Multiple Genes Surrounding Bcl-xL, a Common Retroviral Insertion Site, Can Influence Hematopoiesis Individually or in Concert

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.

Automated summary

Retroviral insertional mutagenesis is a risk in gene therapy and can be used to identify genes that enhance hematopoietic stem cell competitiveness. Studies have shown that a single insertion event may dysregulate distal and multiple genes. Furthermore, multiple genes within a common insertion site can have both positive and negative impacts on hematopoiesis.