Characterization of a bipartite recombinant adeno-associated viral vector for site-specific integration.
Characterization of a bipartite recombinant adeno-associated viral vector for site-specific integration.
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DOI：
10.1089/hum.2007.056
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发表时间：
2007-10
影响因子：
4.2
通讯作者：
C. Zhang;N. Cortez;K. Berns
中科院分区：
文献类型：
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作者：
C. Zhang;N. Cortez;K. Berns
腺相关病毒2型(AAV2)是已知的唯一整合到人类基因组特定位点的病毒。AAVS1位于19号染色体的Q臂上的13.4位。AAV是目前流行的人类基因治疗载体。然而，目前的载体并不包含位点特异性整合所需的两个重要元件，即rep基因或P5启动子，尽管它们确实在人类基因组中的随机位置以低频率整合。我们已经设计了一种将转基因插入AAVS1的双向载体。其中一个成分rAAVSVAV2含有rep基因，由猿猴病毒40早期启动子而不是P5启动子驱动。因此，P5内的整合增强子元件(IEE)已被删除，该元件大大增强了特定部位的整合。另一个组分rAAVP5UF11包含P5IEE加上带有相关调控元件的转基因。我们已经建立了转导的HeLa细胞的克隆，其中大多数似乎将转基因插入到AAVS1中。我们没有检测到任何地方插入了rep的克隆。在最佳感染复数和rAAVSVAV2与rAAVP5UF11的比例下，转基因高效地整合到AAVS1上(60%)。最重要的是，含有AAVS1位点特异性整合绿色荧光蛋白(GFP)的克隆细胞系是健康的，并且在35代中稳定表达GFP。高频率整合于特定位点的AAV载体在体外转导祖细胞和干细胞方面具有显着优势，工程细胞可用于人类基因治疗。AAV定点整合基因治疗为需要长期基因表达的疾病提供了一种新的治疗方法。
Adeno-associated virus type 2 (AAV2) is the only virus known to integrate into a specific locus in the human genome. The locus, AAVS1, is on the q arm of chromosome 19 at position 13.4. AAV is currently a popular vector for human gene therapy. However, current vectors do not contain two important elements needed for site-specific integration, that is, the rep gene or the P5 promoter, although they do integrate with low frequency at random locations in the human genome. We have designed a bipartite vector that does insert the transgene into AAVS1. One component, rAAVSVAV2, contains the rep gene, driven by the simian virus 40 early promoter rather than the P5 promoter. Thus, the integration enhancer element (IEE) within P5, which greatly enhances site-specific integration, has been deleted. The other component, rAAVP5UF11, contains the P5 IEE plus the transgene with associated regulatory elements. We have created clones of transduced HeLa cells, most of which appear to have the transgene inserted in AAVS1. We have not detected any clones that have rep inserted anywhere. With the optimal multiplicity of infection and ratio of rAAVSVAV2 and rAAVP5UF11, the transgene integrated specifically at AAVS1 with high efficiency (>60%). Most importantly, the cloned cell lines with the AAVS1 site-specific integrated green fluorescent protein (GFP) were healthy and stably expressed GFP for 35 passages. An AAV vector that would integrate at a specific site with high frequency could offer significant advantage in the transduction of progenitor cells and stem cells ex vivo and engineered cells could be used for human gene therapy. AAV site-specific integration gene therapy could provide a novel approach for diseases that need long-term gene expression.