ALBERT

Description: In gene therapy with human hematopoietic stem and progenitor cells (HSPCs), each gene-corrected cell and its progeny are marked in a unique way by the integrating vector. This feature enables lineages to be tracked by sampling blood cells and using DNA sequencing to identify the vector integration sites. Here, we studied 5 cell lineages (granulocytes, monocytes, T cells, B cells, and natural killer cells) in patients having undergone HSPC gene therapy for Wiskott-Aldrich syndrome or β hemoglobinopathies. We found that the estimated minimum number of active, repopulating HSPCs (which ranged from 2000 to 50 000) was correlated with the number of HSPCs per kilogram infused. We sought to quantify the lineage output and dynamics of gene-modified clones; this is usually challenging because of sparse sampling of the various cell types during the analytical procedure, contamination during cell isolation, and different levels of vector marking in the various lineages. We therefore measured the residual contamination and corrected our statistical models accordingly to provide a rigorous analysis of the HSPC lineage output. A cluster analysis of the HSPC lineage output highlighted the existence of several stable, distinct differentiation programs, including myeloid-dominant, lymphoid-dominant, and balanced cell subsets. Our study evidenced the heterogeneous nature of the cell lineage output from HSPCs and provided methods for analyzing these complex data.

Description: Background. Wiskott Aldrich Syndrome (WAS) is a rare primary immunodeficiency associated with thrombocytopenia, eczema, severe infectious and autoimmune complications, and lymphomas. Mismatched allogeneic hematopoietic stem cell transplantation (HSCT) is an alternative for patient lacking an HLA-matched donor but is associated with an increased frequency of complications. Moreover low lymphoid and myeloid chimerism is related to a higher rate of autoimmunity and thrombocytopenia. Recent gene therapy (GT) trials showed that gene-corrected autologous CD34+ cells infusion could be an appropriate therapeutic approach for these patients. It has been recently shown that B cell homeostasis is altered in WAS. As the B cell reconstitution participates to the restoration of immunological competence, a comprehensive study of this compartment after GT and the comparison with mismatched allogeneic HSCT is crucial. Objective. To perform a longitudinal study of B cell reconstitution in WAS patients after lentiviral vector-mediated GT, compared to mismatched allogeneic HSCT. Methods. Five patients (age 0.8-15.5 years) underwent GT at our center since 2011(follow-up 1.5-4.2 years) after near-myeloablative and immunosuppressive conditioning regimen with (n=3) or without (n=2) anti-CD20 administration. Patient 2 (P2) died 7 months after GT from a pre-existing infectious complication. Eleven patients undergoing mismatched allogeneic HSCT (age 0.6-10.9 years) at the same center were studied (follow-up 5.1-14.7 years). Longitudinal B cell assessment included B cell count before and after treatment, and the following subsets: switched memory (SM, CD19+ CD27+ IgD-), marginal zone (MZ, CD19+ CD27+ IgD+), naives (CD19+ CD27- IgD+), transitional (CD19+ CD27- IgD+ CD24high CD38high), circulating plasma cells (CD19+ CD27+ IgD- CD27high CD38high) and CD21low B cells (CD19+ CD21low CD38-), a subset abnormally expanded in WAS. Quantification of the B cell replication history was assessed through k-deleting recombination excision circles (KRECs). Analyses were compared to age-matched controls. WAS protein (WASP) expression and vector copy number (VCN) were measured in sorted B cells. Results. All alive GT patients show stable engraftment of functionally corrected lymphoid cells, without adverse events. Transduced B cells number and WASP expression increased progressively after GT. Absolute B cell count attained normal values in all the patients, and correlates with WASP expression and VCN in B cells. IgM levels are below normal ranges in four patients. P3 and P4 attained a B-cell phenotype within normal ranges; P3 discontinued intravenous immunoglobulin (IvIg) replacement. No expansion of CD21low B cells was observed. P1 and P5 (follow-up 18 months) present a variable defect in SM, naives and/or MZ B cells. P1 recently developed autoimmune manifestations; no significant changes were observed concomitantly. A defect in B cell lymphopoiesis was observed before GT as measured by KRECs analysis, normalizing after GT (P1, P3 and P4). Several complications were recorded in patients undergoing mismatched allogeneic HSCT, including dysimmunity, arthritis, developmental deficit and infections. Total B cell count normalized in eight patients, IgM levels were low in three. Among patients with available information, four still remain under IvIg replacement. Four patients developed a mixed lymphoid and myeloid chimerism, variably associated with low B cell count, low IgM and IvIg replacement. A complete B cell assessment for these patients is ongoing. Conclusions. B cell transgene expression is obtained after lentiviral vector-mediated GT in WAS patients and is associated with improved B cell lymphopoiesis. A correct B cell phenotype is observed in two patients who did not receive rituximab prior GT. The question whether this is related to the treatment will need a longer follow-up to be answered. Patients undergoing mismatched allogeneic HSCT present a higher frequency of complications. Although a higher proportion of these patients discontinued IvIg replacement, B cell reconstitution is not optimal. Analysis of patients in particular with mixed chimerism will provide important information in the setting of GT. The analysis of B cell reconstitution after GT and mismatched allogeneic HSCT deserves particular attention in the assessment of immunological reconstitution. Disclosures No relevant conflicts of interest to declare.

Description: The historical model of hematopoiesis, mainly derived from murine studies, is based on the existence of a single hematopoietic stem cell (HSC) capable of generating all blood cell lineages. However this model has been challenged by the proposal of four types of murine HSC, which differ by their relative contribution to the myeloid and lymphoid lineages. Here we have used data from a gene therapy trial to treat Wiskott-Aldrich syndrome (WAS) to explore hematopoiesis in humans. In the trial, the therapeutic vector (lentivirus) integrates into the genome at unique positions in each hematopoietic stem and progenitor cell (HSPC) and is consequently transmitted to all its progeny. Thus hematopoietic ontogeny in humans can be inferred by tracking the appearance of unique integration sites in fractionated blood cell populations. This provides a unique opportunity to model the developmental complexity of the human haematological system. Considerable effort over the last 15 years has been devoted to optimizing retroviral integration sites (RIS) analysis using ligation mediated PCR (LM-PCR), combined with acoustic shearing and high-throughput Illumina sequencing. Acoustic shearing enables more precise quantification of RIS abundance through the enumeration of the various sizes of shear fragments (SonicAbundance) containing a given RIS, which correspond to individual ancestor HSPC and its blood progeny. In four WAS patients treated by gene therapy, we have sorted peripheral blood samples for 5 cell types: myeloid (granulocytes and monocytes) and lymphoid subpopulations (T, B and NK cells), and analysed their RIS profile. Each RIS corresponds to a particular stem/progenitor cell clone, with a particular pattern defined by its presence or absence in each of the 5 lineages. These data are then use to reconstruct aspects of the hematopoietic hierarchy. In order to face the challenging issue of cell sorting contamination we have been using a stringent sort precision mode and we treat residual contamination explicitly in downstream statistical models. Using these approaches, we have characterized up to tens of thousands RIS per patients with a follow up of 4 years. In order to minimize biases due to sparse sampling, we concentrate on the more abundant RIS clones that can be more easily caught and are therefore analysed more reliably. We showed that a significant fraction of RIS clones are detected in a single lineage, while other RIS clones are characterized by different levels of contribution to the myeloid and lymphoid lineages, highlighting the heterogeneity of human HSC. Clones contributing to all 5 lineages are readily recovered but this study also unravels a diversity of inferred hematopoietic programs with various potentials contributing to human blood homeostasis. Longitudinal analysis of clonal dynamics is ongoing, with preliminary results highlighting the maintenance of this heterogeneity of HSPC over time. These new findings provide unique data on human hematopoiesis based on gene corrected WAS patients. Disclosures No relevant conflicts of interest to declare.