Exon 1-targeting miRNA reduces the pathogenic exon 1 HTT protein in Huntington disease models.

Huntington disease (HD) is a fatal neurodegenerative disease caused by a trinucleotide repeat expansion in exon 1 of the huntingtin gene (HTT) resulting in toxic gain-of-function and cell death. Despite its monogenic cause, the pathogenesis of HD is highly complex and increasing evidence indicates that, in addition to the full-length (FL) mutant HTT protein, the expanded exon 1 HTT (HTTexon1) protein that is translated from the HTT1a transcript generated by aberrant splicing is prone to aggregate and may contribute to HD pathology. This finding suggests that reducing the expression of HTT1a may achieve a greater therapeutic benefit than targeting only FL mutant HTT. Conversely, strategies that exclusively target FL HTT may not fully prevent the pathogenesis of HD. We have developed an engineered microRNA targeting the HTT exon 1 sequence (miHTT), delivered via adeno-associated virus serotype 5 (AAV5). The target sequence of miHTT is present in both FL HTT and HTT1a transcripts. Preclinical studies with AAV5-miHTT have demonstrated efficacy in several rodent and large animal models by reducing FL HTT mRNA and protein and rescuing HD-like phenotypes, and have been the rationale for phase I/II clinical studies now ongoing in the US and Europe. In the present study, we evaluated the ability of AAV5-miHTT to reduce the levels of aberrantly spliced HTT1a mRNA and the HTTexon1 protein in the brain of two mouse models of HD (heterozygous zQ175 knock-in mice and humanized Hu128/21 mice). Polyadenylated HTT1a mRNA and HTTexon1 protein were detected in the striatum and cortex of heterozygous zQ175 knock-in mice, but not in wild-type, littermate control mice. Intrastriatal administration of AAV5-miHTT resulted in dose-dependent expression of mature miHTT microRNA in cortical brain regions, accompanied by significant lowering of both FL HTT and HTT1a mRNA expression at two months post-injection. Mutant HTT and HTTexon1 protein levels were also significantly reduced in the striatum and cortex of heterozygous zQ175 knock-in at 2 months after AAV5-miHTT treatment and in humanized Hu128/21 mice 7 months post-treatment. The effects were confirmed in primary Hu128/21 neuronal cultures. These results demonstrate that AAV5-miHTT gene therapy is an effective approach to lower both FL HTT and the pathogenic HTTexon1 levels, which could potentially have an additive therapeutic benefit compared to other HTT-targeting modalities.

Allele-specific quantitation of ATXN3 and HTT transcripts in polyQ disease models.

BACKGROUND: The majority of genes in the human genome is present in two copies but the expression levels of both alleles is not equal. Allelic imbalance is an aspect of gene expression relevant not only in the context of genetic variation, but also to understand the pathophysiology of genes implicated in genetic disorders, in particular, dominant genetic diseases where patients possess one normal and one mutant allele. Polyglutamine (polyQ) diseases are caused by the expansion of CAG trinucleotide tracts within specific genes. Spinocerebellar ataxia type 3 (SCA3) and Huntington's disease (HD) patients harbor one normal and one mutant allele that differ in the length of CAG tracts. However, assessing the expression level of individual alleles is challenging due to the presence of abundant CAG repeats in the human transcriptome, which make difficult the design of allele-specific methods, as well as of therapeutic strategies to selectively engage CAG sequences in mutant transcripts. RESULTS: To precisely quantify expression in an allele-specific manner, we used SNP variants that are linked to either normal or CAG expanded alleles of the ataxin-3 (ATXN3) and huntingtin (HTT) genes in selected patient-derived cell lines. We applied a SNP-based quantitative droplet digital PCR (ddPCR) protocol for precise determination of the levels of transcripts in cellular and mouse models. For HD, we showed that the process of cell differentiation can affect the ratio between endogenous alleles of HTT mRNA. Additionally, we reported changes in the absolute number of the ATXN3 and HTT transcripts per cell during neuronal differentiation. We also implemented our assay to reliably monitor, in an allele-specific manner, the silencing efficiency of mRNA-targeting therapeutic approaches for HD. Finally, using the humanized Hu128/21 HD mouse model, we showed that the ratio of normal and mutant HTT transgene expression in brain slightly changes with the age of mice. CONCLUSIONS: Using allele-specific ddPCR assays, we observed differences in allele expression levels in the context of SCA3 and HD. Our allele-selective approach is a reliable and quantitative method to analyze low abundant transcripts and is performed with high accuracy and reproducibility. Therefore, the use of this approach can significantly improve understanding of allele-related mechanisms, e.g., related with mRNA processing that may be affected in polyQ diseases.

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.

Cerebrospinal fluid mutant huntingtin is a biomarker for huntingtin lowering in the striatum of Huntington disease mice.

Huntington disease (HD) is a neurodegenerative disease caused by a trinucleotide repeat expansion in the HTT gene encoding an elongated polyglutamine tract in the huntingtin (HTT) protein. Expanded mutant HTT (mHTT) is toxic and leads to regional atrophy and neuronal cell loss in the brain, which occurs earliest in the striatum. Therapeutic lowering of mHTT in the central nervous system (CNS) has shown promise in preclinical studies, with multiple approaches currently in clinical development for HD. Quantitation of mHTT in the cerebrospinal fluid (CSF) is being used as a clinical pharmacodynamic biomarker of target engagement in the CNS. We have previously shown that the CNS is a major source of mHTT in the CSF. However, little is known about the specific brain regions and cell types that contribute to CSF mHTT. Therefore, a better understanding of the origins of CSF mHTT and whether therapies targeting mHTT in the striatum would be expected to be associated with significant lowering of mHTT in the CSF is needed. Here, we use complementary pharmacological and genetic-based approaches to either restrict expression of mHTT to the striatum or selectively deplete mHTT in the striatum to evaluate the contribution of this brain region to mHTT in the CSF. We show that viral expression of a mHTT fragment restricted to the striatum leads to detectable mHTT in the CSF. We demonstrate that targeted lowering of mHTT selectively in the striatum using an antisense oligonucleotide leads to a significant reduction of mHTT in the CSF of HD mice. Furthermore, using a transgenic mouse model of HD that expresses full length human mHTT and wild type HTT, we show that genetic inactivation of mHTT selectively in the striatum results in a significant reduction of mHTT in the CSF. Taken together, our data supports the conclusion that the striatum contributes sufficiently to the pool of mHTT in the CSF that therapeutic levels of mHTT lowering in the striatum can be detected by this measure in HD mice. This suggests that CSF mHTT may represent a pharmacodynamic biomarker for therapies that lower mHTT in the striatum.

Gene expression profiles complement the analysis of genomic modifiers of the clinical onset of Huntington disease.

Huntington disease (HD) is a neurodegenerative disorder that is caused by a CAG repeat expansion in HTT. The length of this repeat, however, only explains a proportion of the variability in age of onset in patients. Genome-wide association studies have identified modifiers that contribute toward a proportion of the observed variance. By incorporating tissue-specific transcriptomic information with these results, additional modifiers can be identified. We performed a transcriptome-wide association study assessing heritable differences in genetically determined expression in diverse tissues, with genome-wide data from over 4000 patients. Functional validation of prioritized genes was undertaken in isogenic HD stem cells and patient brains. Enrichment analyses were performed with biologically relevant gene sets to identify the core pathways. HD-associated gene coexpression modules were assessed for associations with neurological phenotypes in an independent cohort and to guide drug repurposing analyses. Transcriptomic analyses identified genes that were associated with age of HD onset and displayed colocalization with gene expression signals in brain tissue (FAN1, GPR161, PMS2, SUMF2), with supporting evidence from functional experiments. This included genes involved in DNA repair, as well as novel-candidate modifier genes that have been associated with other neurological conditions. Further, cortical coexpression modules were also associated with cognitive decline and HD-related traits in a longitudinal cohort. In summary, the combination of population-scale gene expression information with HD patient genomic data identified novel modifier genes for the disorder. Further, these analyses expanded the pathways potentially involved in modifying HD onset and prioritized candidate therapeutics for future study.

A Comprehensive Haplotype-Targeting Strategy for Allele-Specific HTT Suppression in Huntington Disease.

Huntington disease (HD) is a fatal neurodegenerative disorder caused by a gain-of-function mutation in HTT. Suppression of mutant HTT has emerged as a leading therapeutic strategy for HD, with allele-selective approaches targeting HTT SNPs now in clinical trials. Haplotypes associated with the HD mutation (A1, A2, A3a) represent panels of allele-specific gene silencing targets for efficient treatment of individuals with HD of Northern European and indigenous South American ancestry. Here we extend comprehensive haplotype analysis of the HD mutation to key populations of Southern European, South Asian, Middle Eastern, and admixed African ancestry. In each of these populations, the HD mutation occurs predominantly on the A2 HTT haplotype. Analysis of HD haplotypes across all affected population groups enables rational selection of candidate target SNPs for development of allele-selective gene silencing therapeutics worldwide. Targeting SNPs on the A1 and A2 haplotypes in parallel is essential to achieve treatment of the most HD-affected subjects in populations where HD is most prevalent. Current allele-specific approaches will leave a majority of individuals with HD untreated in populations where the HD mutation occurs most frequently on the A2 haplotype. We further demonstrate preclinical development of potent and selective ASOs targeting SNPs on the A2 HTT haplotype, representing an allele-specific treatment strategy for these individuals. On the basis of comprehensive haplotype analysis, we show the maximum proportion of HD-affected subjects that may be treated with three or four allele targets in different populations worldwide, informing current allele-specific HTT silencing strategies.

Potent and sustained huntingtin lowering via AAV5 encoding miRNA preserves striatal volume and cognitive function in a humanized mouse model of Huntington disease.

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.

Intrinsic mutant HTT-mediated defects in oligodendroglia cause myelination deficits and behavioral abnormalities in Huntington disease.

White matter abnormalities are a nearly universal pathological feature of neurodegenerative disorders including Huntington disease (HD). A long-held assumption is that this white matter pathology is simply a secondary outcome of the progressive neuronal loss that manifests with advancing disease. Using a mouse model of HD, here we show that white matter and myelination abnormalities are an early disease feature appearing before the manifestation of any behavioral abnormalities or neuronal loss. We further show that selective inactivation of mutant huntingtin (mHTT) in the NG2+ oligodendrocyte progenitor cell population prevented myelin abnormalities and certain behavioral deficits in HD mice. Strikingly, the improvements in behavioral outcomes were seen despite the continued expression of mHTT in nonoligodendroglial cells including neurons, astrocytes, and microglia. Using RNA-seq and ChIP-seq analyses, we implicate a pathogenic mechanism that involves enhancement of polycomb repressive complex 2 (PRC2) activity by mHTT in the intrinsic oligodendroglial dysfunction and myelination deficits observed in HD. Our findings challenge the long-held dogma regarding the etiology of white matter pathology in HD and highlight the contribution of epigenetic mechanisms to the observed intrinsic oligodendroglial dysfunction. Our results further suggest that ameliorating white matter pathology and oligodendroglial dysfunction may be beneficial for HD.

Rescue of aberrant huntingtin palmitoylation ameliorates mutant huntingtin-induced toxicity.

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG expansion in the HTT gene that codes for an elongated polyglutamine tract in the huntingtin (HTT) protein. HTT is subject to multiple post-translational modifications (PTMs) that regulate its cellular function. Mutating specific PTM sites within mutant HTT (mHTT) in HD mouse models can modulate disease phenotypes, highlighting the key role of HTT PTMs in the pathogenesis of HD. These findings have led to increased interest in developing small molecules to modulate HTT PTMs in order to decrease mHTT toxicity. However, the therapeutic efficacy of pharmacological modulation of HTT PTMs in preclinical HD models remains largely unknown. HTT is palmitoylated at cysteine 214 by the huntingtin-interacting protein 14 (HIP14 or ZDHHC17) and 14-like (HIP14L or ZDHHC13) acyltransferases. Here, we assessed if HTT palmitoylation should be regarded as a therapeutic target to treat HD by (1) investigating palmitoylation dysregulation in rodent and human HD model systems, (2) measuring the impact of mHTT-lowering therapy on brain palmitoylation, and (3) evaluating if HTT palmitoylation can be pharmacologically modulated. We show that palmitoylation of mHTT and some HIP14/HIP14L-substrates is decreased early in multiple HD mouse models, and that mHTT palmitoylation decreases further with aging. Lowering mHTT in the brain of YAC128 mice is not sufficient to rescue aberrant palmitoylation. However, we demonstrate that mHTT palmitoylation can be normalized in COS-7 cells, in YAC128 cortico-striatal primary neurons and HD patient-derived lymphoblasts using an acyl-protein thioesterase (APT) inhibitor. Moreover, we show that modulating palmitoylation reduces mHTT aggregation and mHTT-induced cytotoxicity in COS-7 cells and YAC128 neurons.

Live cell imaging and biophotonic methods reveal two types of mutant huntingtin inclusions.

Huntington's disease (HD) is an autosomal dominant, neurodegenerative disorder that can be characterized by the presence of protein inclusions containing mutant huntingtin within a subset of neurons in the brain. Since their discovery, the relevance of inclusions to disease pathology has been controversial. We show using super-resolution fluorescence imaging and Förster resonance energy transfer (FRET) in live cells, that mutant huntingtin fragments can form two morphologically and conformationally distinct inclusion types. Using fluorescence recovery after photobleaching (FRAP), we demonstrate that the two huntingtin inclusion types have unique dynamic properties. The ability to form one or the other type of inclusion can be influenced by the phosphorylation state of serine residues at amino acid positions 13 and 16 within the huntingtin protein. We can define two types of inclusions: fibrillar, which are tightly packed, do not exchange protein with the soluble phase, and result from phospho-modification at serines 13 and 16 of the N17 domain, and globular, which are loosely packed, can readily exchange with the soluble phase, and are not phosphorylated in N17. We hypothesize that the protective effect of N17 phosphorylation or phospho-mimicry seen in animal models, at the level of protein inclusions with elevated huntingtin levels, is to induce a conformation of the huntingtin amino-terminus that causes fragments to form tightly packed inclusions that do not exit the insoluble phase, and hence exert less toxicity. The identification of these sub-types of huntingtin inclusions could allow for drug discovery to promote protective inclusions of mutant huntingtin protein in HD.

Huntingtin Haplotypes Provide Prioritized Target Panels for Allele-specific Silencing in Huntington Disease Patients of European Ancestry.

Huntington disease (HD) is a dominant neurodegenerative disorder caused by a CAG repeat expansion in the Huntingtin gene (HTT). Heterozygous polymorphisms in cis with the mutation allow for allele-specific suppression of the pathogenic HTT transcript as a therapeutic strategy. To prioritize target selection, precise heterozygosity estimates are needed across diverse HD patient populations. Here we present the first comprehensive investigation of all common target alleles across the HTT gene, using 738 reference haplotypes from the 1000 Genomes Project and 2364 haplotypes from HD patients and relatives in Canada, Sweden, France, and Italy. The most common HD haplotypes (A1, A2, and A3a) define mutually exclusive sets of polymorphisms for allele-specific therapy in the greatest number of patients. Across all four populations, a maximum of 80% are treatable using these three target haplotypes. We identify a novel deletion found exclusively on the A1 haplotype, enabling potent and selective silencing of mutant HTT in approximately 40% of the patients. Antisense oligonucleotides complementary to the deletion reduce mutant A1 HTT mRNA by 78% in patient cells while sparing wild-type HTT expression. By suppressing specific haplotypes on which expanded CAG occurs, we demonstrate a rational approach to the development of allele-specific therapy for a monogenic disorder.

Challenges and advances for huntingtin detection in cerebrospinal fluid: in support of relative quantification.

Huntington disease (HD) is a progressive and devastating neurodegenerative disease caused by expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene above a critical threshold of ~35 repeats resulting in expression of mutant HTT (mHTT). A promising treatment approach being tested in clinical trials is HTT lowering, which aims to reduce levels of the mHTT protein. Target engagement of these therapies in the brain are inferred using antibody-based assays to measure mHTT levels in the cerebrospinal fluid (CSF), which is frequently reported as absolute mHTT concentration based on a monomeric protein standard used to generate a standard curve. However, patient biofluids are a complex milieu of different mHTT protein species, suggesting that absolute quantitation is challenging, and a single, recombinant protein standard may not be sufficient to interpret assay signal as molar mHTT concentration. In this study, we used immunoprecipitation and flow cytometry (IP-FCM) to investigate different factors that influence mHTT detection assay signal. Our results show that HTT protein fragmentation, protein-protein interactions, affinity tag positioning, oligomerization and polyglutamine tract length affect assay signal intensity, indicating that absolute HTT quantitation in heterogeneous biological samples is not possible with current technologies using a single standard protein. We also explore the binding specificity of the MW1 anti-polyglutamine antibody, commonly used in these assays as a mHTT-selective reagent and demonstrate that mHTT binding is preferred but not specific. Furthermore, we find that MW1 depletion is not only incomplete, leaving residual mHTT, but also non-specific, resulting in pull down of some wildtype HTT protein. Based on these observations, we recommend that mHTT detection assays report only relative mHTT quantitation using normalized arbitrary units of assay signal intensity, rather than molar concentrations, in the assessment of central nervous system HTT lowering in ongoing clinical and preclinical studies, and that MW1-depletion not be used a method for quantifying wildtype HTT protein.

Elevated plasma and CSF neurofilament light chain concentrations are stabilized in response to mutant huntingtin lowering in the brains of Huntington's disease mice.

BACKGROUND: Therapeutic approaches aimed at lowering toxic mutant huntingtin (mHTT) levels in the brain can reverse disease phenotypes in animal models of Huntington's disease (HD) and are currently being evaluated in clinical trials. Sensitive and dynamic response biomarkers are needed to assess the efficacy of such candidate therapies. Neurofilament light chain (NfL) is a biomarker of neurodegeneration that increases in cerebrospinal fluid (CSF) and blood with progression of HD. However, it remains unknown whether NfL in biofluids could serve as a response biomarker for assessing the efficacy of disease-modifying therapies for HD. METHODS: Longitudinal plasma and cross-sectional CSF samples were collected from the YAC128 transgenic mouse model of HD and wild-type (WT) littermate control mice throughout the natural history of disease. Additionally, biofluids were collected from YAC128 mice following intracerebroventricular administration of an antisense oligonucleotide (ASO) targeting the mutant HTT transgene (HTT ASO), at ages both before and after the onset of disease phenotypes. NfL concentrations in plasma and CSF were quantified using ultrasensitive single-molecule array technology. RESULTS: Plasma and CSF NfL concentrations were significantly elevated in YAC128 compared to WT littermate control mice from 9 months of age. Treatment of YAC128 mice with either 15 or 50 µg HTT ASO resulted in a dose-dependent, allele-selective reduction of mHTT throughout the brain at a 3-month interval, which was sustained with high-dose HTT ASO treatment for up to 6 months. Lowering of brain mHTT prior to the onset of regional brain atrophy and HD-like motor deficits in this model had minimal effect on plasma NfL at either dose, but led to a dose-dependent reduction of CSF NfL. In contrast, initiating mHTT lowering in the brain after the onset of neuropathological and behavioural phenotypes in YAC128 mice resulted in a dose-dependent stabilization of NfL increases in both plasma and CSF. CONCLUSIONS: Our data provide evidence that the response of NfL in biofluids is influenced by the magnitude of mHTT lowering in the brain and the timing of intervention, suggesting that NfL may serve as a promising exploratory response biomarker for HD.

Systemic delivery of mutant huntingtin lowering antisense oligonucleotides to the brain using apolipoprotein A-I nanodisks for Huntington disease.

Efficient delivery of therapeutics to the central nervous system (CNS) remains a major challenge for the treatment of neurological diseases. Huntington disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion mutation in the HTT gene which codes for a toxic mutant huntingtin (mHTT) protein. Pharmacological reduction of mHTT in the CNS using antisense oligonucleotides (ASO) ameliorates HD-like phenotypes in rodent models of HD, with such therapies being investigated in clinical trials for HD. In this study, we report the optimization of apolipoprotein A-I nanodisks (apoA-I NDs) as vehicles for delivery of a HTT-targeted ASO (HTT ASO) to the brain and peripheral organs for HD. We demonstrate that apoA-I wild type (WT) and the apoA-I K133C mutant incubated with a synthetic lipid, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, can self-assemble into monodisperse discoidal particles with diameters <20 nm that transmigrate across an in vitro blood-brain barrier model of HD. We demonstrate that apoA-I NDs are well tolerated in vivo, and that apoA-I K133C NDs show enhanced distribution to the CNS and peripheral organs compared to apoA-I WT NDs following systemic administration. ApoA-I K133C conjugated with HTT ASO forms NDs (HTT ASO NDs) that induce significant mHTT lowering in the liver, skeletal muscle and heart as well as in the brain when delivered intravenously in the BACHD mouse model of HD. Furthermore, HTT ASO NDs increase the magnitude of mHTT lowering in the striatum and cortex compared to HTT ASO alone following intracerebroventricular administration. These findings demonstrate the potential utility of apoA-I NDs as biocompatible vehicles for enhancing delivery of mutant HTT lowering ASOs to the CNS and peripheral organs for HD.

Limitations of Dual-Single Guide RNA CRISPR Strategies for the Treatment of Central Nervous System Genetic Disorders.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a toxic gain-of-function CAG expansion in the first exon of the huntingtin (HTT) gene. The monogenic nature of HD makes mutant HTT (mHTT) inactivation a promising therapeutic strategy. Single nucleotide polymorphisms frequently associated with CAG expansion have been explored to selectively inactivate mHTT allele using the CRISPR/Cas9 system. One of such allele-selective approaches consists of excising a region flanking the first exon of mHTT by inducing simultaneous double-strand breaks at upstream and downstream positions of the mHTT exon 1. The removal of the first exon of mHTT deletes the CAG expansion and important transcription regulatory sites, leading to mHTT inactivation. However, the frequency of deletion events is yet to be quantified either in vitro or in vivo. Here, we developed accurate quantitative digital polymerase chain reaction-based assays to assess HTT exon 1 deletion in vitro and in fully humanized HU97/18 mice. Our results demonstrate that dual-single guide RNA (sgRNA) strategies are efficient and that 67% of HTT editing events are leading to exon 1 deletion in HEK293T cells. In contrast, these sgRNA actively cleaved HTT in HU97/18 mice, but most editing events do not lead to exon 1 deletion (10% exon 1 deletion). We also showed that the in vivo editing pattern is not affected by CAG expansion but may potentially be due to the presence of multiple copies of wildtype (wt)/mHTT genes HU97/18 mice as well as the slow kinetics of AAV-mediated CRISPR/Cas9 delivery.

Delivery of mutant huntingtin-lowering antisense oligonucleotides to the brain by intranasally administered apolipoprotein A-I nanodisks.

Lowering mutant huntingtin (mHTT) in the central nervous system (CNS) using antisense oligonucleotides (ASOs) is a promising approach currently being evaluated in clinical trials for Huntington disease (HD). However, the therapeutic potential of ASOs in HD patients is limited by their inability to cross the blood-brain barrier (BBB). In non-human primates, intrathecal infusion of ASOs results in limited brain distribution, with higher ASO concentrations in superficial regions and lower concentrations in deeper regions, such as the basal ganglia. To address the need for improved delivery of ASOs to the brain, we are evaluating the therapeutic potential of apolipoprotein A-I nanodisks (apoA-I NDs) as novel delivery vehicles for mHTT-lowering ASOs to the CNS after intranasal administration. Here, we have demonstrated the ability of apoA-I nanodisks to bypass the BBB after intranasal delivery in the BACHD model of HD. Following intranasal administration of apoA-I NDs, apoA-I protein levels were elevated along the rostral-caudal brain axis, with highest levels in the most rostral brain regions including the olfactory bulb and frontal cortex. Double-label immunohistochemistry indicates that both the apoA-I and ASO deposit in neurons. Most importantly, a single intranasal dose of apoA-I ASO-NDs significantly reduces mHTT levels in the brain regions most affected in HD, namely the cortex and striatum. This approach represents a novel non-invasive means for improving delivery and brain distribution of oligonucleotide therapies and enhancing likelihood of efficacy. Improved ASO delivery to the brain has widespread application for treatment of many other CNS disorders.

Cerebrospinal fluid biomarkers for assessing Huntington disease onset and severity.

The identification of molecular biomarkers in CSF from individuals affected by Huntington disease may help improve predictions of disease onset, better define disease progression and could facilitate the evaluation of potential therapies. The primary objective of our study was to investigate novel CSF protein candidates and replicate previously reported protein biomarker changes in CSF from Huntington disease mutation carriers and healthy controls. Our secondary objective was to compare the discriminatory potential of individual protein analytes and combinations of CSF protein markers for stratifying individuals based on the severity of Huntington disease. We conducted a hypothesis-driven analysis of 26 pre-specified protein analytes in CSF from 16 manifest Huntington disease subjects, eight premanifest Huntington disease mutation carriers and eight healthy control individuals using parallel-reaction monitoring mass spectrometry. In addition to reproducing reported changes in previously investigated CSF biomarkers (NEFL, PDYN, and PENK), we also identified novel exploratory CSF proteins (C1QB, CNR1, GNAL, IDO1, IGF2, and PPP1R1B) whose levels were altered in Huntington disease mutation carriers and/or across stages of disease. Moreover, we report strong associations of select CSF proteins with clinical measures of disease severity in manifest Huntington disease subjects (C1QB, CNR1, NEFL, PDYN, PPP1R1B, and TTR) and with years to predicted disease onset in premanifest Huntington disease mutation carriers (ALB, C4B, CTSD, IGHG1, and TTR). Using receiver operating characteristic curve analysis, we identified PENK as being the most discriminant CSF protein for stratifying Huntington disease mutation carriers from controls. We also identified exploratory multi-marker CSF protein panels that improved discrimination of premanifest Huntington disease mutation carriers from controls (PENK, ALB and NEFL), early/mid-stage Huntington disease from premanifest mutation carriers (PPP1R1B, TTR, CHI3L1, and CTSD), and late-stage from early/mid-stage Huntington disease (CNR1, PPP1R1B, BDNF, APOE, and IGHG1) compared with individual CSF proteins. In this study, we demonstrate that combinations of CSF proteins can outperform individual markers for stratifying individuals based on Huntington disease mutation status and disease severity. Moreover, we define exploratory multi-marker CSF protein panels that, if validated, may be used to improve the accuracy of disease-onset predictions, complement existing clinical and imaging biomarkers for monitoring the severity of Huntington disease, and potentially for assessing therapeutic response in clinical trials. Additional studies with CSF collected from larger cohorts of Huntington disease mutation carriers are needed to replicate these exploratory findings.

Functional characterization of variants of unknown significance in a spinocerebellar ataxia patient using an unsupervised machine learning pipeline.

CAG-expanded ATXN7 has been previously defined in the pathogenesis of spinocerebellar ataxia type 7 (SCA7), a polyglutamine expansion autosomal dominant cerebellar ataxia. Pathology in SCA7 occurs as a result of a CAG triplet repeat expansion in excess of 37 in the first exon of ATXN7, which encodes ataxin-7. SCA7 presents clinically with spinocerebellar ataxia and cone-rod dystrophy. Here, we present a novel spinocerebellar ataxia variant occurring in a patient with mutations in both ATXN7 and TOP1MT, which encodes mitochondrial topoisomerase I (top1mt). Using machine-guided, unbiased microscopy image analysis, we demonstrate alterations in ataxin-7 subcellular localization, and through high-fidelity measurements of cellular respiration, bioenergetic defects in association with top1mt mutations. We identify ataxin-7 Q35P and top1mt R111W as deleterious mutations, potentially contributing to disease states. We recapitulate our mutations through Drosophila genetic models. Our work provides important insight into the cellular biology of ataxin-7 and top1mt and offers insight into the pathogenesis of spinocerebellar ataxia applicable to multiple subtypes of the illness. Moreover, our study demonstrates an effective pipeline for the characterization of previously unreported genetic variants at the level of cell biology.

Reliable Resolution of Full-Length Huntingtin Alleles by Quantitative Immunoblotting.

BACKGROUND: Therapeutics that lower mutant huntingtin (mHTT) have shown promise in preclinical studies and are in clinical development for the treatment of Huntington disease (HD). Multiple assays have been developed that either quantify mHTT or total HTT but may not accurately measure levels of wild type HTT (wtHTT) in biological samples. OBJECTIVE: To optimize a method that can be used to resolve, quantify and directly compare levels of full length wtHTT and mHTT in HD samples. METHODS: We provide a detailed quantitative immunoblotting protocol to reproducibly resolve full length wtHTT and mHTT in multiple HD mouse and patient samples. RESULTS: We show that this assay can be modified, depending on the sample, to resolve wtHTT and mHTT with a wide range of polyglutamine length differences (ΔQs 22-179). We also demonstrate that this method can be used to quantify allele-selective lowering of mHTT using an antisense oligonucleotide in HD patient-derived cells. CONCLUSION: This quantitative immunoblotting method can be used to reliably resolve full-length HTT alleles with ΔQs≥22 and allows for direct comparison of wtHTT and mHTT levels in HD samples.

The Interaction of Aging and Cellular Stress Contributes to Pathogenesis in Mouse and Human Huntington Disease Neurons.

Huntington disease (HD) is a fatal, inherited neurodegenerative disorder caused by a mutation in the huntingtin (HTT) gene. While mutant HTT is present ubiquitously throughout life, HD onset typically occurs in mid-life. Oxidative damage accumulates in the aging brain and is a feature of HD. We sought to interrogate the roles and interaction of age and oxidative stress in HD using primary Hu97/18 mouse neurons, neurons differentiated from HD patient induced pluripotent stem cells (iPSCs), and the brains of HD mice. We find that primary neurons must be matured in culture for canonical stress responses to occur. Furthermore, when aging is accelerated in mature HD neurons, mutant HTT accumulates and sensitivity to oxidative stress is selectively enhanced. Furthermore, we observe HD-specific phenotypes in neurons and mouse brains that have undergone accelerated aging, including a selective increase in DNA damage. These findings suggest a role for aging in HD pathogenesis and an interaction between the biological age of HD neurons and sensitivity to exogenous stress.