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

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

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

HIV-1 mediated insertional activation of STAT5B promotes the formation of a viral reservoir in T regulatory cells

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Normalization of clonal diversity in gene therapy studies using shape constrained splines

Viral vectors are used to insert genetic material into semirandom genomic positions of hematopoietic stem cells which, after reinfusion into patients, regenerate the entire hematopoietic system. Hematopoietic cells originating from genetically modified stem cells will harbor insertions in specific genomic positions called integration sites, which represent unique genetic marks of clonal identity. Therefore, the analysis of vector integration sites present in the genomic DNA of circulating cells allows to determine the number of clones in the blood ecosystem. Shannon diversity index is adopted to evaluate the heterogeneity of the transduced population of gene corrected cells. However, this measure can be affected by several technical variables such as the DNA amount used and the sequencing depth of the library analyzed and therefore the comparison across samples may be affected by these confounding factors. We developed an advanced spline-regression approach that leverages on confounding effects to provide a normalized entropy index. Our proposed method was first validated and compared with two state of the art approaches in a specifically designed in vitro assay. Subsequently our approach allowed to observe the expected impact of vector genotoxicity on entropy level decay in an in vivo model of hematopoietic stem cell gene therapy based on tumor prone mice

Characteristics of Recurrent Ischemic Stroke after Embolic Stroke of Undetermined Source: Secondary Analysis of a Randomized Clinical Trial

Importance: The concept of embolic stroke of undetermined source (ESUS) unifies a subgroup of cryptogenic strokes based on neuroimaging, a defined minimum set of diagnostic tests, and exclusion of certain causes. Despite an annual stroke recurrence rate of 5%, little is known about the etiology underlying recurrent stroke after ESUS. Objective: To identify the stroke subtype of recurrent ischemic strokes after ESUS, to explore the interaction with treatment assignment in each category, and to examine the consistency of cerebral location of qualifying ESUS and recurrent ischemic stroke. Design, Setting, and Participants: The NAVIGATE-ESUS trial was a randomized clinical trial conducted from December 23, 2014, to October 5, 2017. The trial compared the efficacy and safety of rivaroxaban and aspirin in patients with recent ESUS (n = 7213). Ischemic stroke was validated in 309 of the 7213 patients by adjudicators blinded to treatment assignment and classified by local investigators into the categories ESUS or non-ESUS (ie, cardioembolic, atherosclerotic, lacunar, other determined cause, or insufficient testing). Five patients with recurrent strokes that could not be defined as ischemic or hemorrhagic in absence of neuroimaging or autopsy were excluded. Data for this secondary post hoc analysis were analyzed from March to June 2019. Interventions: Patients were randomly assigned to receive rivaroxaban, 15 mg/d, or aspirin, 100 mg/d. Main Outcomes and Measures: Association of recurrent ESUS with stroke characteristics. Results: A total of 309 patients (205 men [66%]; mean [SD] age, 68 [10] years) had ischemic stroke identified during the median follow-up of 11 (interquartile range [IQR], 12) months (annualized rate, 4.6%). Diagnostic testing was insufficient for etiological classification in 39 patients (13%). Of 270 classifiable ischemic strokes, 156 (58%) were ESUS and 114 (42%) were non-ESUS (37 [32%] cardioembolic, 26 [23%] atherosclerotic, 35 [31%] lacunar, and 16 [14%] other determined cause). Atrial fibrillation was found in 27 patients (9%) with recurrent ischemic stroke and was associated with higher morbidity (median change in modified Rankin scale score 2 [IQR, 3] vs 0 (IQR, 1]) and mortality (15% vs 1%) than other causes. Risk of recurrence did not differ significantly by subtype between treatment groups. For both the qualifying and recurrent strokes, location of infarct was more often in the left (46% and 54%, respectively) than right hemisphere (40% and 37%, respectively) or brainstem or cerebellum (14% and 9%, respectively). Conclusions and Relevance: In this secondary analysis of randomized clinical trial data, most recurrent strokes after ESUS were embolic and of undetermined source. Recurrences associated with atrial fibrillation were a minority but were more often disabling and fatal. More extensive investigation to identify the embolic source is important toward an effective antithrombotic strategy. Trial Registration: ClinicalTrials.gov Identifier: NCT02313909

Effectiveness of thrombectomy in stroke according to baseline prognostic factors: inverse probability of treatment weighting analysis of a population-based registry

Background and Purpose In real-world practice, the benefit of mechanical thrombectomy (MT) is uncertain in stroke patients with very favorable or poor prognostic profiles at baseline. We studied the effectiveness of MT versus medical treatment stratifying by different baseline prognostic factors. Methods Retrospective analysis of 2,588 patients with an ischemic stroke due to large vessel occlusion nested in the population-based registry of stroke code activations in Catalonia from January 2017 to June 2019. The effect of MT on good functional outcome (modified Rankin Score ≤2) and survival at 3 months was studied using inverse probability of treatment weighting (IPTW) analysis in three pre-defined baseline prognostic groups: poor (if pre-stroke disability, age >85 years, National Institutes of Health Stroke Scale [NIHSS] >25, time from onset >6 hours, Alberta Stroke Program Early CT Score 3), good (if NIHSS <6 or distal occlusion, in the absence of poor prognostic factors), or reference (not meeting other groups’ criteria). Results Patients receiving MT (n=1,996, 77%) were younger, had less pre-stroke disability, and received systemic thrombolysis less frequently. These differences were balanced after the IPTW stratified by prognosis. MT was associated with good functional outcome in the reference (odds ratio [OR], 2.9; 95% confidence interval [CI], 2.0 to 4.4), and especially in the poor baseline prognostic stratum (OR, 3.9; 95% CI, 2.6 to 5.9), but not in the good prognostic stratum. MT was associated with survival only in the poor prognostic stratum (OR, 2.6; 95% CI, 2.0 to 3.3). Conclusions Despite their worse overall outcomes, the impact of thrombectomy over medical management was more substantial in patients with poorer baseline prognostic factors than patients with good prognostic factors