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 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 (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.

中文翻译：

---

基于AAV的MicroRNA基因疗法的发展，用于治疗Machado-Joseph病。

脊髓小脑性共济失调3型（SCA3）或Machado-Joseph病（MJD）是由ATXN3基因中的CAG扩展引起的进行性神经退行性疾病。扩展的CAG重复序列在ataxin-3蛋白中翻译为延长的多谷氨酰胺重复序列，并在内含物中积累，获得毒性，从而导致小脑和脑干退化。在当前研究中，使用人工微RNA研究了非等位基因特异性ATXN3沉默方法，该人工RNA被设计为靶向ATXN3基因（miATXN3）的各个区域。根据miATXN3候选基因在共表达ATXN3的荧光素酶（Luc）报告基因上的沉默效率，在体外筛选候选基因。进一步测试了三种最佳miATXN3候选对象在分化为额脑样神经元的诱导多能干细胞（iPSC）和SCA3敲入小鼠模型中的靶标参与和潜在的脱靶活性。除了ATXN3 mRNA和蛋白质的大量减少外，小的RNA测序还显示出有效的引导链加工过程，而没有产生过客链。我们使用了不同的方法来预测AAV5-miATXN3处理后脱靶基因的改变，但没有发现不良影响的证据。此外，我们在大型动物模型minipig中证明，鞘内递送AAV5可以转导SCA3患者的主要区域。这些结果为进一步研究AAV5-miATXN3在大型动物中的分布，疗效和安全性提供了坚实的基础。