APPLICATION OF GENE TRANSFER TO CORRECT INHERITED ENZYME DEFICIENCIES

含人全长编码序列的全长cDNA的分离 葡萄糖脑苷酶允许在模型研究中使用这种酶 使用重组方法纠正遗传的酶缺陷， 特别是基因转移。特别适合于基因治疗的是那些 紊乱(如高谢病)中储存未降解的 底物限于具有可访问的前体群体的细胞。 在这些情况下，将正常基因转移到骨髓干细胞 既合理又可取。使用源自以下内容的信息 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，以及移植成功的可能性 合理预测了特定的结构和基因整合。虽然 我们已经成功地利用逆转录病毒载体转移和 在宿主鼠和人细胞系中表达葡萄糖脑苷酶， 人类基因治疗实验的先决条件包括持续 转移基因在后续细胞世代中的表达和 不存在对宿主有害的重组事件。这些方面 正在被定义。为了更好地描述基因转移和表达 机制2型高雪氏细胞系被用作受体 逆转录病毒构建物。在这个模型中，单抗8E4不 识别类型2变异的葡萄糖脑苷酶。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，以及 它们允许监测这两种酶的恢复程度 基因转移产生的活性和蛋白质表位。紧随其后的是 显示葡萄糖脑苷酶水平恢复到这些水平以及 在培养的1型和3型细胞系中， 葡萄糖脑苷酶基因对骨髓干细胞的作用将使用 老鼠和非人灵长类动物。本研究的目的是为了更好地应用 这些重组DNA治疗高谢病的策略和 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.