Unraveling the effects of proliferative stress and genotoxicity in hematopoietic stem cells in vivo

Hematopoietic Stem and Progenitor Cells (HSPCs) from patients affected by inherited disorders can be corrected with the use of Gene Therapy (GT), providing long term therapeutic benefit upon reconstitution of the entire hematopoietic system. However, how the replication stress, aging, vector driven oncogene activation and cancer predisposing mutations may impact the processes of hematopoietic reconstitution remains uncertain. Here we characterized the clonal dynamics of hematopoietic reconstitution and the acquisition of somatic mutations of lymphoid and myeloid cells in mice transplanted with wild (WT) type HSPCs transduced either with a lentiviral vector with active long terminal repeats which is highly genotoxic (group WT Genotox N=25) or with the safer self-inactivating long terminal repeats (group WT Non Genotox N=24). Additionally, the same HSPC-GT strategy was applied using mouse HSCPs lacking the tumor suppressor Cdkn2a gene (group Cdkn2a Genotox N=24, group Cdkn2a Non Genotox N=23 and Cdkn2a mock transduced N=20). Blood composition and vector integration sites (IS) of B, T, and myeloid cells were monitored overtime (up to 2.5 years). Somatic mutations were identified analyzing the genomic portion of the mouse genome flanking each IS, and a new Mutation Index (MI) was developed to assess mutation accumulation rates. As expected, the group Cdkn2a Genotox showed an accelerated tumor onset when compared to control groups (p<0.0001), caused by activation of Braf oncogene. Moreover, mice from all groups showed a marked myeloid skewing at the expense of lymphoid lineages at the latest time points, specifically in the group WT Genotox. More than 250,000 IS were identified, corresponding to 9 Gb of sequence genomic information. We found that the MI in both Genotox groups was significantly higher when compared to Non Genotox groups (p<0.001). Notably, myeloid clones exhibited a higher mutation frequency compared to B and T cell lineages. Moreover, the MI of the WT Genotox group in the myeloid compartment was significantly higher than Cdkn2a Genotox (p<0.01). Overall, our data unveils a previously unappreciated effect of genotoxicity by vector insertions which have a profound negative impact on hematopoiesis and accumulation of somatic mutations even in absence of oncogenesis.