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

• 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

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.

外源DNA的靶向整合（TVI：靶向载体整合）对于安全的基因治疗是期望的。腺相关病毒（AAV）具有将其基因组整合到确定的基因座AAVS 1（19q13.3-qter）中的能力。AAV基因组两端的反向末端重复序列（ITR）和Rep蛋白负责这种位点特异性整合。因此，两个AAV组件，Rep和ITR，用于开发TVI系统。缺乏细胞毒性同时保留介导AAVS 1特异性整合的能力的突变Rep蛋白是该系统的有吸引力的分子。我们构建了含有编码突变型Rep蛋白的基因的质粒，其中N-末端区域中的所有带电氨基酸突变为丙氨酸。我们发现与野生型Rep蛋白相比，几种突变体显示出较低的细胞毒性，但不失去位点特异性整合的能力。我们正在进一步筛选更合适的突变体Rep用于AAVS 1指导的基因整合。为了分析Rep介导的整合的详细机制，我们通过使用RT-PCR技术扩增细胞和转基因序列之间的连接区。TVI系统是有价值的，尤其是当分裂细胞如造血细胞被转导并且需要长期转基因表达时。我们已经表明，这种TVI系统可以将转基因导入K562细胞中的AAVS 1。

项目成果

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Ozawa, K., Ueda, Y., Ito, K., Urabe, M., Kume, A., Sakata, T., and Hasagawa, M.: "A novel selective amplifier gene for hematopoietic stem cell gene therapy." Cancer Res.Ther.Contr.7. 27-31 (1998)

Ozawa, K.、Ueda, Y.、Ito, K.、Urabe, M.、Kume, A.、Sakata, T. 和 Hasakawa, M.：“一种用于造血干细胞基因治疗的新型选择性放大器基因。”

Urabe,M.: "Charged-to-alanine scanning mutagenesis of N-terminal half of adeno-associated virus type 2 Rep78 protein" J.Virol.(in press).

Urabe,M.：“腺相关病毒 2 型 Rep78 蛋白 N 末端一半的电荷转丙氨酸扫描诱变”J.Virol.（出版中）。

Fan,D.: "Behavioral recovery in 6-hydroxydopamine-lesioned rats by cotransduction of striatum with tyrosine hydroxylase and aromatic L-amino acid decarboxylase genes using two separate adeno-associated virus vectors" Hum.Gene.Ther.9. 2527-2535 (1998)

Fan,D.：“使用两种独立的腺相关病毒载体，通过纹状体与酪氨酸羟化酶和芳香族 L-氨基酸脱羧酶基因的共转导，使 6-羟基多巴胺损伤的大鼠行为恢复”Hum.Gene.Ther.9。

Matsda,K.M.: "Development of a modified selective amplifier gene for hematopoietic stem cell gene therapy" Gene Ther.(in press).

Matsda,K.M.：“用于造血干细胞基因治疗的改良选择性放大器基因的开发”Gene Ther.（出版中）。