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Development of the new retroviral vector system for stem cell gene therapy.

开发用于干细胞基因治疗的新型逆转录病毒载体系统。

基本信息

批准号：
18591180
负责人：
ONODERA Masafumi
金额：
$ 2.04万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18591180/
关键词：
gene virus immunology translational research regenrative medicine

项目摘要

We have done experiments below to develop the new retroviral transduction system used in stem cell gene therapy clinical trials.1) Cloning of the cytochrome b558 heavy chain (CYBB) gene: The target disease is X-linked chronic granulomatous disease (CGD) in which the CYBB gene is mutated and its coding protein gp9l^<phox> is non-functional, resulting in severe bacterial and fungal infections characterized by formation of granulomas. The open reading frame (ORF) of the gene was amplified by the PCR technique using cDNA reverse-transcribed from mRNA isolated from polynuclear cells of healthy control. The sequence of the ORF (1603bp) was certified.2) Construction of the gene silencing resistant retroviral vector GCDNsap: GCDNsap was constructed by inserting mutations into the clinical retroviral vector Gcsap not to bind to the negative transcription factors such as YY1 and Trim28. The vector allows stable expression of the transduced genes in immature cells such as hematopoietic stem cells or embryonic stem cells.3) Establishment of retroviral producer clones: The vector cloned with the CYBB cDNA was converted to the corresponding retrovirus by transduction into the 293gpg cell that expressed vesicular stomatitis virus derived G protein (VSV-G) under the control of tetracycline (tet off system). The virus titer was approximately 2.0x10^5 ml/I. U. assayed on Jurkat cell.4) Expression of gp91^<phox> in transduced cells: Expression of the gene on the surface or cytoplasm of transduced cells was determined by FACS. Transduction experiments into human CD34^+ cells are under way

我们已经做了下面的实验来开发用于干细胞基因治疗临床试验的新的逆转录病毒转导系统。1)细胞色素b558重链（CYBB）基因的克隆：目标疾病是x连锁慢性肉芽肿病（CGD）， CYBB基因突变，其编码蛋白gp9l^<phox>无功能，导致以肉芽肿形成为特征的严重细菌和真菌感染。利用健康对照多核细胞mRNA反转录cDNA， PCR扩增该基因的开放阅读框（ORF）。ORF序列（1603bp）得到了验证。2)基因沉默耐药逆转录病毒载体GCDNsap的构建：通过在临床逆转录病毒载体Gcsap中插入不与YY1、Trim28等负转录因子结合的突变，构建GCDNsap。该载体允许在未成熟细胞（如造血干细胞或胚胎干细胞）中稳定表达转导基因。3)逆转录病毒产生克隆的建立：在四环素（tet off系统）控制下，将CYBB cDNA克隆的载体转导到表达水疱性口炎病毒衍生G蛋白（VSV-G）的293gpg细胞中，转化为相应的逆转录病毒。病毒滴度约为2.0x10^5 ml/I。在Jurkat细胞上进行分析。4) gp91^<phox>在转导细胞中的表达：通过流式细胞仪检测该基因在转导细胞表面或细胞质上的表达。人类CD34^+细胞的转导实验正在进行中