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

用于基因治疗的理想的基因递送系统应具有几个特征，包括1）以可预测的或位点特异性的方式将递送的基因整合到靶基因组中，2）转基因的长期表达，和3）递送足够大尺寸的DNA序列以包括所有调节元件的能力。一种新的基因传递系统，称为靶向载体整合（TVI）的发展，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），其优先整合到人类基因组中位于染色体19（19q13.3-qter）上的定义的基因座（称为AAVI 1）处。AAV-Rep蛋白被认为介导含有AAV-ITR（反向末端重复）的DNA序列整合到AAVS 1基因座中，通过在ITR内的GAGC重复和AAVS 1基因座中的相似序列之间形成复合物。有4种不同的Rep蛋白（Rep 78、Rep 68、Rep 52和Rep 40）。为了确定赋予ITR连锁基因位点特异性整合能力的Rep，我们构建了表达单个Rep蛋白的各种质粒。将这些质粒与含有侧接ITR的LacZ表达盒的质粒共转染到293细胞中。进行基于PCR的斑点印迹分析、Southern分析和FISH分析以检测位点特异性整合。结果表明，大Rep（78或68）是ITR连锁基因位点特异性整合所必需的。此外，具有R107 A、K136 A或R138 A的突变体Rep蛋白显示与GAGC重复序列的结合降低，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。本研究对开发更精细的TVI系统具有一定的参考价值。

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.