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Adenosine deaminase 2 deficiency (DADA2) is a rare inherited disorder that is caused by autosomal recessive mutations in the ADA2 gene. Clinical manifestations include early-onset lacunar strokes, vasculitis/vasculopathy, systemic inflammation, immunodeficiency, and hematologic defects. Anti–tumor necrosis factor therapy reduces strokes and systemic inflammation. Allogeneic hematopoietic stem/progenitor cell (HSPC) transplantation can ameliorate most disease manifestations, but patients are at risk for complications. Autologous HSPC gene therapy may be an alternative curative option for patients with DADA2. We designed a lentiviral vector encoding ADA2 (LV-ADA2) to genetically correct HSPCs. Lentiviral transduction allowed efficient delivery of the functional ADA2 enzyme into HSPCs from healthy donors. Supranormal ADA2 expression in human and mouse HSPCs did not affect their multipotency and engraftment potential in vivo. The LV-ADA2 induced stable ADA2 expression and corrected the enzymatic defect in HSPCs derived from DADA2 patients. Patients’ HSPCs re-expressing ADA2 retained their potential to differentiate into erythroid and myeloid cells. Delivery of ADA2 enzymatic activity in patients’ macrophages led to a complete rescue of the exaggerated inflammatory cytokine production. Our data indicate that HSPCs ectopically expressing ADA2 retain their multipotent differentiation ability, leading to functional correction of macrophage defects. Altogether, these findings support the implementation of HSPC gene therapy for DADA2.

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, an important number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34(+) cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34(+). cells have a cellular composition that resembles bone marrow (BM), supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPC). To overcome the limit of BM harvest/HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex vivo expansion of HSPC coupled with lentiviral gene transfer. Circulating CD34(+). cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NOD scid gamma common chain (NSG) recipients. Moreover, when CD34(+) cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPC coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by BM fibrosis.

BACKGROUND. The human bone marrow (BM) niche contains a population of mesenchymal stromal cells (MSCs) that provide physical support and regulate hematopoietic stem cell (HSC) homeostasis. β-Thalassemia (BT) is a hereditary disorder characterized by altered hemoglobin beta-chain synthesis amenable to allogeneic HSC transplantation and HSC gene therapy. Iron overload (IO) is a common complication in BT patients affecting several organs. However, data on the BM stromal compartment are scarce. METHODS. MSCs were isolated and characterized from BM aspirates of healthy donors (HDs) and BT patients. The state of IO was assessed and correlated with the presence of primitive MSCs in vitro and in vivo. Hematopoietic supportive capacity of MSCs was evaluated by transwell migration assay and 2D coculture of MSCs with human CD34+ HSCs. In vivo, the ability of MSCs to facilitate HSC engraftment was tested in a xenogenic transplant model, whereas the capacity to sustain human hematopoiesis was evaluated in humanized ossicle models. RESULTS. We report that, despite iron chelation, BT BM contains high levels of iron and ferritin, indicative of iron accumulation in the BM niche. We found a pauperization of the most primitive MSC pool caused by increased ROS production in vitro which impaired MSC stemness properties. We confirmed a reduced frequency of primitive MSCs in vivo in BT patients. We also discovered a weakened antioxidative response and diminished expression of BM niche–associated genes in BT-MSCs. This caused a functional impairment in MSC hematopoietic supportive capacity in vitro and in cotransplantation models. In addition, BT-MSCs failed to form a proper BM niche in humanized ossicle models. CONCLUSION. Our results suggest an impairment in the mesenchymal compartment of BT BM niche and highlight the need for novel strategies to target the niche to reduce IO and oxidative stress before transplantation. FUNDING. This work was supported by the SR-TIGET Core grant from Fondazione Telethon and by Ricerca Corrente.

Activating mutations in the BRAF-MAPK pathway have been reported in histiocytoses, hematological inflammatory neoplasms characterized by multi-organ dissemination of pro-inflammatory myeloid cells. Here, we generate a humanized mouse model of transplantation of human hematopoietic stem and progenitor cells (HSPCs) expressing the activated form of BRAF (BRAFV600E). All mice transplanted with BRAFV600E-expressing HSPCs succumb to bone marrow failure, displaying myeloid-restricted hematopoiesis and multi-organ dissemination of aberrant mononuclear phagocytes. At the basis of this aggressive phenotype, we uncover the engagement of a senescence program, characterized by DNA damage response activation and a senescence-associated secretory phenotype, which affects also non-mutated bystander cells. Mechanistically, we identify TNFα as a key determinant of paracrine senescence and myeloid-restricted hematopoiesis and show that its inhibition dampens inflammation, delays disease onset and rescues hematopoietic defects in bystander cells. Our work establishes that senescence in the human hematopoietic system links oncogene-activation to the systemic inflammation observed in histiocytic neoplasms., BRAF-MAPK activating mutations are reported in histiocytoses—hematological neoplasms with widespread pro-inflammatory myeloid cells. Here, the authors show that an activating mutant BRAF in haematopoietic stem and progenitor cells causes an oncogene-induced senescence response leading to myeloid restricted haematopoiesis, inflammation and histiocytosis.

Human hematopoiesis is a complex and dynamic system where morphologically and functionally diverse mature cell types are generated and maintained throughout life by bone marrow (BM) Hematopoietic Stem/Progenitor Cells (HSPC). Congenital and acquired hematopoietic disorders are often diagnosed through the detection of aberrant frequency or composition of hematopoietic cell populations. We here describe a novel protocol, called “Whole Blood Dissection” (WBD), capable of analyzing in a single test-tube, hematopoietic progenitors and all major mature cell lineages composing either BM or peripheral blood (PB) through a multiparametric flow-cytometry analysis. WBD allows unambiguously identifying in the same tube up to 23 different blood cell types including HSPC subtypes and all the major myeloid and lymphoid lineage compartments at different stages of maturation, through a combination of 17 surface and 1 viability cell markers. We assessed the efficacy of WBD by analyzing BM and PB samples from adult (n = 8) and pediatric (n = 9) healthy donors highlighting age-related shift in cell composition. We also tested the capability of WBD on detecting aberrant hematopoietic cell composition in clinical samples of patients with primary immunodeficiency or leukemia unveiling expected and novel hematopoietic unbalances. Overall, WBD allows unambiguously identifying >99% of the cell subpopulations composing a blood sample in a reproducible, standardized, cost-, and time-efficient manner. This tool has a wide range of potential pre-clinical and clinical applications going from the characterization of hematopoietic disorders to the monitoring of hematopoietic reconstitution in patients after transplant or gene therapy. © 2017 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC.

Chronic granulomatous disease (CGD) is a rare inherited disorder due to loss-of-function mutations in genes encoding the NADPH oxidase subunits. Hematopoietic stem and progenitor cell (HSPC) gene therapy (GT) using regulated lentiviral vectors (LVs) has emerged as a promising therapeutic option for CGD patients. We performed non-clinical Good Laboratory Practice (GLP) and laboratory-grade studies to assess the safety and genotoxicity of LV targeting myeloid-specific Gp91phox expression in X-linked chronic granulomatous disease (XCGD) mice. We found persistence of gene-corrected cells for up to 1 year, restoration of Gp91phox expression and NADPH oxidase activity in XCGD phagocytes, and reduced tissue inflammation after LV-mediated HSPC GT. Although most of the mice showed no hematological or biochemical toxicity, a small subset of XCGD GT mice developed T cell lymphoblastic lymphoma (2.94%) and myeloid leukemia (5.88%). No hematological malignancies were identified in C57BL/6 mice transplanted with transduced XCGD HSPCs. Integration pattern analysis revealed an oligoclonal composition with rare dominant clones harboring vector insertions near oncogenes in mice with tumors. Collectively, our data support the long-term efficacy of LV-mediated HSPC GT in XCGD mice and provide a safety warning because the chronic inflammatory XCGD background may contribute to oncogenesis., Graphical Abstract, In a GLP study, Jofra Hernández and colleagues demonstrate that lentiviral vector-mediated HSPC gene therapy effectively corrects long-term X-linked chronic granulomatous disease in a mouse model of the disease. A small proportion of mice develops hematopoietic tumors originating from rare dominant clones harboring vector insertions near oncogenes.

Lam et al. characterize a novel hematological/autoinflammatory disorder due to a de novo recurrent missense mutation of CDC42. The authors use in silico, in vitro, and in vivo analyses to correlate the molecular mechanisms altering CDC42 function to the observed phenotype., Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. CDC42 mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival., Graphical Abstract

ARPC1B is a key factor for the assembly and maintenance of the ARP2/3 complex that is involved in actin branching from an existing filament. Germline biallelic mutations in ARPC1B have been recently described in 6 patients with clinical features of combined immunodeficiency (CID), whose neutrophils and platelets but not T lymphocytes were studied. We hypothesized that ARPC1B deficiency may also lead to cytoskeleton and functional defects in T cells. We have identified biallelic mutations in ARPC1B in 6 unrelated patients with early onset disease characterized by severe infections, autoimmune manifestations, and thrombocytopenia. Immunological features included T-cell lymphopenia, low numbers of naïve T cells, and hyper–immunoglobulin E. Alteration in ARPC1B protein structure led to absent/low expression by flow cytometry and confocal microscopy. This molecular defect was associated with the inability of patient-derived T cells to extend an actin-rich lamellipodia upon T-cell receptor (TCR) stimulation and to assemble an immunological synapse. ARPC1B-deficient T cells additionally displayed impaired TCR-mediated proliferation and SDF1-α−directed migration. Gene transfer of ARPC1B in patients’ T cells using a lentiviral vector restored both ARPC1B expression and T-cell proliferation in vitro. In 2 of the patients, in vivo somatic reversion restored ARPC1B expression in a fraction of lymphocytes and was associated with a skewed TCR repertoire. In 1 revertant patient, memory CD8+ T cells expressing normal levels of ARPC1B displayed improved T-cell migration. Inherited ARPC1B deficiency therefore alters T-cell cytoskeletal dynamics and functions, contributing to the clinical features of CID.

Hematopoietic stem and progenitor cells (HSPC) are endowed with the role of generating and maintaining lifelong the extremely diverse pool of blood cells(1). Clinically, transplantation of human HSPC from an allogeneic healthy donor or infusion of autologous gene-corrected HSPC can effectively replenish defective blood cell production caused by congenital or acquired disorders(2-9). However, due to methodological and ethical constraints that have limited the study of human HSPC primarily to in vitro assays(10) or xenotransplantation models(11,12), the in vivo activity of HSPC has to date remained relatively unexplored in humans(13-16). Here we report a comprehensive study of the frequencies, dynamics and output of seven HSPC subtypes in humans that was performed by tracking 148,093 individual clones in six patients treated with lentiviral gene therapy using autologous HSPC transplantation and followed for up to 5 years. We discovered that primitive multipotent progenitor and hematopoietic stem cell (HSC) populations have distinct roles during the initial reconstitution after transplant, compared with subsequent steady-state phases. Furthermore, we showed that a fraction of in vitro-activated HSC are resilient and undergo a defined delayed activation period upon transplant. Finally, our data support the concept that early lymphoid-biased progenitors might be capable of long-term survival, such that they can be maintained independently of their continuous production from HSC. Overall, this study provides comprehensive data on HSPC dynamics after autologous transplantation and gene therapy in humans.

Summary Hematopoietic stem/progenitor cells (HSPCs) are capable of supporting the lifelong production of blood cells exerting a wide spectrum of functions. Lentiviral vector HSPC gene therapy generates a human hematopoietic system stably marked at the clonal level by vector integration sites (ISs). Using IS analysis, we longitudinally tracked >89,000 clones from 15 distinct bone marrow and peripheral blood lineages purified up to 4 years after transplant in four Wiskott-Aldrich syndrome patients treated with HSPC gene therapy. We measured at the clonal level repopulating waves, populations' sizes and dynamics, activity of distinct HSPC subtypes, contribution of various progenitor classes during the early and late post-transplant phases, and hierarchical relationships among lineages. We discovered that in-vitro-manipulated HSPCs retain the ability to return to latency after transplant and can be physiologically reactivated, sustaining a stable hematopoietic output. This study constitutes in vivo comprehensive tracking in humans of hematopoietic clonal dynamics during the early and late post-transplant phases., Graphical Abstract, Highlights • Hematopoietic reconstitution occurs in two distinct clonal waves • A few thousand HSPC clones stably sustain multilineage blood cell production • Steady-state hematopoiesis after transplant is maintained by both HSCs and MPPs • Natural killer clones have closer relationships to myeloid cells than to lymphoid cells, Biasco et al. report a clonal tracking study on the dynamics and nature of hematopoietic reconstitution in humans after transplant. Using integration sites as molecular tags, they measured, in gene therapy patients, repopulating waves, population size and dynamics, activity of progenitor subtypes during the early and late post-transplant phases, and hierarchical relationships among lineages.

Gene-corrected cells in Gene Therapy (GT) treated patients can be tracked in vivo by means of vector integration site (IS) analysis, since each engineered clone becomes univocally and stably marked by an individual IS. As the proper IS identification and quantification is crucial to accurately perform clonal tracking studies, we designed a customizable and tailored pipeline to analyze LAM-PCR amplicons sequenced by Illumina MiSeq/HiSeq technology. The sequencing data are initially processed through a series of quality filters and cleaned from vector and Linker Cassette (LC) sequences with customizable settings. Demultiplexing is then performed according to the recognition of specific barcodes combination used upon library preparation and the sequences are aligned to the reference genome. Importantly, the human genome assembly Hg19 is composed of 93 contigs, among which the mitochondrial genome, unlocalized and unplaced contigs and some alternative haplotypes of chr6. While previous approaches aligned IS sequences only to the standard 24 human chromosomes, using the whole assembled genome allowed improving alignment accuracy and concomitantly increased the amount of detectable ISs. To date, we have processed 28 independent human sample sets retrieving 260,994 ISs from 189,270,566 sequencing reads. Although, sequencing read counts at each IS have been widely used to estimate the relative IS abundance, this method carries inherent accuracy constraints due to the rounds of exponential amplification required by LAM-PCR that might generate unbalances on the original clonal representation. More recently, a method based on genomic sonication has been proposed exploiting shear site counts to tag the number of original fragments belonging to each IS before PCR amplification. However, the number of cells composing a given clone could far exceed the number of fragments of different lengths that can be generated upon fragmentation in proximity of that given IS. This would rapidly saturate the available diversity of shear sites and progressively generate more and more same-site shearing on independent genomes. In order to overcome the described biases and reliably quantify ISs, we designed and tested a new LC encoding random barcodes. The new LC is composed of a known sequence of 29nt used as binding site for the primers upon amplification steps, a 6nt-random barcode, a fixed-anchor sequence of 6nt, a second 6nt-random barcode and a final known sequence of 22nt containing sticky ends for the three main restriction enzymes in use (MluI, HpyCH4IV and AciI). This peculiar design allowed increasing the accuracy of clonal diversity estimation since the fixed-anchor sequence acts as a control for sequencing reliability in the barcode area. The theoretical number of different available barcodes per clone (412=16,777,216) far exceeds the requirements for not saturating the original diversity of the analyzed sample (on average composed by around 50.000 cells). We validated this novel approach by performing assays on serial dilutions of individual clones carrying known ISs. The precision rate obtained was averagely around 99.3%, while the worst error rate reaches at most the 1.86%, confirming the reliability of IS quantification. We successfully applied the barcoded-LC system to the analysis of clinical samples from a Wiskott Aldrich Syndrome GT patient, collecting to date 50,215 barcoded ISs from 94,052,785 sequencing reads.