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Development of an AAV-based microRNA gene therapy to treat Machado-Joseph disease
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 non-allele 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 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 (iPSC) differentiated into frontal brain-
like neurons and in a SCA3 knock-in mouse model. Besides a strong reduction of ATXN3...Show moreSpinocerebellar 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 non-allele 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 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 (iPSC) differentiated into frontal brain-
like neurons and in a SCA3 knock-in 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.
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- All authors
- Martier, R.; Sogorb Gonzalez, M.; Stricker-Shaver, J.; Hubener-Schmid, J.; Keskin, S.; Klima, J.; Toonen, L.J.; Juhas, S.; Juhasova, J.; Ellederova, Z.; Motlik, J.; Haas, E.; Deventer, S. van; Konstantinova, P.; Nguyen, H.P.; Evers, M.M.
- Date
- 2019-12-13
- Volume
- 15
- Pages
- 343 - 358