Development of an AAV-Based MicroRNA Gene Therapy to Treat Machado-Joseph Disease

Abstract

Spinocerebellar ataxia type 3 (SCA3), or Machado-Joseph disease (MJD), is a progressive neurodegenerative disorder caused by a CAG expansion in the ATXN3 gene. The expanded CAG repeat is translated into a prolonged polyglutamine repeat in the ataxin-3 protein and accumulates within inclusions, acquiring toxic properties, which results in degeneration of the cerebellum and brain stem. In the current study, a nonallele-specific ATXN3 silencing approach was investigated using artificial microRNAs engineered to target various regions of the ATXN3 gene (miATXN3). The miATXN3 candidates were screened in vitro based on their silencing efficacy on a luciferase (Luc) reporter co-expressing ATXN3. The three best miATXN3 candidates were further tested for target engagement and potential off-target activity in induced pluripotent stem cells (iPSCs) differentiated into frontal brain-like neurons and in a SCA3 knockin mouse model. Besides a strong reduction of ATXN3 mRNA and protein, small RNA sequencing revealed efficient guide strand processing without passenger strands being 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 a large animal model, the minipig, that intrathecal delivery of AAV5 can transduce the main areas affected in SCA3 patients. These results proved a strong basis to move forward to investigate distribution, efficacy, and safety of AAV5-miATXN3 in large animals.

PolyQ-expanded ataxin-3 protein levels in peripheral blood mononuclear cells correlate with clinical parameters in SCA3: a pilot study

“…For SCA3, it was previously shown that targeting the expanded ataxin-3-allele by exon-skipping or ataxin-3 gene suppression using antisense oligonucleotides (ASO) in SCA3 mouse models result in lower polyQ-expanded ataxin-3 levels and in a reduction of aggregation [20][21][22]. Similar results are shown if the down-regulation of the disease protein ataxin-3 via specific micro-RNA (miRNA) is induced [23][24][25][26]. To get prepared for ataxin-3 lowering therapies in further clinical trials, our study aimed to establish sensitive methods to measure total full-length and polyQ-expanded ataxin-3 protein in PBMCs, an easily accessible human biomaterial.…”

“…Therefore, measurements of ataxin-3 in PBMCs or—by more sensitive assays—in serum, plasma or CSF can pave the way for further clinical trials to track the disease progression and therapeutic response longitudinally. Our immunoassays are able to analyze ataxin-3 protein amounts under protein-lowering therapies like ASO or miRNA approaches as demonstrated by a miRNA approach using our SCA3 knock-in mouse model [ 26 ], thus representing promising pharmacodynamics/response biomarker which capture target-engagement in easily accessible peripheral blood.…”

A novel Ataxin-3 knock-in mouse model mimics the human SCA3 disease phenotype including neuropathological, behavioral, and transcriptional abnormalities

“…First studies have already used this KI model to prove antisense oligonucleotide (ASOs) therapy and longitudinal biomarker studies. Martier et al (2019) could recently show that they can reduce the amount of expanded Atxn3 mRNA in these KI mice by delivering microRNAs in an adenoassociated-vector system. Moreover, Wilke and colleagues are using this model to investigate the progression of neurofilament light chain (NfL) and phosphorylated neurofilament heavy chain (pNfH) as potential easily accessible serum biomarkers in a longitudinal study with mice in the age range from 2 to 24 months.…”

Gene Therapy for Neurodegenerative Diseases: Slowing Down the Ticking Clock

“…The highest CAG repeats were embryonically lethal while the shorter CAG repeats were tolerated ( Wang et al, 2011 ; Nalavade et al, 2013 ). Since the mutated ATXN3 gene produces a protein with a toxic gain of function, it is considered a promising target for gene silencing approaches such as ASOs and RNAi-based gene therapy ( Evers et al, 2014 ; Toonen et al, 2017 ; Martier et al, 2019c ).…”