Melvin M. Evers

Antisense oligonucleotides in therapy for neurodegenerative disorders.

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.

Targeting Several CAG Expansion Diseases by a Single Antisense Oligonucleotide

To date there are 9 known diseases caused by an expanded polyglutamine repeat, with the most prevalent being Huntingtons disease. Huntingtons 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 Huntingtons 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 Huntingtons 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.

Ataxin-3 protein modification as a treatment strategy for spinocerebellar ataxia type 3: removal of the CAG containing exon.

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.

Ataxin-3 protein and RNA toxicity in spinocerebellar ataxia type 3: current insights and emerging therapeutic strategies.

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.

Antisense-mediated RNA targeting: versatile and expedient genetic manipulation in the brain.

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.

In vivo proof-of-concept of removal of the huntingtin caspase cleavage motif-encoding exon 12 approach in the YAC128 mouse model of Huntington's disease

Huntingtons 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.

A07 A comparative study on blood and brain hd signatures: comparing mouse and human hd gene expression data

Background While the genetic cause of Huntington’s Disease (HD) is known since 1993, still no cure exists. Therapeutic development would benefit from a method to monitor disease progression and treatment efficacy, ideally using blood biomarkers. We previously showed that HD specific functional signatures in human blood adequately represent signatures in human brain and hence could be used as biomarkers. Aims Since potential drugs are first screened in rodent models, we aimed to determine whether the previously identified human signatures are also present in the YAC128 HD mouse model. Methods We isolated and sequenced RNA from blood collected at 12 and 20 months and four end stage brain regions from 8 YAC128 mice and 8 wild type mice. Differential gene expression analysis was applied to identify genes differentially expressed (DE) and weighted gene coexpression network analysis to identify groups of genes strongly co-expressed (modules). To technically validate RNAseq results, qPCR and western blot were performed. Results RNAseq data was validated by qPCR and western blot, confirming our gene expression analysis. Early stage blood displayed modest changes related to immune response(7 DE genes; 2 modules). At 20 months, an intermediate pathology was detected in blood (162 DE genes; 22 modules), including additional processes such as autophagy, protein transport and modification and DNA repair. In terms of differential gene expression, cortex and brainstem exhibited a mild phenotype (33 and 60 DE genes respectively), while cerebellum and striatum showed intermediate to moderate changes (145 and 101 DE genes respectively). Cerebellum and striatum showed respectively 26 and 11 modules significantly associated with HD, while this was 16 for cortex and 14 for brainstem. Representative annotations presented by all four brain regions were immune response, DNA repair, protein transport, chromatin modification and myelination. Conclusions Similar modules were present in blood and brain gene expression data from mouse and human related to immune response, protein transport and chromatin remodelling. Our next step is to statistically determine similarities between blood and brain signatures in mouse with a computational randomization experiment.

Ameliorating Huntington's Disease by Targeting Huntingtin mRNA

To date there are 9 known neurological diseases caused by an expanded polyglutamine (polyQ) repeat, with the most prevalent being Huntington’s Disease (HD) (Cummings & Zoghbi, 2000). HD is a progressive autosomal dominant disorder. It is caused by a CAG repeat expansion in the HTT gene, which results in an expansion of a polyQ stretch at the Nterminal end of the huntingtin (htt) protein. This polyQ expansion plays a central role in the disease and results in the accumulation of cytoplasmic and nuclear aggregates. In this chapter we will discuss wild-type htt function and the gain of toxic function of mutant htt in HD. Currently no treatment is available to delay onset or slow disease progression. However, recently developed RNA modulating therapies have great potential to lower mutant htt levels in HD. Already promising results in animal and human studies for other neurodegenerative disorders have been obtained, from which HD research can learn.

P01 Antisense oligonucleotide mediated transcript reduction and modulation—the European approach to develop a therapy for Huntington disease

There has been a recent surge in research using antisense oligonucleotides (AONs) to reduce huntingtin transcript levels and thus huntingtin protein levels both in vitro an in vivo. This can be done in a non-allele specific manner by targeting both mutant and normal huntingtin transcripts but preferred would be an allele specific approach targeting mutant huntingtin transcripts through SNP-specific AONs or by use of triplet-repeat AONs complementary towards the (CAG)n expansion. Studies in our group and others have confirmed the feasibility of this approach. The disadvantage however is that there is a reduction in huntingtin protein levels and lowering huntingtin levels too much will cause unwanted side effects. Fortunately, AONs are a versatile tool that can also be exploited to induce inclusion or exclusion of target exons, thereby modulating the translated protein product. It is known that caspase 6 cleavage at aa 586 gives rise to a toxic N-terminal huntingtin fragment, and mutation of this site in the YAC128 mouse model (C6R-YAC128 by the group of Michael Hayden) provides protection from neuronal dysfunction and neurodegeneration. This (and other) caspase cleavage sites are encoded by exon 12 of the HTT gene. By skipping this exon with AONs, a shorter huntingtin protein will be formed lacking the caspase cleavage sites, without altering overall huntingtin protein levels. We propose a combinatorial AON approach for Huntington disease, making use of the transcript reducing properties of (CUG)7 AONs that preferentially targets mutant huntingtin transcripts, as well as the transcript modifying AONs that remove important caspase cleavage sites from the huntingtin protein.

P03 Reducing toxic N-terminal huntingtin fragments in HD using exon skipping

Background Several studies have implicated the importance of proteolytic cleavage of mutant huntingtin in HD pathogenesis (Thornberry et al 1997). Huntingtin fragments within the striatum of human HD brains clearly differ from those of control brains (Mende-Mueller et al 2001), suggesting cleavage is disease specific. HD cell models with caspase-3 and caspase-6 resistant neuronal and non-neuronal cells showed reduced toxicity and were found to be less prone to aggregate formation (Wellington et al 2000). In YAC128 HD mice, expressing human genomic mutant huntingtin containing 128 glutamines, the HD phenotype was prevented by blocking caspase 6-specific cleavage by mutating the caspase-6 cleavage motif (Graham et al 2006; Pouladi et al 2009). This suggests that caspase-6 cleavage at position 586 is a key player in neuronal dysfunction and neurodegeneration. Aims For our study we make use of 2’O-methyl modified antisense oligonucleotides (AONs) with a phosphorothioate (PS) backbone to induce in-frame exon skipping in huntingtin of the exons in which the proteolytic cleavage motifs are located. Methods/techniques Patient derived fibroblast cells were transfected with AONs, RNA was isolated 1-day after transfection and skipping efficiency was determined. To assess the formation of a shorter, skipped protein, total protein lysates were obtained 3 days after transfection and shown by Western blot. A caspase-6 assay was used to determine the caspase-resistance of the truncated huntingtin protein. Results/outcome Patient derived fibroblast cells transfected with an AON binding to the 3’ part of exon 12, resulted in a partial skip of 135 base pairs. This partial exclusion of the 3’ part of huntingtin exon 12 can be explained by the existence of an cryptic 5’ splice site AG|GTCAG (Zhang et al 1998). The in-frame skip results in a slightly shorter huntingtin protein resistant to proteolytic cleavage at the caspase-6 cleavage site. In-frame multi-exon skipping of exon 12 and 13 from huntingtin pre-mRNA resulted in a shorter huntingtin protein resistant to proteolytic cleavage at the caspase-3 and caspase-6 cleavage sites. Currently we are testing whether skipping of this caspase-6 motif results in a reduction of N-terminal huntingtin fragments and thus reduced toxicity. Conclusions Above described preliminary results suggest a novel therapeutic approach to reduce toxic N-terminal huntingtin fragments using exon skipping while maintaining huntingtin protein levels. Currently we are testing in vivo whether a single bilateral injection of AONs in the mice striatum would result in skipping of the caspase motifs.