Nicole A. Datson

Huntington’s disease (HD) is caused by a CAG repeat expansion in the huntingtin ( HTT ) gene, encoding an elongated polyglutamine stretch in the corresponding mutant HTT (mHTT) protein. HD pathogenesis is driven by multiple neurotoxic mHTT protein species, including HTT1a and repeat-associated non-AUG translation (RAN) proteins, which increase in expression as mutant CAG repeats lengthen due to somatic expansion. VO659 is an investigational CAG repeat-targeting antisense oligonucleotide (ASO) that lowers mHTT protein levels by blocking translation. Here, we show that VO659 allele-preferentially lowers levels of multiple neurotoxic mHTT protein species in a CAG repeat length-dependent manner, relatively sparing wild-type HTT, while becoming more efficacious as somatic expansion occurs. VO659 reduced HTT1a mRNA levels in HD fibroblasts with an ultra-long CAG repeat, blocked the translation of HTT1a and polyalanine-containing RAN proteins derived from HTT exon 1 overexpression constructs, and displayed a long tissue half-life in mouse brain, supporting an infrequent dosing regimen. A first-in-human, open-label phase 1/2a clinical trial ( NCT05822908 ) is currently ongoing in HD patients. VO659’s unique CAG repeat-dependent and allele-preferential mechanism of action holds promise for its therapeutic effect in HD patients by targeting multiple core neurotoxic proteins derived from (somatically) expanded mHTT CAG repeats, while sparing wild-type HTT expression.

Polyglutamine (polyQ) diseases are monogenic fatal neurodegenerative disorders caused by a CAG repeat expansion that is translated into a toxic polyQ tract. There are nine polyQ diseases: Huntington’s disease (HD), spinocerebellar ataxias 1, 2, 3, 6, 7 and 17 (SCA1, 2, 3, 6, 7, 17), dentatorubral-pallidoluysian atrophy (DRPLA) and spinal and bulbar muscular atrophy (SBMA). Although no disease-modifying therapies are available, lowering levels of the causative mutant polyQ protein is a promising potential treatment. Preclinically, the efficacy of polyQ protein-lowering compounds is often assessed using time-consuming Western blots (WB), which can produce variable results. Therefore, to improve throughput and accuracy of polyQ protein level quantification, Simple Western (SW) capillary immunoassays were developed. A panel of antibodies was screened for reactivity to the polyQ proteins on SW. The most promising antibodies were selected for further assay development. This resulted in optimised SW immunoassays for huntingtin (HTT), ataxin 1, 2 and 3 (ATXN1, 2, 3), atrophin 1 (ATN1) and androgen receptor (AR). Additionally, size-separation of the wild-type and polyQ-expanded mutant protein isoforms on SW was shown for ATXN1, ATXN3 and ATN1, allowing for their separate quantification. To facilitate size-separation of the larger HTT protein (≥ 348 kDa), a novel caspase 3-based assay was developed to generate N-terminal wild-type and mutant HTT fragments that could be separately quantified on SW in contrast to full-length HTT. In conclusion, SW capillary immunoassays were developed for polyQ proteins to improve preclinical research and aid the development of polyQ-lowering therapies for polyQ diseases.

Objective Huntington’s disease (HD) is an autosomal dominant, fully penetrant, neurodegenerative disease that most commonly affects middle-aged adults. HD is caused by a CAG repeat expansion in the HTT gene, resulting in the expression of mutant huntingtin (mHTT). Our aim was to detect and quantify mHTT in tear fluid, which, to our knowledge, has never been measured before.Methods We recruited 20 manifest and 13 premanifest HD gene expansion carriers, and 20 age-matched controls. All patients underwent detailed assessments, including the Unified Huntington’s Disease Rating Scale (UHDRS) total motor score (TMS) and total functional capacity (TFC) score. Tear fluid was collected using paper Schirmer’s strips. The level of tear mHTT was determined using single-molecule counting SMCxPRO technology.Results The average tear mHTT levels in manifest (67,223 ± 80,360 fM) and premanifest patients (55,561 ± 45,931 fM) were significantly higher than those in controls (1,622 ± 2,179 fM). We noted significant correlations between tear mHTT levels and CAG repeat length, “estimated years to diagnosis,” disease burden score and UHDRS TMS and TFC. The receiver operating curve demonstrated an almost perfect score (area under the curve [AUC] = 0.9975) when comparing controls to manifest patients. Similarly, the AUC between controls and premanifest patients was 0.9846. The optimal cutoff value for distinguishing between controls and manifest patients was 4,544 fM, whereas it was 6,596 fM for distinguishing between controls and premanifest patients.Conclusion Tear mHTT has potential for early and noninvasive detection of alterations in HD patients and could be integrated into both clinical trials and clinical diagnostics.

Duchenne muscular dystrophy (DMD) is a severe childhood muscle disease primarily caused by the lack of functional dystrophin at the muscle fiber membranes. Multiple therapeutic approaches are currently in (pre)clinical development, aimed at restoring expression of (truncated) dystrophin. Key questions in this phase relate to route of drug administration, dose regimen, and levels of dystrophin required to improve muscle function. A series of studies applying antisense oligonucleotides (AONs) in the mdx mouse model for DMD has been reported over the last two decades, claiming a variable range of exon skipping and increased dystrophin levels correlated to some functional improvement. The aim of this study was to compare the efficacy of subcutaneous (SC) versus intravenous (IV) dosing routes of an mdx -specific AON at both the molecular and functional level, using state-of-the-art quantitative technologies, including digital droplet polymerase chain reaction, capillary Western immunoassay, magnetic resonance imaging, and automated kinematic analysis. The majority of all readouts we quantified, both molecular and functional, showed that IV dosing of the AON had a more pronounced beneficial effect than SC dosing in mdx mice. Last, but not least, the more quantitative molecular and functional data obtained in this study suggest that low levels of dystrophin protein of at least 2.5% of wild type may already have a beneficial effect on muscle leakiness and may improve motor performance of mdx mice.

Duchenne muscular dystrophy is a lethal disease caused by lack of dystrophin. Skipping of exons adjacent to out-of-frame deletions has proven to restore dystrophin expression in Duchenne patients. Exon 51 has been the most studied target in both preclinical and clinical settings and the availability of standardized procedures to quantify exon skipping would be advantageous for the evaluation of preclinical and clinical data. To compare methods currently used to quantify antisense oligonucleotide–induced exon 51 skipping in the DMD transcript and to provide guidance about the method to use. Six laboratories shared blinded RNA samples from Duchenne patient-derived muscle cells treated with different amounts of exon 51 targeting antisense oligonucleotide. Exon 51 skipping levels were quantified using five different techniques: digital droplet PCR, single PCR assessed with Agilent bioanalyzer, nested PCR with agarose gel image analysis by either ImageJ or GeneTools software and quantitative real-time PCR. Differences in mean exon skipping levels and dispersion around the mean were observed across the different techniques. Results obtained by digital droplet PCR were reproducible and showed the smallest dispersion. Exon skipping quantification with the other methods showed overestimation of exon skipping or high data variation. Our results suggest that digital droplet PCR was the most precise and quantitative method. The quantification of exon 51 skipping by Agilent bioanalyzer after a single round of PCR was the second-best choice with a 2.3-fold overestimation of exon 51 skipping levels compared to digital droplet PCR.

Duchenne muscular dystrophy (DMD) is a severe, neuromuscular disorder caused by mutations in the DMD gene, precluding synthesis of functional dystrophin protein. Antisense oligonucleotide (AON)-mediated exon skipping has been developed as a method to restore the reading frame, which allows the synthesis of internally truncated, but partially functional dystrophin proteins, as found in the less severe Becker muscular dystrophy (BMD). This approach is species specific, since AONs targeting human exons often will not have full homology to mouse exons. As such, mouse models with mutations in the murine Dmd gene are of limited use to study human specific AONs in vivo. However, our del52hDMD/mdx mouse model contains mutated copies of both the mouse (nonsense mutation in exon 23) and human (deletion of exon 52) dystrophin-encoding genes. This model allows for testing effects of treatment with human specific exon 51 or 53 targeting AONs on RNA, protein, histological, and functional levels. Therefore, the model can be used to optimize human specific AONs, e.g., by comparing dystrophin protein and exon skipping levels. Absolute quantification of exon skipping levels can be obtained by digital droplet PCR (ddPCR). This method compartmentalizes samples into thousands of droplets that represent individual micro PCR reactions, and can be either positive or negative after amplification depending on whether there was a template molecule present or not. This allows for precise determination of the copy numbers of template molecules. The protocol described here uses probes binding to exon–exon junctions (EEJs) of human DMD transcripts with and without skipping of exon 51. We report that this method is specific for human transcripts so that exon skipping levels can be quantified accurately by ddPCR in del52hDMD/mdx mice.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease characterized by progressive weakness of the skeletal and cardiac muscles. This X-linked disorder is caused by open reading frame disrupting mutations in the DMD gene, resulting in strong reduction or complete absence of dystrophin protein. In order to use dystrophin as a supportive or even surrogate biomarker in clinical studies on investigational drugs aiming at correcting the primary cause of the disease, the ability to reliably quantify dystrophin expression in muscle biopsies of DMD patients pre- and post-treatment is essential. Here we demonstrate the application of the ProteinSimple capillary immunoassay (Wes) method, a gel- and blot-free method requiring less sample, antibody and time to run than conventional Western blot assay. We optimized dystrophin quantification by Wes using 2 different antibodies and found it to be highly sensitive, reproducible and quantitative over a large dynamic range. Using a healthy control muscle sample as a reference and α-actinin as a protein loading/muscle content control, a panel of skeletal muscle samples consisting of 31 healthy controls, 25 Becker Muscle dystrophy (BMD) and 17 DMD samples was subjected to Wes analysis. In healthy controls dystrophin levels varied 3 to 5-fold between the highest and lowest muscle samples, with the reference sample representing the average of all 31 samples. In BMD muscle samples dystrophin levels ranged from 10% to 90%, with an average of 33% of the healthy muscle average, while for the DMD samples the average dystrophin level was 1.3%, ranging from 0.7% to 7% of the healthy muscle average. In conclusion, Wes is a suitable, efficient and reliable method for quantification of dystrophin expression as a biomarker in DMD clinical drug development.

Clinical efficacy of antisense oligonucleotides (AONs) for the treatment of neuromuscular disorders depends on efficient cellular uptake and proper intracellular routing to the target. Selection of AONs with highest in vitro efficiencies is usually based on chemical or physical methods for forced cellular delivery. Since these methods largely bypass existing natural mechanisms for membrane passage and intracellular trafficking, spontaneous uptake and distribution of AONs in cells are still poorly understood. Here, we report on the unassisted uptake of naked AONs, so-called gymnosis, in muscle cells in culture. We found that gymnosis works similarly well for proliferating myoblasts as for terminally differentiated myotubes. Cell biological analyses combined with microscopy imaging showed that a phosphorothioate backbone promotes efficient gymnosis, that uptake is clathrin mediated and mainly results in endosomal-lysosomal accumulation. Nuclear localization occurred at a low level, but the gymnotically delivered AONs effectively modulated the expression of their nuclear RNA targets. Chloroquine treatment after gymnotic delivery helped increase nuclear AON levels. In sum, we demonstrate that gymnosis is feasible in proliferating and non-proliferating muscle cells and we confirm the relevance of AON chemistry for uptake and intracellular trafficking with this method, which provides a useful means for bio-activity screening of AONs in vitro.

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.

In the limbic brain, mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) both function as receptors for the naturally occurring glucocorticoids (corticosterone/cortisol) but mediate distinct effects on cellular physiology via transcriptional mechanisms. The transcriptional basis for specificity of these MR- vs GR-mediated effects is unknown. To address this conundrum, we have identified the extent of MR/GR DNA-binding selectivity in the rat hippocampus using chromatin immunoprecipitation followed by sequencing. We found 918 and 1450 nonoverlapping binding sites for MR and GR, respectively. Furthermore, 475 loci were co-occupied by MR and GR. De novo motif analysis resulted in a similar binding motif for both receptors at 100% of the target loci, which matched the known glucocorticoid response element (GRE). In addition, the Atoh/NeuroD consensus sequence was found in co-occurrence with all MR-specific binding sites but was absent for GR-specific or MR-GR overlapping sites. Basic helix-loop-helix family members Neurod1, Neurod2, and Neurod6 showed hippocampal expression and were hypothesized to bind the Atoh motif. Neurod2 was detected at rat hippocampal MR binding sites but not at GR-exclusive sites. All three NeuroD transcription factors acted as DNA-binding–dependent coactivators for both MR and GR in reporter assays in heterologous HEK293 cells, likely via indirect interactions with the receptors. In conclusion, a NeuroD family member binding to an additional motif near the GRE seems to drive specificity for MR over GR binding at hippocampal binding sites.