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Huntington disease is caused by a CAG repeat expansion in exon 1 of the huntingtin gene (HTT) that is translated into a polyglutamine stretch in the huntingtin protein (HTT). We previously showed that HTT mRNA carrying an expanded CAG repeat was incompletely spliced to generate HTT1a, an exon 1 only transcript, which was translated to produce the highly aggregation-prone and pathogenic exon 1 HTT protein. This occurred in all knock-in mouse models of Huntington’s disease and could be detected in patient cell lines and post-mortem brains. To extend these findings to a model system expressing human HTT, we took advantage of YAC128 mice that are transgenic for a yeast artificial chromosome carrying human HTT with an expanded CAG repeat. We discovered that the HTT1a transcript could be detected throughout the brains of YAC128 mice. We implemented RNAscope to visualize HTT transcripts at the single molecule level and found that full-length HTT and HTT1a were retained together in large nuclear RNA clusters, as well as being present as single transcripts in the cytoplasm. Homogeneous time-resolved fluorescence analysis demonstrated that the HTT1a transcript had been translated to produce the exon 1 HTT protein. The levels of exon 1 HTT in YAC128 mice, correlated with HTT aggregation, supportive of the hypothesis that exon 1 HTT initiates the aggregation process. Huntingtin-lowering strategies are a major focus of therapeutic development for Huntington’s disease. These approaches often target full-length HTT alone and would not be expected to reduce pathogenic exon 1 HTT levels. We have established YAC128 mouse embryonic fibroblast lines and shown that, together with our QuantiGene multiplex assay, these provide an effective screening tool for agents that target HTT transcripts. The effects of current targeting strategies on nuclear RNA clusters are unknown, structures that may have a pathogenic role or alternatively could be protective by retaining HTT1a in the nucleus and preventing it from being translated. In light of recently halted antisense oligonucleotide trials, it is vital that agents targeting HTT1a are developed, and that the effects of HTT-lowering strategies on the subcellular levels of all HTT transcripts and their various HTT protein isoforms are understood.

Antisense oligonucleotides (ASOs) dosed into cerebrospinal fluid (CSF) distribute broadly throughout the central nervous system (CNS). By modulating RNA, they hold the promise of targeting root molecular causes of disease and hold potential to treat myriad CNS disorders. Realization of this potential requires that ASOs must be active in the disease-relevant cells, and ideally, that monitorable biomarkers also reflect ASO activity in these cells. The biodistribution and activity of such centrally delivered ASOs have been deeply characterized in rodent and non-human primate (NHP) models, but usually only in bulk tissue, limiting our understanding of the distribution of ASO activity across individual cells and across diverse CNS cell types. Moreover, in human clinical trials, target engagement is usually monitorable only in a single compartment, CSF. We sought a deeper understanding of how individual cells and cell types contribute to bulk tissue signal in the CNS, and how these are linked to CSF biomarker outcomes. We employed single nucleus transcriptomics on tissue from mice treated with RNase H1 ASOs against Prnp and Malat1 and NHPs treated with an ASO against PRNP. Pharmacologic activity was observed in every cell type, though sometimes with substantial differences in magnitude. Single cell RNA count distributions implied target RNA suppression in every single sequenced cell, rather than intense knockdown in only some cells. Duration of action up to 12 weeks post-dose differed across cell types, being shorter in microglia than in neurons. Suppression in neurons was generally similar to, or more robust than, the bulk tissue. In macaques, PrP in CSF was lowered 40% in conjunction with PRNP knockdown across all cell types including neurons, arguing that a CSF biomarker readout is likely to reflect ASO pharmacodynamic effect in disease-relevant cells in a neuronal disorder. Our results provide a reference dataset for ASO activity distribution in the CNS and establish single nucleus sequencing as a method for evaluating cell type specificity of oligonucleotide therapeutics and other modalities.

Prion protein (PrP) concentration controls the kinetics of prion replication and is a genetically and pharmacologically validated therapeutic target for prion disease. In order to evaluate PrP concentration as a pharmacodynamic biomarker and assess its contribution to known prion disease risk factors, we developed and validated a plate-based immunoassay reactive for PrP across 6 species of interest and applicable to brain and cerebrospinal fluid (CSF). PrP concentration varied dramatically across different brain regions in mice, cynomolgus macaques, and humans. PrP expression did not appear to contribute to the known risk factors of age, sex, or common PRNP genetic variants. CSF PrP was lowered in the presence of rare pathogenic PRNP variants, with heterozygous carriers of P102L displaying 55%, and D178N just 31%, of the CSF PrP concentration of mutation-negative controls. In rodents, pharmacologic reduction of brain Prnp RNA was reflected in brain parenchyma PrP and, in turn in CSF PrP, validating CSF as a sampling compartment for the effect of PrP-lowering therapy. Our findings support the use of CSF PrP as a pharmacodynamic biomarker for PrP-lowering drugs and suggest that relative reduction from individual baseline CSF PrP concentration may be an appropriate marker for target engagement.

Prion protein (PrP) concentration controls the kinetics of prion replication and is a genetically and pharmacologically validated therapeutic target for prion disease. In order to evaluate PrP concentration as a pharmacodynamic biomarker and assess its contribution to known prion disease risk factors, we developed and validated a plate-based immunoassay reactive for PrP across six species of interest and applicable to brain and cerebrospinal fluid (CSF). PrP concentration varies dramatically between different brain regions in mice, cynomolgus macaques, and humans. PrP expression does not appear to contribute to the known risk factors of age, sex, or common PRNP genetic variants. CSF PrP is lowered in the presence of rare pathogenic PRNP variants, with heterozygous carriers of P102L displaying 55% and of D178N just 31% the CSF PrP concentration of mutation-negative controls. In rodents, pharmacologic reduction of brain Prnp RNA is reflected in brain parenchyma PrP, and in turn in CSF PrP, validating CSF as a sampling compartment for the effect of PrP-lowering therapy. Our findings support the use of CSF PrP as a pharmacodynamic biomarker for PrP-lowering drugs, and suggest that relative reduction from individual baseline CSF PrP concentration may be an appropriate marker for target engagement.

The genetic basis for most inherited neurodegenerative diseases has been identified, yet there are limited disease modifying therapies for these patients. A new class of drugs - antisense oligonucleotides (ASOs) - are showing promise as a therapeutic platform for treating neurological diseases. ASOs are designed to bind to the RNAs encoded by a target gene, thereby suppressing expression by catalyzing degradation of those RNAs or to elevate expression by correcting faulty RNA splicing. Following delivery by intrathecal injection into the cerebral spinal fluid that surrounds the brain and spinal cord, antisense agents distribute broadly into nervous tissues where they produce long-term effects. The recent approval of nusinersen as a treatment for spinal muscular atrophy validated antisense technology as a platform for neurodegenerative and other neurological diseases. Nusinersen demonstrated that effective treatments for neurodegenerative disease can be identified and that treatment not only slows disease progression but improves some symptoms. Antisense drugs are currently in development for amyotrophic lateral sclerosis, Huntington’s disease, Alzheimer’s disease, Parkinson’s, disease and Angelman syndrome. Several additional drugs are in late stage research for the treatment of spinocerebellar ataxias, sporadic forms of amyotrophic lateral sclerosis, infantile seizure disorders, and neurodevelopmental disorders, as well as multiple programs in drug discovery for the treatment of additional neurological diseases. This review will highlight the advances in antisense technology as potential treatments for neurological diseases.

Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing the field to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal, and non-neuronal cells. Therefore, it is critically important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and robust activity throughout the CNS in neurons, oligodendrocytes, astrocytes, and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.

Expanded trinucleotide repeats cause many human diseases, including Huntington’s disease (HD). Recent studies indicate that somatic instability of these repeats contributes to pathogenesis in several expansion disorders. We find that lowering huntingtin protein (HTT) levels reduces somatic instability of both the Htt and Atxn2 CAG tracts in knockin mouse models, and the HTT CAG tract in human iPSC-derived neurons, revealing an unexpected role for HTT in regulating somatic instability.

SummaryProgressive striatal gene expression changes and epigenetic alterations are a prominent feature of Huntington’s disease (HD), but direct relationships between the huntingtin (HTT) protein and chromatin remain poorly described. Here, using chromatin immunoprecipitation and sequencing (ChIP-seq), we show that HTT reproducibly occupies specific locations in the mouse genome, including thousands of genomic loci that are differentially occupied in striatal tissue from a knock-in mouse model of HD (B6.HttQ111/+) versus wildtype controls. ChIP-seq of histone modifications, generated in parallel, revealed genotype-specific colocalization of HTT with trimethylation of histone 3 lysine 27 (H3K27me3), a repressive chromatin mark. Near genes that are differentially regulated in HD, greater HTT occupancy in HttQ111/+ vs. wildtype mice predicted increased H3K27me3, reduced histone 3 lysine 4 (H3K4me3), a marker of poised and active promoters, and down-regulated gene expression. Altered huntingtin-chromatin interactions may therefore play a direct role in driving transcriptional dysregulation in HD.

Lowering of prion protein (PrP) expression in the brain is a genetically validated therapeutic hypothesis in prion disease. We recently showed that antisense oligonucleotide (ASO)-mediated PrP suppression extends survival and delays disease onset in intracerebrally prion-infected mice in both prophylactic and delayed dosing paradigms. Here, we examine the efficacy of this therapeutic approach across diverse paradigms, varying the dose and dosing regimen, prion strain, treatment timepoint, and examining symptomatic, survival, and biomarker readouts. We recapitulate our previous findings with additional PrP-targeting ASOs, and demonstrate therapeutic benefit against four additional prion strains. We demonstrate that

BackgroundThe intrathecal (IT) dosing route introduces drugs directly into the CSF to bypass the blood-brain barrier and gain direct access to the CNS. We evaluated the use of convective forces acting on the cerebrospinal fluid as a means for increasing rostral delivery of IT dosed radioactive tracer molecules and antisense oligonucleotides (ASO) in the monkey CNS. We also measured the cerebral spinal fluid (CSF) volume in a group of cynomolgus monkeys.MethodsThere are three studies presented, in each of which cynomolgus monkeys were injected into the IT space with radioactive tracer molecules and/or ASO by lumbar puncture in either a low or high volume. The first study used the radioactive tracer 64Cu-DOTA and PET imaging to evaluate the effect of the convective forces. The second study combined the injection of the radioactive tracer 99mTc-DTPA and ASO, then used SPECT imaging and ex vivo tissue analysis of the effects of convective forces to bridge between the tracer and the ASO distributions. The third experiment evaluated the effects of different injection volumes on the distribution of an ASO. In the course of performing these studies we also measured the CSF volume in the subject monkeys by Magnetic Resonance Imaging.ResultsIt was consistently found that larger bolus dose volumes produced greater rostral distribution along the neuraxis. Thoracic percussive treatment also increased rostral distribution of low volume injections. There was little added benefit on distribution by combining the thoracic percussive treatment with the high-volume injection. The CSF volume of the monkeys was found to be 11.9 ± 1.6 cm3.ConclusionsThese results indicate that increasing convective forces after IT injection increases distribution of molecules up the neuraxis. In particular, the use of high IT injection volumes will be useful to increase rostral CNS distribution of therapeutic ASOs for CNS diseases in the clinic.

There is an urgent need for better treatments for chronic pain, which affects more than 1 billion people worldwide. Antisense oligonucleotides (ASOs) have proven successful in treating children with spinal muscular atrophy, a severe infantile neurological disorder, and several ASOs are currently being tested in clinical trials for various neurological disorders. Here, we characterize the pharmacodynamic activity of ASOs in spinal cord and dorsal root ganglia (DRG), key tissues for pain signaling. We demonstrate that activity of ASOs lasts up to 2 months after a single intrathecal bolus dose. Interestingly, comparison of subcutaneous, intracerebroventricular, and intrathecal administration shows that DRGs are targetable by systemic and central delivery of ASOs, while target reduction in the spinal cord is achieved only after direct central delivery. Upon detailed characterization of ASO activity in individual cell populations in DRG, we observe robust target suppression in all neuronal populations, thereby establishing that ASOs are effective in the cell populations involved in pain propagation. Furthermore, we confirm that ASOs are selective and do not modulate basal pain sensation. We also demonstrate that ASOs targeting the sodium channel Nav1.7 induce sustained analgesia up to 4 weeks. Taken together, our findings support the idea that ASOs possess the required pharmacodynamic properties, along with a long duration of action beneficial for treating pain.

Knockout of the memory suppressor gene histone deacetylase 2 (Hdac2) in mice elicits cognitive enhancement, and drugs that block HDAC2 have potential as therapeutics for disorders affecting memory. Currently available HDAC2 catalytic activity inhibitors are not fully isoform specific and have short half-lives. Antisense oligonucleotides (ASOs) are drugs that elicit extremely long-lasting, specific inhibition through base pairing with RNA targets. We utilized an ASO to reduce Hdac2 messenger RNA (mRNA) in mice and determined its longevity, specificity, and mechanism of repression. A single injection of the Hdac2-targeted ASO in the central nervous system produced persistent reduction in HDAC2 protein and Hdac2 mRNA levels for 16 weeks. It enhanced object location memory for 8 weeks. RNA sequencing (RNA-seq) analysis of brain tissues revealed that the repression was specific to Hdac2 relative to related Hdac isoforms, and Hdac2 reduction caused alterations in the expression of genes involved in extracellular signal-regulated kinase (ERK) and memory-associated immune signaling pathways. Hdac2-targeted ASOs also suppress a nonpolyadenylated Hdac2 regulatory RNA and elicit direct transcriptional suppression of the Hdac2 gene through stalling RNA polymerase II. These findings identify transcriptional suppression of the target gene as a novel mechanism of action of ASOs. Keywords: antisense oligonucleotide, Hdac2, memory, autism, Alzheimer's disease, extra-coding RNA, transcription

Antisense oligonucleotides (ASOs) designed to lower prion protein (PrP) expression in the brain through RNase H1-mediated degradation of PrP RNA are in development as prion disease therapeutics. ASOs were previously reported to sequence-independently interact with PrP and inhibit prion accumulation in cell culture, yet in vivo studies using a new generation of ASOs found that only PrP-lowering sequences were effective at extending survival. Cerebrospinal fluid (CSF) PrP has been proposed as a pharmacodynamic biomarker for trials of such ASOs, but is only interpretable if PrP lowering is indeed the relevant mechanism of action in vivo and if measurement of PrP is unconfounded by any PrP&ndash, ASO interaction. Here, we examine the PrP-binding and antiprion properties of ASOs in vitro and in cell culture. Binding parameters determined by isothermal titration calorimetry were similar across all ASOs tested, indicating that ASOs of various chemistries bind full-length recombinant PrP with low- to mid-nanomolar affinity in a sequence-independent manner. Nuclear magnetic resonance, dynamic light scattering, and visual inspection of ASO&ndash, PrP mixtures suggested, however, that this interaction is characterized by the formation of large aggregates, a conclusion further supported by the salt dependence of the affinity measured by isothermal titration calorimetry. Sequence-independent inhibition of prion accumulation in cell culture was observed. The inefficacy of non-PrP-lowering ASOs against prion disease in vivo may be because their apparent activity in vitro is an artifact of aggregation, or because the concentration of ASOs in relevant compartments within the central nervous system (CNS) quickly drops below the effective concentration for sequence-independent antiprion activity after bolus dosing into CSF. Measurements of PrP concentration in human CSF were not impacted by the addition of ASO. These findings support the further development of PrP-lowering ASOs and of CSF PrP as a pharmacodynamic biomarker.

Parkinson’s disease (PD) is a prevalent neurodegenerative disease with no approved disease-modifying therapies. Multiplications, mutations, and single nucleotide polymorphisms in the SNCA gene, encoding alpha-synuclein protein (aSyn), either cause or increase risk for PD. Intracellular accumulations of aSyn are pathological hallmarks of PD. Taken together, reduction of aSyn production may provide a disease-modifying therapy for PD. We show that antisense oligonucleotides (ASOs) reduce production of aSyn in rodent pre-formed fibril (PFF) models of PD. Reduced aSyn production leads to prevention and removal of established aSyn pathology and prevents dopaminergic cell dysfunction. In addition, we address the translational potential of the approach through characterization of human SNCA targeting ASOs that efficiently suppress the human SNCA transcript in vivo. We demonstrate broad activity and distribution of the human SNCA ASOs throughout the non-human primate brain and a corresponding decrease in aSyn cerebral spinal fluid (CSF) levels. Taken together, these data suggest that by inhibiting production of aSyn it may be possible to reverse established pathology and thus supports the development of SNCA ASOs as a potentially disease modifying therapy for PD and related synucleinopathies.SummaryAntisense oligonucleotides designed against SNCA, which are progressing to the clinic, have the potential to be a disease modifying therapeutic for Parkinson’s disease patients.

Repression of the memory suppressor genehistonedeacetylase 2 (Hdac2) in mice elicits cognitive enhancement, and drugs that block HDAC2 catalytic activity are being investigated for treating disorders affecting memory. Currently available compounds that target HDAC2 are not specific to the HDAC2 isoform, and have short half-lives. Antisense oligonucleotides (ASOs) are a class of drugs that base pair with RNA targets and exhibit extremely long-lasting, specific inhibition relative to small molecule drugs. We utilized an ASO to reduceHdac2messenger RNA (mRNA) quantities, and explored its longevity, specificity, and mechanism of repression. A single injection of theHdac2-targeted ASO in the central nervous system diminishedHdac2mRNA levels for at least 4 months in the brain, and knockdown of this factor resulted in significant memory enhancement for 8 weeks in mice. RNA-seq analysis of brain tissues revealed that the ASO repression was specific to theHdac2isoform relative to other classicalHdacgenes, and caused alterations in levels of other memory-associated mRNAs. In cultured neurons, we observed that theHdac2-targeted ASO suppressedHdac2mRNA and anHdac2non-coding regulatory extra-coding RNA (ecRNA). The ASO not only triggered a reduction in mRNA levels, but also elicited direct transcriptional suppression of theHdac2gene through blocking RNA polymerase II elongation. These findings suggest transcriptional suppression of the target gene as a novel mechanism of action of ASOs, and opens up the possibility of using ASOs to achieve lasting gene silencing in the brain without altering the nucleotide sequence of a gene.

Summary Human haploinsufficiency of the transcription factor Tcf4 leads to a rare autism spectrum disorder called Pitt-Hopkins syndrome (PTHS), which is associated with severe language impairment and development delay. Here, we demonstrate that Tcf4 haploinsufficient mice have deficits in social interaction, ultrasonic vocalization, prepulse inhibition, and spatial and associative learning and memory. Despite learning deficits, Tcf4 (+/−) mice have enhanced long-term potentiation in the CA1 area of the hippocampus. In translationally oriented studies, we found that small-molecule HDAC inhibitors normalized hippocampal LTP and memory recall. A comprehensive set of next-generation sequencing experiments of hippocampal mRNA and methylated DNA isolated from Tcf4 -deficient and WT mice before or shortly after experiential learning, with or without administration of vorinostat, identified "memory-associated" genes modulated by HDAC inhibition and dysregulated by Tcf4 haploinsufficiency. Finally, we observed that Hdac2 isoform-selective knockdown was sufficient to rescue memory deficits in Tcf4 (+/−) mice.

Huntington disease (HD) is caused by a cytosine-adenine-guanine trinucleotide repeat expansion in the huntingtin gene, HTT, that results in expression of variant (mutant) huntingtin protein (HTT). Therapeutic strategies that reduce HTT levels are currently being pursued to slow or stop disease progression in people with HD. These approaches are supported by robust preclinical data indicating that reducing variant huntingtin protein is associated with decreased HD pathology. However, the risk-benefit profile of reducing either variant HTT or both variant and wild-type HTT is currently an open question that is being addressed in ongoing clinical trials. This review aims to examine the current data available regarding altered HTT in humans, normal animals, and animal models of HD. Studies indexed in PubMed were searched using the MeSH term Huntington disease or the text words huntington or huntingtin from August 31, 1999, to August 31, 2019, with no language restrictions. Additional studies were included from the reference lists of relevant studies and the authors' personal files. Articles describing at least 1 aspect of HTT reduction were included, prioritizing those published within the last 10 years. In vivo studies were also prioritized, with a focus on studies that examined the consequences of wild-type HTT reduction in adults. In a recently completed phase 1/2a study of RG6042 in 46 adults with early manifest HD, antisense oligonucleotide-mediated partial reduction of HTT was reported to be generally safe and well tolerated over the course of 4-monthly RG6042 doses. In case studies of people with rare genetic variations in huntingtin alleles, the loss of 1 wild-type allele was not associated with HD. People with homozygous cytosine-adenine-guanine expansions developed normally until the onset of HD, although they may have experienced a more aggressive disease course. In mouse models of HD, partial reduction of HTT was beneficial, with improvements in motor, cognitive, and behavioral phenotypes. The partial reduction of wild-type HTT in normal adult rodents and nonhuman primates was generally safe and well tolerated. The body of evidence reviewed in this article indicates a positive risk-benefit profile for the partial reduction of either variant HTT alone or both variant and wild-type HTT. These strategies target the underlying cause of HD and are currently being tested in several investigational clinical trials.

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited ataxia caused by expansion of a translated CAG repeat encoding a glutamine tract in the ataxin-1 (ATXN1) protein. Despite advances in understanding the pathogenesis of SCA1, there are still no therapies to alter its progressive fatal course. RNA-targeting approaches have improved disease symptoms in preclinical rodent models of several neurological diseases. Here, we investigated the therapeutic capability of an antisense oligonucleotide (ASO) targeting mouse Atxn1 in Atxn1154Q/2Q-knockin mice that manifest motor deficits and premature lethality. Following a single ASO treatment at 5 weeks of age, mice demonstrated rescue of these disease-associated phenotypes. RNA-sequencing analysis of genes with expression restored to WT levels in ASO-treated Atxn1154Q/2Q mice was used to demonstrate molecular differences between SCA1 pathogenesis in the cerebellum and disease in the medulla. Finally, select neurochemical abnormalities detected by magnetic resonance spectroscopy in vehicle-treated Atxn1154Q/2Q mice were reversed in the cerebellum and brainstem (a region containing the pons and the medulla) of ASO-treated Atxn1154Q/2Q mice. Together, these findings support the efficacy and therapeutic importance of directly targeting ATXN1 RNA expression as a strategy for treating both motor deficits and lethality in SCA1.

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation in the huntingtin (HTT) protein, resulting in acquisition of toxic functions. Previous studies have shown that lowering mutant HTT has the potential to be broadly beneficial. We previously identified HTT single-nucleotide polymorphisms (SNPs) tightly linked to the HD mutation and developed antisense oligonucleotides (ASOs) targeting HD-SNPs that selectively suppress mutant HTT. We tested allele-specific ASOs in a mouse model of HD. Both early and late treatment reduced cognitive and behavioral impairments in mice. To determine the translational potential of the treatment, we examined the effect of ASO administration on HTT brain expression in nonhuman primates. The treatment induced robust HTT suppression throughout the cortex and limbic system, areas implicated in cognition and psychiatric function. The results suggest that ASOs specifically targeting mutated HTT might have therapeutic effects on HD-mediated cognitive impairments.

Objectives Clinical trials for progressive neurodegenerative disorders such as Alzheimer's Disease and Amyotrophic Lateral Sclerosis have been hindered due to the absence of effective pharmacodynamics markers to assay target engagement. We tested whether measurements of new protein production would be a viable pharmacodynamics tool for RNA‐targeted therapies. Methods Transgenic animal models expressing human proteins implicated in neurodegenerative disorders – microtubule‐associated protein tau (hTau) or superoxide dismutase‐1 (hSOD1) – were treated with antisense oligonucleotides (ASOs) delivered to the central nervous system to target these human mRNA transcripts. Simultaneously, animals were administered 13C6‐leucine via drinking water to measure new protein synthesis after ASO treatment. Measures of new protein synthesis and protein concentration were assayed at designated time points after ASO treatment using targeted proteomics. Results ASO treatment lowered hTau mRNA and protein production (measured by 13C6‐leucine‐labeled hTau protein) earlier than total hTau protein concentration in transgenic mouse cortex. In the CSF of hSOD1 transgenic rats, ASO treatment lowered newly generated hSOD1 protein driven by decreases in newly synthesized hSOD1 protein, not overall protein concentration, 30 days after treatment. At later time points, decreases in newly generated protein were still observed after mRNA lowering reached a steady state after ASO treatment. Interpretation Measures of newly generated protein show earlier pharmacodynamics changes for RNA‐lowering therapeutics compared with total protein concentration. Early in ASO treatment, decreases in newly generated protein are driven by changes in newly synthesized protein. Measuring new protein production in CSF may be a promising early pharmacodynamics marker for RNA‐targeted therapeutics.

OBJECTIVE: Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is the most common dominantly inherited ataxia. Despite advances in understanding this CAG repeat/polyglutamine expansion disease, there are still no therapies to alter its progressive fatal course. Here we investigate whether an antisense oligonucleotide (ASO) targeting the SCA3 disease gene, ATXN3, can prevent molecular, neuropathological, electrophysiological and behavioral features of the disease in a mouse model of SCA3. METHODS: The top ATXN3-targeting ASO from an in vivo screen was injected intracerebroventricularly into early symptomatic transgenic SCA3 mice that express the full human disease gene and recapitulate key disease features. Following a single ASO treatment at 8 weeks of age, mice were evaluated longitudinally for ATXN3 suppression and rescue of disease-associated pathological changes. Mice receiving an additional repeat injection at 21 weeks were evaluated longitudinally up to 29 weeks for motor performance. RESULTS: The ATXN3-targeting ASO achieved sustained reduction of polyglutamine-expanded ATXN3 up to 8 weeks after treatment and prevented oligomeric and nuclear accumulation of ATXN3 up to at least 14 weeks after treatment. Longitudinal ASO therapy rescued motor impairment in SCA3 mice, and this rescue was associated with a recovery of defects in Purkinje neuron firing frequency and afterhyperpolarization. INTERPRETATION: This preclinical study established efficacy of ATXN3-targeted ASOs as a disease-modifying therapeutic strategy for SCA3. These results support further efforts to develop ASOs for human clinical trials in this polyglutamine disease as well as in other dominantly inherited disorders caused by toxic gain of function.

Advances in molecular biology and genetics have been used to elucidate the fundamental genetic mechanisms underlying central nervous system (CNS) diseases, yet disease-modifying therapies are currently unavailable for most CNS conditions. Antisense oligonucleotides (ASOs) are synthetic single stranded chains of nucleic acids that bind to a specific sequence on ribonucleic acid (RNA) and regulate posttranscriptional gene expression. Decreased gene expression with ASOs might be able to reduce production of the disease-causing protein underlying dominantly inherited neurodegenerative disorders. Huntington's disease (HD), which is caused by a CAG repeat expansion in exon 1 of the huntingtin (HTT) gene and leads to the pathogenic expansion of a polyglutamine (PolyQ ) tract in the N terminus of the huntingtin protein (Htt), is a prime candidate for ASO therapy.State-of-the art translational science techniques can be applied to the development of an ASO targeting HTT RNA, allowing for a data-driven, stepwise progression through the drug development process. A deep and wide-ranging understanding of the basic, preclinical, clinical, and epidemiologic components of drug development will improve the likelihood of success. This includes characterizing the natural history of the disease, including evolution of biomarkers indexing the underlying pathology; using predictive preclinical models to assess the putative gain-of-function of mutant Htt protein and any loss-of-function of the wild-type protein; characterizing toxicokinetic and pharmacodynamic effects of ASOs in predictive animal models; developing sensitive and reliable biomarkers to monitor target engagement and effects on pathology that translate from animal models to patients with HD; establishing a drug delivery method that ensures reliable distribution to relevant CNS tissue; and designing clinical trials that move expeditiously from proof of concept to proof of efficacy. This review focuses on the translational science techniques that allow for efficient and informed development of an ASO for the treatment of HD.

Mutations in superoxide dismutase 1 (SOD1) are responsible for 20% of familial ALS. Given the gain of toxic function in this dominantly inherited disease, lowering SOD1 mRNA and protein is predicted to provide therapeutic benefit. An early generation antisense oligonucleotide (ASO) targeting SOD1 was identified and tested in a phase I human clinical trial, based on modest protection in animal models of SOD1 ALS. Although the clinical trial provided encouraging safety data, the drug was not advanced because there was progress in designing other, more potent ASOs for CNS application. We have developed next-generation SOD1 ASOs that more potently reduce SOD1 mRNA and protein and extend survival by more than 50 days in SOD1G93A rats and by almost 40 days in SOD1G93A mice. We demonstrated that the initial loss of compound muscle action potential in SOD1G93A mice is reversed after a single dose of SOD1 ASO. Furthermore, increases in serum phospho-neurofilament heavy chain levels, a promising biomarker for ALS, are stopped by SOD1 ASO therapy. These results define a highly potent, new SOD1 ASO ready for human clinical trial and suggest that at least some components of muscle response can be reversed by therapy.

SummaryThe primary cause of Huntington's disease (HD) is expression of huntingtin with a polyglutamine expansion. Despite an absence of consensus on the mechanism(s) of toxicity, diminishing the synthesis of mutant huntingtin will abate toxicity if delivered to the key affected cells. With antisense oligonucleotides (ASOs) that catalyze RNase H-mediated degradation of huntingtin mRNA, we demonstrate that transient infusion into the cerebrospinal fluid of symptomatic HD mouse models not only delays disease progression but mediates a sustained reversal of disease phenotype that persists longer than the huntingtin knockdown. Reduction of wild-type huntingtin, along with mutant huntingtin, produces the same sustained disease reversal. Similar ASO infusion into nonhuman primates is shown to effectively lower huntingtin in many brain regions targeted by HD pathology. Rather than requiring continuous treatment, our findings establish a therapeutic strategy for sustained HD disease reversal produced by transient ASO-mediated diminution of huntingtin synthesis.

Cross-linking and immunoprecipitation coupled with high-throughput sequencing was used to identify binding sites within 6,304 genes as the brain RNA targets for TDP-43, an RNA binding protein which when mutated causes Amyotrophic Lateral Sclerosis (ALS). Use of massively parallel sequencing and splicing-sensitive junction arrays revealed that levels of 601 mRNAs are changed (including Fus/Tls, progranulin, and other transcripts encoding neurodegenerative disease-associated proteins) and 965 altered splicing events are detected (including in sortilin, the receptor for progranulin), following depletion of TDP-43 from mouse adult brain with antisense oligonucleotides. RNAs whose levels are most depleted by reduction in TDP-43 are derived from genes with very long introns and which encode proteins involved in synaptic activity. Lastly, TDP-43 was found to auto-regulate its synthesis, in part by directly binding and enhancing splicing of an intron within the 3′ untranslated region of its own transcript, thereby triggering nonsense mediated RNA degradation. (147 words)

It has become widely accepted that along with its ability to directly regulate gene expression, estradiol also influences cell signaling and brain function via rapid membrane-initiated events. Many of these novel signaling processes are dependent on estrogen receptors (ERs) localized to the neuronal membrane. However, the mechanism(s) by which ERs are able to trigger cell signaling when targeted to the neuronal membrane surface has yet to be determined. In hippocampal neurons, we find that caveolin proteins are essential for the regulation of CREB (cAMP response element-binding protein) phosphorylation after estradiol activation of metabotropic glutamate receptor (mGluR) signaling. Furthermore, caveolin-1 (CAV1) and CAV3 differentially regulate the ability of estradiol to activate two discrete signaling pathways. ERα activation of mGluR1a is dependent on CAV1, whereas CAV3 is necessary for ERα and ERβ activation of mGluR2/3. These results are consistent with previous reports in non-neuronal cells, implicating the importance of caveolin proteins in rapid estrogen signaling. In addition, the functional isolation of distinct estrogen-sensitive signaling pathways by different caveolin proteins suggests novel mechanisms through which the membrane-initiated effects of estradiol are orchestrated.

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disorder caused by abnormal expansions of a trinucleotide CAG repeat in exon 47 of the CACNA1A gene, which encodes the alpha1A subunit of the P/Q-type voltage-gated calcium channel. The CAG repeat expansion is translated into an elongated polyglutamine tract in the carboxyl terminus of the alpha1A subunit. The alpha1A subunit is the main pore-forming subunit of the P/Q-type calcium channel. Patients with SCA6 suffer from a severe form of progressive ataxia and cerebellar dysfunction. Design of treatments for this disorder will depend on better definition of the mechanism of disease. As a disease arising from a mutation in an ion channel gene, SCA6 may behave as an ion channelopathy, and may respond to attempts to modulate or correct ion channel function. Alternatively, as a disease in which the mutant protein contains an expanded polyglutamine tract, SCA6 may respond to the targets of drug therapies developed for Huntington's disease and other polyglutamine disorders. In this review we will compare SCA6 to other polyglutamine diseases and channelopathies, and we will highlight recent advances in our understanding of alpha1A subunits and SCA6 pathology. We also propose a mechanism for how two seemingly divergent hypotheses can be combined into a cohesive model for disease progression.

Accumulation of hyperphosphorylated tau directly correlates with cognitive decline in Alzheimer’s disease and other primary tauopathies. One therapeutic strategy may be to reduce total tau expression. We identified antisense oligonucleotides (ASOs) that selectively decreased human tau mRNA and protein in mice expressing mutant P301S human tau. Following reduction of human tau in this mouse model of tauopathy, fewer tau inclusions developed and pre-existing phosphorylated tau and thioflavin S pathology was reversed. The resolution of tau pathology was accompanied by the prevention of hippocampal volume loss, neuronal death, and nesting deficits. In addition, mouse survival was extended and pathological tau seeding was reversed. In non-human primates, tau ASOs distributed throughout the brain and spinal cord and reduced tau mRNA and protein in the brain, spinal cord, and cerebrospinal fluid. These data support investigation of a tau lowering therapy in human patients who have tau-positive inclusions, even after pathological tau deposition has begun.

P/Q-type voltage-gated calcium channels are regulated, in part, through the cytoplasmic C-terminus of their alpha1A subunit. Genetic absence or alteration of the C-terminus leads to abnormal channel function and neurological disease. Here, we show that the terminal 60-75 kDa of the endogenous alpha1A C-terminus is cleaved from the full-length protein and is present in cell nuclei. Antiserum to the C-terminus (CT-2) labels both wild-type mouse and human Purkinje cell nuclei, but not leaner mouse cerebellum. Human embryonic kidney cells stably expressing beta3 and alpha2delta subunits and transiently transfected with full-length human alpha1A contain a 75 kDa CT-2 reactive peptide in their nuclear fraction. Primary granule cells transfected with C-terminally Green fluorescent protein (GFP)-tagged alpha1A exhibit GFP nuclear labeling. Nuclear translocation depends partly on the presence of three nuclear localization signals within the C-terminus. The C-terminal fragment bears a polyglutamine tract which, when expanded (Q33) as in spinocerebellar ataxia type 6 (SCA6), is toxic to cells. Moreover, polyglutamine-mediated toxicity is dependent on nuclear localization. Finally, in the absence of flanking sequence, the Q33 expansion alone does not kill cells. These results suggest a novel processing of the P/Q-type calcium channel and a potential mechanism for the pathogenesis of SCA6.

Transactivating response region DNA binding protein (TDP-43) is the major protein component of ubiquitinated inclusions found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitinated inclusions. Two ALS-causing mutants (TDP-43(Q331K) and TDP-43(M337V)), but not wild-type human TDP-43, are shown here to provoke age-dependent, mutant-dependent, progressive motor axon degeneration and motor neuron death when expressed in mice at levels and in a cell type-selective pattern similar to endogenous TDP-43. Mutant TDP-43-dependent degeneration of lower motor neurons occurs without: (i) loss of TDP-43 from the corresponding nuclei, (ii) accumulation of TDP-43 aggregates, and (iii) accumulation of insoluble TDP-43. Computational analysis using splicing-sensitive microarrays demonstrates alterations of endogenous TDP-43-dependent alternative splicing events conferred by both human wild-type and mutant TDP-43(Q331K), but with high levels of mutant TDP-43 preferentially enhancing exon exclusion of some target pre-mRNAs affecting genes involved in neurological transmission and function. Comparison with splicing alterations following TDP-43 depletion demonstrates that TDP-43(Q331K) enhances normal TDP-43 splicing function for some RNA targets but loss-of-function for others. Thus, adult-onset motor neuron disease does not require aggregation or loss of nuclear TDP-43, with ALS-linked mutants producing loss and gain of splicing function of selected RNA targets at an early disease stage.

Mutant huntingtin (HTT) protein causes Huntington’s Disease (HD), an incurable neurological disorder. Silencing mutant HTT using nucleic acids would eliminate the root cause of HD. Developing nucleic acid drugs is challenging, and an ideal clinical approach to gene silencing would combine the simplicity of single-stranded antisense oligonucleotides with the efficiency of RNAi. Here we describe RNAi by single-stranded silencing RNAs (ss-siRNAs). ss-siRNAs are potent (>100-fold more than unmodified RNA) and allele-selective (>30-fold) inhibitors of mutant HTT expression in cells derived from HD patients. Strategic placement of mismatched bases mimics micro-RNA recognition and optimizes discrimination between mutant and wild-type alleles. ss-siRNAs require argonaute protein and function through the RNAi pathway. Intraventricular infusion of ss-siRNA produced selective silencing of the mutant HTT allele throughout the brain in a mouse HD model. These data demonstrate that chemically modified ss-siRNAs function through the RNAi pathway and provide allele-selective compounds for clinical development.

The identification of the genes and proteins involved in many neurodegenerative diseases 1 offers the exciting possibility of modifying those disease-linked proteins to develop novel, targeted therapies for diseases such as amyotrophic lateral sclerosis (ALS) or Huntington disease. In many of these diseases, the simplest modification-decreasing the amount of the offending protein-may represent a potent therapy. This realization, coupled with great strides in the techniques for decreasing specific proteins, has set the stage for moving these new therapies from animal models to clinical trials in the near future.

Episodic ataxia type 2 (EA2) is an autosomal dominant disorder arising from CACNA1A mutations, which commonly predict heterozygous expression of Ca(v)2.1 calcium channels with truncated alpha(1)2.1 pore subunits. We hypothesized that alpha(1)2.1 truncations in EA2 exert dominant-negative effects on the function of wild-type subunits. Wild-type and truncated alpha(1)2.1 subunits with fluorescent protein tags were transiently co-expressed in cells stably expressing Ca(v) auxiliary beta subunits, which facilitate alpha1 subunit functional expression through high-affinity interactions with the alpha interaction domain (AID). Co-expression of wild-type subunits with truncations often resulted in severely reduced whole-cell currents compared to expression of wild-type subunits alone. Cellular image analyses revealed that current suppression was not due to reduced wild-type expression levels. Instead, the current suppression depended on truncations terminating distal to the AID. Moreover, only AID-bearing alpha(1)2.1 proteins co-immunoprecipitated with Ca(v) beta subunits. These results indicate that Ca(v) beta subunits may play a prominent role in EA2 disease pathogenesis.