CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors
CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors
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DOI：
10.1038/gt.2011.70
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发表时间：
2012-01-01
期刊：
影响因子：
5.1
通讯作者：
Mermod, N.
中科院分区：
文献类型：
--
作者：
Gaussin, A.;Modlich, U.;Mermod, N.
基于基因转移的治疗方法极大地受益于一些病毒载体有效整合在细胞基因组内并确保新获得的转基因持续传递到靶细胞后代的能力。然而，整合的前病毒已与表观遗传学的影响，可能会影响表达的转基因和细胞基因接近载体整合位点。遗传绝缘子元件的开发可以通过其作为限制转基因沉默的屏障和/或作为增强子阻断剂防止载体增强子激活内源基因的能力来克服这两个问题。我们建立了基于质粒的定量分析系统来筛选增强子-阻断剂和屏障遗传元件。短的合成绝缘子，结合到核因子-I蛋白家族转录因子被确定为发挥增强子阻滞剂和屏障功能，并进行了比较的绝缘子蛋白CTCF（CCCTC结合因子）的结合位点。包围这些绝缘子元件的γ-逆转录病毒载体以与其非绝缘对应物相似的滴度产生，并且在体外永生化测定中证明具有较低的遗传毒性，产生Evi 1癌基因表达的较低激活和骨髓细胞的克隆扩增减少。Gene Therapy（2012）19，15-24; doi：10.1038/gt.2011.70; 2011年5月12日在线发表
Gene transfer-based therapeutic approaches have greatly benefited from the ability of some viral vectors to efficiently integrate within the cell genome and ensure persistent transmission of newly acquired transgenes to the target cell progeny. However, integration of provirus has been associated with epigenetic repercussions that may influence the expression of both the transgene and cellular genes close to vector integration loci. The exploitation of genetic insulator elements may overcome both issues through their ability to act as barriers that limit transgene silencing and/or as enhancer-blockers preventing the activation of endogenous genes by the vector enhancer. We established quantitative plasmid-based assay systems to screen enhancer-blocker and barrier genetic elements. Short synthetic insulators that bind to nuclear factor-I protein family transcription factors were identified to exert both enhancer-blocker and barrier functions, and were compared to binding sites for the insulator protein CTCF (CCCTC-binding factor). Gamma-retroviral vectors enclosing these insulator elements were produced at titers similar to their non-insulated counterparts and proved to be less genotoxic in an in vitro immortalization assay, yielding lower activation of Evi1 oncogene expression and reduced clonal expansion of bone marrow cells. Gene Therapy (2012) 19, 15-24; doi:10.1038/gt.2011.70; published online 12 May 2011