APPLICATION OF GENE TRANSFER TO CORRECT INHERITED ENZYME DEFICIENCIES

人源性大肠杆菌cDNA的分离 葡糖脑苷脂酶允许在模型研究中使用这种酶， 使用重组方法纠正遗传性酶缺陷， 特别是基因转移。 特别适合基因治疗的是那些 疾病（如戈谢病），其中储存的未降解的 基底被限制在具有可接近的前体群体的细胞中。 在这些情况下，正常基因转移到骨髓干细胞， 既合理又可取。 利用来自 蛋白质研究，突变细胞中基因转移的产物可以是 与正常细胞相比， 合理预测特定构建体和基因整合。 虽然 我们已经成功地利用逆转录病毒载体转移， 在宿主小鼠和人细胞系中表达葡糖脑苷脂酶， 人类基因治疗实验的先决条件包括 在随后的细胞世代中表达转移的基因， 不存在对宿主有害的重组事件。 这些方面 正在被定义。 为了更好地表征基因转移和表达， 2型Gaucher细胞系被用作受体。 逆转录病毒构建体。 在该模型中，单克隆抗体8E4不 识别2型葡萄糖脑苷脂酶变体。 因此，这些细胞提供 缺乏8E4识别的正常酶表位的宿主细胞系，和 它们允许监测两种酶的恢复程度 活性和由基因转移产生的蛋白质表位。 后 证明葡萄糖脑苷脂酶水平恢复至这些水平， 培养物中的1型和3型细胞系， 骨髓干细胞中的葡糖脑苷脂酶基因将使用 小鼠和非人类灵长类动物。 本研究的目的是应用 高雪氏病的重组DNA治疗策略， 其他遗传性疾病。

The isolation of cDNA containing the full sequence encoding human glucocerebrosidase has permitted the use of this enzyme in model studies to correct inherited enzyme deficiences using recombinant methodologies, specifically gene transfer. Particularly suited for gene therapy are those disorders (such as Gaucher's disease) where the storage of undegraded substrate is confined to cells having an accessible precursor population. In these cases, the transfer of normal genes to stem cells in bone morrow would be both rational and desirable. Using information derived from protein studies, the products of gene transfer in mutant cells can be compared to that of normal cells, and the likelihood for success of a particular construct and gene integration rationally predicted. Although we have been successful in utilizing retroviral vectors to transfer and express glucocerebrosidase in host mouse and human cell lines, prerequisites for human gene therapy experiments include sustained expression of the transferred gene during subsequent cell generations and the absence of recombination events detrimental to the host. These aspects are being defined. To better characterize the gene transfer and expression mechanisms Type 2 Gaucher cell lines were utilized as recipients of the retroviral constructs. In this model, monoclonal antibody 8E4 does not recognize the Type 2 variant glucocerebrosidase. Thus, these cells provide a host cell line lacking the normal enzyme epitope recognized by 8E4, and they allow the monitoring of the degree of restoration of both enzyme activity and protein epitopes resulting from gene transfer. Following the demonstration of restored glucocerebrosidase levels to these as well as Type 1 and Type 3 cell lines in culture, the transfer of the glucocerebrosidase gene to bone marrow stem cells will be evaluated using mice and non-human primates. The goal of this research is the application of these recombinant DNA therapeutic strategies to Gaucher's disease and other genetic disorders.