目的:设计一种新型有效的scAAV-Gusb病毒载体,用于治疗由β-Glu基因(Gusb等位基因)突变引起的VII型粘多糖贮积症(MPSVII)。
方法:用培养的鼠Gusb成纤维细胞测试单链AAV-Gusb(ssAAV-Gusb)和自身互补AAV(scAAV-Gusb)载体的β-Glu表达。在进一步的研究中选择scAAV-Gusb载体,通过新生儿肝内注射和成人基质内注射来延长寿命和治疗Gusb小鼠的角膜病理学。分别。使用HRT2体内共聚焦显微镜和小鼠角膜的组织化学研究了角膜病理学。
结果:ssAAV-Gusb和scAAV-Gusb载体均在培养的Gusb成纤维细胞中表达鼠β-Glu。scAAV-Gusb载体的转导效率高于ssAAV-Gusb载体。为了延长Gusb小鼠的寿命,新生儿(3日龄)通过肝内注射给予scAAV-Gusb病毒.该治疗提高了Gusb小鼠的存活率,将中位生存率从22.5周(未治疗)延长至50周(治疗)。此后,我们确定了scAAV-Gusb病毒改善老年Gusb小鼠角膜混浊的疗效.角膜混浊度和基质厚度均下降,并且在接受scAAV-Gusb病毒的Gusb角膜中存在β-Glu酶活性,与未处理的变形虫基质细胞的形态变化有关,与scAAV-Gusb病毒注射4-12周后的特征性树突状角膜细胞形态有关。
结论:肝内注射scAAV-Gusb可有效延长Gusb小鼠的寿命,基质内注射可以改善角膜表型。两种策略都可以适用于治疗其他MPS。
OBJECTIVE: To design a novel efficacious scAAV-Gusb viral vector for treating Mucopolysaccharidosis Type VII (MPS VII) caused by a mutation in the β-Glu gene (Gusb allele).
METHODS: β-Glu expression of single-stranded AAV-Gusb (ssAAV-Gusb) and self-complementary AAV (scAAV-Gusb) vectors are tested with cultured murine Gusb fibroblasts. The scAAV-Gusb vector was chosen in further studies to prolong the life span and treat corneal pathology of Gusb mice via intrahepatic injection of neonates and intrastromal injection in adults, respectively. Corneal pathology was studied using HRT2 in vivo confocal microscope and histochemistry in mice corneas.
RESULTS: Both ssAAV-Gusb and scAAV-Gusb vectors expressed murine β-Glu in cultured Gusb fibroblasts. The scAAV-Gusb vector had higher transduction efficiency than the ssAAV-Gusb vector. To prolong the life span of Gusb mice, neonates (3 days old) were administered with scAAV-Gusb virus via intrahepatic injection. The treatment improves the survival rate of Gusb mice, prolonging the median survival rate from 22.5 weeks (untreated) to 50 weeks (treated). Thereafter, we determined the efficacy of the scAAV-Gusb virus in ameliorating corneal cloudiness observed in aged Gusb mice. Both corneal cloudiness and stroma thickness decreased, and there was the presence of β-Glu enzyme activity in the Gusb corneas receiving scAAV-Gusb virus associated with morphology change of amoeboid stromal cells in untreated to characteristic dendritic keratocytes morphology after 4-12 weeks of scAAV-Gusb virus injection.
CONCLUSIONS: Intrahepatic injection of scAAV-Gusb is efficacious in prolonging the life span of Gusb mice, and intrastromal injection can ameliorate corneal phenotypes. Both strategies can be adapted for treating other MPS.