Development of the method for chromosomal site-specific integration of transgenes using AAV and its application to hematopoietic cells

AAV转基因染色体位点特异性整合方法的开发及其在造血细胞中的应用

基本信息

批准号：
10470213
负责人：
OZAWA Keiya
金额：
$ 6.59万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10470213/
关键词：
Gene theray Gene transfer Integration AAV AAVSI locus Rep ITR TVI method

项目摘要

Targeted integration of foreign DNA (TVI:targeted vector integration) is desirable for safe gene therapy. Adeno-associated virus (AAV) has the ability to integrate its genome into a defined locus, AAVS1 (19q13.3-qter). The inverted terminal repeat (ITR) at both ends of the AAV genome and Rep proteins are responsible for this site-specific integration. Therefore, two AAV components, Rep and ITR, are utilized to develop the TVI system. A mutant Rep protein that lacks cytotoxicity while retaining the ability to mediate AAVS1-specific integration is an attractive molecule for this system. We constructed plasmids containing the genes encoding mutant Rep proteins, in which all the charged amino acids in the N-terminal region were mutated to alanine. We found several mutants showed lower cytotoxicity, compared to the wild type Rep protein, without losing the ability of the site-specific integration. We are further screening a more suitable mutant Rep for the AAVS1-directed integration of genes. To analyze detailed mechanism of Rep-mediated integration, we amplified junctional regions between cellular and transgene sequences by using an Alu-PCR technique. The TVI system is valuable especially when dividing cells such as hematopoietic cells are transduced and long-term transgene expression is needed. We have showed that this TVI system could introduce a transgene into AAVS1 in K562 cells.

对于安全的基因治疗，需要靶向外国DNA（TVI：靶向载体整合）的靶向整合。腺相关病毒（AAV）具有将其基因组整合到定义的基因座AAVS1（19q13.3-qter）中的能力。 AAV基因组和REP蛋白两端的倒末端重复（ITR）是该位点特异性集成的原因。因此，将两个AAV组件（REP和ITR）用于开发TVI系统。缺乏细胞毒性的突变体REP蛋白，同时保持介导AAVS1特异性整合的能力是该系统的有吸引力的分子。我们构建了包含编码突变体REP蛋白的基因的质粒，其中N末端区域的所有电荷氨基酸都被突变为丙氨酸。我们发现，与野生型REP蛋白相比，几个突变体显示出较低的细胞毒性，而不会失去位点特异性整合的能力。我们正在进一步筛选一个更合适的突变体REP，以供基因的AAVS1定向整合。为了分析REP介导的整合的详细机制，我们使用ALU-PCR技术扩增了细胞和转基因序列之间的连接区域。 TVI系统是有价值的，尤其是在转导造血细胞（例如造血细胞）并需要长期转基因表达时。我们已经表明，该TVI系统可以将转基因引入K562细胞中的AAVS1。