Megan S. Keiser

CRISPR/Cas9 Editing of the Mutant Huntingtin Allele In Vitro and In Vivo

Huntington disease (HD) is a fatal dominantly inherited neurodegenerative disorder caused by CAG repeat expansion (>36 repeats) within the first exon of the huntingtin gene. Although mutant huntingtin (mHTT) is ubiquitously expressed, the brain shows robust and early degeneration. Current RNA interference-based approaches for lowering mHTT expression have been efficacious in mouse models, but basal mutant protein levels are still detected. To fully mitigate expression from the mutant allele, we hypothesize that allele-specific genome editing can occur via prevalent promoter-resident SNPs in heterozygosity with the mutant allele. Here, we identified SNPs that either cause or destroy PAM motifs critical for CRISPR-selective editing of one allele versus the other in cells from HD patients and in a transgenic HD model harboring the human allele.

Broad Therapeutic Benefit After RNAi Expression Vector Delivery to Deep Cerebellar Nuclei: Implications for Spinocerebellar Ataxia Type 1 Therapy

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant, late-onset neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the ataxin-1 protein, which causes progressive neurodegeneration in cerebellar Purkinje cells and brainstem nuclei. Here, we tested if reducing mutant ataxin-1 expression would significantly improve phenotypes in a knock-in (KI) mouse model that recapitulates spatial and temporal aspects of SCA1. Adeno-associated viruses (AAVs), expressing inhibitory RNAs targeting ataxin-1, were injected into the deep cerebellar nuclei (DCN) of KI mice. This approach induced ataxin-1 suppression in the cerebellar cortex and in brainstem neurons. RNA interference (RNAi) of ataxin-1 preserved cerebellar lobule integrity and prevented disease-related transcriptional changes for over a year. Notably, RNAi therapy also preserved rotarod performance and neurohistology. These data suggest that delivery of AAVs encoding RNAi sequences against ataxin-1, to DCN alone, may be sufficient for SCA1 therapy.

Altered Purkinje cell miRNA expression and SCA1 pathogenesis

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disorder caused by polyglutamine repeat expansions in Ataxin-1. Recent evidence supports a role for microRNAs (miRNAs) deregulation in SCA1 pathogenesis. However, the extent to which miRNAs may modulate the onset, progression or severity of SCA1 remains largely unknown. In this study, we used a mouse model of SCA1 to determine if miRNAs are misregulated in pre- and post-symptomatic SCA1 cerebellum. We found a significant alteration in the steady-state levels of numerous miRNAs prior to and following phenotypic onset. In addition, we provide evidence that increased miR-150 levels in SCA1 Purkinje neurons may modulate disease pathogenesis by targeting the expression of Rgs8 and Vegfa.

RNAi or overexpression: Alternative therapies for Spinocerebellar Ataxia Type 1

Spinocerebellar Ataxia Type 1 (SCA1) is an autosomal dominant late onset neurodegenerative disease caused by an expanded polyglutamine tract in ataxin-1. Here, we compared the protective effects of overexpressing ataxin-1-like using recombinant AAVs, or reducing expression of mutant ataxin-1 using virally delivered RNA interference (RNAi), in a transgenic mouse model of SCA1. For the latter, we used an artificial microRNA (miR) design that optimizes potency, efficacy and safety to suppress ataxin-1 expression (miS1). Delivery of either ataxin-1-like or miS1 viral vectors to SCA1 mice cerebella resulted in widespread cerebellar Purkinje cell transduction and improved behavioral and histological phenotypes. Our data indicate the utility of either approach as a possible therapy for SCA1 patients.

Recent Advances in RNA Interference Therapeutics for CNS Diseases

Over the last decade, RNA interference technology has shown therapeutic promise in rodent models of dominantly inherited brain diseases, including those caused by polyglutamine repeat expansions in the coding region of the affected gene. For some of these diseases, proof-of concept studies in model organisms have transitioned to safety testing in larger animal models, such as the nonhuman primate. Here, we review recent progress on RNA interference-based therapies in various model systems. We also highlight outstanding questions or concerns that have emerged as a result of an improved (and ever advancing) understanding of the technologies employed.

Broad distribution of ataxin 1 silencing in rhesus cerebella for spinocerebellar ataxia type 1 therapy

Spinocerebellar ataxia type 1 is one of nine polyglutamine expansion diseases and is characterized by cerebellar ataxia and neuronal degeneration in the cerebellum and brainstem. Currently, there are no effective therapies for this disease. Previously, we have shown that RNA interference mediated silencing of ATXN1 mRNA provides therapeutic benefit in mouse models of the disease. Adeno-associated viral delivery of an engineered microRNA targeting ATXN1 to the cerebella of well-established mouse models improved motor phenotypes, neuropathy, and transcriptional changes. Here, we test the translatability of this approach in adult rhesus cerebella. Nine adult male and three adult female rhesus macaque were unilaterally injected with our therapeutic vector, a recombinant adeno-associated virus type 1 (rAAV1) expressing our RNAi trigger (miS1) and co-expressing enhanced green fluorescent protein (rAAV1.miS1eGFP) into the deep cerebellar nuclei using magnetic resonance imaging guided techniques combined with a Stealth Navigation system (Medtronics Inc.). Transduction was evident in the deep cerebellar nuclei, cerebellar Purkinje cells, the brainstem and the ventral lateral thalamus. Reduction of endogenous ATXN1 messenger RNA levels were ≥30% in the deep cerebellar nuclei, the cerebellar cortex, inferior olive, and thalamus relative to the uninjected hemisphere. There were no clinical complications, and quantitative and qualitative analyses suggest that this therapeutic intervention strategy and subsequent reduction of ATXN1 is well tolerated. Collectively the data illustrate the biodistribution and tolerability of rAAV1.miS1eGFP administration to the adult rhesus cerebellum and are supportive of clinical application for spinocerebellar ataxia type 1.

RNAi prevents and reverses phenotypes induced by mutant human ataxin-1

Spinocerebellar ataxia type 1 is an autosomal dominant fatal neurodegenerative disease caused by a polyglutamine expansion in the coding region of ATXN1. We showed previously that partial suppression of mutant ataxin‐1 (ATXN1) expression, using virally expressed RNAi triggers, could prevent disease symptoms in a transgenic mouse model and a knockin mouse model of the disease, using a single dose of virus. Here, we set out to test whether RNAi triggers targeting ATXN1 could not only prevent, but also reverse disease readouts when delivered after symptom onset.

RNA Interference of Human α-Synuclein in Mouse

α-Synuclein is postulated to play a key role in the pathogenesis of Parkinson’s disease (PD). Aggregates of α-synuclein contribute to neurodegeneration and cell death in humans and in mouse models of PD. Here, we use virally mediated RNA interference to knockdown human α-synuclein in mice. We used an siRNA design algorithm to identify eight siRNA sequences with minimal off-targeting potential. One RNA-interference sequence (miSyn4) showed maximal protein knockdown potential in vitro. We then designed AAV vectors expressing miSyn4 and injected them into the mouse substantia nigra. miSyn4 was robustly expressed and did not detectably change dopamine neurons, glial proliferation, or mouse behavior. We then injected AAV2-miSyn4 into Thy1-hSNCA mice over expressing α-synuclein and found decreased human α-synuclein (hSNCA) in both midbrain and cortex. In separate mice, co-injection of AAV2-hSNCA and AAV2-miSyn4 demonstrated decreased hSNCA expression and rescue of hSNCA-mediated behavioral deficits. These data suggest that virally mediated RNA interference can knockdown hSNCA in vivo, which could be helpful for future therapies targeting human α-synuclein.

The long non-coding RNA NEAT1 is elevated in polyglutamine repeat expansion diseases and protects from disease gene-dependent toxicities

&NA; Polyglutamine (polyQ) repeat diseases are a class of neurodegenerative disorders caused by CAG‐repeat expansion. There are diverse cellular mechanisms behind the pathogenesis of polyQ disorders, including transcriptional dysregulation. Interestingly, we find that levels of the long isoform of nuclear paraspeckle assembly transcript 1 (Neat1L) are elevated in the brains of mouse models of spinocerebellar ataxia types 1, 2, 7 and Huntingtons disease (HD). Neat1L was also elevated in differentiated striatal neurons derived from HD knock‐in mice and in HD patient brains. The elevation was mutant Huntingtin (mHTT) dependent, as knockdown of mHTT in vitro and in vivo restored Neat1L to normal levels. In additional studies, we found that Neat1L is repressed by methyl CpG binding protein 2 (MeCP2) by RNA‐protein interaction but not by occupancy of MeCP2 at its promoter. We also found that NEAT1L overexpression protects from mHTT‐induced cytotoxicity, while reducing it enhanced mHTT‐dependent toxicity. Gene set enrichment analysis of previously published RNA sequencing data from mouse embryonic fibroblasts and cells derived from HD patients shows that loss of NEAT1L impairs multiple cellular functions, including pathways involved in cell proliferation and development. Intriguingly, the genes dysregulated in HD human brain samples overlap with pathways affected by a reduction in NEAT1, confirming the correlation of NEAT1L and HD‐induced perturbations. Cumulatively, the role of NEAT1L in polyQ disease model systems and human tissues suggests that it may play a protective role in CAG‐repeat expansion diseases.

583. Translating RNAi Therapy for Spinocerebellar Ataxia 1 to the Clinic

Spinocerebellar ataxia 1 (SCA1) is among a group polyglutamine expansion diseases and is characterized by cerebellar ataxia and neuronal degeneration in the cerebellum and brainstem. Currently, there are no effective treatment strategies for this disease. RNA interference (RNAi) is a naturally occurring process that mediates gene silencing and is currently being investigated as a therapy for dominant diseases such as SCA1. Previously, we used AAV vectors to deliver RNAi triggers to transgenic and knock-in mouse models of SCA1 and noted improved neuropathological, motor phenotypes and transcriptional changes. We have also completed studies in non-human primates (NHPs) evaluating the biodistribution, safety and efficacy of vector delivery to the deep cerebellar nuclei (DCN) in NHPs. We next performed dosing studies in pre-and post-symptomatic mice to identify the lowest efficacious dose, the highest tolerated dose. For this, groups of pre-symptomatic mice were given 1 of 4 doses at 5 weeks of age and motor function assayed after symptom onset for untreated mice (34 weeks of age) and immediately sacrificed for post-necropsy analysis. We identified a ceiling dose that conferred toxicity, a low dose that had no effect, and two doses that prevented phenotypic rotarod deficits relative to control injected SCA1 littermates. Concurrently, post-symptomatic mice were injected at 12 weeks of age at 3 escalating doses. Motor function tests at rotarod at 20 weeks of age identified a dose that not only prevented further deficit but significantly improved performance relative to baseline performance. Thus, our AAV-mediated delivery of RNAi to the SCA1 model can reverse motor impairment in mice, and is scalable to nonhuman primates, two important considerations in advancing this therapy to the clinic.