Liu Xiaoming, Yan Ziying, Luo Meihui, Zak Roman, Li Ziyi, Driskell Ryan R, Huang Yumao, Tran Nam, Engelhardt John F
Department of Anatomy and Cell Biology and the Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
J Virol. 2004 Apr;78(8):4165-75. doi: 10.1128/jvi.78.8.4165-4175.2004.
Recombinant adeno-associated virus (rAAV) vectors possess the unique ability to introduce genetic alterations at sites of homology in genomic DNA through a mechanism thought to predominantly involve homologous recombination. We have investigated the efficiency of this approach using a mutant enhanced green fluorescent protein (eGFP) fluorescence recovery assay that facilitates detection of gene correction events in living cells under nonselective conditions. Our data demonstrate that rAAV infection can correct a mutant eGFP transgene at an efficiency of 0.1% in 293 cells, as determined by fluorescence-activated cell-sorting analysis. Gene repair was also confirmed using clonal expansion of GFP-positive cells and sequencing of the eGFP transgene. These results support previous findings demonstrating the efficacy of rAAV for gene targeting. In an effort to improve gene-targeting efficiencies, we evaluated several agents known to increase rAAV transduction (i.e., expression of an expressed gene), including genotoxic stress and proteasome inhibitors, but observed no correlation between the level of gene repair and rAAV transduction. Interestingly, however, our results demonstrated that enrichment of G(1)/S-phase cells in the target population through the addition of thymidine moderately (approximately 2-fold) increased gene correction compared to cells in other cell cycle phases, including G(0)/G1, G(1), and G(2)/M. These results suggest that the S phase of the cell cycle may more efficiently facilitate gene repair by rAAV. Transgenic mice expressing the mutant GFP were used to evaluate rAAV targeting efficiencies in primary fetal fibroblast and tibialis muscles. However, targeting efficiencies in primary mouse fetal fibroblasts were significantly lower (approximately 0.006%) than in 293 cells, and no correction was seen in tibialis muscles following rAAV infection. To evaluate the molecular structures of rAAV genomes that might be responsible for gene repair, single-cell injection studies were performed with purified viral DNA in a mutant eGFP target cell line. However, the failure of direct cytoplasm- or nucleus-injected rAAV DNA to facilitate gene repair suggests that some aspect of intracellular viral processing may be required to prime recombinant viral genomes for gene repair events.
重组腺相关病毒（rAAV）载体具有独特的能力，可通过一种主要涉及同源重组的机制，在基因组DNA的同源位点引入基因改变。我们使用突变增强绿色荧光蛋白（eGFP）荧光恢复测定法研究了这种方法的效率，该测定法有助于在非选择性条件下检测活细胞中的基因校正事件。我们的数据表明，通过荧光激活细胞分选分析确定，rAAV感染可在293细胞中以0.1%的效率校正突变的eGFP转基因。使用GFP阳性细胞的克隆扩增和eGFP转基因测序也证实了基因修复。这些结果支持了先前关于rAAV基因靶向有效性的研究结果。为了提高基因靶向效率，我们评估了几种已知可增加rAAV转导（即表达基因的表达）的试剂，包括基因毒性应激和蛋白酶体抑制剂，但未观察到基因修复水平与rAAV转导之间的相关性。然而，有趣的是，我们的结果表明，与包括G(0)/G1、G(1)和G(2)/M在内的其他细胞周期阶段的细胞相比，通过添加胸苷适度（约2倍）富集靶细胞群体中的G(1)/S期细胞可增加基因校正。这些结果表明，细胞周期的S期可能更有效地促进rAAV介导的基因修复。表达突变GFP的转基因小鼠用于评估rAAV在原代胎儿成纤维细胞和胫前肌中的靶向效率。然而，原代小鼠胎儿成纤维细胞中的靶向效率明显低于293细胞（约0.006%），rAAV感染后胫前肌中未观察到校正。为了评估可能负责基因修复的rAAV基因组的分子结构，在突变的eGFP靶细胞系中对纯化的病毒DNA进行了单细胞注射研究。然而，直接细胞质或细胞核注射的rAAV DNA未能促进基因修复，这表明可能需要细胞内病毒加工的某些方面来启动重组病毒基因组进行基因修复事件。
