Experimental design of mouse treatment groups.

Mucopolysaccharidosis type IIID (MPS IIID; Sanfilippo D) is caused by biallelic pathogenic variants in N-acetylglucosamine-6-sulfatase (GNS), which participates in catabolism of heparan sulfate (HS) glycosaminoglycans. Characterization of MPS IIID disease at a cellular level has not been robustly achieved. We used unbiased quantitative proteomics to establish a cellular phenotype for MPS IIID mice. Recombinant human GNS (rhGNS), a variant of which previously demonstrated single dose efficacy in MPS IIID human fibroblasts and in MPS IIID neonatal mice, was used to establish a repeat dosing schedule to treat MPS IIID mice. Adult Gns KO mice or heterozygous carriers were treated via intracerebroventricular (ICV) injections and received 3, 30, or 200 μg rhGNS in 4 doses over 2 weeks or vehicle. Twenty-four hours after the final dose, HS in brain and CSF showed dose-dependent reductions, reaching carrier levels in the higher dose groups. Furthermore, the proteomic perturbations that we described were corrected by rhGNS treatment. Next, Gns KO or carrier adult mice were treated via ICV and received 3, 30 or 200 μg rhGNS or vehicle once every two weeks (Day 1, 15, 29, 43, 57, 71, 85) and were euthanized on day 91. Following treatment, total HS and MPS IIID-specific HS (GlcNAc6S) showed dose-dependent reductions in brain and CSF and markers of neuroinflammation were substantially reduced. ICV enzyme replacement therapy with rhGNS restores CNS pathology of adult MPS IIID mice even with treatment at 14-day intervals, demonstrating preclinical efficacy for MPS IIID.

粘多糖贮积症IIID型（Mucopolysaccharidosis type IIID, MPS IIID；Sanfilippo D型）系由N-乙酰葡糖胺-6-硫酸酯酶（N-acetylglucosamine-6-sulfatase, GNS）的双等位致病变异所致，该酶参与硫酸乙酰肝素（heparan sulfate, HS）糖胺聚糖的分解代谢。目前学界尚未在细胞层面对MPS IIID疾病完成充分的特征表征。本研究采用无偏倚定量蛋白质组学技术，构建了MPS IIID模型小鼠的细胞表型。重组人GNS（recombinant human GNS, rhGNS）——其此前的变体已在MPS IIID人成纤维细胞及新生MPS IIID小鼠中展现出单剂给药疗效——被用于确立重复给药方案，以治疗MPS IIID模型小鼠。成年Gns基因敲除（knockout, KO）小鼠或杂合携带者经脑室内（intracerebroventricular, ICV）注射给药，在2周内接受4次剂量分别为3μg、30μg或200μg的rhGNS或溶剂对照处理。末次给药24小时后，脑组织与脑脊液中的HS水平呈剂量依赖性降低，高剂量组的HS水平降至杂合携带者水平。此外，本研究观测到的蛋白质组学扰动可通过rhGNS治疗得到纠正。随后，我们对成年Gns基因敲除或杂合携带者小鼠采用脑室内给药方案，每2周（第1、15、29、43、57、71、85天）给予3μg、30μg或200μg的rhGNS或溶剂对照，并于第91天实施安乐死。给药结束后，脑组织与脑脊液中的总HS及MPS IIID特异性HS（GlcNAc6S）均呈剂量依赖性降低，神经炎症标志物水平也显著下调。采用rhGNS的脑室内酶替代疗法，即便以14天为给药间隔，仍可恢复成年MPS IIID模型小鼠的中枢神经系统病理表型，这证实了该疗法针对MPS IIID的临床前疗效。