Fluorescence Imaging of Huntingtin mRNA Knockdown (original) (raw)
Related papers
Alternative processing of human HTT mRNA with implications for Huntington’s disease therapeutics
Brain, 2022
Huntington disease is caused by a CAG repeat expansion in exon 1 of the huntingtin gene (HTT) that is translated into a polyglutamine stretch in the huntingtin protein (HTT). We previously showed that HTT mRNA carrying an expanded CAG repeat was incompletely spliced to generate HTT1a, an exon 1 only transcript, which was translated to produce the highly aggregation-prone and pathogenic exon 1 HTT protein. This occurred in all knock-in mouse models of Huntington's disease and could be detected in patient cell lines and post-mortem brains. To extend these findings to a model system expressing human HTT, we took advantage of YAC128 mice that are transgenic for a yeast artificial chromosome carrying human HTT with an expanded CAG repeat. We discovered that the HTT1a transcript could be detected throughout the brains of YAC128 mice. We implemented RNAscope to visualize HTT transcripts at the single molecule level and found that full-length HTT and HTT1a were retained together in large nuclear RNA clusters, as well as being present as single transcripts in the cytoplasm. Homogeneous time-resolved fluorescence analysis demonstrated that the HTT1a transcript had been translated to produce the exon 1 HTT protein. The levels of exon 1 HTT in YAC128 mice, correlated with HTT aggregation, supportive of the hypothesis that exon 1 HTT initiates the aggregation process. Huntingtin-lowering strategies are a major focus of therapeutic development for Huntington's disease. These approaches often target full-length HTT alone and would not be expected to reduce pathogenic exon 1 HTT levels. We have established YAC128 mouse embryonic fibroblast lines and shown that, together with our QuantiGene multiplex assay, these provide an effective screening tool for agents that target HTT transcripts. The effects of current targeting strategies on nuclear RNA clusters are unknown, structures that may have a pathogenic role or alternatively could be protective by retaining HTT1a in the nucleus and preventing it from being translated. In light of recently halted antisense oligonucleotide trials, it is vital that agents targeting HTT1a are developed, and that the effects of HTT-lowering strategies on the subcellular levels of all HTT transcripts and their various HTT protein isoforms are understood.
Proceedings of the National Academy of Sciences, 2008
Huntington's disease is a dominant autosomal neurodegenerative disorder caused by an expansion of polyglutamines in the huntingtin (Htt) protein, whose cellular function remains controversial. To gain insight into Htt function, we purified epitope-tagged Htt and identified Argonaute as associated proteins. Colocalization studies demonstrated Htt and Ago2 to be present in P bodies, and depletion of Htt showed compromised RNA-mediated gene silencing. Mouse striatal cells expressing mutant Htt showed fewer P bodies and reduced reporter gene silencing activity compared with wild-type counterparts. These data suggest that the previously reported transcriptional deregulation in HD may be attributed in part to mutant Htt's role in post-transcriptional processes.
Huntington's disease (HD) is a fatal progressive neurodegenerative disorder caused by a mutation in the huntingtin (HTT) gene. To date, there is no treatment to halt or reverse the course of HD. Lowering of either total or only the mutant HTT expression is expected to have therapeutic benefit. This can be achieved by engineered micro (mi)RNAs targeting HTT transcripts and delivered by an adeno-associated viral (AAV) vector. We have previously showed a miHTT construct to induce total HTT knock-down in Hu128/21 HD mice, while miSNP50T and miSNP67T constructs induced allele-selective HTT knock-down in vitro. In the current preclinical study, the mechanistic efficacy and gene specificity of these selected constructs delivered by an AAV serotype 5 (AAV5) vector was addressed using an acute HD rat model. Our data demonstrated suppression of mutant HTT messenger RNA, which almost completely prevented mutant HTT aggregate formation, and ultimately resulted in suppression of DARPP-32-associated neuronal dysfunction. The AAV5-miHTT construct was found to be the most efficient, although AAV5-miSNP50T demonstrated the anticipated mutant HTT allele selectivity and no passenger strand expression. Ultimately, AAV5-delivered-miRNA-mediated HTT lowering did not cause activation of microglia or astrocytes suggesting no immune response to the AAV5 vector or therapeutic precursor sequences. These preclinical results suggest that using gene therapy to knock-down HTT may provide important therapeutic benefit for HD patients and raised no safety concerns, which supports our ongoing efforts for the development of an RNA interference-based gene therapy product for HD.
Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain
Molecular Therapy—Nucleic Acids, 2015
Applications of RNA interference for neuroscience research have been limited by a lack of simple and efficient methods to deliver oligonucleotides to primary neurons in culture and to the brain. Here, we show that primary neurons rapidly internalize hydrophobically modified siRNAs (hsiRNAs) added directly to the culture medium without lipid formulation. We identify functional hsiRNAs targeting the mRNA of huntingtin, the mutation of which is responsible for Huntington's disease, and show that direct uptake in neurons induces potent and specific silencing in vitro. Moreover, a single injection of unformulated hsiRNA into mouse brain silences Htt mRNA with minimal neuronal toxicity. Thus, hsiRNAs embody a class of therapeutic oligonucleotides that enable simple and straightforward functional studies of genes involved in neuronal biology and neurodegenerative disorders in a native biological context.
The aim of these studies was to demonstrate the therapeutic capacity of an antisense oligonucleotide with the sequence (CUG)7 targeting the expanded CAG repeat in huntingtin (HTT) mRNA in vivo in the R6/2 N-terminal fragment and Q175 knock-in Huntington's disease (HD) mouse models. In a first study, R6/2 mice received six weekly intracerebroventricular infusions with a low and high dose of (CUG)7 and were sacrificed 2 weeks later. A 15-60% reduction of both soluble and aggregated mutant HTT protein was observed in striatum, hippocampus and cortex of (CUG)7-treated mice. This correction at the molecular level resulted in an improvement of performance in multiple motor tasks, increased whole brain and cortical volume, reduced levels of the gliosis marker myo-inositol, increased levels of the neuronal integrity marker N-aceyl aspartate and increased mRNA levels of the striatal marker Darpp-32. These neuroanatomical and neurochemical changes, together with the improved motor performance, suggest that treatment with (CUG)7 ameliorates basal ganglia dysfunction. The HTT-lowering was confirmed by an independent study in Q175 mice using a similar (CUG)7 AON dosing regimen, further demonstrating a lasting reduction of mutant HTT protein in striatum, hippocampus and cortex for up to 18 weeks post last infusion along with an increase in motor activity. Based on these encouraging results, (CUG)7 may thus offer an interesting alternative HTT-lowering strategy for HD.
CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression
Nucleic acids research, 2017
Huntington's disease (HD) is a fatal, neurodegenerative disorder in which patients suffer from mobility, psychological and cognitive impairments. Existing therapeutics are only symptomatic and do not significantly alter the disease progression or increase life expectancy. HD is caused by expansion of the CAG trinucleotide repeat region in exon 1 of the Huntingtin gene (HTT), leading to the formation of mutant HTT transcripts (muHTT). The toxic gain-of-function of muHTT protein is a major cause of the disease. In addition, it has been suggested that the muHTT transcript contributes to the toxicity. Thus, reduction of both muHTT mRNA and protein levels would ideally be the most useful therapeutic option. We herein present a novel strategy for HD treatment using oligonucleotides (ONs) directly targeting the HTT trinucleotide repeat DNA. A partial, but significant and potentially long-term, HTT knock-down of both mRNA and protein was successfully achieved. Diminished phosphorylation...
PLoS Currents, 2010
A means for measuring levels of soluble huntingtin proteins in clinical samples is essential for assessing the biological effects of potential mutant huntingtin (mtHtt) modifying treatments being developed for Huntington's disease (HD). We have optimized a previously described cell-based Homogeneous Time Resolved Fluorescence method that can measure soluble mtHtt and its ratio to the total Htt (tHtt) in blood buffy coats . The results of the optimization and assay qualification indicate the assay to be specific for mtHtt in HD compared to Control subjects, highly sensitive, and technically and biologically reproducible. We therefore generated a Good Laboratory Practice Standard Operating Procedure which we validated, using 30 HD and 8 control buffy coat samples in which significant differences in mtHtt levels were found. We intend to deploy the assay to evaluate sample sets from observational and therapeutic studies enrolling HD subjects to further validate soluble mtHtt measurement by HTRF as a biomarker for HD and to explore its potential uses. Other Sections▼ o AbstractReferencesIntroduction The proximate cause of neuronal dysfunction and death in Huntington Disease (HD) is the presence of soluble mutant huntingtin ( mt Htt) protein or its cleavage products [1][[2],[3].
Journal of Nuclear Medicine Official Publication Society of Nuclear Medicine, 2002
Disease-specific genes of unknown function can be imaged in vivo with antisense radiopharmaceuticals, providing the transcellular transport of these molecules is enabled with drug-targeting technology. The current studies describe the production of 16-mer peptide nucleic acid (PNA) that is antisense around the methionine initiation codon of the huntingtin gene of Huntington's disease (HD). Methods: The PNA is biotinylated, which allows for rapid capture by a conjugate of streptavidin and the rat 8D3 monoclonal antibody (mAb) to the mouse transferrin receptor (TfR), and contains a tyrosine residue, which enables radiolabeling with 125 I. The reformulated PNA antisense radiopharmaceutical that is conjugated to the 8D3 mAb is designated 125 I-PNA/8D3. This form of the PNA is able to access endogenous transferrin transport pathways at both the bloodbrain barrier and the brain cell membrane and undergoes both import from the blood to the brain and export from the brain to the blood through the TfR. Results: The ability of the PNA to hybridize to the target huntingtin RNA, despite conjugation to the mAb, was shown both with cell-free translation assays and with ribonuclease protection assays. The 125 I-PNA/8D3 conjugate was administered intravenously to either littermate control mice or to R6/2 transgenic mice, which express the exon 1 of the human HD gene. The mice were sacrificed 6 h later for frozen sectioning of the brain and quantitative autoradiography. The studies showed a 3-fold increase in sequestration of the 125 I-PNA/8D3 antisense radiopharmaceutical in the brains of the HD transgenic mice in vivo, consistent with the selective expression of the HD exon-1 messenger RNA in these animals. Conclusion: These results support the hypothesis that gene expression in vivo can be quantitated with antisense radiopharmaceuticals, providing these molecules are reformulated with drug-targeting technology. Drug targeting enables access of the antisense agent to endogenous transport pathways, which permits passage across the cellular barriers that separate blood and intracellular compartments of target tissues.