LENTIVIRAL VECTOR GENE TRANSFER TO THE CNS IN MPS VII

粘多糖沉积症（MPS）疾病是由遗传性酶缺乏引起的多器官病变。发病通常在儿童时期;智力迟钝是一个共同的特点。这些疾病之一MPS VII是由缺乏溶酶体酶β-葡萄糖醛酸酶（GUSB）引起的。MPS VII的小鼠同源物的可用性和检测GUSB活性的各种方法已成为基因转移实验的有价值的工具，因为基因转移的程度和治疗功效都可以在相同的小鼠中进行评估。基于我们新的初步数据，我们提出了实验，严格测试的最有前途的工具之一，基因转移到中枢神经系统，慢病毒载体，使用四个精心设计的载体在体内和体外实验。本修订研究提案的具体目的是：（1a）定量慢病毒载体转导的培养神经元中的GUSB酶活性;（1b）定量载体校正前后培养MPS VII神经元中GAG转换的生化变化;（2a）定量体内四种慢病毒载体的表达并鉴定转导细胞类型;（2b）评估是否可以用慢病毒载体实现长期CNS转基因表达;和（2c）确定MPS VII小鼠脑中编码GUSB的慢病毒载体对病理学的校正程度。

The mucopolysaccharidosis (MPS) diseases are caused by hereditary enzyme deficiencies which result in multi-organ pathology. Onset is usually in childhood; mental retardation is a common feature. One of these diseases, MPS VII, is caused by a lack of the lysosomal enzyme beta- glucuronidase (GUSB). The avail-ability of a mouse homologue of MPS VII and a variety of methods to detect GUSB activity have been valuable tools for gene transfer experiments because both the degree of gene transfer and the therapeutic efficacy can be assessed in the same mice. Based on our new preliminary data, we propose experiments to rigorously test one of the most promising tools for gene transfer into the CNS, the lentiviral vector, using four carefully designed vectors for in vivo and in vitro experiments. The Specific Aims of this revised research proposal are to: (1a) Quantitate GUSB enzyme activity in cultured neurons transduced by lentiviral vectors; (1b) Quantitate biochemical changes in GAG turnover in cultured MPS VII neurons before and after vector correction; (2a) Quantitate expression from four lentiviral vectors in vivo and identify transduced cell types; (2b) Evaluate whether long-term CNS transgene expression can be achieved with lentiviral vectors; and (2c) Determine the extent of correction of pathology by GUSB-encoding lentiviral vectors in the MPS VII mouse brain.