Cesana Daniela, Ranzani Marco, Volpin Monica, Bartholomae Cynthia, Duros Caroline, Artus Alexandre, Merella Stefania, Benedicenti Fabrizio, Sergi Sergi Lucia, Sanvito Francesca, Brombin Chiara, Nonis Alessandro, Serio Clelia Di, Doglioni Claudio, von Kalle Christof, Schmidt Manfred, Cohen-Haguenauer Odile, Naldini Luigi, Montini Eugenio
San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.
1] San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy [2] Vita Salute San Raffaele University, Milan, Italy.
Mol Ther. 2014 Apr;22(4):774-85. doi: 10.1038/mt.2014.3. Epub 2014 Jan 20.
Self-inactivating (SIN) lentiviral vectors (LV) have an excellent therapeutic potential as demonstrated in preclinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could still pose a significant risk in clinical applications. Taking advantage of novel in vivo genotoxicity assays, we tested a battery of LV constructs, including some with clinically relevant designs, and found that oncogene activation by promoter insertion is the most powerful mechanism of early vector-induced oncogenesis. SIN LVs disabled in their capacity to activate oncogenes by promoter insertion were less genotoxic and induced tumors by enhancer-mediated activation of oncogenes with efficiency that was proportional to the strength of the promoter used. On the other hand, when enhancer activity was reduced by using moderate promoters, oncogenesis by inactivation of tumor suppressor gene was revealed. This mechanism becomes predominant when the enhancer activity of the internal promoter is shielded by the presence of a synthetic chromatin insulator cassette. Our data provide both mechanistic insights and quantitative readouts of vector-mediated genotoxicity, allowing a relative ranking of different vectors according to these features, and inform current and future choices of vector design with increasing biosafety.
自失活（SIN）慢病毒载体（LV）在临床前研究和临床试验中已显示出优异的治疗潜力。然而，较弱的插入诱变机制在临床应用中仍可能构成重大风险。利用新型体内遗传毒性检测方法，我们测试了一系列LV构建体，包括一些具有临床相关设计的构建体，发现启动子插入导致的癌基因激活是早期载体诱导肿瘤发生的最主要机制。通过启动子插入激活癌基因能力丧失的SIN慢病毒载体遗传毒性较低，通过增强子介导的癌基因激活诱导肿瘤的效率与所用启动子的强度成正比。另一方面，当使用中等强度启动子降低增强子活性时，会揭示出肿瘤抑制基因失活导致的肿瘤发生。当内部启动子的增强子活性被合成染色质绝缘子盒屏蔽时，这种机制变得占主导地位。我们的数据提供了载体介导的遗传毒性的机制见解和定量读数，允许根据这些特征对不同载体进行相对排序，并为提高生物安全性的当前和未来载体设计选择提供参考。
