Wang Hongjie, Lieber André
Division of Medical Genetics, University of Washington, Box 357720, Seattle, WA 98195, USA.
J Virol. 2006 Dec;80(23):11699-709. doi: 10.1128/JVI.00779-06. Epub 2006 Sep 20.
Random integration of viral gene therapy vectors and subsequent activation or disruption of cellular genes poses safety risks. Major efforts in the field are aimed toward targeting vector integration to specific sites in the host genome. The adeno-associated virus (AAV) Rep78 protein is able to target AAV integration to a specific site on human chromosome 19, called AAVS1. We studied whether this ability could be harnessed to achieve site-specific integration of a 27-kb transgene cassette into a model cell line for human hematopoietic cells (Mo7e). To deliver rep78 and the transgene to Mo7e cells, we used helper-dependent adenovirus (Ad) vectors containing Ad serotype 35 fiber knob domains (HD-Ad). An HD-Ad vector containing the rep78 gene under the control of the globin locus control region (LCR) (Ad.LCR-rep78) conferred Rep78 expression on Mo7e cells. Upon coinfection of Ad.LCR-rep78 with an HD-Ad vector containing a 27-kb globin-LCR-green fluorescent protein (GFP) transgene cassette flanked by AAV inverted terminal repeats (ITRs) (Ad.AAV-LCR-GFP), transduced cells were cloned and expanded (without selection pressure), and vector integration was analyzed in clones with more than 30% GFP-positive cells. Vector integration into the AAVS1 region was seen in 30% of analyzed integration sites, and GFP expression from these integrants was stable over time. Of the remaining integration sites, 25% were within the genomic globin LCR. In almost 90% of sites, transgene integration occurred via the Ad ITR. This indicates that rescue of the AAV ITR-flanked transgene cassette from Ad.AAV-LCR-GFP is not required for Rep78-mediated integration into AAVS1 and that free ends within the vector genome can be created by breaks within the Ad ITRs, whose structure is apparently recognized by cellular "nicking" enzymes. The finding that 55% of all analyzed integration sites were either within the AAVS1 or globin LCR region demonstrates that a high frequency of targeted integration of a large transgene cassette can be achieved in human hematopoietic stem cell lines.
病毒基因治疗载体的随机整合以及随后细胞基因的激活或破坏会带来安全风险。该领域的主要努力方向是将载体整合靶向到宿主基因组的特定位点。腺相关病毒（AAV）的Rep78蛋白能够将AAV整合靶向到人类19号染色体上的一个特定位点，称为AAVS1。我们研究了是否可以利用这种能力将一个27kb的转基因盒定点整合到一种人类造血细胞模型细胞系（Mo7e）中。为了将rep78和转基因递送至Mo7e细胞，我们使用了含有Ad血清型35纤维纽扣结构域的辅助依赖型腺病毒（Ad）载体（HD-Ad）。一种在珠蛋白基因座控制区（LCR）控制下含有rep78基因的HD-Ad载体（Ad.LCR-rep78）在Mo7e细胞中赋予了Rep78表达。当Ad.LCR-rep78与一种含有由AAV反向末端重复序列（ITR）侧翼的27kb珠蛋白-LCR-绿色荧光蛋白（GFP）转基因盒的HD-Ad载体（Ad.AAV-LCR-GFP）共感染后，对转导的细胞进行克隆并扩增（无选择压力），并在GFP阳性细胞超过30%的克隆中分析载体整合情况。在30%的分析整合位点中观察到载体整合到AAVS1区域，并且这些整合体的GFP表达随时间稳定。在其余的整合位点中，25%位于基因组珠蛋白LCR内。在几乎90%的位点，转基因整合是通过Ad ITR发生的。这表明从Ad.AAV-LCR-GFP中拯救AAV ITR侧翼的转基因盒对于Rep78介导的整合到AAVS1中不是必需的，并且载体基因组内的游离末端可以由Ad ITR内的断裂产生，其结构显然被细胞“切口”酶识别。所有分析的整合位点中有55%要么在AAVS1内，要么在珠蛋白LCR区域内，这一发现表明在人类造血干细胞系中可以实现大型转基因盒的高频定点整合。
