Guo Jingwei, Zou Qian, Xu Jiawei, Lei Jieqiong, Yin Xin, Li Botao, Fu Jinyu, Mi Junjie, Wang Yanbo, Huang Huan, Zhang Chen-Yu, Chen Xi
Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, Jiangsu 210023, China.
The Second People's Hospital of Changzhou, The Third Affiliated Hospital of Nanjing Medical University, Changzhou Medical Center, Nanjing Medical University, Changzhou, Jiangsu 213003, China.
Brain. 2025 Aug 8. doi: 10.1093/brain/awaf291.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the death of both upper and lower motor neurons. Approximately 20% of familial ALS cases are associated with mutations in the superoxide dismutase type 1 (SOD1) gene. Developing a specific strategy to characteristically silence the pathogenic SOD1 gene remains a crucial goal amidst significant challenges. In this study, we developed a synthetic biology strategy to reprogram the liver as a tissue chassis for the in vivo self-assembly of small extracellular vesicles (sEVs)-encapsulated SOD1-siRNA, aiming to target spinal neurons and silence mutant SOD1 specifically in Tg(SOD1G93A) transgenic mice. We designed a CMV promoter-directed synthetic construct to encode a SOD1-siRNA along with a neuron-targeting rabies virus glycoprotein (RVG) tagged on sEV surface. Theoretically, upon liver uptake, this construct reprograms liver cells to generate and self-assemble SOD1-siRNAs into RVG-tagged sEVs. Subsequently, the sEV-encapsulated SOD1-siRNAs are transported via the endogenous sEV circulation and guided by the RVG tag to the spinal neurons. Experimental results illustrated that intravenous administration of this synthetic construct effectively facilitated in vivo self-assembly of SOD1-siRNAs into circulating sEVs. The functional delivery of SOD1-siRNAs to the spinal cord and cerebral cortex was confirmed through in vivo tracking of sEVs and sEV-encapsulated siRNAs. Treatment of Tg(SOD1G93A) transgenic mice with this construct significantly reduced mutant SOD1 protein levels in the spinal cord and cerebral cortex. Consequently, the characteristic symptoms of ALS, including decreased body weight, shortened lifespan, compromised motor function, muscle atrophy, neuroinflammation, motor neuron loss, and neuromuscular junction degeneration, were substantially ameliorated by the synthetic construct. Furthermore, an AAV-based strategy was devised for the enduring self-assembly of sEV-encapsulated SOD1-siRNA, whereby a single injection led to substantial and sustained inhibition of mutant SOD1 and significant symptom amelioration in transgenic mice. Overall, this study established an effective and convenient therapeutic approach for mitigating muscle atrophy and denervation in animal model, presenting a promising solution for future ALS treatment.
肌萎缩侧索硬化症（ALS）是一种神经退行性疾病，其特征是上运动神经元和下运动神经元均死亡。大约20%的家族性ALS病例与超氧化物歧化酶1（SOD1）基因突变有关。在面临重大挑战的情况下，制定一种特异性沉默致病性SOD1基因的策略仍然是一个关键目标。在本研究中，我们开发了一种合成生物学策略，将肝脏重新编程为一个组织底盘，用于体内自组装包裹SOD1-siRNA的小细胞外囊泡（sEV），旨在靶向脊髓神经元并在Tg（SOD1G93A）转基因小鼠中特异性沉默突变型SOD1。我们设计了一种由巨细胞病毒（CMV）启动子指导的合成构建体，用于编码SOD1-siRNA以及标记在sEV表面的靶向神经元的狂犬病病毒糖蛋白（RVG）。理论上，肝脏摄取该构建体后，会将肝细胞重新编程，以生成SOD1-siRNA并将其自组装成带有RVG标记的sEV。随后，包裹在sEV中的SOD1-siRNA通过内源性sEV循环进行运输，并在RVG标记的引导下到达脊髓神经元。实验结果表明，静脉注射这种合成构建体有效地促进了SOD1-siRNA在体内自组装成循环sEV。通过对sEV和包裹在sEV中的siRNA进行体内追踪，证实了SOD1-siRNA向脊髓和大脑皮层的功能性递送。用该构建体治疗Tg（SOD1G93A）转基因小鼠可显著降低脊髓和大脑皮层中突变型SOD1蛋白水平。因此，该合成构建体显著改善了ALS的特征性症状，包括体重减轻、寿命缩短、运动功能受损、肌肉萎缩、神经炎症、运动神经元丧失和神经肌肉接头退化。此外，还设计了一种基于腺相关病毒（AAV）的策略，用于sEV包裹的SOD1-siRNA的持久自组装，单次注射可导致转基因小鼠中突变型SOD1的大量持续抑制和症状的显著改善。总体而言，本研究建立了一种有效且便捷的治疗方法，用于减轻动物模型中的肌肉萎缩和失神经支配，为未来ALS治疗提供了一个有前景的解决方案。
