Development of a model system for somatic cell gene therapy using hematopoietic stem cells and lymphoid cells.

使用造血干细胞和淋巴细胞开发体细胞基因治疗模型系统。

基本信息

批准号：
04557034
负责人：
NAKAUCHI Hiromitsu
金额：
$ 10.11万
依托单位：
University of Tsukuba (1994)The Institute of Physical and Chemical Research (1992-1993)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

1)Establishment of a mouse model system of a somatic cell gene therapy with an immunological safety system.Mouse fibroblasts were transfected with an expression vector containing human insulin cDNA.When one million of these fibroblasts were injected i.p.into streptozocin-induced diabetic mice, we observed a significant improvement of the blood glucose concentrations. To ensure the safety of somatic cell gene therapy, the proinsulin-producing fibroblasts were further transfected with an expression vector containing CD8.2, a gene encoding a cell surface antigen.Somatic gene therapy with these doubly transfected cells followed by the administration of a monoclonal antibody to CD8.2 resulted in an initial decrease of blood glucose concentrations followed by the permanent recurrence of hyperglycemia, thus proving the complete removal of the transplanted cells.2)Purification of the hematopoietic stem cells in adult mouse bone marrow.Using monoclonal antibodies and FACS,we established a system by which the hematopoietic stem cells can be isolated. The results indicate that c-kit+Sca-1+CD34-Lin-cells constituting only 0.008% of bone marrow mononuclear cells are highly enriched for long-term marrow repopulating ability. We were able to reconstitute bone marrow of a lethally irradiated mouse by an injection of a single c-kit+Sca-1+CD34-Lin-cell. We also demonstrated that short-term culture of enriched stem cells under certain cytokines increase the efficiency of retroviral transdution into the hematopoietic stem cells.

1）建立具有免疫安全系统的体细胞基因疗法的小鼠模型系统。小鼠成纤维细胞用含有人类胰岛素cDNA的表达载体转染。将其中一百万个这些成纤维细胞注入I.P.Into链球菌诱导的糖尿病诱导的糖尿病小鼠时，我们观察到血糖浓度的显着改善。为了确保体细胞基因疗法的安全性，用含有CD8.2的表达载体进一步转染了产生促蛋白的成纤维细胞，该载体是编码细胞表面抗原的基因。使用这些双重转染的细胞进行单克线粒体的基因疗法。 CD8.2的抗体导致血糖浓度的初始降低，然后是高血糖的永久性复发，从而证明了移植细胞的完全去除。2）纯化成年小鼠骨髓中造血干细胞的纯化。 FACS，我们建立了一个可以分离造血干细胞的系统。结果表明，仅构成0.008％的骨髓单核细胞的C-KIT+SCA-1+CD34-LIN细胞高度富含长期的骨髓再植物能力。我们能够通过注射单个C-KIT+SCA-1+CD34-LIN细胞来重建致命辐照小鼠的骨髓。我们还证明，在某些细胞因子下，富集的干细胞的短期培养增加了逆转录病毒转导向造血干细胞的效率。