/15

REPOSITORIES: GEO

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RNA-Targeting CRISPR/Cas13d System Alleviates Disease-Related Phenotypes in Pre-clinical Models of Huntington’s Disease (Mouse).

Project description:Huntington's disease (HD) is a fatal, dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in exon 1 of the huntingtin (HTT) gene. Although the pathogenesis of HD remains complex, the CAG-expanded (CAGEX) HTT mRNA and protein ultimately causes disease through a toxic gain-of-function mechanism. As the reduction of pathogenic mutant HTT mRNA is beneficial as a treatment, we developed a mutant allele-sensitive CAGEX RNA-targeting CRISPR-Cas13d system (Cas13d/CAGEX) that eliminates toxic CAGEX RNA in HD patient-derived fibroblasts and iPSC-derived striatal neurons. We show that intrastriatal delivery of Cas13d/CAGEX via a single adeno-associated viral vector, serotype 9 (AAV9) mediates significant and selective reduction of mutant HTT mRNA and protein levels within the striatum of heterozygous zQ175 mice, an established mouse model of HD. Moreover, the reduction of mutant HTT mRNA renders a sustained partial reversal of HD phenotypes, including improved motor coordination, attenuated striatal atrophy, and reduction of mutant HTT protein aggregates. Importantly, phenotypic improvements were durable for at least 8 months without gross or behavioral adverse effects, and with minimal off-target interactions of Cas13d/CAGEX in the mouse transcriptome. Taken together, we demonstrate a proof-of-principle of an RNA-targeting CRISPR/Cas13d system as a therapeutic approach for HD, a strategy with broad implications for the treatment of other dominantly inherited neurodegenerative disorders.

2022-11-15 | GSE214109 | GEO

Specificity assessment of allele selective Zinc Finger Protein Repressors in hESC-derived HD neurons

Project description:Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range, while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and at least 9 months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological, and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.

2019-06-30 | GSE127821 | GEO

Specificity assessment of allele selective Zinc Finger Protein Repressors in HD fibroblasts

2019-06-30 | GSE127820 | GEO

Integrated genomics and proteomics to define huntingtin CAG length-dependent molecular networks in HD mouse brains

Project description:To gain insight into how mutant Huntingtin (mHTT) CAG repeat length may modify Huntington’s disease (HD) pathogenesis, we profiled mRNA in over 600 brain and peripheral tissue samples from HD knock-in mice with increasing CAG repeat length. We find repeat length dependent transcriptional signatures are prominent in the striatum, less so in cortex, and minimal in the liver. Co-expression network analyses reveal 13 striatal and 5 cortical modules that are highly correlated with CAG length and age, and that are preserved in HD models and some in the patients. Top striatal modules implicate mHTT CAG length and age in graded impairment of striatal medium spiny neuron identity gene expression and in dysregulation of cAMP signaling, cell death, and protocadherin genes. Importantly, we used proteomics to confirm 790 genes and 5 striatal modules with CAG length-dependent dysregulation at both RNA and protein levels and validated 21 striatal module genes as modifiers of mHtt toxicities in vivo.

2016-02-16 | PXD003442 | Pride

Huntington's disease brain-derived small RNAs recapitulate associated neuropathology in mice [human-miRNA]

Project description:Progressive motor alterations and selective death of striatal medium spiny neurons (MSNs) are key pathological hallmarks of Huntington's disease (HD), a neurodegenerative condition caused by a CAG trinucleotide repeat expansion in the coding region of the huntingtin (HTT) gene. Most research has focused on the pathogenic effects of the resultant protein product(s); however, growing evidence indicates that expanded CAG repeats within mutant HTT mRNA and derived small CAG repeat RNAs (sCAG) participate in HD pathophysiology. The individual contribution of protein versus RNA toxicity to HD pathophysiology remains largely uncharacterized and the role of other classes of small RNAs (sRNA) that are strongly perturbed in HD is uncertain. Here, have injected vehicle, CTL-sRNA (obtained from non-affected individuals) or HD-sRNA (obtained from patients with HD) from different brain areas (putamen, PT; cortex, CTX and cerebellum, CB) into the striatum of wild type (WT) mice and demonstrate that sRNA produced in the putamen and cortex of HD patients are sufficient to induce HD pathology in vivo. We have performed small RNA (sRNA) sequencing of the human samples used for injection. Here we describe the procedure to obtain and sequence human sRNA isolated from the different brain areas.

2021-02-16 | GSE165663 | GEO

In vivo cell-autonomous transcriptional abnormalities revealed in mice expressing mutant huntingtin in striatal but not cortical neurons

Project description:Huntington’s disease (HD), caused by a CAG repeat expansion in the huntingtin (HTT) gene, is characterized by abnormal protein aggregates and motor and cognitive dysfunction. Htt protein is ubiquitously expressed, but the striatal medium spiny neuron (MSN) is most susceptible to neuronal dysfunction and death. Abnormal gene expression represents a core pathogenic feature of HD, but the relative roles of cell-autonomous and non-cell-autonomous effects on transcription remain unclear. To determine the extent of cell-autonomous dysregulation in the striatum in vivo, we examined genome-wide RNA expression in symptomatic D9-N171-98Q (a.k.a. DE5) transgenic mice in which the forebrain expression of the first 171 amino acids of human Htt with a 98Q repeat expansion is limited to MSNs. Microarray data generated from these mice were compared to those generated on the identical array platform from a pan-neuronal HD mouse model, R6/2, carrying two different CAG repeat lengths, and a relatively high degree of overlap of changes in gene expression was revealed. We further focused on known canonical pathways associated with excitotoxicity, oxidative stress, mitochondrial dysfunction, dopamine signaling and trophic support, among others. While genes related to excitotoxicity, dopamine signaling and trophic support, were altered in both DE5 and R6/2 transgenic mice, which may be either cell-autonomous or non-cell-autonomous,, genes related to mitochondrial dysfunction, oxidative stress and the peroxisome proliferator-activated receptor are primarily affected in DE5 transgenic mice, indicating cell autonomous mechanisms Overall, our results demonstrate that HD-induced dysregulation of the striatal transcriptome can be largely attributed to intrinsic effects of mutant Htt,,such that mutant Htt-induced effects in cortical neurons is not necessary for striatal dysfunction/degeneration. Striatum from DE5 transgenic mice vs. wt littermate controls

2011-01-01 | E-GEOD-25232 | biostudies-arrayexpress

Genome-scale chromatin interaction data for huntingtin from mouse striatal tissue

Project description:Transcriptional changes are an early feature of Huntington's disease (HD). We profiled genome-wide interaction sites for the huntingtin protein (HTT) using ChIP-sequencing from mouse striatal tissue at 4 months of age. We include replicate samples from CAG-expanded murine Htt (heterozygous Q111/+) and wildtype littermate controls.

2018-12-01 | GSE102750 | GEO

A genome-wide screening in pluripotent cells identifies Mtf1 as a suppressor of mutant huntingtin toxicity

Project description:Huntington’s disease (HD) is an inherited and incurable neurodegenerative disorder caused by CAG repeat expansions in the huntingtin (HTT) gene. The resulting mutant HTT protein alters cellular physiology at multiple levels, inducing toxicity and cell death. The mechanisms directly causing toxicity are partially understood, thus precluding the development of effective therapeutic strategies. Here we identified novel suppressors of mutant HTT toxicity by performing a genome-wide screen in pluripotent stem cells, followed by hit validation both in zebrafish and mouse HD models. The candidate suppressors identified were strongly enriched for HD-associated processes. We focused on Metal regulatory transcription factor 1 (Mtf1), a transcription factor controlling metal homeostasis in the cell. Forced expression of Mtf1 counteracts cell death, oxidative stress and transcriptional alterations caused by mutant HTT in pluripotent cells. In zebrafish, Mtf1 reduces malformations and apoptosis induced by mutant HTT, while Mtf1 delivery by Adeno-Associated viral (AAV) vectors ablated motor defects observed in an HD mouse model. Our data reveal that delivery of Mtf1 might act as a novel therapeutic strategy against HD and potentially other neurodegenerative disorders.

2023-05-30 | GSE166567 | GEO

Huntington's disease brain-derived small RNAs recapitulate associated neuropathology in mice [Mouse RNA-seq]

Project description:Progressive motor alterations and selective death of striatal medium spiny neurons (MSNs) are key pathological hallmarks of Huntington's disease (HD), a neurodegenerative condition caused by a CAG trinucleotide repeat expansion in the coding region of the huntingtin (HTT) gene. Most research has focused on the pathogenic effects of the resultant protein product(s); however, growing evidence indicates that expanded CAG repeats within mutant HTT mRNA and derived small CAG repeat RNAs (sCAG) participate in HD pathophysiology. The individual contribution of protein versus RNA toxicity to HD pathophysiology remains largely uncharacterized and the role of other classes of small RNAs (sRNA) that are strongly perturbed in HD is uncertain. Here, have injected vehicle (VEH), CTL-sRNA-PT (obtained from the putamen of non-affected individuals) or HD-sRNA-PT (obtained from the putamen of patients with HD) into the striatum of wild type (WT) mice, and demonstrate that sRNA produced in the putamen and cortex of HD patients are sufficient to induce HD pathology in vivo, including transcriptional alterations. To reveal the transcriptional changes produced by sCAG, we co-injected human sRNAs with a locked nucleic acid (LNA) modified anti-sense oligonucleotide complementary to sCAG (HD-sRNA-PT + LNA-CTG) or an analogous scrambled LNA (HD-sRNA-PT + LNA-SCB). We have performed mRNA-seq in the different experimental groups: Vehicle, CTL-sRNA-PT, HD-sRNA-PT, HD-sRNA-PT + LNA-CTG, and HD-sRNA-PT + LNA-SCB. Here we describe the procedure to obtain and sequence human sRNA dataset isolated from the different brain areas.

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2011-01-01 | GSE25232 | GEO