Metachromatic leukodystrophy (MLD) is a rare, inherited, demyelinating lysosomal storage disorder caused by mutations in the arylsulfatase-A gene (ARSA). In patients, levels of functional ARSA enzyme are diminished and lead to deleterious accumulation of sulfatides. Herein, we demonstrate that intravenous administration of HSC15/ARSA restored the endogenous murine biodistribution of the corresponding enzyme, and overexpression of ARSA corrected disease biomarkers and ameliorated motor deficits in Arsa KO mice of either sex. In treated Arsa KO mice, when compared with intravenously administered AAV9/ARSA, significant increases in brain ARSA activity, transcript levels, and vector genomes were observed with HSC15/ARSA. Durability of transgene expression was established in neonate and adult mice out to 12 and 52 weeks, respectively. Levels and correlation between changes in biomarkers and ARSA activity required to achieve functional motor benefit was also defined. Finally, we demonstrated blood–nerve, blood–spinal and blood–brain barrier crossing as well as the presence of circulating ARSA enzyme activity in the serum of healthy nonhuman primates of either sex. Together, these findings support the use of intravenous delivery of HSC15/ARSA-mediated gene therapy for the treatment of MLD.

SIGNIFICANCE STATEMENT Herein, we describe the method of gene therapy adeno-associated virus (AAV) capsid and route of administration selection leading to an efficacious gene therapy in a mouse model of metachromatic leukodystrophy. We demonstrate the therapeutic outcome of a new naturally derived clade F AAV capsid (AAVHSC15) in a disease model and the importance of triangulating multiple end points to increase the translation into higher species via ARSA enzyme activity and biodistribution profile (with a focus on the CNS) with that of a key clinically relevant biomarker.

异染性脑白质营养不良 (MLD) 是一种罕见的遗传性脱髓鞘性溶酶体贮积症，由芳基硫酸酯酶 A 基因 ( ARSA ) 突变引起。在患者中，功能性 ARSA 酶的水平降低并导致脑硫苷脂的有害积累。在此，我们证明静脉注射 HSC15/ ARSA可恢复相应酶的内源性小鼠生物分布，并且ARSA的过度表达可纠正疾病生物标志物并改善任一性别Arsa KO 小鼠的运动缺陷。在治疗的Arsa KO 小鼠中，与静脉注射 AAV9/ ARSA 相比， HSC15/ ARSA观察到脑 ARSA 活性、转录水平和载体基因组显着增加。在新生小鼠和成年小鼠中分别建立了 12 周和 52 周的转基因表达持久性。还定义了实现功能性运动益处所需的生物标志物变化和 ARSA 活性之间的水平和相关性。最后，我们证明了血液-神经、血液-脊髓和血-脑屏障的穿越，以及健康非人灵长类动物血清中循环 ARSA 酶活性的存在。总之，这些发现支持使用静脉注射 HSC15/ ARSA介导的基因疗法来治疗 MLD。

意义声明在此，我们描述了基因治疗腺相关病毒（AAV）衣壳的方法以及在异染性脑白质营养不良小鼠模型中产生有效基因治疗的给药途径选择。我们展示了疾病模型中新的自然衍生进化枝 F AAV 衣壳 (AAVHSC15) 的治疗结果，以及三角测量多个终点以通过 ARSA 酶活性和生物分布概况（重点关注中枢神经系统）增加向高等物种的翻译的重要性）与关键的临床相关生物标志物的值。