Unraveling the effect of proliferative stress in vivo in hematopoietic stem cell gene therapy mouse study

The hematopoietic system of patients enrolled in hematopoietic stem cells (HSC) gene therapy (GT) treatments is fully reconstituted upon autologous transplantation of engineered stem cells. HSCs highly proliferate up to full restoration of homeostasis and compete for niche homing and engraftment. The impact of the proliferation stress in HSC on genetic instability remains an open question that cured patients advocate for characterizing long-term safety and efficacy. The accumulation of somatic mutations has been widely used as a sensor of proliferative stress. Vector integration site (IS) can be used as a molecular tool for clonal identity, inherited by all HSC progeny, to uncover lineage dynamics in vivo at single-cell level. Here we characterized at single-clone granularity the proliferative stress of HSCs and their progeny over time by measuring the accumulation of mutations from the DNA of each IS. To test the feasibility of the approach, we set-up an experimental framework that combines tumor-prone Cdkn2a-/- and wild type (WT) mouse models of HSC-GT and molecular analyses on different hematopoietic cell lineages after transplantation of HSCs transduced with genotoxic LV (LV.SF.LTR) or GT-like non-genotoxic LV (SIN.LV.PGK). The Cdkn2a-/- mouse model provided the experimental conditions to detect the accumulation of somatic mutations, since the absence of p16INK4A and p19ARF enhances the proliferative potential of cells that have acquired oncogenic mutations. As expected, mice transplanted with Cdkn2a-/- Lin- cells marked with LV.SF.LTR (N=24) developed tumors significantly earlier compared to mock (N=20, p<0.0001), while mice treated with SIN. LV.PGK (N=23) did not. On the other side, mice that received WT Lin- cells treated with LV.SF.LTR (N=25) or SIN.LV.PGK (N=24) vector have not developed tumors. Given this scenario, we expect that Cdkn2a-/- Lin- cells transduced with LV.SF.LTR are associated with higher mutation rates compared to the SIN.LV.PGK group and wild type control mice. The composition of peripheral blood, lymphoid (B and T) and myeloid compartments was assessed by FACS on samples collected every 4 weeks and IS identification. More than 200,000 IS have been recovered. To identify the presence of somatic mutations, the genomic portions of sequencing reads flanking each different IS were analyzed with VarScan2. The accumulation rates of mutations have been evaluated by our new Mutation Index (MI) which normalizes the number of mutations by clones and coverage. Considering that a large portion of IS has been discarded since not covered by a minimum number of 5 unique reads (genomes), the remaining number of IS contained >90% of reads in each group. The MI increased over time in both LV.SF.LTR groups, with higher values for the Cdkn2a-/-. On the other hand, treatment with SIN.LV.PGK resulted in lower MI in both groups compared to LV.SF.LTR groups, reflecting the higher clonal composition of the cells treated with the SIN.LV.PGK and the phenomenon of insertional mutagenesis in the LV.SF.LTR. Moreover, the higher MI values of the SIN.LV.PGK Cdkn2a-/- group compared with the WT group proved the induction of DNA fragility. Our results showed that the analysis of the accumulation of somatic mutations at single clone unraveled HSC proliferation stress in vivo, combining for the first time the analysis of acquired mutations with IS. We are now applying our model to different clinical trials, and studying HSCs sub- clonal trees by symmetric divisions, previously indistinguishable by IS only. Our study will open the doors to in vivo long-term non-invasive studies of HSC stability in patients

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

Acquisition of somatic mutations after hematopoietic stem cell gene therapy varies among cell lineages and is modulated by vector genotoxicity and the activity of key cellular senescence gene

The hematopoietic system of patients undergoing Hematopoietic Stem and Progenitor Cell (HSPC) Gene Therapy (GT) is fully restored when autologous engineered HSPCs are reinfused into the patient. During this process, HSPCs go through a high level of proliferation until the hematopoietic reconstitution is complete. The impact of proliferation in HSPCs on cellular fitness and safety remains an open question. Moreover, the accumulation of somatic mutations in vivo could show differences in different hematopoietic lineages depending on their susceptibility to the negative effects elicited by the DNA damage response. Furthermore, oncogene activation in human HSPCs has been shown to trigger a chronic inflammatory response leading to hematopoietic decay. Here we studied the clonality and the accumulation of somatic mutations in different hematopoietic lineages and during hematopoietic reconstitution in mice subjected to HSPC-GT. Indeed, wild type C57 mice were transplanted with bone marrow-derived lineage negative (Lin-) cells from WT mice or tumor-prone Cdkn2a-/- mice which lack p16INK4A and p19ARF proteins and thus have no barriers against proto-oncogene activation. Moreover, to evaluate if genotoxic integrations may increase the probability of acquiring somatic mutation upon oncogene activation, Lin- cells were transduced with a genotoxic LV harboring the strong retroviral enhancer/promoter Spleen Focus Forming Virus in the LTR (LV.SF.LTR) or the safer GTlike non-genotoxic LV (SIN.LV.PGK). Mice receiving WT Lin- cells treatedwith LV.SF.LTR (N= 25) or SIN.LV.PGK (N= 24) did not develop tumors, while mice transplanted with Cdkn2a/LV.SF.LTR-marked cells (N = 24) developed tumors significantly earlier compared to mock (N = 20, p < 0.0001) and mice receiving Cdkn2a/SIN.LV.PGK-treated cells (N = 23, p < 0.0001). To evaluate the clonal dynamics of hematopoietic reconstitution, vector integration sites (IS)were identified by by Sonication Mediated Integration Site (SLiM) PCR from peripheral blood, lymphoid (B and T) and myeloid cells collected every 4 weeks post transplantation. Somatic mutations were identified by analyzing the mouse genomic portion flanking each IS using VarScan2. Overall, we detected >200,000 IS, corresponding to more than 135 Mb of genomic sequence information. We introduced a new Mutation Index (MI), which normalizes the number of mutations by clones and coverage to assess mutation accumulation rates. By this approach, we found that the MI increased over time in LV.SF.LTR-treated mice and was significantly higher when compared to SIN.LV.PGK-treated mice (p < 0.001). Notably, myeloid clones exhibited a higher frequency of mutation accumulation compared to T and B cell lineages. This phenomenon was further exacerbated in Cdkn2a/LV.SF.LTR-marked cells, indicating that the absence of barriers to proto-oncogene activation and the presence of genotoxic insertions result in progressive somatic mutation accumulation and insertional mutagenesis. These results demonstrate for the first time that by combining the assessment of acquired mutations with IS analysis at the single clone level we can identify differential accumulations of somatic mutations in different hematopoietic lineages in vivo which depend on the genotoxic potential of the vector used and the ability of the genetically modified cells to sense and react to genotoxic lesions

The failed liberalisation of Algeria and the international context: a legacy of stable authoritarianism

The paper attempts to challenge the somewhat marginal role of international factors in the study of transitions to democracy. Theoretical and practical difficulties in proving causal mechanisms between international variables and domestic outcomes can be overcome by defining the international dimension in terms of Western dominance of world politics and by identifying Western actions towards democratising countries. The paper focuses on the case of Algeria, where international factors are key in explaining the initial process of democratisation and its following demise. In particular, the paper argues that direct Western policies, the pressures of the international system and external shocks influence the internal distribution of power and resources, which underpins the different strategies of all domestic actors. The paper concludes that analysis based purely on domestic factors cannot explain the process of democratisation and that international variables must be taken into more serious account and much more detailed

Molecular Characterization of the Region 7q22.1 in Splenic Marginal Zone Lymphomas

Splenic marginal zone lymphomas (SMZL) are an uncommon type of B-cell non-Hodgkin's lymphoma (NHL-B) in which no specific chromosomal translocations have been described. In contrast, the most frequent cytogenetic abnormality is the loss of the long arm of chromosome 7 (7q). Previous reports have located this loss in the 7q32 region. In order to better characterize the genomic imbalances in SMZL, molecular studies were carried out in 73 patients with SMZL. To gain insight into the mapping at 7q a tiling array was also used. The results confirmed the loss of 7q as the most frequent change. In addition, several abnormalities, including 4q22.1, 1q21.3–q22, 6q25.3, 20q13.33, 3q28, 2q23.3–q24.1 and 17p13, were also present. A loss of 7q22.1 at 99925039–101348479 bp was observed in half of the cases. The region of 7q22.1 has not previously been characterised in SMZL. Our results confirmed the presence of a new region of loss on chromosome 7 in these NHL