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.

人芳基硫酸酯酶 A (hASA) 缺乏会导致异染性脑白质营养不良 (MLD)，这是一种溶酶体贮积病，其特征是脑硫苷脂积聚和中枢神经系统 (CNS) 脱髓鞘。通过 hASA 基因工程分别提高其活性和穿过血脑屏障 (BBB) 的转移，从而提高酶替代疗法 (ERT) 的疗效。为了进一步提高该酶在中枢神经系统中的生物利用度，我们对组织蛋白酶裂解热点进行了突变，获得了半衰期大幅延长的 hASA。然后，我们将超稳定交换 E424A 与活性促进三重取代 M202V/T286L/R291N 以及用于 BBB 转移的 ApoEII 标签组合在称为 SuPerTurbo-ASA 的三峰修饰新酶中。与野生型hASA相比，半衰期、活性和M6P独立摄取分别增加了7倍以上、约3倍和100倍以上。对野生型 hASA 具有免疫耐受性的 MLD 小鼠模型的 ERT 不会诱导抗体形成，表明不存在新表位。与野生型 hASA 相比，SuPerTurbo-ASA 在减少大脑和脊髓硫苷脂储存方面的效率分别提高了 8 倍和 12 倍。在这两种组织中，储存量减少了约 60%，之前野生型 hASA 累积剂量增加 65 倍时，清除率大约增加了一倍。由于其增强的治疗潜力，SuPerTurbo-ASA 可能是 MLD 的 ERT 和基因治疗的决定性进步。