Rybarikova Margareta, Rey Maria, Hasanovic Ed, Sipion Mélanie, Rambousek Lukas, Déglon Nicole
Lausanne University Hospital (CHUV) and University of Lausanne (UNIL), Department of Clinical Neurosciences (DNC), Laboratory of Cellular and Molecular Neurotherapies, Lausanne, Switzerland.
Lausanne University Hospital (CHUV) and University of Lausanne (UNIL), Neuroscience Research Center (CRN), Laboratory of Cellular and Molecular Neurotherapies (LCMN), Lausanne, Switzerland.
Gene Ther. 2025 Jul 28. doi: 10.1038/s41434-025-00557-2.
Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by a CAG expansion of the ataxin-3 gene (ATXN3). SCA3 patients suffer from ataxia, spasticity and dystonia in mid-adulthood, with spinocerebellar dysfunction and degeneration. As a monogenic disease for which only symptomatic treatment is available, ATXN3 is an attractive target for gene editing. We used the KamiCas9, a self-inactivating gene editing system, to explore gene editing strategies suitable for all SCA3 patients. We first tested the deletion of exon 10 or the introduction of a premature stop codon into exon 9. High editing events were observed in vitro, but efficiency was very low in SCA3 transgenic mice. We then evaluated an ablate-and-replace strategy. The ablate experiments resulted in 55 ± 18% cerebellar editing of the ATXN3 gene. A human ATXN3L paralog, expressed in the brains of SCA3 patients, may act as a natural, CRISPR-resistant replacement gene. In a proof-of-principle study, ablate and ablate-and-replace strategies were evaluated in SCA3 transgenic mice. Two months after injection, similar editing efficiencies were obtained in the ablate and ablate-and-replace groups. Immunofluorescence and RT-qPCR analyses of cerebellar markers support the development of this strategy for SCA3 treatment.
3型脊髓小脑共济失调（SCA3）是一种由ataxin-3基因（ATXN3）的CAG重复扩增引起的罕见神经退行性疾病。SCA3患者在成年中期会出现共济失调、痉挛和肌张力障碍，并伴有脊髓小脑功能障碍和退化。作为一种仅能进行对症治疗的单基因疾病，ATXN3是基因编辑的一个有吸引力的靶点。我们使用自失活基因编辑系统KamiCas9来探索适用于所有SCA3患者的基因编辑策略。我们首先测试了外显子10的缺失或在外显子9中引入提前终止密码子。在体外观察到了高编辑事件，但在SCA3转基因小鼠中的效率非常低。然后我们评估了一种切除并替换的策略。切除实验导致ATXN3基因在小脑中有55±18%的编辑。在SCA3患者大脑中表达的人ATXN3L旁系同源基因可能作为一种天然的、抗CRISPR的替代基因。在一项原理验证研究中，在SCA3转基因小鼠中评估了切除和切除并替换策略。注射两个月后，切除组和切除并替换组获得了相似的编辑效率。对小脑标志物的免疫荧光和RT-qPCR分析支持了这种用于SCA3治疗策略的开发。
