Retroviral insertional mutagenesis (RIM) is both a relevant risk in gene therapy and a powerful tool for identifying genes that enhance the competitiveness of repopulating hematopoietic stem and progenitor cells (HSPCs). However, focusing only on the gene closest to the retroviral vector insertion site (RVIS) may underestimate the effects of RIM, as dysregulation of distal and/or multiple genes by a single insertion event was reported in several studies. As a proof of concept, we examined the common insertion site (CIS) Bcl-x_L, which revealed seven genes located within ±150 kb from the RVIS for our study. We confirmed that Bcl-x_L enhanced the competitiveness of HSPCs, whereas the Bcl-x_L neighbor Id1 hindered HSPC long-term repopulation. This negative influence of Id1 could be counteracted by co-expressing Bcl-x_L. Interestingly, >90% of early reconstituted myeloid cells were found to originate from transduced HSPCs upon simultaneous overexpression of Bcl-x_L and Id1, which implies that Bcl-x_L and Id1 can collaborate to rapidly replenish the myeloid compartment under stress conditions. To directly compare the competitiveness of HSPCs conveyed by multiple transgenes, we developed a multiple competitor competitive repopulation (MCCR) assay to simultaneously screen effects on HSPC repopulating capacity in a single mouse. The MCCR assay revealed that multiple genes within a CIS can have positive or negative impact on hematopoiesis. Furthermore, these data highlight the importance of studying multiple genes located within the proximity of an insertion site to understand complex biological effects, especially as the number of gene therapy patients increases.

中文翻译：

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Bcl-xL 周围的多个基因，一个常见的逆转录病毒插入位点，可以单独或共同影响造血

逆转录病毒插入诱变 (RIM) 既是基因治疗的相关风险，也是识别增强造血干细胞和祖细胞 (HSPC) 竞争力的强大工具。然而，仅关注最接近逆转录病毒载体插入位点 (RVIS) 的基因可能会低估 RIM 的影响，因为在几项研究中报告了单个插入事件导致远端和/或多个基因的失调。作为概念验证，我们检查了共同插入位点 (CIS) Bcl-x _L，它揭示了位于 RVIS ±150 kb 范围内的七个基因用于我们的研究。我们证实Bcl-x _L增强了 HSPCs 的竞争力，而Bcl-x _L邻居Id1阻碍了 HSPC 的长期增殖。Id1的这种负面影响可以通过共同表达Bcl-x _L来抵消。有趣的是，在Bcl-x _L和Id1同时过表达后，发现 > 90% 的早期重组骨髓细胞源自转导的 HSPC ，这意味着Bcl-x _L和Id1可以协作在压力条件下快速补充骨髓隔室。为了直接比较多个转基因携带的 HSPC 的竞争力，我们开发了一种多竞争者竞争性再增殖 (MCCR) 测定法，以同时筛选对单个小鼠中 HSPC 再增殖能力的影响。MCCR 分析显示 CIS 内的多个基因对造血有积极或消极的影响。此外，这些数据强调了研究位于插入位点附近的多个基因以了解复杂的生物学效应的重要性，尤其是随着基因治疗患者数量的增加。