Research Department Cell and Gene Therapy, Clinic for Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.
Exp Hematol. 2013 Jan;41(1):28-38.e3. doi: 10.1016/j.exphem.2012.09.003. Epub 2012 Sep 16.
Retroviral gene marking has been used successfully in preclinical and clinical transplantation settings. Highly sensitive techniques for vector insertion-site determination, such as linear amplification-mediated polymerase chain reaction (LAM-PCR) in conjunction with next-generation sequencing, have been introduced to assess the composition of gene-marked hematopoiesis at a single-cell level. Here we used these novel techniques for directly comparing clonal reconstitution kinetics in mice transplanted with bone-marrow-derived stem cells genetically marked with either a standard, spleen focus-forming virus long terminal repeat (LTR)-driven γ-retroviral, or a lentiviral self-inactivating vector containing an identical but internal spleen focus-forming virus-derived enhancer/promoter. We observed that the use of the lentiviral self-inactivating vector for gene marking was associated with a broader repertoire of differently marked hematopoietic clones. More importantly, we found a significantly higher probability of insertions in growth-promoting, clonal-dominance-associated genes in the spleen focus-forming virus LTR-driven γ-retroviral vector at later time points of analysis. Based on our data, we suggest that the combined use of LAM-PCR and next-generation sequencing represents a potent tool for the analysis of clonal reconstitution kinetics in the context of gene marking with integrated vectors. At the same time, our findings prove that the use of multiple restriction enzymes for LAM-PCR is indispensable to detect most or ideally all individual stem cell clones contributing to hematopoiesis. We have also found that techniques such as quantitative PCR can be helpful to retrospectively analyze reconstitution kinetics for individual hematopoietic stem cell clones. Finally, our results confirm the notion that marking with lentiviral self-inactivating vectors is associated with a lower risk of genotoxicity as compared with γ-retroviral LTR vectors.
逆转录病毒基因标记已成功应用于临床前和临床移植环境中。已经引入了用于载体插入位点确定的高度敏感技术，例如线性扩增介导的聚合酶链反应（LAM-PCR）与下一代测序相结合，以评估单个细胞水平的基因标记造血的组成。在这里，我们使用这些新的技术直接比较了用骨髓源性干细胞移植的小鼠的克隆重建动力学，这些干细胞经过基因标记，使用的是标准的、脾焦点形成病毒长末端重复（LTR）驱动的γ-逆转录病毒，或含有相同但内部脾焦点形成病毒衍生增强子/启动子的慢病毒自我失活载体。我们观察到，使用慢病毒自我失活载体进行基因标记与不同标记的造血克隆的更大范围有关。更重要的是，我们发现，在分析的后期时间点，在脾焦点形成病毒 LTR 驱动的γ-逆转录病毒载体中，与生长促进、克隆优势相关的基因中的插入的可能性显著增加。基于我们的数据，我们建议 LAM-PCR 和下一代测序的组合使用是分析整合载体基因标记的克隆重建动力学的有力工具。同时，我们的发现证明，对于 LAM-PCR 来说，使用多种限制酶是必不可少的，以检测对造血有贡献的大多数或理想上所有单个干细胞克隆。我们还发现，定量 PCR 等技术可以帮助回顾性地分析单个造血干细胞克隆的重建动力学。最后，我们的结果证实了这样的观点，即与γ-逆转录病毒 LTR 载体相比，使用慢病毒自我失活载体进行标记与较低的遗传毒性风险相关。
