Huntington disease (HD) is a fatal, neurodegenerative genetic disorder with aggregation of mutant Huntingtin protein (mutHTT) in the brain as a key pathological mechanism. There are currently no... Show moreHuntington disease (HD) is a fatal, neurodegenerative genetic disorder with aggregation of mutant Huntingtin protein (mutHTT) in the brain as a key pathological mechanism. There are currently no disease modifying therapies for HD; however, HTT-lowering therapies hold promise. Recombinant adeno-associated virus serotype 5 expressing a microRNA that targets HTT mRNA (AAV5-miHTT) is in development for the treatment of HD with promising results in rodent and minipig HD models. To support a clinical trial, toxicity studies were performed in non-human primates (NHP, Macaca fascicularis) and Sprague-Dawley rats to evaluate the safety of AAV5-miHTT, the neurosurgical administration procedure, vector delivery and expression of the miHTT transgene during a 6-month observation period. For accurate delivery of AAV5-miHTT to the striatum, real-time magnetic resonance imaging (MRI) with convection-enhanced delivery (CED) was used in NHP. Catheters were successfully implanted in 24 NHP, without neurological symptoms, and resulted in tracer signal in the target areas. Widespread vector DNA and miHTT transgene distribution in the brain was found, particularly in areas associated with HD pathology. Intrastriatal administration of AAV5-miHTT was well tolerated with no clinically relevant changes in either species. These studies demonstrate the excellent safety profile of AAV5-miHTT, the reproducibility and tolerability of intrastriatal administration, and the delivery of AAV5-miHTT to the brain, which support the transition of AAV5-miHTT into clinical studies. Show less
The development of gene therapies for central nervous system disorders is challenging because it is difficult to translate preclinical data from current in vitro and in vivo models to the clinic.... Show moreThe development of gene therapies for central nervous system disorders is challenging because it is difficult to translate preclinical data from current in vitro and in vivo models to the clinic. Therefore, we developed induced pluripotent stem cell (iPSC)-derived cerebral organoids as a model for recombinant adeno-associated virus (rAAV) capsid selection and for testing efficacy of AAV-based gene therapy in a human context. Cerebral organoids are physiological 3D structures that better recapitulate the human brain compared with 2D cell lines. To validate the model, we compared the transduction efficiency and distribution of two commonly used AAV serotypes (rAAV5 and rAAV9). In cerebral organoids, transduction with rAAV5 led to higher levels of vector DNA, transgenic mRNA, and protein expression as compared with rAAV9. The superior transduction of rAAV5 was replicated in iPSC-derived neuronal cells. Furthermore, rAAV5-mediated delivery of a human sequence-specific engineered microRNA to cerebral organoids led to a lower expression of its target ataxin-3. Our studies provide a new tool for selecting and deselecting AAV serotypes, and for demonstrating therapeutic efficacy of transgenes in a human context. Implementing cerebral organoids during gene therapy development could reduce the usage of animal models and improve translation to the clinic. Show less
Huntington disease (HD) is a fatal neurodegenerative genetic disorder, thought to reflect a toxic gain of function in huntingtin (Htt) protein. Adeno-associated viral vector serotype 5 (AAV5)-... Show moreHuntington disease (HD) is a fatal neurodegenerative genetic disorder, thought to reflect a toxic gain of function in huntingtin (Htt) protein. Adeno-associated viral vector serotype 5 (AAV5)- microRNA targeting huntingtin (miHTT) is a HD gene-therapy candidate that efficiently lowers HTT using RNAi. This study analyzed the efficacy and potential for offtarget effects with AAV5-miHTT in neuronal and astrocyte cell cultures differentiated from induced pluripotent stem cells (iPSCs) from two individuals with HD (HD71 and HD180). One-time AAV5-miHTT treatment significantly reduced human HTT mRNA by 57% and Htt protein by 68% in neurons. Small RNA sequencing showed that mature miHTT was processed correctly without off-target passenger strand. No cellular microRNAs were dysregulated, indicating that endogenous RNAi machinery was unaffected by miHTT overexpression. qPCR validation of in silico-predicted off-target transcripts, next-generation sequencing, and pathway analysis confirmed absence of dysregulated genes due to sequence homology or seed-sequence activity of miHTT. Minor effects on gene expression were observed in both AAV5-miHTT and AAV5-GFP-treated samples, suggesting that they were due to viral transduction rather than miHTT. This study confirms the efficacy of AAV5-miHTT in HD patient iPSC-derived neuronal cultures and lack of off-target effects in gene expression and regulation in neuronal cells and astrocytes. Show less
Spinocerebellar ataxia type 3 (SCA3) or Machado-Joseph disease (MJD) is a progressiveneurodegenerative disorder caused by a CAG expansion in the ATXN3 gene. The expanded CAGrepeat is translated... Show moreSpinocerebellar ataxia type 3 (SCA3) or Machado-Joseph disease (MJD) is a progressiveneurodegenerative disorder caused by a CAG expansion in the ATXN3 gene. The expanded CAGrepeat is translated into a prolonged polyglutamine repeat in the ataxin-3 protein and accumulateswithin inclusions, acquiring toxic properties, which results in degeneration of the cerebellum and brainstem.In the current study, a non-allele specific ATXN3 silencing approach was investigated using artificialmicroRNAs engineered to target various regions of the ATXN3 gene (miATXN3). The miATXN3candidates were screened in vitro based on their silencing efficacy on a luciferase reporter co-expressing ATXN3. The three best miATXN3 candidates were further tested for target engagement andpotential off-target activity in induced-pluripotent stem cells (iPSC) differentiated into frontal brain-like neurons and in a SCA3 knock-in mouse model. Besides a strong reduction of ATXN3 mRNA andprotein, small RNA sequencing revealed efficient guide strand processing without passenger strandsbeing produced. We used different methods to predict alteration of off-target genes upon AAV5-miATXN3 treatment and found no evidence for unwanted effects. Furthermore, we demonstrated in alarge animal model, the minipig, that intrathecal delivery of AAV5 can transduce the main areasaffected in SCA3 patients. These results proved a strong basis to move forward to investigatedistribution, efficacy and safety of AAV5-miATXN3 in large animals. Show less
Huntington disease (HD) is a fatal neurodegenerative disorder caused by an autosomal dominant CAG repeat expansion in the huntingtin (HTT) gene. The translated expanded polyglutamine repeat in the... Show moreHuntington disease (HD) is a fatal neurodegenerative disorder caused by an autosomal dominant CAG repeat expansion in the huntingtin (HTT) gene. The translated expanded polyglutamine repeat in the HTT protein is known to cause toxic gain of function. We showed previously that strong HTT lowering prevented neuronal dysfunction in HD rodents and minipigs after single intracranial injection of adeno-associated viral vector serotype 5 expressing a microRNA targeting human HTT (AAV5-miHTT). To evaluate long-term efficacy, AAV5-miHTT was injected into the striatum of knockin Q175 HD mice, and the mice were sacrificed 12 months post-injection. AAV5-miHTT caused a dose-dependent and sustained HTT protein reduction with subsequent suppression of mutant HTT aggregate formation in the striatum and cortex. Functional proof of concept was shown in transgenic R6/2 HD mice. Eight weeks after AAV5-miHTT treatment, a significant improvement in motor coordination on the rotarod was observed. Survival analysis showed that a single AAV5-miHTT treatment resulted in a significant 4-week increase in median survival compared with vehicle-treated R6/2 HD mice. The combination of long-term HTT lowering, reduction in aggregation, prevention of neuronal dysfunction, alleviation of HD-like symptoms, and beneficial survival observed in HD rodents treated with AAV5-miHTT supports the continued development of HTT-lowering gene therapies for HD. Show less
Martier, R.; Liefhebber, J.M.; Garcia-Osta, A.; Miniarikova, J.; Cuadrado-Tejedor, M.; Espelosin, M.; ... ; Konstantinova, P. 2019
A hexanucleotide GGGGCC expansion in intron 1 of chromosome 9open reading frame 72 (C9orf72) gene is the most frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia ... Show moreA hexanucleotide GGGGCC expansion in intron 1 of chromosome 9open reading frame 72 (C9orf72) gene is the most frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The corresponding repeat-containing sense and antisense transcripts cause a gain of toxicity through the accumulation of RNA foci in the nucleus and deposition of dipeptide-repeat (DPR) proteins in the cytoplasm of the affected cells. We have previously reported on the potential of engineered artificial anti-C9orf72-targeting miRNAs (miC) targeting C9orf72 to reduce the gain of toxicity caused by the repeat-containing transcripts. In the current study, we tested the silencing efficacy of adeno-associated virus (AAV)5-miC in human-derived induced pluripotent stem cell (iPSC) neurons and in an ALS mouse model. We demonstrated that AAV5-miC transduces different types of neuronal cells and can reduce the accumulation of repeat-containing C9orf72 transcripts. Additionally, we demonstrated silencing of C9orf72 in both the nucleus and cytoplasm, which has an added value for the treatment of ALS and/or FTD patients. A proof of concept in an ALS mouse model demonstrated the significant reduction in repeat-containing C9orf72 transcripts and RNA foci after treatment. Taken together, these findings support the feasibility of a gene therapy for ALS and FTD based on the reduction in toxicity caused by the repeat-containing C9orf72 transcripts. Show less
Martier, R.; Liefhebber, J.M.; Miniarikova, J.; Zon, T. van der; Snapper, J.; Kolder, I.; ... ; Konstantinova, P. 2019
The most common pathogenic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic GGGGCC (G4C2) repeat in the chromosome 9open reading frame 72 (C9orf72) ... Show moreThe most common pathogenic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic GGGGCC (G4C2) repeat in the chromosome 9open reading frame 72 (C9orf72) gene. Cellular toxicity due to RNA foci and dipeptide repeat (DPR) proteins produced by the sense and antisense repeat-containing transcripts is thought to underlie the pathogenesis of both diseases.RNA sequencing (RNA-seq) data of C9orf72-ALS patients and controls were analyzed to better understand the sequence conservation of C9orf72 in patients. MicroRNAs were developed in conserved regions to silence C9orf72 (miC), and the feasibility of different silencing approaches was demonstrated in reporter overexpression systems. In addition, we demonstrated the feasibility of a bidirectional targeting approach by expressing two concatenated miC hairpins. The efficacy of miC was confirmed by the reduction of endogenously expressed C9orf72 mRNA, in both nucleus and cytoplasm, and an ∼50% reduction of nuclear RNA foci in (G4C2)44-expressing cells. Ultimately, two miC candidates were incorporated in adeno-associated virus vector serotype 5 (AAV5), and silencing of C9orf72 was demonstrated in HEK293T cells and induced pluripotent stem cell (iPSC)-derived neurons. These data support the feasibility of microRNA (miRNA)-based and AAV-delivered gene therapy that could alleviate the gain of toxicity seen in ALS and FTD patients. Show less
Toonen, L.J.A.; Overzier, M.; Evers, M.M.; Leon, L.G.; Zeeuw, S.A.J. van der; Mei, H.L.; ... ; Roon-Mom, W.M.C. van 2018
Background: Spinocerebellar ataxia type 3 (SCA3) is a progressive neurodegenerative disorder caused by expansion of the polyglutamine repeat in the ataxin-3 protein. Expression of mutant ataxin-3... Show moreBackground: Spinocerebellar ataxia type 3 (SCA3) is a progressive neurodegenerative disorder caused by expansion of the polyglutamine repeat in the ataxin-3 protein. Expression of mutant ataxin-3 is known to result in transcriptional dysregulation, which can contribute to the cellular toxicity and neurodegeneration. Since the exact causative mechanisms underlying this process have not been fully elucidated, gene expression analyses in brains of transgenic SCA3 mouse models may provide useful insights.Methods: Here we characterised the MJD84.2 SCA3 mouse model expressing the mutant human ataxin-3 gene using a multi-omics approach on brain and blood. Gene expression changes in brainstem, cerebellum, striatum and cortex were used to study pathological changes in brain, while blood gene expression and metabolites/lipids levels were examined as potential biomarkers for disease.Results: Despite normal motor performance at 17.5 months of age, transcriptional changes in brain tissue of the SCA3 mice were observed. Most transcriptional changes occurred in brainstem and striatum, whilst cerebellum and cortex were only modestly affected. The most significantly altered genes in SCA3 mouse brain were Tmc3, Zfp488, Cart, and Chdh. Based on the transcriptional changes, a-adrenergic and CREB pathways were most consistently altered for combined analysis of the four brain regions. When examining individual brain regions, axon guidance and synaptic transmission pathways were most strongly altered in striatum, whilst brainstem presented with strongest alterations in the pi-3 k cascade and cholesterol biosynthesis pathways. Similar to other neurodegenerative diseases, reduced levels of tryptophan and increased levels of ceramides, di- and triglycerides were observed in SCA3 mouse blood.Conclusions: The observed transcriptional changes in SCA3 mouse brain reveal parallels with previous reported neuropathology in patients, but also shows brain region specific effects as well as involvement of adrenergic signalling and CREB pathway changes in SCA3. Importantly, the transcriptional changes occur prior to onset of motor- and coordination deficits. Show less
Toonen, L.J.A.; Overzier, M.; Evers, M.M.; Leon, L.G.; Zeeuw, S.A.J. van der; Mei, H.L.; ... ; Roon-Mom, W.M.C. van 2018
In this thesis various genetic therapies to reduce polyglutamine-induced toxicity are discussed. Although polyglutamine disorders are caused by CAG triplet repeat expansions in different genes,... Show moreIn this thesis various genetic therapies to reduce polyglutamine-induced toxicity are discussed. Although polyglutamine disorders are caused by CAG triplet repeat expansions in different genes, they all result in gain of toxic polyglutamine protein function and subsequently neurodegeneration. The polyglutamine disorders have a monogenic cause and thus far no therapies are available to delay the age of onset or slow the disease progression. These expanded polyglutamine proteins are known to undergo proteolytic processing and this results in polyglutamine-containing protein fragments that are considered to be the main toxic entities in polyglutamine disorders. In this thesis various novel genetic therapies for polyglutamine disorders are proposed. By targeting the polyglutamine-encoding transcripts, translation of mutant polyQ protein is reduced or the polyglutamine protein is modified. This is achieved by targeting the CAG repeat directly (chapter 3), removing the motifs that are implicated in the formation of polyglutamine fragments (chapter 4), or by removal of the CAG repeat-encoding exon (chapter 5). Show less
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... Show moreA 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. Show less
