Mucopolysaccharidosis IIIB is a paediatric lysosomal storage disease caused by deficiency of the enzyme α-N-acetylglucosaminidase (NAGLU), involved in the degradation of the glycosaminoglycan heparan sulphate. Absence of NAGLU leads to accumulation of partially degraded heparan sulphate within lysosomes and the extracellular matrix, giving rise to severe CNS degeneration with progressive cognitive impairment and behavioural problems. There are no therapies. Haematopoietic stem cell transplant shows great efficacy in the related disease mucopolysaccharidosis I, where donor-derived monocytes can transmigrate into the brain following bone marrow engraftment, secrete the missing enzyme and cross-correct neighbouring cells. However, little neurological correction is achieved in patients with mucopolysaccharidosis IIIB. We have therefore developed an ex vivo haematopoietic stem cell gene therapy approach in a mouse model of mucopolysaccharidosis IIIB, using a high-titre lentiviral vector and the myeloid-specific CD11b promoter, driving the expression of NAGLU (LV.NAGLU). To understand the mechanism of correction we also compared this with a poorly secreted version of NAGLU containing a C-terminal fusion to IGFII (LV.NAGLU-IGFII). Mucopolysaccharidosis IIIB haematopoietic stem cells were transduced with vector, transplanted into myeloablated mucopolysaccharidosis IIIB mice and compared at 8 months of age with mice receiving a wild-type transplant. As the disease is characterized by increased inflammation, we also tested the anti-inflammatory steroidal agent prednisolone alone, or in combination with LV.NAGLU, to understand the importance of inflammation on behaviour. NAGLU enzyme was substantially increased in the brain of LV.NAGLU and LV.NAGLU-IGFII-treated mice, with little expression in wild-type bone marrow transplanted mice. LV.NAGLU treatment led to behavioural correction, normalization of heparan sulphate and sulphation patterning, reduced inflammatory cytokine expression and correction of astrocytosis, microgliosis and lysosomal compartment size throughout the brain. The addition of prednisolone improved inflammatory aspects further. Substantial correction of lysosomal storage in neurons and astrocytes was also achieved in LV.NAGLU-IGFII-treated mice, despite limited enzyme secretion from engrafted macrophages in the brain. Interestingly both wild-type bone marrow transplant and prednisolone treatment alone corrected behaviour, despite having little effect on brain neuropathology. This was attributed to a decrease in peripheral inflammatory cytokines. Here we show significant neurological disease correction is achieved using haematopoietic stem cell gene therapy, suggesting this therapy alone or in combination with anti-inflammatories may improve neurological function in patients.

中文翻译：

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巨噬细胞酶和减少的炎症通过干细胞基因疗法推动粘多糖贮积症IIIB的大脑校正。

粘多糖贮积症IIIB是由酶α- N缺乏引起的小儿溶酶体贮积病-乙酰氨基葡糖苷酶（NAGLU），参与糖胺聚糖硫酸乙酰肝素的降解。缺乏NAGLU会导致部分降解的硫酸乙酰肝素在溶酶体和细胞外基质中蓄积，从而导致严重的CNS变性，并伴有进行性认知障碍和行为问题。没有疗法。造血干细胞移植在相关疾病粘多糖贮积病I中显示出巨大的功效，其中供体来源的单核细胞可在骨髓植入后迁移到大脑中，分泌缺失的酶并交叉校正邻近细胞。但是，在黏多糖贮积症IIIB患者中几乎没有神经矫正。因此，我们开发了一种离体使用高滴度慢病毒载体和髓样特异性CD11b启动子在粘液多糖病IIIB小鼠模型中进行造血干细胞基因治疗，从而驱动NAGLU（LV.NAGLU）的表达。为了了解校正的机制，我们还将其与分泌较弱的NAGLU进行了比较，该NAGLU包含与IGFII（LV.NAGLU-IGFII）的C端融合体。用载体转导粘多糖贮积症IIIB造血干细胞，移植到骨髓变性粘多糖贮积症IIIB小鼠中，并在8个月大时与接受野生型移植的小鼠进行比较。由于该疾病的特征在于炎症增加，因此我们还单独或与LV.NAGLU联合测试了抗炎甾体强的松龙，以了解炎症对行为的重要性。在LV.NAGLU和LV.NAGLU-IGFII处理的小鼠的大脑中，NAGLU酶显着增加，在野生型骨髓移植小鼠中几乎没有表达。LV.NAGLU治疗导致行为矫正，硫酸乙酰肝素正常化和硫酸化模式，炎性细胞因子表达降低以及星形胶质细胞增多症，小胶质细胞增生和溶酶体区室大小在整个大脑中的校正。泼尼松龙的添加进一步改善了炎症方面。LV.NAGLU-IGFII处理的小鼠中，尽管从大脑中植入的巨噬细胞分泌的酶有限，但仍可对神经元和星形胶质细胞中的溶酶体贮藏进行实质性校正。有趣的是，尽管对脑神经病理学影响不大，但野生型骨髓移植和泼尼松龙治疗都能单独纠正行为。这归因于外周炎症细胞因子的减少。在这里，我们显示使用造血干细胞基因疗法可实现明显的神经系统疾病矫正，表明该疗法单独使用或与抗炎药联合使用可改善患者的神经功能。