Our previous study delivered zinc finger nucleases to treat mice with mucopolysaccharidosis type I (MPS I), resulting in a phase I/II clinical trial (ClinicalTrials.gov: NCT02702115). However, in the clinical trial, the efficacy needs to be improved due to the low transgene expression level. To this end, we designed a proprietary system (PS) gene editing approach with CRISPR to insert a promoterless α-l-iduronidase (IDUA) cDNA sequence into the albumin locus of hepatocytes. In this study, adeno-associated virus 8 (AAV8) vectors delivering the PS gene editing system were injected into neonatal and adult MPS I mice. IDUA enzyme activity in the brain significantly increased, while storage levels were normalized. Neurobehavioral tests showed that treated mice had better memory and learning ability. Also, histological analysis showed efficacy reflected by the absence of foam cells in the liver and vacuolation in neuronal cells. No vector-associated toxicity or increased tumorigenesis risk was observed. Moreover, no off-target effects were detected through the unbiased genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) analysis. In summary, these results showed the safety and efficacy of the PS in treating MPS I and paved the way for clinical studies. Additionally, as a therapeutic platform, the PS has the potential to treat other lysosomal diseases.

我们之前的研究使用锌指核酸酶来治疗 I 型粘多糖贮积症 (MPS I) 小鼠，并进行了 I/II 期临床试验 (ClinicalTrials.gov: NCT02702115)。但在临床试验中，由于转基因表达水平较低，疗效有待提高。为此，我们设计了一种使用 CRISPR 的专有系统 (PS) 基因编辑方法，将无启动子的 α- l -艾杜糖醛酸酶 (IDUA) cDNA 序列插入肝细胞的白蛋白基因座中。在这项研究中，将传递 PS 基因编辑系统的腺相关病毒 8 (AAV8) 载体注射到新生和成年 MPS I 小鼠中。大脑中的 IDUA 酶活性显着增加，而储存水平则正常化。神经行为测试表明，接受治疗的小鼠具有更好的记忆和学习能力。此外，组织学分析表明，肝脏中泡沫细胞的缺失和神经元细胞中空泡的存在反映了功效。没有观察到载体相关的毒性或增加的肿瘤发生风险。此外，通过测序（GUIDE-seq）分析实现的双链断裂的无偏见全基因组无偏见识别，没有检测到脱靶效应。总之，这些结果表明了PS治疗MPS I的安全性和有效性，并为临床研究铺平了道路。此外，作为一种治疗平台，PS 具有治疗其他溶酶体疾病的潜力。