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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该有几个特点，包括1)以可预测的或特定位点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)能够传递足够大的DNA序列以包含所有调控元件。一种新型基因传递系统的开发，称为靶向载体整合（TVI），目前正在进行中，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的第19号染色体（19q13.3-qter）上一个称为AAVI1的特定位点。AAV-Rep蛋白被认为介导含有AAV-ITR（反向末端重复序列）的DNA序列整合到AAVS1位点，通过在ITR内的GAGC重复序列与AAVS1位点的类似序列之间形成复合物。有4种不同的Rep蛋白（Rep78、Rep68、Rep52和Rep40）。为了确定Rep (s)，它赋予itr连接基因的位点特异性整合能力，我们构建了表达单个Rep蛋白的各种质粒。将这些质粒与含有LacZ表达盒的质粒共转染293细胞。采用基于pcr的点印迹分析、Southern分析和FISH分析来检测位点特异性整合。结果表明，大Rep（78 ~ 68）是itr连锁基因位点特异性整合所必需的。此外，R107A、K136A和R138A突变体Rep蛋白与GAGC重复序列的结合减少，无切口活性。这些突变体也失去了位点特异性整合活性。本文的研究对开发更完善的TVI系统具有一定的参考价值。