Biology subjects

Dion, V.

Publications and source records attributed to Dion, V..

12 recordsLinked to original sources

bioRxiv2026

Single-AAV Delivery of the SpCas9 Nickase Improves CAG/CTG Repeat contraction in a mouse model of Huntington's disease

The best studied Cas9 enzyme, from Streptococcus pyogenes, is thought to be too large to be efficiently packaged into a single adeno-associated virus (AAV) together with a single-guide (sg) RNA cassette, requiring a dual-vector system. Here we have circumvented this limitation and packaged the full CRISPR system into a single AAV by using a single promoter driving the expression of both the Cas9 enzyme and a sgRNA. We compared the ability of the dual and single vector systems to induce contraction of CAG/CTG repeats in the striatum of a Huntington's disease model. We show that the all-in-one system led to larger contractions of the CAG/CTG repeats. This approach provides a more efficient and less costly approach for in vivo gene editing and improves the translational potential of AAV-mediated delivery of Cas9.

neuroscience↗

bioRxiv2026

Targeted sequencing of expanded tandem repeats: Identifying interruptions and errors

Expanded repeat disorders remain without a disease modifying treatment. Some of the most important modifiers of disease severity include inherited repeat size, their somatic mosaicism, and whether they contain interruptions. Targeted sequencing approaches are becoming the gold standard on how these parameters are measured despite repeats being notoriously challenging to sequence. Here we developed CHARLIE (Comprehensive High-throughput Analysis of Repeat Length, Interruptions and Expansions) to identify and position interruptions within expanded repeats. We used samples from Huntingtons disease and myotonic dystrophy type 1 sequenced with Illumina MiSeq and PacBios Single-Molecule Real-Time Sequencing. We validated the pipeline against previous methods for identifying germline-inherited interruptions. One challenge that can potentially mask the identification of true interruptions and sequence variation is the accuracy of sequencing platforms, which, when applied to expanded repeats, is largely unknown. Our results suggest that each sequencing platform produces a distinct error profile and we show that PCR-free library preparation for SMRT sequencing improves sequencing accuracy. CHARLIE, therefore, provides a method that can help differentiate between trivial sequencing errors and those interruptions that modify disease presentation, which can be inherited or somatic in origin.

bioinformatics↗

bioRxiv2026

Cell-specific transcription dysregulation in human Huntington's disease-positive developing striatum

Huntingtons disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG-repeat expansion in the HTT gene. Progressive loss of striatal projection neurons leads to cognitive, psychiatric, and motor impairments that typically manifest in midlife, despite the presence of the expansion from conception. Increasing evidence supports a neurodevelopmental component to HD; however, authentic human developing HD striatal tissue has not previously been characterised. We analysed an HD positive human fetal striatal sample alongside an age- and sex-matched control. CAG-repeat length was determined, and single-cell RNA sequencing was used to investigate gene expression. We compared the fetal HD transcriptional signature with publicly available datasets from postmortem adult HD brain tissue. We identified 2,032 differentially expressed genes and defined nine cellular clusters, each exhibiting distinct transcriptional profiles. Gene enrichment analysis revealed disruption of key biological processes across the developing HD striatum, with pathway-level dysregulation varying between clusters. There was overlap in gene expression changes between fetal and adult HD striatal tissues. Together, these findings demonstrate that molecular features of HD pathology are present during early human striatal development, supporting the concept that disease mechanisms are established decades prior to clinical onset.

neuroscience↗

bioRxiv2026

SCIA: A fast and widely applicable pipeline for measuring expanded repeat instability

The expansion of short tandem repeats is a feature of over 60 different human diseases. Ongoing somatic instability throughout a patients lifetime can influence disease progression and has emerged as a therapeutic target. Understanding its mechanism is essential for the identification of both drug targets and therapeutic interventions. A major obstacle towards this translational goal has been to measure changes in repeat size distribution given that these are complex datasets. To address this, here we provide a new analysis method, and accompanying software, that generates delta plots, extracts the instability frequency from targeted long-read sequencing data, the bias towards expansion or contraction, and the average size of the changes. It further provides statistical analysis for comparison between treatments. We show its applicability to non-dividing cells, and in vivo datasets. Moreover, we have developed a streamlined experimental design for dividing cells, Single Clone-based Instability Assay (SCIA), that saves weeks in assessing the effect of a gene knockout on repeat instability and is ideal for an initial screen. We have validated the approach using FAN1, PMS1, and MLH1 knockouts. Using SCIA, we find that although FAN1 knockout clones showed increased frequency of expansions, the size of the expansions were smaller. This highlights the wealth of information that can be extracted and the potential for novel insights into the mechanism of repeat instability.

neuroscience↗

bioRxiv2024

The TRX assay for triplet repeat expansions

Expansion mutations of triplet repeat sequences cause numerous inherited neurological diseases. In some diseases, affected individuals display somatic expansions in affected tissues which have been linked to accelerated disease onset and progression. There is currently considerable interest in developing therapies to slow somatic repeat expansions to delay or block disease onset. In vitro assays are particularly important to evaluate potential therapeutic interventions. Current assays typically use physical methods to monitor triplet repeat lengths within a population of cells. While useful, most of these assays are relatively slow ([~]six weeks) and are somewhat limited in sensitivity to rare events. Here, a new assay, called TRX, is described to monitor CAG*CTG triplet repeat expansions more rapidly and with better sensitivity. TRX uses human tissue culture cells expressing two fluorescent proteins. Red fluorescent protein TagRFP658 is constitutively expressed and serves as an internal control. GFP is expressed in a CAG*CTG repeat length-dependent manner, with longer repeat lengths predicted to give higher green fluorescence intensity. Standard flow cytometry allows quantification of changes in fluorescent signal as a simple readout with <2% sensitivity. Two independently derived cell lines with 63 or 59 CAG repeats yielded similar rates of TRX activity. Cells with increased green fluorescence were observed within one to two weeks of culture, with longer times leading to additional signal. The appearance of green fluorescence was partly dependent on MutS{beta}, the DNA MSH2-MSH3 complex, based on siRNA knockdown of MSH3. However, physical analysis of the CAG*CTG repeat tracts by MiSeq deep sequencing or capillary electrophoresis showed limited changes in the length of the repeat tracts. We conclude that the TRX assay is a promising new tool for monitoring CAG*CTG repeat expansions but that further development of the assay is needed to make it fully useful.

genetics↗

bioRxiv2024

Cas9 nickase-mediated contraction of CAG/CTG repeatsat multiple disease loci

Expanded CAG/CTG repeats cause over 15 different diseases that all remain without a disease-modifying treatment. Because repeat length accounts for most of the variation in disease severity, contracting them presents an attractive therapeutic avenue. Here, we show that the CRISPR-Cas9 nickase targeted to CAG/CTG repeats leads to efficient contractions in Huntingtons disease patient-derived neurons and astrocytes, and in myotonic dystrophy type 1 patient-derived neurons. The approach is allele-selective and free of detectable off-target mutations. Striatal injection of the Cas9 nickase in a mouse model for Huntingtons disease using adeno-associated viral vectors led to contractions in over half the infected cells. Upon injection, we observed a reduction in the number of inclusion bodies, improved transcriptome, and ameliorated locomotion. The effects were greater than expected from the contractions induced and suggest that non-cell autonomous mechanisms may be involved. Our results provide the proof-of-concept that correction of CAG/CTG repeats can improve Huntingtons disease phenotypes in vivo. One sentence summaryThe Cas9 nickase contracts CAG/CTG repeats at multiple disease loci in patient-derived cells and improves molecular and behavioral phenotypes in HD.

neuroscience↗

bioRxiv2023

Developing small Cas9 hybrids using molecular modeling

The RNA-guided CRISPR-Cas9 from Streptococcus pyogenes is the best characterized enzyme for gene editing. Its large size, however, precludes it from being packaged together with its single guide (sg)RNA into a single adeno-associated virus, limiting in vivo applications. Here, we developed smaller Cas9 hybrids, made of the PAM interacting domain (PID) of S. pyogenes and the catalytic domains of the smaller Cas9 orthologues, as well as sgRNA cognate hybrids. Molecular modeling revealed that the presence of a sgRNA stabilizes Cas9. Making the D10A mutation to turn Cas9 into a nickase dramatically alters its binding energy to the sgRNA, showing that the approach can identify functionally relevant changes. However, we found that the four Cas9/sgRNA hybrid pairs tested in human cells failed to edit target sequences. We conclude that in silico approaches can identify functional changes caused by point mutations but are not sufficient for designing Cas9/sgRNA hybrids.

bioengineering↗

bioRxiv2023

Differences in white matter detected by ex vivo 9.4T MRI are associated with axonal changes in the R6/1 model of Huntington's Disease

White matter (WM) volume loss has been reported in people with Huntingtons disease (HD), but the cellular basis of this deficit remains to be elucidated. To address this, we assessed ex vivo WM microstructure in the transgenic R6/1 mouse model of HD with magnetic resonance imaging (MRI) and studied the neurobiological basis of the MRI brain signals with histological and electron microscopy analyses in a separate cohort of age- and sex-matched mice. Differences in the macromolecular proton fraction (MPF) from quantitative magnetization transfer (qMT) as a proxy myelin measure, and the intra-axonal signal fraction (FR) from the composite hindered and restricted model of diffusion (CHARMED) as a proxy marker of axon density, were assessed alongside diffusion tensor imaging (DTI) parameters. A tractometry approach was employed to inspect region-specific differences across the corpus callosum (CC). Furthermore, voxel-based morphometry (VBM) and tract-based spatial statistics (TBSS) were used to explore brain-wise WM macro- and microstructure abnormalities. To gain insight into disease-associated impairments in attentional and visuospatial processing, a third cohort of age-matched mice was assessed with the 5-choice serial reaction time task (5-CSRTT). We report cognitive impairments in R6/1 mice and, by evaluating MRI and light and electron microscopy results, we show that this HD mouse model presents disruptions in axonal morphology (i.e. less complex, thinner axons) and organization (i.e. more densely packed axons). Furthermore, we show that, at least early in disease progression, R6/1 mice present a reduction in the expression or content of myelin-associated proteins without significant alterations in the structure of myelin sheaths. Finally, our findings indicate that neuroinflammation-driven glial and axonal swelling might also affect this mouse model early in disease progression. Crucially, we demonstrate the potential of FR, an in vivo estimate of axon density, as a novel MRI biomarker of HD-associated changes in WM microstructure.

neuroscience↗

bioRxiv2022

Repeat Detector: accurate, efficient, and flexible sizing of expanded CAG/CTG repeats from targeted DNA sequencing

Targeted DNA sequencing approaches will improve how the size of short tandem repeats is measured for diagnostic tests and pre-clinical studies. The expansion of these sequences causes dozens of disorders, with longer tracts generally leading to a more severe disease. Interrupted alleles are sometimes present within repeats and can alter disease manifestation. Determining repeat size mosaicism and identifying interruptions in targeted sequencing datasets remains a major challenge. This is in part because standard alignment tools are ill-suited for repetitive and unstable sequences. To address this, we have developed Repeat Detector (RD), a deterministic profile weighting algorithm for counting repeats in targeted sequencing data. We tested RD using blood-derived DNA samples from Huntingtons disease and Fuchs endothelial corneal dystrophy patients sequenced using either Illumina MiSeq or Pacific Biosciences single-molecule, real-time sequencing platforms. RD was highly accurate in determining repeat sizes of 609 blood-derived samples from Huntingtons disease individuals and did not require prior knowledge of the flanking sequences. Furthermore, RD can be used to identify alleles with interruptions and provide a measure of repeat instability within an individual. RD is therefore highly versatile and may find applications in the diagnosis of expanded repeat disorders and the development of novel therapies.

neuroscience↗

bioRxiv2019

Three-dimensional chromatin interactions remain stable upon CAG/CTG repeat expansion

Expanded CAG/CTG repeats underlie thirteen neurological disorders, including myotonic dystrophy (DM1) and Huntingtons disease (HD). Upon expansion, CAG/CTG repeat loci acquire heterochromatic characteristics. This observation raises the hypothesis that repeat expansion provokes changes to higher order chromatin folding and thereby affects both gene expression in cis and the genetic instability of the repeat tract. Here we tested this hypothesis directly by performing 4C sequencing at the DMPK and HTT loci from DM1 and HD patient-derived cells. Surprisingly, chromatin contacts remain unchanged upon repeat expansion at both loci. This was true for loci with different DNA methylation levels and CTCF binding. Repeat sizes ranging from 15 to 1,700 displayed strikingly similar chromatin interaction profiles. Our findings argue that extensive changes in heterochromatic properties are not enough to alter chromatin folding at expanded CAG/CTG repeat loci. Moreover, the ectopic insertion of an expanded repeat tract did not change three-dimensional chromatin contacts. We conclude that expanded CAG/CTG repeats have little to no effect on chromatin conformation.

neuroscience↗

bioRxiv2018

Uncovering the interplay between epigenome editing efficiency and sequence context using a novel inducible targeting system

Expanded CAG/CTG repeat disorders affect over 1 in 2500 individuals worldwide. Potential therapeutic avenues include gene silencing and modulation of repeat instability. However, there are major mechanistic gaps in our understanding of these processes, which prevent the rational design of an efficient treatment. To address this, we developed a novel system, ParB/ANCHOR-mediated Inducible Targeting (PInT), in which any protein can be recruited at will to a GFP reporter containing an expanded CAG/CTG repeat. Using PInT, we found no evidence that the histone deacetylase HDAC5 or the DNA methyltransferase DNMT1 modulate repeat instability upon targeting to the expanded repeat, suggesting that their effect is independent of local chromatin structure. Unexpectedly, we found that expanded CAG/CTG repeats reduce the effectiveness of gene silencing mediated by HDAC5 or DNMT1 targeting. The repeat-length effect in gene silencing by HDAC5 was abolished by a small molecule inhibitor of HDAC3. Our results have important implications on the design of epigenome editing approaches for expanded CAG/CTG repeat disorders. PInT is a versatile synthetic system to study the effect of any sequence of interest on epigenome editing.

molecular biology↗

bioRxiv2017

Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications

Expanded CAG/CTG repeats underlie the aetiology of 14 neurological and neuromuscular disorders. The size of the repeat tract determines in large part the severity of these disorders with longer tracts causing more severe phenotypes. Expanded CAG/CTG repeats are also unstable in somatic tissues, which is thought to modify disease progression. Routine molecular biology applications involving these repeats, including quantifying their instability, are plagued by low PCR yields. This leads to the need for setting up more PCRs of the same locus, thereby increasing the risk of carry-over contamination. Here we aimed to reduce this risk by pre-treating the samples with a Uracil N-Glycosylase (Ung) and using dUTP instead of dTTP in PCRs. We successfully applied this method to the PCR amplification of expanded CAG/CTG repeats, their sequencing, and their molecular cloning. In addition, we optimized the gold-standard method for measuring repeat instability, small-pool PCR, such that it can be used together with Ung and dUTP-containing PCRs, without compromising data quality. We expect that the protocols herein to be applicable for molecular diagnostics of expanded repeat disorders and to manipulate other tandem repeats.

molecular biology↗