CAG Trinucleotide Repeat Expansion Research Articles

CAG repeat instability in embryonic stem cells and derivative spermatogenic cells of transgenic Huntington’s disease monkey

The expansion of CAG (glutamine; Q) trinucleotide repeats (TNRs) predominantly occurs through male lineage in Huntington's disease (HD). As a result, offspring will have larger CAG repeats compared to their fathers, which causes an earlier onset of the disease called genetic anticipation. This study aims to develop a novel in vitro model to replicate CAG repeat instability in early spermatogenesis and demonstrate the biological process of genetic anticipation by using the HD stem cell model for the first time. HD rhesus monkey embryonic stem cells (rESCs) were cultured in vitro for an extended period. Male rESCs were used to derive spermatogenic cells in vitro with a 10-day differentiation. The assessment of CAG repeat instability was performed by GeneScan and curve fit analysis. Spermatogenic cells derived from rESCs exhibit progressive expansion of CAG repeats with high daily expansion rates compared to the extended culture of rESCs. The expansion of CAG repeats is cell type-specific and size-dependent. Here, we report a novel stem cell model that replicates genome instability and CAG repeat expansion in in vitro derived HD monkey spermatogenic cells. The in vitro spermatogenic cell model opens a new opportunity for studying TNR instability and the underlying mechanism of genetic anticipation, not only in HD but also in other TNR diseases.

Huntington's disease brain-derived small RNAs recapitulate associated neuropathology in mice.

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, we demonstrate that sRNA produced in the putamen of HD patients (HD-sRNA-PT) are sufficient to induce HD pathology in vivo. Mice injected with HD-sRNA-PT show motor abnormalities, decreased levels of striatal HD-related proteins, disruption of the indirect pathway, and strong transcriptional abnormalities, paralleling human HD pathology. Importantly, we show that the specific blockage of sCAG mitigates HD-sRNA-PT neurotoxicity only to a limited extent. This observation prompted us to identify other sRNA species enriched in HD putamen with neurotoxic potential. We detected high levels of tRNA fragments (tRFs) in HD putamen, and we validated the neurotoxic potential of an Alanine derived tRF in vitro. These results highlight that HD-sRNA-PT are neurotoxic, and suggest that multiple sRNA species contribute to striatal dysfunction and general transcriptomic changes, favoring therapeutic strategies based on the blockage of sRNA-mediated toxicity.

The phasor FLIM method reveals a link between a change in energy metabolism and mHtt protein spread in healthy Mammalian cells when co-cultured with Huntington diseased cells

Huntington Disease (HD) is a late-onset autosomal neurodegenerative disease characterized by the aggregations of mutant Huntingtin proteins (mHTT). A glutamine stretch (PolyQ) at the N-terminal of the Huntingtin protein is generated by the abnormal expansion of CAG trinucleotide repeats in exon 1 of the HTT gene. While the resulting polyQ aggregates are the predominate feature of HD, the intercellular spread of the expanded protein and the effect upon this transfer inside healthy cells have not yet fully understood. Here, we have employed the phasor Fluorescence Lifetime Imaging Microscopy (FLIM) method to measure NADH fluorescence lifetime change after the internalization of the PolyQ protein. Based on our analysis, we have found a significant decrease in the fraction of bound NADH in both cytoplasmic and nucleus regions when cells are co-cultured or when healthy cells uptake the supernatant containing polyQ proteins and aggregates. Overall, our FLIM study combined with confocal fluorescence imaging visualizes the absorption of the mutant Htt protein aggregates which results in a distinct NADH fluorescence lifetime between control cells and acceptor cells. These studies show, for the first time, the influence of how neighboring cells expressing the expanded Htt protein can regulate energy metabolism in healthy cells.

Targeting Mitochondrial Network Disorganization is Protective in C. elegans Models of Huntington's Disease.

Huntington’s disease (HD) is an adult-onset neurodegenerative disease caused by a trinucleotide CAG repeat expansion in the HTT gene. While the pathogenesis of HD is incompletely understood, mitochondrial dysfunction is thought to be a key contributor. In this work, we used C. elegans models to elucidate the role of mitochondrial dynamics in HD. We found that expression of a disease-length polyglutamine tract in body wall muscle, either with or without exon 1 of huntingtin, results in mitochondrial fragmentation and mitochondrial network disorganization. While mitochondria in young HD worms form elongated tubular networks as in wild-type worms, mitochondrial fragmentation occurs with age as expanded polyglutamine protein forms aggregates. To correct the deficit in mitochondrial morphology, we reduced levels of DRP-1, the GTPase responsible for mitochondrial fission. Surprisingly, we found that disrupting drp-1 can have detrimental effects, which are dependent on how much expression is decreased. To avoid potential negative side effects of disrupting drp-1, we examined whether decreasing mitochondrial fragmentation by targeting other genes could be beneficial. Through this approach, we identified multiple genetic targets that rescue movement deficits in worm models of HD. Three of these genetic targets, pgp-3, F25B5.6 and alh-12, increased movement in the HD worm model and restored mitochondrial morphology to wild-type morphology. This work demonstrates that disrupting the mitochondrial fission gene drp-1 can be detrimental in animal models of HD, but that decreasing mitochondrial fragmentation by targeting other genes can be protective. Overall, this study identifies novel therapeutic targets for HD aimed at improving mitochondrial health.

Mutant Huntingtin Is Cleared from the Brain via Active Mechanisms in Huntington Disease.

Huntington disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Therapeutics that lower HTT have shown preclinical promise and are being evaluated in clinical trials. However, clinical assessment of brain HTT lowering presents challenges. We have reported that mutant HTT (mHTT) in the CSF of HD patients correlates with clinical measures, including disease burden as well as motor and cognitive performance. We have also shown that lowering HTT in the brains of HD mice results in correlative reduction of mHTT in the CSF, prompting the use of this measure as an exploratory marker of target engagement in clinical trials. In this study, we investigate the mechanisms of mHTT clearance from the brain in adult mice of both sexes to elucidate the significance of therapy-induced CSF mHTT changes. We demonstrate that, although neurodegeneration increases CSF mHTT concentrations, mHTT is also present in the CSF of mice in the absence of neurodegeneration. Importantly, we show that secretion of mHTT from cells in the CNS followed by glymphatic clearance from the extracellular space contributes to mHTT in the CSF. Furthermore, we observe secretion of wild type HTT from healthy control neurons, suggesting that HTT secretion is a normal process occurring in the absence of pathogenesis. Overall, our data support both passive release and active clearance of mHTT into CSF, suggesting that its treatment-induced changes may represent a combination of target engagement and preservation of neurons.SIGNIFICANCE STATEMENT: Changes in CSF mutant huntingtin (mHTT) are being used as an exploratory endpoint in HTT lowering clinical trials for the treatment of Huntington disease (HD). Recently, it was demonstrated that intrathecal administration of a HTT lowering agent leads to dose-dependent reduction of CSF mHTT in HD patients. However, little is known about how HTT, an intracellular protein, reaches the extracellular space and ultimately the CSF. Our findings that HTT enters CSF by both passive release and active secretion followed by glymphatic clearance may have significant implications for interpretation of treatment-induced changes of CSF mHTT in clinical trials for HD.

Role of mosaic aneuploidy in the development and progression of Huntington's disease

AbstractBackgroundHuntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion mutation in the huntingtin (HTT) gene. HTT encodes the huntingtin protein, which has been shown to localize to spindle poles and is required for proper mitotic spindle orientation, chromosome segregation, and cell cycle regulation. Mutations in HTT lead to the production of a mature huntingtin protein with an expanded polyglutamine region (>35 repeats) at its N terminus. Previous studies have shown that mouse models of HD exhibit defects in microtubule function and in cell cycle regulation. HD is a progressive condition characterized by decline in cognitive and executive functions. Over the years, studies from our laboratory and others have shown elevated levels of aneuploidy in many neurodegenerative disorders that are characterized by cognitive deficits, including Alzheimer’s disease (AD) and frontotemporal lobar degeneration (FTLD). Here, we investigated whether chromosome segregation defects that lead to mosaic aneuploidy and consequent apoptosis may also contribute to neuronal loss in HD.MethodSingle cell suspensions were prepared from human brain samples (cortex and cerebellum) and fibroblast cell lines from HD donors and age‐matched controls and were processed for FISH analysis of human chromosome 21, NeuN immunostaining, and/or TUNEL staining. Single cell suspensions from cerebellum were prepared from mouse models of HD and age‐matched controls for FISH analysis of mouse chromosome 5 or 16, which is syntenic to human chromosome 21.ResultWe observed increased levels of mosaic aneuploidy and associated apoptosis in brain tissues and in cultured fibroblasts from HD donors compared to age‐matched controls. We also observed increased levels of mosaic aneuploidy in brain tissue from HD mice compared to age‐matched controls.ConclusionTogether with our previous studies of AD and FTLD, these data provide evidence that chromosome segregation defects that lead to genomic instability may serve as a shared mechanism underlying many neurodegenerative disorders. Our findings highlight the need to further investigate the biological mechanism(s) underlying HD pathology and set the stage for novel therapeutic approaches to HD that may also be applied to other neurodegenerative disorders.

Inconsistencies in histone acetylation patterns among different HD model systems and HD post-mortem brains

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in exon 1 of the huntingtin gene. Emerging evidence shows that additional epigenetic factors can modify disease phenotypes. Harnessing the ability of the epigenome to modify the disease for therapeutic purposes is therefore of interest. Epigenome modifiers, such as histone deacetylase inhibitors (HDACi), have improved pathology in a range of HD models. Yet in clinical trials, HDACi have failed to alleviate HD symptoms in patients. This study investigated potential reasons for the lack of translation of the therapeutic benefits of HDACi from lab to clinic. We analysed histone acetylation patterns of immuno-positive nuclei from brain sections and tissue microarrays from post-mortem human control and HD cases alongside several well-established HD models (OVT73 transgenic HD sheep, YAC128 mice, and an in vitro cell model expressing 97Q mutant huntingtin). Significant increases in histone H4 acetylation were observed in post-mortem HD cases, OVT73 transgenic HD sheep and in vitro models; these changes were absent in YAC128 mice. In addition, nuclear labelling for acetyl-histone H4 levels were inversely proportional to mutant huntingtin aggregate load in HD human cortex. Our data raise concerns regarding the utility of HDACi for the treatment of HD when regions of pathology exhibit already elevated histone acetylation patterns and emphasize the importance of searching for alternative epigenetic targets in future therapeutic strategies aiming to rescue HD phenotypes.

Molecular pathogenesis of spinal bulbar muscular atrophy (Kennedy's disease) and avenues for treatment.

The aim of this study was to illustrate the current understanding and avenues for developing treatment in spinal and bulbar muscular atrophy (SBMA), an inherited neuromuscular disorder caused by a CAG trinucleotide repeat expansion in the androgen receptor (AR) gene. Important advances have been made in characterizing the molecular mechanism of the disease, including the disruption of protein homeostasis, intracellular trafficking and signalling pathways. Biomarkers such as MRI quantification of muscle volume and fat fraction have been used to track disease progression, and will be useful in future clinical studies. Therapies tested and under development have been based on diverse strategies, including targeting mutant AR gene expression, stability and activity, and pathways that mitigate disease toxicity. We provide an overview of the recent advances in understanding the SBMA disease mechanism and highlight efforts to translate these insights into well tolerated and effective therapy.

Epigenomic Remodeling in Huntington's Disease-Master or Servant?

In light of our aging population, neurodegenerative disorders are becoming a tremendous challenge, that modern societies have to face. They represent incurable, progressive conditions with diverse and complex pathological features, followed by catastrophic occurrences of massive neuronal loss at the later stages of the diseases. Some of these disorders, like Huntington’s disease (HD), rely on defined genetic factors. HD, as an incurable, fatal hereditary neurodegenerative disorder characterized by its mid-life onset, is caused by the expansion of CAG trinucleotide repeats coding for glutamine (Q) in exon 1 of the huntingtin gene. Apart from the genetic defect, environmental factors are thought to influence the risk, onset and progression of HD. As epigenetic mechanisms are known to readily respond to environmental stimuli, they are proposed to play a key role in HD pathogenesis. Indeed, dynamic epigenomic remodeling is observed in HD patients and in brains of HD animal models. Epigenetic signatures, such as DNA methylation, histone variants and modifications, are known to influence gene expression and to orchestrate various aspects of neuronal physiology. Hence, deciphering their implication in HD pathogenesis might open up new paths for novel therapeutic concepts, which are discussed in this review.

Preclinical Nanomedicines for Polyglutamine-Based Neurodegenerative Diseases.

Polyglutamine (polyQ) diseases, such as Huntington's disease and several types of spinocerebellar ataxias, are dominantly inherited progressive neurodegenerative disorders and characterized by the presence of expanded CAG trinucleotide repeats in the respective disease locus of the patient genomes. Patients with polyQ diseases currently need to rely on symptom-relieving treatments because disease-modifying therapeutic interventions remain scarce. Many disease-modifying therapeutic agents are now under clinical testing for treating polyQ diseases, but their delivery to the brain is often too invasive (e.g., intracranial injection) or inefficient, owing to in vivo degradation and clearance by physiological barriers (e.g., oral and intravenous administration). Nanoparticles provide a feasible solution for improving drug delivery to the brain, as evidenced by an increasing number of preclinical studies that document the efficacy of nanomedicines for polyQ diseases over the past 5-6 years. In this review, we present the pathogenic mechanisms of polyQ diseases, the common animal models of polyQ diseases for evaluating the efficacy of nanomedicines, and the common administration routes for delivering nanoparticles to the brain. Next, we summarize the recent preclinical applications of nanomedicines for treating polyQ diseases and improving neurological conditions in vivo, placing emphasis on antisense oligonucleotides, small peptide inhibitors, and small molecules as the disease-modifying agents. We conclude with our perspectives of the burgeoning field of "nanomedicines for polyQ diseases", including the use of inorganic nanoparticles and potential drugs as next-generation nanomedicines, development of higher-order animal models of polyQ diseases, and importance of "brain-nano" interactions.

A Patient with Spinocerebellar Ataxia 2 Presenting with Multiple System Atrophy

Spinocerebellar ataxia type-2 (SCA2) is an autosomal dominant cerebellar ataxia that occurs due to expanded CAG trinucleotide repeats in the ATXN2 gene. Clinical features of parkinsonism in SCA2 vary from phenotypes of levodopa-responsive parkinsonism to multiple system atrophy. We described a patient with SCA2 presenting typical clinical manifestations of multiple system atrophy-c type with levodopa responsive parkinsonism whose dopamine transporter (DAT) image showed atypically reduced DAT uptake in in the striatum.

Case report and literature review of Huntington disease with intermediate CAG expansion

BackgroundHuntington disease (HD) is a genetically inherited neurodegenerative disorder that classically involves a trinucleotide CAG repeat expansion on chromosome 4, with 36 repeats or greater being disease identifying. It generally...

Neurodegenerative diseases and cancer: sharing common mechanisms in complex interactions.

Several epidemiological studies support low cancer rates in patients with neurodegenerative disorders, including Parkinson's disease, Huntington's disease, and Alzheimer's disease. Different mechanisms were raised as possible causes, from mutated tumor suppressor genes (PARKIN, PINK1) to small interfering RNA based on the CAG trinucleotide repeat expansions located in introns or untranslated regions. However, as every rule has an exception, some tumors have an increased incidence in these neurodegenerative diseases such as breast and skin cancer (melanoma). This mini-review aims to establish the epidemiology between these neurodegenerative disorders and cancer to determine the possible mechanisms involved and therefore set eventual therapeutic applications. According to our findings, we conclude the presence of an inverse relationship among most cancers and the aforementioned neurodegenerative disorders. However, this concept needs to be considered cautiously considering specific genetic and extra-genetic linkage factors for particular tumors.

Loss of Ataxin-1 potentiates Alzheimer's pathogenesis by elevating cerebral BACE1 transcription

Summary Expansion of CAG trinucleotide repeats in ATXN1 causes spinocerebellar ataxia type 1 (SCA1), a neurodegenerative disease that impairs coordination and cognition. While ATXN1 is associated with increased Alzheimer’s disease (AD) risk, CAG repeat number in AD patients is not changed. Here, we investigated the consequences of ataxin-1 loss of function and discovered that knockout of Atxn1 reduced CIC-ETV4/5-mediated inhibition of Bace1 transcription, leading to increased BACE1 levels and enhanced amyloidogenic cleavage of APP, selectively in AD-vulnerable brain regions. Elevated BACE1 expression exacerbated Aβ deposition and gliosis in AD mouse models and impaired hippocampal neurogenesis and olfactory axonal targeting. In SCA1 mice, polyglutamine-expanded mutant ataxin-1 led to the increase of BACE1 post-transcriptionally, both in cerebrum and cerebellum, and caused axonal-targeting deficit and neurodegeneration in the hippocampal CA2 region. These findings suggest that loss of ataxin-1 elevates BACE1 expression and Aβ pathology, rendering it a potential contributor to AD risk and pathogenesis.

Loss of Ataxin-1 Potentiates Alzheimer's Pathogenesis by Elevating Cerebral BACE1 Transcription.

Expansion of CAG trinucleotide repeats in ATXN1 causes spinocerebellar ataxia type 1 (SCA1), a neurodegenerative disease that impairs coordination and cognition. While ATXN1 is associated with increased Alzheimer's disease (AD) risk, CAG repeat number in AD patients is not changed. Here, we investigated the consequences of ataxin-1 loss of function and discovered that knockout of Atxn1 reduced CIC-ETV4/5-mediated inhibition of Bace1 transcription, leading to increased BACE1 levels and enhanced amyloidogenic cleavage of APP, selectively in AD-vulnerable brain regions. Elevated BACE1 expression exacerbated Aβ deposition and gliosis in AD mouse models and impaired hippocampal neurogenesis and olfactory axonal targeting. InSCA1 mice, polyglutamine-expanded mutant ataxin-1 led to the increase of BACE1 post-transcriptionally, both in cerebrum and cerebellum, and caused axonal-targeting deficit and neurodegeneration in the hippocampal CA2 region. These findings suggest that loss of ataxin-1 elevates BACE1 expression and Aβ pathology, rendering it a potential contributor to AD risk and pathogenesis.

A soluble truncated tau species related to cognitive dysfunction and caspase-2 is elevated in the brain of Huntington\u2019s disease patients

Huntington’s disease (HD) is a progressive neurodegenerative disease. Involuntary movements, cognitive impairment and psychiatric disturbance are the major clinical manifestations, and gradual atrophy and selective neuronal loss in the striatum and cerebral cortex are the pathologic hallmarks. HD is caused by expanded CAG trinucleotide repeats at the N-terminus of IT15 that encodes the huntingtin (HTT) protein, though the molecular mechanisms through which the mutant HTT (mHTT) exerts toxic effects remain obscure. Members of the caspase family, including caspase-2 (Casp2), play an important role in HD pathogenesis. Genetic ablation of Casp2 ameliorates cognitive and motor deficits of HD mice, though the molecular targets of Casp2 are still unclear. It is well established that the microtubule-associated protein tau potentiates cognitive dysfunction in a variety of neurodegenerative disorders, including HD. Our recent study indicates that Casp2-catalyzed tau cleavage at aspartate 314 (tau 2N4R isoform numbering system) mediates synaptotoxicity, cognitive deficits and neurodegeneration in cellular and mouse models of frontotemporal dementia; further, levels of Δtau314, the soluble, N-terminal cleavage product, are elevated in individuals with mild cognitive impairment and Alzheimer’s disease, compared with cognitively normal individuals. Here, we identified the presence of Δtau314 proteins in the striatum (caudate nucleus) and prefrontal cortex (Brodmann’s area 8/9) of human subjects, and showed that in both structures, levels of Casp2 and Δtau314 proteins correlate well, and both proteins are higher in HD patients than non-HD individuals. Our findings advance our understanding of the contribution of Casp2-mediated Δtau314 production to HD pathogenesis.

CRISPR-Cas9-Mediated Genome Editing Increases Lifespan and Improves Motor Deficits in a Huntington’s Disease Mouse Model

Huntington’s disease (HD) is a currently incurable and, ultimately, fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion within exon 1 of the huntingtin (HTT) gene, which results in the production of a mutant protein that forms inclusions and selectively destroys neurons in the striatum and other adjacent structures. The RNA-guided Cas9 endonuclease from CRISPR-Cas9 systems is a versatile technology for inducing DNA double-strand breaks that can stimulate the introduction of frameshift-inducing mutations and permanently disable mutant gene function. Here, we show that the Cas9 nuclease from Staphylococcus aureus, a small Cas9 ortholog that can be packaged alongside a single guide RNA into a single adeno-associated virus (AAV) vector, can be used to disrupt the expression of the mutant HTT gene in the R6/2 mouse model of HD following its in vivo delivery to the striatum. Specifically, we found that CRISPR-Cas9-mediated disruption of the mutant HTT gene resulted in a ∼50% decrease in neuronal inclusions and significantly improved lifespan and certain motor deficits. These results thus illustrate the potential for CRISPR-Cas9 technology to treat HD and other autosomal dominant neurodegenerative disorders caused by a trinucleotide repeat expansion via in vivo genome editing.

Full sequence of mutant huntingtin 3'-untranslated region and modulation of its gene regulatory activity by endogenous microRNA.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in the first exon of the huntingtin gene (HTT). Since the entire course of the disease starts from this dominant gain-of-function mutation, lowering total or mutant huntingtin mRNA/protein has emerged as an appealing therapeutic strategy. We reasoned that endogenous mechanisms underlying HTT gene regulation may inform strategies to target the source of the disease. As part of our investigation to understand how the expression of HTT is controlled, we performed (1) complete sequencing analysis for mutant HTT 3'-UTR and (2) unbiased screening assays to identify naturally-occurring miRNAs that could lower the HTT mRNA levels. By sequencing HD families inheriting the major European mutant haplotype, we determined the full sequence of HTT 3'-UTRs of the most frequent mutant (i.e., hap.01) and normal (i.e., hap.08) haplotypes, revealing 5 sites with alternative alleles. In subsequent miRNA activity assays using the full-length hap.01 and hap.08 3'-UTR reporter vectors and follow-up validation experiments, hsa-miR-4324 and hsa-miR-4756-5p significantly reduced HTT 3'-UTR reporter activity and endogenous HTT protein levels. However, those miRNAs did not show strong haplotype-specific effects. Nevertheless, our data highlighting full sequences of HTT 3'-UTR haplotypes, effects of miRNAs on HTT levels, and potential interaction sites provide rationale and promising targets for total and mutant-specific HTT lowering intervention strategies using endogenous and artificial miRNAs, respectively.

Targeting Huntingtin Expression in Patients with Huntington’s Disease

BackgroundHuntington’s disease is an autosomal-dominant neurodegenerative disease caused by CAG trinucleotide repeat expansion in HTT, resulting in a mutant huntingtin protein. IONIS-HTTRx (hereafter, HTTRx) is an antisense oligonucleotide designed to inhibit HTT messenger RNA and thereby reduce concentrations of mutant huntingtin.MethodsWe conducted a randomized, double-blind, multiple-ascending-dose, phase 1–2a trial involving adults with early Huntington’s disease. Patients were randomly assigned in a 3:1 ratio to receive HTTRx or placebo as a bolus intrathecal administration every 4 weeks for four doses. Dose selection was guided by a preclinical model in mice and nonhuman primates that related dose level to reduction in the concentration of huntingtin. The primary end point was safety. The secondary end point was HTTRx pharmacokinetics in cerebrospinal fluid (CSF). Prespecified exploratory end points included the concentration of mutant huntingtin in CSF.ResultsOf the 46 patients who were enrolled in the trial, 34 were randomly assigned to receive HTTRx (at ascending dose levels of 10 to 120 mg) and 12 were randomly assigned to receive placebo. Each patient received all four doses and completed the trial. Adverse events, all of grade 1 or 2, were reported in 98% of the patients. No serious adverse events were seen in HTTRx-treated patients. There were no clinically relevant adverse changes in laboratory variables. Predose (trough) concentrations of HTTRx in CSF showed dose dependence up to doses of 60 mg. HTTRx treatment resulted in a dose-dependent reduction in the concentration of mutant huntingtin in CSF (mean percentage change from baseline, 10% in the placebo group and −20%, −25%, −28%, −42%, and −38% in the HTTRx 10-mg, 30-mg, 60-mg, 90-mg, and 120-mg dose groups, respectively).ConclusionsIntrathecal administration of HTTRx to patients with early Huntington’s disease was not accompanied by serious adverse events. We observed dose-dependent reductions in concentrations of mutant huntingtin. (Funded by Ionis Pharmaceuticals and F. Hoffmann–La Roche; ClinicalTrials.gov number, NCT02519036.)

Focused ultrasound-induced blood brain-barrier opening enhanced vascular permeability for GDNF delivery in Huntington's disease mouse model

BackgroundHuntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the gene encoding the huntingtin (Htt) protein, which results in a protein containing an abnormally expanded polyglutamine (polyQ) sequence. The expanded polyQ in the Htt protein is toxic to brain cells. No therapy exists to delay disease progression. MethodsThis study describes a gene-liposome system that synergistically applied focused ultrasound (FUS)-blood-brain barrier (BBB) opening for rescuing motor and neuropathological impairments when administered from pre to post-symptomatic transgenic mouse models of HD. DPPC liposomes (LPs) are designed to carry glia cell line-derived neurotrophic factor (GDNF) plasmid DNA (GDNFp) to form a GDNFp-liposome (GDNFp-LPs) complex. Pulsed FUS exposure with microbubbles (MBs) was used to induce BBB opening for non-viral, non-invasive, and targeted gene delivery into the central nervous system (CNS) for therapeutic purposes. ResultsFUS-gene therapy significantly improved motor performance with GDNFp-LPs + FUS treated HD mice equilibrating longer periods in the animal behavior. Reflecting the improvements observed in motor function, GDNF overexpression results in significantly decreased formation of polyglutamine-expanded aggregates, reduced oxidative stress and apoptosis, promoted neurite outgrowth, and improved neuronal survival. Immunoblotting and histological staining further confirmed the neuroprotective effect from delivery of GDNF genes to neuronal cells. ConclusionsThis study suggests that the GDNFp-LPs plus FUS sonication can provide an effective gene therapy to achieve local extravasation and triggered gene delivery for non-invasive in vivo treatment of CNS diseases.