PDF(Pubmed)
Abstract：
A deficiency of human arylsulfatase A (hASA) causes metachromatic leukodystrophy (MLD), a lysosomal storage disease characterized by sulfatide accumulation and central nervous system (CNS) demyelination. Efficacy of enzyme replacement therapy (ERT) is increased by genetic engineering of hASA to elevate its activity and transfer across the blood-brain barrier (BBB), respectively. To further improve the enzyme\'s bioavailability in the CNS, we mutated a cathepsin cleavage hot spot and obtained hASAs with substantially increased half-lives. We then combined the superstabilizing exchange E424A with the activity-promoting triple substitution M202V/T286L/R291N and the ApoEII-tag for BBB transfer in a trimodal modified neoenzyme called SuPerTurbo-ASA. Compared with wild-type hASA, half-life, activity, and M6P-independent uptake were increased more than 7-fold, about 3-fold, and more than 100-fold, respectively. ERT of an MLD-mouse model with immune tolerance to wild-type hASA did not induce antibody formation, indicating absence of novel epitopes. Compared with wild-type hASA, SuPerTurbo-ASA was 8- and 12-fold more efficient in diminishing sulfatide storage of brain and spinal cord. In both tissues, storage was reduced by ∼60%, roughly doubling clearance achieved with a 65-fold higher cumulative dose of wild-type hASA previously. Due to its enhanced therapeutic potential, SuPerTurbo-ASA might be a decisive advancement for ERT and gene therapy of MLD.
摘要：
人芳基硫酸酯酶A（hASA）的缺乏会导致异染性脑白质营养不良（MLD），一种溶酶体贮积病，以硫酸脂积累和中枢神经系统脱髓鞘为特征。酶替代疗法（ERT）的功效通过基因工程hASA来提高其活性和跨血脑屏障（BBB）的转移，分别。为了进一步提高酶在中枢神经系统中的生物利用度，我们突变了组织蛋白酶裂解热点，并获得了半衰期显着增加的hASAs。然后，我们将超稳定交换E424A与促进活性的三重取代M202V/T286L/R291N和ApoEII标签结合起来，用于在三峰修饰的新酶SuPerTurbo-ASA中进行BBB转移。与野生型HASA相比，半衰期,活动,与M6P无关的摄取增加了7倍以上，大约3倍，超过100倍，分别。对野生型hASA免疫耐受的MLD小鼠模型的ERT不诱导抗体形成，表明不存在新的表位。与野生型HASA相比，SuPerTurbo-ASA在减少大脑和脊髓的硫酸盐储存方面的效率分别提高了8倍和12倍。在这两个组织中，储存减少了~60%,以前野生型hASA的累积剂量提高了65倍,达到了大约两倍的清除率.由于其增强的治疗潜力，SuPerTurbo-ASA可能是ERT和MLD基因治疗的决定性进展。
