Lentiviral gene therapy for mucopolysaccharidosis

描述(申请人提供)：粘多糖病II型(MPS II，Hunter综合征)是一种X连锁隐性遗传性疾病，由于缺乏艾杜酸-2-硫酸酯酶，导致糖胺葡聚糖、硫酸肝素和硫酸皮肤素的全身积聚。受影响的个体患有骨骼异常、器官肿大、危及生命的阻塞性呼吸道疾病，并在严重的酶缺乏形式下，神经系统退化，并在15岁前死亡。尽管造血干细胞移植和植入在治疗一些MPS疾病方面显示出有效性，但异基因移植的MPSII患者迄今尚未表现出改善神经功能的结果。在这个项目中，我们假设MPSII的同种异体移植是无效的，因为移植细胞产生的IDS酶不充分，并且通过基因工程使供体细胞表达高水平的IDS将克服这一不足，并提供有效的代谢交叉校正，包括疾病的神经学表现。Lentigen是开发用于治疗人类疾病的慢病毒载体的领先公司。在这个第一阶段的STTR项目中，我们建议将Lentigen的慢病毒载体技术与明尼苏达大学在溶酶体储存疾病的细胞治疗方面的经验相结合，开发一种治疗Hunter综合征的体外转导方法，MPS II。该计划的具体目的是：(I)构建和测试以绿色荧光蛋白为细胞标记的转导人类入侵检测系统基因的慢病毒载体。载体构建将基于双启动子、双顺反子和融合蛋白策略生成，并使用Lentigen的专有LentiMax平台进行包装。(Ii)通过体外慢病毒转导人类入侵系统基因到MSPII小鼠的造血干细胞来纠正代谢和神经疾病。将携带入侵检测系统基因的慢病毒载体导入携带入侵检测系统缺陷小鼠的骨髓，移植到携带入侵防御系统缺陷小鼠的受者体内，作为体外基因治疗亨特综合征的靶向造血干细胞的模型。经过处理的动物将接受移植和转导的测试，血浆和组织中的IDS酶的表达，尿液和组织中储存物质的清除，以及学习和运动功能的神经行为测试中的改善表现。这些研究的总体目标是提供临床前数据，以支持对MPS II进行基因治疗的最直接和最可行的方法，并对未来慢病毒基因治疗其他溶酶体储存疾病的发展具有指导意义。

DESCRIPTION (provided by applicant): Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X-linked recessive inherited disorder caused by absence of iduronate-2-sulfatase, resulting in systemic accumulation of glycosaminoglycans heparan sulphate and dermatan sulphate. Affected individuals suffer from skeletal abnormalities, organomegaly, life- threatening obstructive airway disease, and, in the severely enzyme deficient form, neurologic degeneration and death by age 15. While transplantation and engraftment of hematopoietic stem cells has shown efficacy in the treatment of some MPS diseases, allografted MPSII patients have thus far not exhibited improved neurologic outcomes. In this project, we hypothesize that allotransplant for MPSII is ineffective due to insufficient generation of IDS enzyme from engrafted cells, and that genetic engineering of donor cells to express high levels of IDS will overcome this insufficiency and provide effective metabolic cross- correction that includes neurologic manifestations of the disease. Lentigen is a leading company in the development of lentiviral vectors for treatment of human disease. In this Phase I STTR project, we propose to combine Lentigen's lentiviral vector technology with the University of Minnesota's experience in cellular therapies for lysosomal storage diseases by developing an ex vivo transduction approach for the treatment of Hunter syndrome, MPS II. The Specific Aims of the proposal are: (i) To construct and test lentiviral vectors for transduction of the human IDS gene along with green fluorescent protein as a cellular marker. Vector constructs will be generated based on dual promoter, bicistronic and fusion protein strategies, and packaged using Lentigen's proprietary LentiMax platform. (ii) Correction of metabolic and neurologic disease by ex vivo lentiviral transduction of the human IDS gene into hematopoietic stem cells of MSPII mice. Marrow from IDS deficient mice will be transduced with lentiviral vector carrying the IDS gene and transplanted into IDS deficient recipients as a model for ex vivo gene therapy of Hunter syndrome targeting hematopoietic stem cells. Treated animals will be tested for engraftment and transduction of donor cells, IDS enzyme expression in plasma and tissues, clearing of storage materials in urine and in tissues, and improved performance in neurobehavioral tests of learning and motor function. The overall goal of the proposed studies is to provide preclinical data to support the most straightforward and feasible approach for implementation of gene therapy for MPS II, with implications for the development of lentiviral gene therapies for other lysosomal storage diseases in the future.