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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE127821 | GEO | 2019/06/30

REPOSITORIES: GEO

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

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 | GSE127820 | GEO

Neuronal and astrocytic contributions to Huntington’s disease dissected following mutant Huntingtin (mHTT) lowering

Project description:Huntington’s disease (HD) is caused by expanded CAG repeats in the Huntingtin gene (HTT) that results in expression of mutant HTT proteins (mHTT) with extended polyglutamine tracts in diverse cells of the body, including striatal neurons and astrocytes. The relative contributions of neurons and astrocytes to disease pathogenesis are unknown for HD and other neurodegenerative diseases. This is a critical issue to address since it has been proposed that neuronal loss in HD and other major neurodegenerative diseases is downstream of astrocytic dysfunction that drives neuronal death. Moreover, astrocyte-to-neuron conversion is considered a promising approach in HD and other neurodegenerative diseases with little reported detriment with regards to normal astrocytic physiological functions, which are varied and extensive in health and disease. Thus, astrocytes are considered both causative and also largely replaceable in neurodegeneration, and their basic contributions to disease pathophysiology relative to neurons remain undefined in vivo. We used genetically encoded and cell-specific zinc finger protein (ZFP) transcriptional repressors to lower mHTT and molecularly dissected neuronal and astrocytic influences on HD pathophysiology at molecular, cellular, and behavioral levels. We found that the major disease drivers were in fact neurons, with astrocytes displaying lesser loss of essential functions such as cholesterol metabolism that were downstream of greater neuronal dysfunction, which encompassed neuromodulation, synaptic, and intracellular signaling. We thus dissected the cell autonomous and non-cell autonomous mechanistic and causal contributions of both neurons and astrocytes to a complex neurodegenerative disease in vivo with wider implications for rescue and repair strategies.

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Polyglutamine-mediated ribotoxicity disrupts proteostasis and stress responses in Huntington’s Disease

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| EGAS00001006289 | EGA

Mutant huntingtin stalls ribosomes and represses protein synthesis independent of fragile X mental retardation protein

Project description:Ribosomes translocate one codon at a time in mRNA during protein synthesis, but the regulators of ribosome translocation and their impact on neurodegenerative disease remain poorly understood. Here, we show that huntingtin (Htt) and its poly-Q expanded form, mHtt, a cause of Huntington disease (HD), promotes ribosome stalling and suppresses protein synthesis in vivo and in vitro. We found that fragile mental retardation protein (FMRP), a known promoter of ribosome stalling, is upregulated in HD cells and patients’ tissue. Unexpectedly, FMRP depletion fails to reverse mHtt-mediated ribosome stalling in HD cells. mHtt binds directly to ribosomes in a RNase-sensitive manner and interacts with ribosomal proteins. Combining high-resolution ribosome footprint profiling (Ribo-Seq) and RNA-Seq, we provide a global snapshot of stalling on mRNA transcripts, with a shift in ribosome occupancy toward the 5’ and 3’ end and single-codon unique pauses in HD cells. We also found ribosomes that appear to have stalled in mHtt mRNA before CAG repeat expansion. Thus, Htt regulates protein synthesis via ribosome stalling mechanisms and in turn may affect mRNA elongation, which may be exploited for HD therapeutics.

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Reduced Representation Bisulfite Sequencing from HD knock-in mouse model

Project description:We generated two types of DNA methylation datasets (Reduced Representation Bisulfite Sequencing [RRBS] and custom methylation array) from a heterozygous HD knock-in mouse model. The heterozygous Htt knock-in line expressed one wildtype endogenous Htt allele and a second Htt allele with knock-in of human mHTT exon 1 with either approximately 190 CAG repeats (Q175) or 20 CAG repeats (Q20). For each genotype (Q175 and Q20) we analyzed two brain regions (striatum and cerebellum) from 8 mice each on the RRBS platform (N=32 RRBS samples). Our EWAS of mutant Htt gene status (i.e. Q175 status) across the two brain regions identified two genome-wide significant CpGs in Htt region. contributor: CHDI foundation

2020-07-02 | GSE147156 | GEO

Site-Specific Ubiquitination of Pathogenic Huntingtin Attenuates its Deleterious Effects

Project description:Huntington’s disease (HD) is a progressive incurable neurodegenerative disorder characterized by motor and neuropsychiatric symptoms. It is caused by expansion of a CAG triplet in the N-terminal domain of exon 1 in the huntingtin (HTT) gene that codes for an expanded polyglutamine (polyQ) stretch in the protein product which becomes aggregation-prone. The mutant Htt (mHtt) aggregates are associated with components of the Ubiquitin-Proteasome System (UPS), suggesting that mHtt is marked for proteasomal degradation and that for reasons still debated are not properly degraded. We used a novel HD rat model, proteomic analysis, and long-term live neuronal imaging to characterize the effects of ubiquitination on aggregation of mHtt and its subsequent cellular response. We identified two lysine residues - 6 and 9 - in the first exon of mHtt that are specifically ubiquitinated in striatal and cortical brain tissues of mHtt-transgenic animals. Expression of mHtt exon 1 lacking these ubiquitination sites in cortical neurons and cultured cells was found to slow aggregate appearance rates and reduce their size, but at the same time increase the number of much smaller and less visible ones. Importantly, expression of this form of mHtt was associated with elevated death rates. Furthermore, proteomic analysis indicated that compared to cells expressing the protein that can be modified by ubiquitin, cells expressing the lysine-less protein did not activate protein synthetic pathways that enable the cell to cope with stress. Taken together, the findings suggest a novel role for ubiquitination - attenuation of the pathogenic effect of mHtt.

2020-06-30 | PXD019553 | Pride

Site-Specific Ubiquitination of Pathogenic Huntingtin Attenuates its Deleterious Effects- PART2

2020-06-30 | PXD019552 | Pride

Analysis of striatal zinc finger protein (ZFP) repression of mutant Huntingtin (HTT) in the zQ175DN knock-in mouse model of Huntington's disease (bulk RNA-seq)

Project description:We assessed the relative efficacy of selectively lowering mHTT in the striatum at different intervention times in a human HTT exon 1 knock-in mouse model of Huntington’s disease, zQ175DN KI, using a virally delivered zinc finger protein transcriptional repressor directed against the expanded CAG repeat of the mutant HTT gene (Zeitler et al. Nat Med. 2019 Jul;25(7):1131-1142).

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