Papanikolaou Eleni, Paruzynski Anna, Kasampalidis Ioannis, Deichmann Annette, Stamateris Evangelos, Schmidt Manfred, von Kalle Christof, Anagnou Nicholas P
1] Laboratory of Biology, University of Athens School of Medicine, Athens, Greece [2] Laboratory of Cell and Gene Therapy, Centre for Basic Research ΙΙ, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
Mol Ther. 2015 Apr;23(4):683-96. doi: 10.1038/mt.2014.246. Epub 2014 Dec 19.
Gene therapy utilizing lentiviral-vectors (LVs) is postulated as a dynamic therapeutic alternative for monogenic diseases. However, retroviral gene transfer may cause insertional mutagenesis. Although, such risks had been originally estimated as extremely low, several reports of leukemias or clonal dominance, have led to a re-evaluation of the mechanisms operating in insertional mutagenesis. Therefore, unraveling the mechanism of retroviral integration is mandatory toward safer gene therapy applications. In the present study, we undertook an experimental approach which enabled direct correlation of the cell cycle stage of the target cell with the integration profile of LVs. CD34(+) cells arrested at different stages of cell cycle, were transduced with a GFP-LV. LAM-PCR was employed for integration site detection, followed by microarray analysis to correlate transcribed genes with integration sites. The results indicate that ~10% of integration events occurred in actively transcribed genes and that the cell cycle stage of target cells affects integration pattern. Specifically, use of thymine promoted a safer profile, since it significantly reduced integration within cell cycle-related genes, while we observed increased possibility for integration into genes related to development, and decreased possibility for integration within cell cycle and cancer-related genes, when transduction occurs during mitosis.
利用慢病毒载体（LVs）的基因治疗被认为是治疗单基因疾病的一种动态治疗选择。然而，逆转录病毒基因转移可能会导致插入诱变。尽管最初估计这种风险极低，但几例白血病或克隆优势的报道促使人们对插入诱变的作用机制进行重新评估。因此，阐明逆转录病毒整合机制对于更安全的基因治疗应用至关重要。在本研究中，我们采用了一种实验方法，能够将靶细胞的细胞周期阶段与慢病毒载体的整合图谱直接关联起来。将停滞在细胞周期不同阶段的CD34(+)细胞用绿色荧光蛋白慢病毒载体（GFP-LV）进行转导。采用连接介导的PCR（LAM-PCR）检测整合位点，随后进行微阵列分析，以将转录基因与整合位点相关联。结果表明，约10%的整合事件发生在活跃转录的基因中，并且靶细胞的细胞周期阶段会影响整合模式。具体而言，使用胸腺嘧啶可促进更安全的图谱，因为它显著减少了在细胞周期相关基因内的整合，而当在有丝分裂期间进行转导时，我们观察到整合到与发育相关基因中的可能性增加，而整合到细胞周期和癌症相关基因内的可能性降低。
