Development of a novel gene therapy technology for site-specific integration of large-sized genes

开发用于大尺寸基因位点特异性整合的新型基因治疗技术

基本信息

批准号：
08457280
负责人：
OZAWA Keiya
金额：
$ 4.54万
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：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457280/
关键词：
Gene therapy Gene transfer integration AAV AVVS1 locus Rep ITR TVI method AAVS1

项目摘要

The desirable gene-delivery system for gene therapy should have several features, including 1) integration of the delivered gene in a predictable or site-specific manner into the target genome, 2) long-term expression of the transgene, and 3) the ability to deliver DNA sequences of sufficiently large size to include all the regulatory elements. The development of a novel gene delivery system, termed targeted vector integration (TVI), is currently in progress to fulfilll the above prerequisites. The system is based on adeno-associated virus (AAV) which preferentially integrates into the human genome at a defined locus, called AAVI1, on chromosome 19 (19q13.3-qter). The AAV-Rep proteins are considered to mediate integration of DNA sequence containing AAV-ITR (inverted terminal repeat) into AAVS1 locus, through the formation of a complex between GAGC repeats within ITR and a similar sequence in AAVS1 locus. There are 4 different Rep proteins (Rep78, Rep68, Rep52, and Rep40). Aiming at determining the Rep (s), which confer the ability of site-specific integration of ITR-linked genes, we constructed various plasmids that express individual Rep proteins. These plasmids were co-transfected into 293 cells with the plasmid containing a LacZ expression cassette flanked by ITRs. A PCR-based dot blot assay, Southern analysis, and FISH analysis were conducted to detect site-specific integration. The results showed that the large Rep (78 ot 68) is necessary for the site-specific integration of ITR-linked genes. In addition, mutant Rep proteins with R107A,K136A,or R138A showed the decreased binding to GAGC repeat and no nicking activity. These mutants also lost the site-specific integration activity. The present study will be valuable to develop the more refined TVI system.

用于基因治疗的理想基因传递系统应具有几个特征，包括 1) 以可预测或位点特异性的方式将传递的基因整合到目标基因组中，2) 转基因的长期表达，以及 3)提供足够大的 DNA 序列以包含所有调控元件。目前正在开发一种称为靶向载体整合（TVI）的新型基因传递系统，以满足上述先决条件。该系统基于腺相关病毒 (AAV)，该病毒优先整合到人类基因组中 19 号染色体 (19q13.3-qter) 上的一个定义位点（称为 AAVI1）上。 AAV-Rep 蛋白被认为通过在 ITR 内的 GAGC 重复序列与 AAVS1 基因座中的相似序列之间形成复合物，介导包含 AAV-ITR（反向末端重复序列）的 DNA 序列整合到 AAVS1 基因座中。有 4 种不同的 Rep 蛋白（Rep78、Rep68、Rep52 和 Rep40）。为了确定具有 ITR 相关基因位点特异性整合能力的 Rep，我们构建了表达各个 Rep 蛋白的各种质粒。这些质粒与含有侧翼为 ITR 的 LacZ 表达盒的质粒共转染至 293 细胞中。进行基于 PCR 的斑点印迹测定、Southern 分析和 FISH 分析来检测位点特异性整合。结果表明，大Rep (78 ot 68)对于ITR连锁基因的位点特异性整合是必需的。此外，具有 R107A、K136A 或 R138A 的突变 Rep 蛋白显示出与 GAGC 重复序列的结合减少，并且没有切口活性。这些突变体也失去了位点特异性整合活性。本研究对于开发更精细的 TVI 系统具有重要意义。