Oligonucleotide-mediated gene targeting in human hepatocytes: Implications of mismatch repair
Oligonucleotide-mediated gene targeting in human hepatocytes: Implications of mismatch repair
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DOI：
10.1089/oli.2008.0120
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发表时间：
2008-06-01
期刊：
影响因子：
--
通讯作者：
Rubin, Emanuel
中科院分区：
文献类型：
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作者：
Igoucheva, Olga;Alexeev, Vitali;Rubin, Emanuel
使用病毒载体治疗肝病，特别是先天性疾病，尤其是对病毒抗原的免疫反应是困难的。因此，在肝细胞中进行基因转移的非病毒方法也得到了探索。小分子人工合成的单链寡核苷酸(ODN)可在哺乳动物细胞基因组中产生靶向性改变，在非病毒基因治疗方面具有巨大的潜力。为了验证ODN介导的人肝细胞染色体DNA内基因修复的可行性，建立了两个稳定整合突变型报告基因的新细胞系，即新霉素和增强型绿色荧光蛋白。用专门为修复设计的ODN靶向这些细胞，导致报告基因的单点突变的定点和永久性基因转化。此外，基因改变的频率高度依赖于细胞的有丝分裂活性，表明增殖状态是人肝细胞靶向成功的重要因素。不同细胞培养条件下DNA修复基因的表达谱结合RNA干扰分析表明，活跃生长的肝细胞中的错配修复(MMR)为ODN介导的有效基因修复设置了强大的障碍。针对MSH2 mRNA的短发夹状RNA(ShRNAs)抑制了肝细胞中MSH2的活性，导致基因修复率增加25-30倍，提示MMR对ODN介导的基因修复有负面影响。综上所述，这些数据表明，在适当的条件下，非病毒染色体靶向可能是一种可行的肝病基因治疗方法。
Gene therapy using viral vectors for liver diseases, particularly congenital disorders, is besought with difficulties, particularly immunologic reactions to viral antigens. As a result, nonviral methods for gene transfer in hepatocytes have also been explored. Gene repair by small synthetic single-stranded oligodeoxynucleotides (ODNs) produces targeted alterations in the genome of mammalian cells and represents a great potential for nonviral gene therapy. To test the feasibility of ODN-mediated gene repair within chromosomal DNA in human hepatocytes, two new cell lines with stably integrated mutant reporter genes, namely neomycin and enhanced green fluorescent protein were established. Targeting theses cells with ODNs specifically designed for repair resulted in site-directed and permanent gene conversion of the single-point mutation of the reporter genes. Moreover, the frequency of gene alteration was highly dependent on the mitotic activity of the cells, indicating that the proliferative status is an important factor for successful targeting in human hepatocytes. cDNA array expression profiling of DNA repair genes under different cell culture conditions combined with RNA interference assay showed that mismatch repair (MMR) in actively growing hepatocytes imposes a strong barrier to efficient gene repair mediated by ODNs. Suppression of MSH2 activity in hepatocytes transduced with short hairpin RNAs (shRNAs) targeted to MSH2 mRNA resulted in 25- to 30-fold increase in gene repair rate, suggesting a negative effect of MMR on ODN-mediated gene repair. Taken together, these data suggest that under appropriate conditions nonviral chromosomal targeting may represent a feasible approach to gene therapy in liver disease.