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M Leontien van der Bent, Melvin M Evers, Astrid Vallès uniQure biopharma B.V., Department of Research and Development, Amsterdam, the NetherlandsCorrespondence: Astrid Vallès, uniQure biopharma B.V, Postbus 22506, Amsterdam, 1100 DA, the Netherlands, Tel +31 20 240 6000, Email a.vallessanchez@uniqure.comAbstract: Huntington’s disease is a devastating heritable neurodegenerative disorder that is caused by the presence of a trinucleotide CAG repeat expansion in the Huntingtin gene, leading to a polyglutamine tract in the protein. Various mechanisms lead to the production of N-terminal Huntingtin protein fragments, which are reportedly more toxic than the full-length protein. In this review, we summarize the current knowledge on the production and toxicity of N-terminal Huntingtin protein fragments. Further, we expand on various therapeutic strategies targeting N-terminal Huntingtin on the protein, RNA and DNA level. Finally, we compare the therapeutic approaches that are clinically most advanced, including those that do not target N-terminal Huntingtin, discussing differences in mode of action and translational applicability.Keywords: Huntingtin, N-terminal fragments, proteolysis, aberrant splicing, exon1 fragment, Huntington’s disease therapeutics

Little is known of the palliative care needs and experiences of older adults. This study explored whether these needs differed from those of younger patients. We performed a retrospective data analysis of 1184 palliative care consultations in a major teaching hospital. There were statistically significant differences across age groups in patient demographic and clinical characteristics, advance care planning, and service utilization. Patients over age 80 had a reduced prevalence of cancer, a higher prevalence of dementia and incapacity, more frequent decisions to withhold or withdraw life-sustaining treatments, and fewer interventions for symptom management. The palliative care needs of older adults appear to be substantially different from those of younger patients. Dementia and incapacity profoundly influence decision-making, requiring more time and communication with patients and families. [ABSTRACT FROM AUTHOR]

Huntingtin (HTT)-lowering therapies show great promise in treating Huntington's disease. We have developed a microRNA targeting human HTT that is delivered in an adeno-associated serotype 5 viral vector (AAV5-miHTT), and here use animal behaviour, MRI, non-invasive proton magnetic resonance spectroscopy and striatal RNA sequencing as outcome measures in preclinical mouse studies of AAV5-miHTT. The effects of AAV5-miHTT treatment were evaluated in homozygous Q175FDN mice, a mouse model of Huntington's disease with severe neuropathological and behavioural phenotypes. Homozygous mice were used instead of the more commonly used heterozygous strain, which exhibit milder phenotypes. Three-month-old homozygous Q175FDN mice, which had developed acute phenotypes by the time of treatment, were injected bilaterally into the striatum with either formulation buffer (phosphate-buffered saline + 5% sucrose), low dose (5.2 × 109 genome copies/mouse) or high dose (1.3 × 1011 genome copies/mouse) AAV5-miHTT. Wild-type mice injected with formulation buffer served as controls. Behavioural assessments of cognition, T1-weighted structural MRI and striatal proton magnetic resonance spectroscopy were performed 3 months after injection, and shortly afterwards the animals were sacrificed to collect brain tissue for protein and RNA analysis. Motor coordination was assessed at 1-month intervals beginning at 2 months of age until sacrifice. Dose-dependent changes in AAV5 vector DNA level, miHTT expression and mutant HTT were observed in striatum and cortex of AAV5-miHTT-treated Huntington's disease model mice. This pattern of microRNA expression and mutant HTT lowering rescued weight loss in homozygous Q175FDN mice but did not affect motor or cognitive phenotypes. MRI volumetric analysis detected atrophy in four brain regions in homozygous Q175FDN mice, and treatment with high dose AAV5-miHTT rescued this effect in the hippocampus. Like previous magnetic resonance spectroscopy studies in Huntington's disease patients, decreased total N-acetyl aspartate and increased myo-inositol levels were found in the striatum of homozygous Q175FDN mice. These neurochemical findings were partially reversed with AAV5-miHTT treatment. Striatal transcriptional analysis using RNA sequencing revealed mutant HTT-induced changes that were partially reversed by HTT lowering with AAV5-miHTT. Striatal proton magnetic resonance spectroscopy analysis suggests a restoration of neuronal function, and striatal RNA sequencing analysis shows a reversal of transcriptional dysregulation following AAV5-miHTT in a homozygous Huntington's disease mouse model with severe pathology. The results of this study support the use of magnetic resonance spectroscopy in HTT-lowering clinical trials and strengthen the therapeutic potential of AAV5-miHTT in reversing severe striatal dysfunction in Huntington's disease., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Enterovirus D68 (EV-D68) is an RNA virus that can cause outbreaks of acute flaccid paralysis (AFP), a polio-like disease. Before 2010, EV-D68 was a rare pathogen associated with mild respiratory symptoms, but the recent EV-D68 related increase in severe respiratory illness and outbreaks of AFP is not yet understood. An explanation for the rise in severe disease is that it may be due to changes in the viral genome resulting in neurotropism. In this regard, in addition to sialic acid, binding to heparan sulfate proteoglycans (HSPGs) has been identified as a feature for viral entry of some EV-D68 strains in cell lines. Studies in human primary organotypic cultures that recapitulate human physiology will address the relevance of these HSPG-binding mutations for EV-D68 infection in vivo. Therefore, in this work, we studied the replication and neurotropism of previously determined sialic acid-dependent and HSPG-dependent strains using primary human airway epithelial (HAE) cultures and induced human pluripotent stem cell (iPSC)-derived brain organoids. All three strains (B2/2042, B2/947, and A1/1348) used in this study infected HAE cultures and human brain organoids (shown for the first time). Receptor-blocking experiments in both cultures confirm that B2/2042 infection is solely dependent on sialic acid, while B2/947 and A1/1348 (HSPG to a lesser extent) binds to sialic acid and HSPG for cell entry. Our data suggest that HSPG-binding can be used by EV-D68 for entry in human physiological models but offers no advantage for EV-D68 infection of brain cells. IMPORTANCE Recent outbreaks of enterovirus D68, a nonpolio enterovirus, is associated with a serious neurological condition in young children, acute flaccid myelitis (AFM). As there is no antiviral treatment or vaccine available for EV-D68 it is important to better understand how EV-D68 causes AFM and why only recent outbreaks are associated with AFM. We investigated if a change in receptor usage of EV-D68 increases the virulence of EV-D68 in the airway or the central nervous system and thus could explain the increase in AFM cases. We studied this using physiologically relevant human airway epithelium and cerebral organoid cultures that are physiologically relevant human models. Our data suggest that heparan sulfate proteoglycans can be used by EV-D68 as an additional entry receptor in human physiological models but offers no advantage for EV-D68 infection of brain cells, and our data show the potential of these 46 innovative models for virology.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by the expansion of a CAG repeat in the ATXN3 gene. This mutation leads to a toxic gain of function of the ataxin-3 protein, resulting in neuronal dysfunction and atrophy of specific brain regions over time. As ataxin-3 is a dispensable protein in rodents, ataxin-3 knockdown by gene therapy may be a powerful approach for the treatment of SCA3. In this study, we tested the feasibility of an adeno-associated viral (AAV) vector carrying a previously described artificial microRNA against ATXN3 in a striatal mouse model of SCA3. Striatal injection of the AAV resulted in good distribution throughout the striatum, with strong dose-dependent ataxin-3 knockdown. The hallmark intracellular ataxin-3 inclusions were almost completely alleviated by the microRNA-induced ATXN3 knockdown. In addition, the striatal lesion of dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32) in the SCA3 mice was rescued by ATXN3 knockdown, indicating functional rescue of neuronal signaling and health upon AAV treatment. Together, these data suggest that microRNA-induced ataxin-3 knockdown is a promising therapeutic strategy in the treatment of SCA3.

Pathogenesis of viral infections of the central nervous system (CNS) is poorly understood, and this is partly due to the limitations of currently used preclinical models. Brain organoid models can overcome some of these limitations, as they are generated from human derived stem cells, differentiated in three dimensions (3D), and can mimic human neurodevelopmental characteristics. Therefore, brain organoids have been increasingly used as brain models in research on various viruses, such as Zika virus, severe acute respiratory syndrome coronavirus 2, human cytomegalovirus, and herpes simplex virus. Brain organoids allow for the study of viral tropism, the effect of infection on organoid function, size, and cytoarchitecture, as well as innate immune response; therefore, they provide valuable insight into the pathogenesis of neurotropic viral infections and testing of antivirals in a physiological model. In this review, we summarize the results of studies on viral CNS infection in brain organoids, and we demonstrate the broad application and benefits of using a human 3D model in virology research. At the same time, we describe the limitations of the studies in brain organoids, such as the heterogeneity in organoid generation protocols and age at infection, which result in differences in results between studies, as well as the lack of microglia and a blood brain barrier.

Huntingtin (HTT)-lowering therapies hold promise to slow down neurodegeneration in Huntington's disease (HD). Here, we assessed the translatability and long-term durability of recombinant adeno-associated viral vector serotype 5 expressing a microRNA targeting human HTT (rAAV5-miHTT) administered by magnetic resonance imaging-guided convention-enhanced delivery in transgenic HD minipigs. rAAV5-miHTT (1.2 × 10 13 vector genome (VG) copies per brain) was successfully administered into the striatum (bilaterally in caudate and putamen), using age-matched untreated animals as controls. Widespread brain biodistribution of vector DNA was observed, with the highest concentration in target (striatal) regions, thalamus, and cortical regions. Vector DNA presence and transgene expression were similar at 6 and 12 months after administration. Expression of miHTT strongly correlated with vector DNA, with a corresponding reduction of mutant HTT (mHTT) protein of more than 75% in injected areas, and 30 to 50% lowering in distal regions. Translational pharmacokinetic and pharmacodynamic measures in cerebrospinal fluid (CSF) were largely in line with the effects observed in the brain. CSF miHTT expression was detected up to 12 months, with CSF mHTT protein lowering of 25 to 30% at 6 and 12 months after dosing. This study demonstrates widespread biodistribution, strong and durable efficiency of rAAV5-miHTT in disease-relevant regions in a large brain, and the potential of using CSF analysis to determine vector expression and efficacy in the clinic., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The preclinical development of microRNA-based gene therapies for inherited neurodegenerative diseases is accompanied by translational challenges. Due to the inaccessibility of the brain to periodically evaluate therapy effects, accessible and reliable biomarkers indicative of dosing, durability and therapeutic efficacy in the central nervous system are very much needed. This is particularly important for viral vector-based gene therapies, in which a one-time administration results in long-term expression of active therapeutic molecules in the brain. Recently, extracellular vesicles have been identified as carriers of RNA species, including microRNAs, and proteins in all biological fluids, whilst becoming potential sources of biomarkers for diagnosis. In this study, we investigated the secretion and potential use of circulating miRNAs associated with extracellular vesicles as suitable sources to monitor the expression and durability of gene therapies in the brain. Neuronal cells derived from induced pluripotent stem cells were treated with adeno-associated viral vector serotype 5 carrying an engineered microRNA targeting huntingtin or ataxin3 gene sequences, the diseases-causing genes of Huntington disease and spinocerebellar ataxia type 3, respectively. After treatment, the secretion of mature engineered microRNA molecules was confirmed, with extracellular microRNA levels correlating with viral dose and cellular microRNA expression in neurons. We further investigated the detection of engineered microRNAs over time in the CSF of non-human primates after a single intrastriatal injection of adeno-associated viral vector serotype 5 carrying a huntingtin -targeting engineered microRNA. Quantifiable engineered microRNA levels enriched in extracellular vesicles were detected in the CSF up to 2 years after brain infusion. Altogether, these results confirm the long-term expression of adeno-associated viral vector serotype 5-delivered microRNAs and support the use of extracellular vesicle-associated microRNAs as novel translational pharmacokinetic markers in ongoing clinical trials of gene therapies for neurodegenerative diseases., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Huntington disease (HD) is a fatal, neurodegenerative genetic disorder with aggregation of mutant Huntingtin protein (mutHTT) in the brain as a key pathological mechanism. There are currently no disease modifying therapies for HD; however, HTT -lowering therapies hold promise. Recombinant adeno-associated virus serotype 5 expressing a microRNA that targets HTT mRNA (AAV5-miHTT) is in development for the treatment of HD with promising results in rodent and minipig HD models. To support a clinical trial, toxicity studies were performed in non-human primates (NHP, Macaca fascicularis ) and Sprague-Dawley rats to evaluate the safety of AAV5-miHTT, the neurosurgical administration procedure, vector delivery and expression of the mi HTT transgene during a 6-month observation period. For accurate delivery of AAV5-miHTT to the striatum, real-time magnetic resonance imaging (MRI) with convection-enhanced delivery (CED) was used in NHP. Catheters were successfully implanted in 24 NHP, without neurological symptoms, and resulted in tracer signal in the target areas. Widespread vector DNA and mi HTT transgene distribution in the brain was found, particularly in areas associated with HD pathology. Intrastriatal administration of AAV5-miHTT was well tolerated with no clinically relevant changes in either species. These studies demonstrate the excellent safety profile of AAV5-miHTT, the reproducibility and tolerability of intrastriatal administration, and the delivery of AAV5-miHTT to the brain, which support the transition of AAV5-miHTT into clinical studies.

Animal models and cell lines are invaluable for virology research and host-pathogen interaction studies. However, it is increasingly evident that these models are not sufficient to fully understand human viral diseases. With the advent of three-dimensional organotypic cultures, it is now possible to study viral infections in the human context. This perspective explores the potential of these organotypic cultures, also known as organoids, for virology research, antiviral testing, and shaping the virology landscape.

The development of gene therapies for central nervous system disorders is challenging because it is difficult to translate preclinical data from current in vitro and in vivo models to the clinic. Therefore, we developed induced pluripotent stem cell (iPSC)-derived cerebral organoids as a model for recombinant adeno-associated virus (rAAV) capsid selection and for testing efficacy of AAV-based gene therapy in a human context. Cerebral organoids are physiological 3D structures that better recapitulate the human brain compared with 2D cell lines. To validate the model, we compared the transduction efficiency and distribution of two commonly used AAV serotypes (rAAV5 and rAAV9). In cerebral organoids, transduction with rAAV5 led to higher levels of vector DNA, transgenic mRNA, and protein expression as compared with rAAV9. The superior transduction of rAAV5 was replicated in iPSC-derived neuronal cells. Furthermore, rAAV5-mediated delivery of a human sequence-specific engineered microRNA to cerebral organoids led to a lower expression of its target ataxin-3. Our studies provide a new tool for selecting and deselecting AAV serotypes, and for demonstrating therapeutic efficacy of transgenes in a human context. Implementing cerebral organoids during gene therapy development could reduce the usage of animal models and improve translation to the clinic., (© 2020 The Author(s).)

Huntington disease (HD) is a fatal neurodegenerative disease caused by a pathogenic expansion of a CAG repeat in the huntingtin (HTT) gene. There are no disease-modifying therapies for HD. Artificial microRNAs targeting HTT transcripts for degradation have shown preclinical promise and will soon enter human clinical trials. Here, we examine the tolerability and efficacy of non-selective HTT lowering with an AAV5 encoded miRNA targeting human HTT (AAV5-miHTT) in the humanized Hu128/21 mouse model of HD. We show that intrastriatal administration of AAV5-miHTT results in potent and sustained HTT suppression for at least 7 months post-injection. Importantly, non-selective suppression of huntingtin was generally tolerated, however high dose AAV5-miHTT did induce astrogliosis. We observed an improvement of select behavioural and modest neuropathological HD-like phenotypes in Hu128/21 mice, suggesting a potential therapeutic benefit of miRNA-mediated non-selective HTT lowering. Finally, we also observed that potent reduction of wild type HTT (wtHTT) in Hu21 control mice was tolerated up to 7 months post-injection but may induce impairment of motor coordination and striatal atrophy. Taken together, our data suggests that in the context of HD, the therapeutic benefits of mHTT reduction may outweigh the potentially detrimental effects of wtHTT loss following non-selective HTT lowering., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Spinocerebellar ataxia type 3 (SCA3), or Machado-Joseph disease (MJD), is a progressive neurodegenerative disorder caused by a CAG expansion in the ATXN3 gene. The expanded CAG repeat is translated into a prolonged polyglutamine repeat in the ataxin-3 protein and accumulates within inclusions, acquiring toxic properties, which results in degeneration of the cerebellum and brain stem. In the current study, a non-allele-specific ATXN3 silencing approach was investigated using artificial microRNAs engineered to target various regions of the ATXN3 gene (miATXN3). The mi ATXN3 candidates were screened in vitro based on their silencing efficacy on a luciferase (Luc) reporter co-expressing ATXN3 . The three best miATXN3 candidates were further tested for target engagement and potential off-target activity in induced pluripotent stem cells (iPSCs) differentiated into frontal brain-like neurons and in a SCA3 knockin mouse model. Besides a strong reduction of ATXN3 mRNA and protein, small RNA sequencing revealed efficient guide strand processing without passenger strands being produced. We used different methods to predict alteration of off-target genes upon AAV5-mi ATXN3 treatment and found no evidence for unwanted effects. Furthermore, we demonstrated in a large animal model, the minipig, that intrathecal delivery of AAV5 can transduce the main areas affected in SCA3 patients. These results proved a strong basis to move forward to investigate distribution, efficacy, and safety of AAV5-mi ATXN3 in large animals., (© 2019 The Author(s).)

Huntington disease (HD) is a fatal neurodegenerative genetic disorder, thought to reflect a toxic gain of function in huntingtin (Htt) protein. Adeno-associated viral vector serotype 5 (AAV5)- microRNA targeting huntingtin (miHTT) is a HD gene-therapy candidate that efficiently lowers HTT using RNAi. This study analyzed the efficacy and potential for off-target effects with AAV5-miHTT in neuronal and astrocyte cell cultures differentiated from induced pluripotent stem cells (iPSCs) from two individuals with HD (HD71 and HD180). One-time AAV5-miHTT treatment significantly reduced human HTT mRNA by 57% and Htt protein by 68% in neurons. Small RNA sequencing showed that mature miHTT was processed correctly without off-target passenger strand. No cellular microRNAs were dysregulated, indicating that endogenous RNAi machinery was unaffected by miHTT overexpression. qPCR validation of in silico -predicted off-target transcripts, next-generation sequencing, and pathway analysis confirmed absence of dysregulated genes due to sequence homology or seed-sequence activity of miHTT. Minor effects on gene expression were observed in both AAV5-miHTT and AAV5-GFP-treated samples, suggesting that they were due to viral transduction rather than miHTT. This study confirms the efficacy of AAV5-miHTT in HD patient iPSC-derived neuronal cultures and lack of off-target effects in gene expression and regulation in neuronal cells and astrocytes., (© 2019 The Author(s).)

A hexanucleotide GGGGCC expansion in intron 1 of chromosome 9 open reading frame 72 (C9orf72) gene is the most frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The corresponding repeat-containing sense and antisense transcripts cause a gain of toxicity through the accumulation of RNA foci in the nucleus and deposition of dipeptide-repeat (DPR) proteins in the cytoplasm of the affected cells. We have previously reported on the potential of engineered artificial anti-C9orf72-targeting miRNAs (miC) targeting C9orf72 to reduce the gain of toxicity caused by the repeat-containing transcripts. In the current study, we tested the silencing efficacy of adeno-associated virus (AAV)5-miC in human-derived induced pluripotent stem cell (iPSC) neurons and in an ALS mouse model. We demonstrated that AAV5-miC transduces different types of neuronal cells and can reduce the accumulation of repeat-containing C9orf72 transcripts. Additionally, we demonstrated silencing of C9orf72 in both the nucleus and cytoplasm, which has an added value for the treatment of ALS and/or FTD patients. A proof of concept in an ALS mouse model demonstrated the significant reduction in repeat-containing C9orf72 transcripts and RNA foci after treatment. Taken together, these findings support the feasibility of a gene therapy for ALS and FTD based on the reduction in toxicity caused by the repeat-containing C9orf72 transcripts., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Huntington disease (HD) is a fatal neurodegenerative disorder caused by an autosomal dominant CAG repeat expansion in the huntingtin ( HTT ) gene. The translated expanded polyglutamine repeat in the HTT protein is known to cause toxic gain of function. We showed previously that strong HTT lowering prevented neuronal dysfunction in HD rodents and minipigs after single intracranial injection of adeno-associated viral vector serotype 5 expressing a microRNA targeting human HTT (AAV5-miHTT). To evaluate long-term efficacy, AAV5-miHTT was injected into the striatum of knockin Q175 HD mice, and the mice were sacrificed 12 months post-injection. AAV5-miHTT caused a dose-dependent and sustained HTT protein reduction with subsequent suppression of mutant HTT aggregate formation in the striatum and cortex. Functional proof of concept was shown in transgenic R6/2 HD mice. Eight weeks after AAV5-miHTT treatment, a significant improvement in motor coordination on the rotarod was observed. Survival analysis showed that a single AAV5-miHTT treatment resulted in a significant 4-week increase in median survival compared with vehicle-treated R6/2 HD mice. The combination of long-term HTT lowering, reduction in aggregation, prevention of neuronal dysfunction, alleviation of HD-like symptoms, and beneficial survival observed in HD rodents treated with AAV5-miHTT supports the continued development of HTT-lowering gene therapies for HD.

The most common pathogenic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic GGGGCC (G 4 C 2 ) repeat in the chromosome 9 open reading frame 72 (C9orf72) gene. Cellular toxicity due to RNA foci and dipeptide repeat (DPR) proteins produced by the sense and antisense repeat-containing transcripts is thought to underlie the pathogenesis of both diseases. RNA sequencing (RNA-seq) data of C9orf72-ALS patients and controls were analyzed to better understand the sequence conservation of C9orf72 in patients. MicroRNAs were developed in conserved regions to silence C9orf72 (miC), and the feasibility of different silencing approaches was demonstrated in reporter overexpression systems. In addition, we demonstrated the feasibility of a bidirectional targeting approach by expressing two concatenated miC hairpins. The efficacy of miC was confirmed by the reduction of endogenously expressed C9orf72 mRNA, in both nucleus and cytoplasm, and an ∼50% reduction of nuclear RNA foci in (G 4 C 2 ) 44 -expressing cells. Ultimately, two miC candidates were incorporated in adeno-associated virus vector serotype 5 (AAV5), and silencing of C9orf72 was demonstrated in HEK293T cells and induced pluripotent stem cell (iPSC)-derived neurons. These data support the feasibility of microRNA (miRNA)-based and AAV-delivered gene therapy that could alleviate the gain of toxicity seen in ALS and FTD patients., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Previously, we showed strong huntingtin reduction and prevention of neuronal dysfunction in HD rodents using an engineered microRNA targeting human huntingtin, delivered via adeno-associated virus (AAV) serotype 5 vector with a transgene encoding an engineered miRNA against HTT mRNA (AAV5-miHTT). One of the challenges of rodents as a model of neurodegenerative diseases is their relatively small brain, making successful translation to the HD patient difficult. This is particularly relevant for gene therapy approaches, where distribution achieved upon local administration into the parenchyma is likely dependent on brain size and structure. Here, we aimed to demonstrate the translation of huntingtin-lowering gene therapy to a large-animal brain. We investigated the feasibility, efficacy, and tolerability of one-time intracranial administration of AAV5-miHTT in the transgenic HD (tgHD) minipig model. We detected widespread dose-dependent distribution of AAV5-miHTT throughout the tgHD minipig brain that correlated with the engineered microRNA expression. Both human mutant huntingtin mRNA and protein were significantly reduced in all brain regions transduced by AAV5-miHTT. The combination of widespread vector distribution and extensive huntingtin lowering observed with AAV5-miHTT supports the translation of a huntingtin-lowering gene therapy for HD from preclinical studies into the clinic., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Spinocerebellar ataxia type 3 (SCA3) is a progressive neurodegenerative disorder caused by expansion of the polyglutamine repeat in the ataxin-3 protein. Expression of mutant ataxin-3 is known to result in transcriptional dysregulation, which can contribute to the cellular toxicity and neurodegeneration. Since the exact causative mechanisms underlying this process have not been fully elucidated, gene expression analyses in brains of transgenic SCA3 mouse models may provide useful insights., Methods: Here we characterised the MJD84.2 SCA3 mouse model expressing the mutant human ataxin-3 gene using a multi-omics approach on brain and blood. Gene expression changes in brainstem, cerebellum, striatum and cortex were used to study pathological changes in brain, while blood gene expression and metabolites/lipids levels were examined as potential biomarkers for disease., Results: Despite normal motor performance at 17.5 months of age, transcriptional changes in brain tissue of the SCA3 mice were observed. Most transcriptional changes occurred in brainstem and striatum, whilst cerebellum and cortex were only modestly affected. The most significantly altered genes in SCA3 mouse brain were Tmc3, Zfp488, Car2, and Chdh. Based on the transcriptional changes, α-adrenergic and CREB pathways were most consistently altered for combined analysis of the four brain regions. When examining individual brain regions, axon guidance and synaptic transmission pathways were most strongly altered in striatum, whilst brainstem presented with strongest alterations in the pi-3 k cascade and cholesterol biosynthesis pathways. Similar to other neurodegenerative diseases, reduced levels of tryptophan and increased levels of ceramides, di- and triglycerides were observed in SCA3 mouse blood., Conclusions: The observed transcriptional changes in SCA3 mouse brain reveal parallels with previous reported neuropathology in patients, but also shows brain region specific effects as well as involvement of adrenergic signalling and CREB pathway changes in SCA3. Importantly, the transcriptional changes occur prior to onset of motor- and coordination deficits.

The single mutation underlying the fatal neuropathology of Huntington's disease (HD) is a CAG triplet expansion in exon 1 of the huntingtin (HTT) gene, which gives rise to a toxic mutant HTT protein. There have been a number of not yet successful therapeutic advances in the treatment of HD. The current excitement in the HD field is due to the recent development of therapies targeting the culprit of HD either at the DNA or RNA level to reduce the overall mutant HTT protein. In this review, we briefly describe short-term and long-term HTT-lowering strategies targeting HTT transcripts. One of the most advanced HTT-lowering strategies is a microRNA (miRNA)-based gene therapy delivered by a single administration of an adeno-associated viral (AAV) vector to the HD patient. We outline the outcome measures for the miRNA-based HTT-lowering therapy in the context of preclinical evaluation in HD animal and cell models. We highlight the strengths and ongoing queries of the HTT-lowering gene therapy as an HD intervention with a potential disease-modifying effect. This review provides a perspective on the fast-developing HTT-lowering therapies for HD and their translation to the clinic based on existing knowledge in preclinical models., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Huntington's disease (HD) is a progressive autosomal dominant disease, caused by a CAG repeat expansion in the HTT gene, resulting in an expanded polyglutamine stretch at the N-terminal of the huntingtin protein. An important event in HD pathogenesis appears to be the proteolysis of the mutant protein, which forms N-terminal huntingtin fragments. These fragments form insoluble aggregates and are found in nuclei and cytoplasm of affected neurons where they interfere with normal cell functioning. Important cleavage sites are encoded by exon 12 of HTT. A novel approach is Htt protein modification through exon skipping, which has recently been proven effective both in vitro and in vivo. Here we report proof-of-concept of AON 12.1 in vivo using the YAC128 mouse model of HD. Our results support and encourage future longitudinal studies exploring the therapeutic effects of sustained infusions in the YAC128 mouse model., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Antisense oligonucleotides are synthetic single stranded strings of nucleic acids that bind to RNA and thereby alter or reduce expression of the target RNA. They can not only reduce expression of mutant proteins by breakdown of the targeted transcript, but also restore protein expression or modify proteins through interference with pre-mRNA splicing. There has been a recent revival of interest in the use of antisense oligonucleotides to treat several neurodegenerative disorders using different approaches to prevent disease onset or halt disease progression and the first clinical trials for spinal muscular atrophy and amyotrophic lateral sclerosis showing promising results. For these trials, intrathecal delivery is being used but direct infusion into the brain ventricles and several methods of passing the blood brain barrier after peripheral administration are also under investigation., (Copyright © 2015. Published by Elsevier B.V.)

Background: Huntington disease (HD) is an autosomal dominant neurodegenerative disorder, characterized by motor, psychiatric and cognitive symptoms. HD is caused by a CAG repeat expansion in the first exon of the HTT gene, resulting in an expanded polyglutamine tract at the N-terminus of the huntingtin protein. Typical disease onset is around mid-life (adult-onset HD) whereas onset below 21 years is classified as juvenile HD. While much research has been done on the underlying HD disease mechanisms, little is known about regulation and expression levels of huntingtin RNA and protein., Results: In this study we used 15 human post-mortem HD brain samples to investigate the expression of wild-type and mutant huntingtin mRNA and protein. In adult-onset HD brain samples, there was a small but significantly lower expression of mutant huntingtin mRNA compared to wild-type huntingtin mRNA, while wild-type and mutant huntingtin protein expression levels did not differ significantly. Juvenile HD subjects did show a lower expression of mutant huntingtin protein compared to wild-type huntingtin protein. Our results in HD brain and fibroblasts suggest that protein aggregation does not affect levels of huntingtin RNA and protein. Additionally, we did not find any evidence for a reduced expression of huntingtin antisense in fibroblasts derived from a homozygous HD patient., Conclusions: We found small differences in allelic huntingtin mRNA levels in adult-onset HD brain, with significantly lower mutant huntingtin mRNA levels. Wild-type and mutant huntingtin protein were not significantly different in adult-onset HD brain samples. Conversely, in juvenile HD brain samples mutant huntingtin protein levels were lower compared with wild-type huntingtin, showing subtle differences between juvenile HD and adult-onset HD. Since most HD model systems harbor juvenile repeat expansions, our results suggest caution with the interpretation of huntingtin mRNA and protein studies using HD cell and animal models with such long repeats. Furthermore, our huntingtin antisense results in homozygous HD cells do not support reduced huntingtin antisense expression due to an expanded CAG repeat.

Ataxin-3 is a ubiquitously expressed deubiqutinating enzyme with important functions in the proteasomal protein degradation pathway and regulation of transcription. The C-terminus of the ataxin-3 protein contains a polyglutamine (PolyQ) region that, when mutationally expanded to over 52 glutamines, causes the neurodegenerative disease spinocerebellar ataxia 3 (SCA3). In spite of extensive research, the molecular mechanisms underlying the cellular toxicity resulting from mutant ataxin-3 remain elusive and no preventive treatment is currently available. It has become clear over the last decade that the hallmark intracellular ataxin-3 aggregates are likely not the main toxic entity in SCA3. Instead, the soluble PolyQ containing fragments arising from proteolytic cleavage of ataxin-3 by caspases and calpains are now regarded to be of greater influence in pathogenesis. In addition, recent evidence suggests potential involvement of a RNA toxicity component in SCA3 and other PolyQ expansion disorders, increasing the pathogenic complexity. Herein, we review the functioning of ataxin-3 and the involvement of known protein and RNA toxicity mechanisms of mutant ataxin-3 that have been discovered, as well as future opportunities for therapeutic intervention.

Huntington's disease (HD) is a progressive autosomal dominant disorder, caused by a CAG repeat expansion in the HTT gene, which results in expansion of a polyglutamine stretch at the N-terminal end of the huntingtin protein. Several studies have implicated the importance of proteolytic cleavage of mutant huntingtin in HD pathogenesis and it is generally accepted that N-terminal huntingtin fragments are more toxic than full-length protein. Important cleavage sites are encoded by exon 12 of HTT. Here we report proof of concept using antisense oligonucleotides to induce skipping of exon 12 in huntingtin pre-mRNA, thereby preventing the formation of a 586 amino acid N-terminal huntingtin fragment implicated in HD toxicity. In vitro studies showed successful exon skipping and appearance of a shorter huntingtin protein. Cleavage assays showed reduced 586 amino acid N-terminal huntingtin fragments in the treated samples. In vivo studies revealed exon skipping after a single injection of antisense oligonucleotides in the mouse striatum. Recent advances to inhibit the formation of mutant huntingtin using oligonucleotides seem promising therapeutic strategies for HD. Nevertheless, huntingtin is an essential protein and total removal has been shown to result in progressive neurodegeneration in vivo. Our proof of concept shows a completely novel approach to reduce mutant huntingtin toxicity not by reducing its expressing levels, but by modifying the huntingtin protein.

Spinocerebellar ataxia type 3 is caused by a polyglutamine expansion in the ataxin-3 protein, resulting in gain of toxic function of the mutant protein. The expanded glutamine stretch in the protein is the result of a CAG triplet repeat expansion in the penultimate exon of the ATXN3 gene. Several gene silencing approaches to reduce mutant ataxin-3 toxicity in this disease aim to lower ataxin-3 protein levels, but since this protein is involved in deubiquitination and proteasomal protein degradation, its long-term silencing might not be desirable. Here, we propose a novel protein modification approach to reduce mutant ataxin-3 toxicity by removing the toxic polyglutamine repeat from the ataxin-3 protein through antisense oligonucleotide-mediated exon skipping while maintaining important wild type functions of the protein. In vitro studies showed that exon skipping did not negatively impact the ubiquitin binding capacity of ataxin-3. Our in vivo studies showed no toxic properties of the novel truncated ataxin-3 protein. These results suggest that exon skipping may be a novel therapeutic approach to reduce polyglutamine-induced toxicity in spinocerebellar ataxia type 3., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Na(v)1.5, the pore forming α-subunit of the voltage-dependent cardiac Na(+) channel, is an integral membrane protein involved in the initiation and conduction of action potentials. Mutations in the gene-encoding Na(v)1.5, SCN5A, have been associated with a variety of arrhythmic disorders, including long QT, Brugada, and sick sinus syndromes as well as progressive cardiac conduction defect and atrial standstill. Moreover, alterations in the Na(v)1.5 expression level and/or sodium current density have been frequently noticed in acquired cardiac disorders, such as heart failure. The molecular mechanisms underlying these alterations are poorly understood, but are considered essential for conception of arrhythmogenesis and the development of therapeutic strategies for prevention or treatment of arrhythmias. The unravelling of such mechanisms requires critical molecular insight into the biology of Na(v)1.5 expression and function. Therefore, the aim of this review is to provide an up-to-date account of molecular determinants of normal Na(v)1.5 expression and function. The parts of the Na(v)1.5 life cycle that are discussed include (i) regulatory aspects of the SCN5A gene and transcript structure, (ii) the nature, molecular determinants, and functional consequences of Na(v)1.5 post-translational modifications, and (iii) the role of Na(v)1.5 interacting proteins in cellular trafficking. The reviewed studies have provided valuable information on how the Na(v)1.5 expression level, localization, and biophysical properties are regulated, but also revealed that our understanding of the underlying mechanisms is still limited.

A limiting factor in brain research still is the difficulty to evaluate in vivo the role of the increasing number of proteins implicated in neuronal processes. We discuss here the potential of antisense-mediated RNA targeting approaches. We mainly focus on those that manipulate splicing (exon skipping and exon inclusion), but will also briefly discuss mRNA targeting. Classic knockdown of expression by mRNA targeting is only one possible application of antisense oligonucleotides (AON) in the control of gene function. Exon skipping and inclusion are based on the interference of AONs with splicing of pre-mRNAs. These are powerful, specific and particularly versatile techniques, which can be used to circumvent pathogenic mutations, shift splice variant expression, knock down proteins, or to create molecular models using in-frame deletions. Pre-mRNA targeting is currently used both as a research tool, e.g., in models for motor neuron disease, and in clinical trials for Duchenne muscular dystrophy and amyotrophic lateral sclerosis. AONs are particularly promising in relation to brain research, as the modified AONs are taken up extremely fast in neurons and glial cells with a long residence, and without the need for viral vectors or other delivery tools, once inside the blood brain barrier. In this review we cover (1). The principles of antisense-mediated techniques, chemistry, and efficacy. (2) The pros and cons of AON approaches in the brain compared to other techniques of interfering with gene function, such as transgenesis and short hairpin RNAs, in terms of specificity of the manipulation, spatial, and temporal control over gene expression, toxicity, and delivery issues. (3) The potential applications for Neuroscience. We conclude that there is good evidence from animal studies that the central nervous system can be successfully targeted, but the potential of the diverse AON-based approaches appears to be under-recognized.

To date there are 9 known diseases caused by an expanded polyglutamine repeat, with the most prevalent being Huntington's disease. Huntington's disease is a progressive autosomal dominant neurodegenerative disorder for which currently no therapy is available. It is caused by a CAG repeat expansion in the HTT gene, which results in an expansion of a glutamine stretch at the N-terminal end of the huntingtin protein. This polyglutamine expansion plays a central role in the disease and results in the accumulation of cytoplasmic and nuclear aggregates. Here, we make use of modified 2'-O-methyl phosphorothioate (CUG)n triplet-repeat antisense oligonucleotides to effectively reduce mutant huntingtin transcript and protein levels in patient-derived Huntington's disease fibroblasts and lymphoblasts. The most effective antisense oligonucleotide, (CUG)(7), also reduced mutant ataxin-1 and ataxin-3 mRNA levels in spinocerebellar ataxia 1 and 3, respectively, and atrophin-1 in dentatorubral-pallidoluysian atrophy patient derived fibroblasts. This antisense oligonucleotide is not only a promising therapeutic tool to reduce mutant huntingtin levels in Huntington's disease but our results in spinocerebellar ataxia and dentatorubral-pallidoluysian atrophy cells suggest that this could also be applicable to other polyglutamine expansion disorders as well.

Objective: To assess the prevalence, diagnosis and treatment of depression among dementia patients and normal controls in chronic care facilities in the last six months of life., Method: We reviewed perimortal data concerning dementia severity, depressive symptoms and diagnoses, and medication use for 279 dementia patients and 24 normal controls brought to autopsy through an Alzheimer's Disease Resource Center., Results: Major depression was highly prevalent among both dementia patients and normal controls in chronic care facilities in the last six months of life. This depression was under-diagnosed by physicians. Documentation of depressive symptoms by medical support staff has improved over time. However, physician diagnosis of depression has not improved. Recognition of depression was significantly lower for patients with severe dementia. Depression was under-treated in both dementia patients and normal controls, although treatment rates may be increasing. Anxiolytics and hypnotics were often used in lieu of, or in addition to, antidepressant therapy., Conclusions: Major depression was highly prevalent in both dementia patients and normal controls, indicating that depression is an important issue for the elderly in the last six months of life irrespective of cognitive status. Under-diagnosis of depression may be an important clinical issue. As physician diagnosis of depression has not improved with time, further physician training and/or awareness initiatives may be warranted. Depression, a treatable cause of excess morbidity and mortality, was undertreated in all groups studied. However, treatment rates may be improving. The prevalent use of anxiolytics and hypnotics for depressed patients is problematic., (Copyright 2002 John Wiley & Sons, Ltd.)

Objectives: The goals of this study were to establish the frequency of palliative and aggressive treatment measures among patients with and without dementia during the last six months of life, to identify relationships between the severity of dementia and aggressive and palliative care, and to determine whether treatment patterns have changed over time., Methods: Antemortem data for 279 patients with dementia and 24 control patients who were brought for autopsy in chronic care facilities between 1985 and 2000 were reviewed. The severity of dementia was defined by scores on the Clinical Dementia Rating scale. Data on use of systemic antibiotics (designated as an aggressive treatment measure) and on use of narcotic and nonnarcotic pain medications and nasal oxygen (defined as palliative measures) were collected from medical charts., Results: Fifty-three percent of the patients with dementia and 46 percent of those without dementia had received systemic antibiotics. Fourteen percent of the patients with dementia and 38 percent of those without dementia had received narcotic pain medications. The prevalence of aggressive and palliative measures did not vary significantly with the severity of dementia. Eleven percent of the patients with dementia who died between 1991 and 1995 and 18 percent of those who died between 1996 and 2000 had received narcotic pain medications in the last six months of their lives., Conclusions: Use of systemic antibiotics is prevalent in the treatment of patients with end-stage dementia, despite the limited utility and discomfort associated with the use of these agents. That patients with severe dementia and those with milder cognitive impairment received similar treatment may be contrary to good clinical practice, given the poor prognosis of patients with severe dementia.