Lentiviral-Mediated Gene Therapy in Fanconi Anemia-A Mice Reveals Long-Term Engraftment and Continuous Turnover of Corrected HSCs
Lentiviral-Mediated Gene Therapy in Fanconi Anemia-A Mice Reveals Long-Term Engraftment and Continuous Turnover of Corrected HSCs
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DOI：
10.2174/1566523215666150929110903
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发表时间：
2015-01-01
影响因子：
3.6
通讯作者：
Schmidt, Manfred
中科院分区：
文献类型：
--
作者：
Javier Molina-Estevez, F.;Nowrouzi, Ali;Schmidt, Manfred
范可尼贫血是一种 DNA 修复缺陷综合征，主要特征是癌症易感性和骨髓衰竭。为了试图恢复这些患者的造血功能，最近提出了慢病毒载体介导的基因治疗试验。然而，由于迄今为止尚未针对范可尼贫血等 DNA 修复缺陷综合征进行插入肿瘤发生研究，因此我们在对范卡 (-/-) 造血干细胞 (HSC) 进行基因校正后，使用 LAM-PCR 和 454-焦磷酸测序对慢病毒插入位点进行了全基因组筛选。我们的研究首先证明，用专为临床应用设计的慢病毒载体转导 Fanca(-/-) HSC 可有效纠正范可尼贫血再生细胞的表型，且没有任何毒性迹象。对初级和次级受体中超过 6,500 个插入位点的鉴定显示出多克隆重建模式，以及校正的 Fanca(-/-) HSC 克隆的连续更新，但没有针对特定常见整合位点的选择证据。综上所述，我们的数据首次表明，在 DNA 修复缺陷综合征中，慢病毒载体介导的基因治疗可有效纠正受影响 HSC 的表型，并促进体内克隆更新的健康模式。这些研究将对受 DNA 修复综合征（尤其是范可尼贫血）患者的新基因治疗试验的发展产生特殊影响。
Fanconi anemia is a DNA repair-deficiency syndrome mainly characterized by cancer pre-disposition and bone marrow failure. Trying to restore the hematopoietic function in these patients, lentiviral vector-mediated gene therapy trials have recently been proposed. However, because no insertional oncogenesis studies have been conducted so far in DNA repair-deficiency syndromes such as Fanconi anemia, we have carried out a genome-wide screening of lentiviral insertion sites after the gene correction of Fanca(-/-) hematopoietic stem cells (HSCs), using LAM-PCR and 454-pyrosequencing. Our studies first demonstrated that transduction of Fanca(-/-) HSCs with a lentiviral vector designed for clinical application efficiently corrects the phenotype of Fanconi anemia repopulating cells without any sign of toxicity. The identification of more than 6,500 insertion sites in primary and secondary recipients showed a polyclonal pattern of reconstitution, as well as a continuous turnover of corrected Fanca(-/-) HSC clones, without evidences of selection towards specific common integration sites. Taken together our data show, for the first time in a DNA repair-deficiency syndrome, that lentiviral vector-mediated gene therapy efficiently corrects the phenotype of affected HSCs and promotes a healthy pattern of clonal turnover in vivo. These studies will have a particular impact in the development of new gene therapy trials in patients affected by DNA repair syndromes, particularly in Fanconi anemia.