Cellular and transcriptional dynamics of human neutrophils at steady state and upon stress.

Traditionally viewed as poorly plastic, neutrophils are now recognized as functionally diverse; however, the extent and determinants of neutrophil heterogeneity in humans remain unclear. We performed a comprehensive immunophenotypic and transcriptome analysis, at a bulk and single-cell level, of neutrophils from healthy donors and patients undergoing stress myelopoiesis upon exposure to growth factors, transplantation of hematopoietic stem cells (HSC-T), development of pancreatic cancer and viral infection. We uncover an extreme diversity of human neutrophils in vivo, reflecting the rates of cell mobilization, differentiation and exposure to environmental signals. Integrated control of developmental and inducible transcriptional programs linked flexible granulopoietic outputs with elicitation of stimulus-specific functional responses. In this context, we detected an acute interferon (IFN) response in the blood of patients receiving HSC-T that was mirrored by marked upregulation of IFN-stimulated genes in neutrophils but not in monocytes. Systematic characterization of human neutrophil plasticity may uncover clinically relevant biomarkers and support the development of diagnostic and therapeutic tools.

Circulating hematopoietic stem/progenitor cell subsets contribute to human hematopoietic homeostasis.

ABSTRACT: In physiological conditions, few circulating hematopoietic stem/progenitor cells (cHSPCs) are present in the peripheral blood, but their contribution to human hematopoiesis remain unsolved. By integrating advanced immunophenotyping, single-cell transcriptional and functional profiling, and integration site (IS) clonal tracking, we unveiled the biological properties and the transcriptional features of human cHSPC subpopulations in relationship to their bone marrow (BM) counterpart. We found that cHSPCs reduced in cell count over aging and are enriched for primitive, lymphoid, and erythroid subpopulations, showing preactivated transcriptional and functional state. Moreover, cHSPCs have low expression of multiple BM-retention molecules but maintain their homing potential after xenotransplantation. By generating a comprehensive human organ-resident HSPC data set based on single-cell RNA sequencing data, we detected organ-specific seeding properties of the distinct trafficking HSPC subpopulations. Notably, circulating multi-lymphoid progenitors are primed for seeding the thymus and actively contribute to T-cell production. Human clonal tracking data from patients receiving gene therapy (GT) also showed that cHSPCs connect distant BM niches and participate in steady-state hematopoietic production, with primitive cHSPCs having the highest recirculation capability to travel in and out of the BM. Finally, in case of hematopoietic impairment, cHSPCs composition reflects the BM-HSPC content and might represent a biomarker of the BM state for clinical and research purposes. Overall, our comprehensive work unveiled fundamental insights into the in vivo dynamics of human HSPC trafficking and its role in sustaining hematopoietic homeostasis. GT patients' clinical trials were registered at ClinicalTrials.gov (NCT01515462 and NCT03837483) and EudraCT (2009-017346-32 and 2018-003842-18).

Early bone marrow alterations in patients with adenosine deaminase 2 deficiency across disease phenotypes and severities.

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) is a complex monogenic disease caused by recessive mutations in the ADA2 gene. DADA2 exhibits a broad clinical spectrum encompassing vasculitis, immunodeficiency, and hematologic abnormalities. Yet, the impact of DADA2 on the bone marrow (BM) microenvironment is largely unexplored. OBJECTIVE: This study comprehensively examined the BM and peripheral blood of pediatric and adult patients with DADA2 presenting with rheumatologic/immunologic symptoms or severe hematologic manifestations. METHODS: Immunophenotyping of hematopoietic stem cells (HSCs), progenitor cells, and mature cell populations was performed for 18 patients with DADA2. We also conducted a characterization of mesenchymal stromal cells. RESULTS: Our study revealed a significant decrease in primitive HSCs and progenitor cells, alongside their reduced clonogenic capacity and multilineage differentiation potential. These BM defects were evident in patients with both severe and nonsevere hematologic manifestations, including pediatric patients, demonstrating that BM disruption can emerge silently and early on, even in patients who do not show obvious hematologic symptoms. Beyond stem cells, there was a reduction in mature cell populations in the BM and peripheral blood, affecting myeloid, erythroid, and lymphoid populations. Furthermore, BM mesenchymal stromal cells in patients with DADA2 exhibited reduced clonogenic and proliferation capabilities and were more prone to undergo cellular senescence marked by elevated DNA damage. CONCLUSIONS: Our exploration into the BM landscape of patients with DADA2 sheds light on the critical hematologic dimension of the disease and emphasizes the importance of vigilant monitoring, even in the case of subclinical presentation.

Mesenchymal stromal cells improve the transplantation outcome of CRISPR-Cas9 gene-edited human HSPCs.

Mesenchymal stromal cells (MSCs) have been employed in vitro to support hematopoietic stem and progenitor cell (HSPC) expansion and in vivo to promote HSPC engraftment. Based on these studies, we developed an MSC-based co-culture system to optimize the transplantation outcome of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene-edited (GE) human HSPCs. We show that bone marrow (BM)-MSCs produce several hematopoietic supportive and anti-inflammatory factors capable of alleviating the proliferation arrest and mitigating the apoptotic and inflammatory programs activated in GE-HSPCs, improving their expansion and clonogenic potential in vitro. The use of BM-MSCs resulted in superior human engraftment and increased clonal output of GE-HSPCs contributing to the early phase of hematological reconstitution in the peripheral blood of transplanted mice. In conclusion, our work poses the biological bases for a novel clinical use of BM-MSCs to promote engraftment of GE-HSPCs and improve their transplantation outcome.

A Novel Assay in Whole Blood Demonstrates Restoration of Mitochondrial Activity in Phagocytes After Successful HSCT in Hyperinflamed X-Linked Chronic Granulomatous Disease.

X-linked chronic granulomatous disease is a rare disease caused by mutations in the CYBB gene. While more extensive knowledge is available on genetics, pathogenesis, and possible therapeutic options, mitochondrial activity and its implications on patient monitoring are still not well-characterized. We have developed a novel protocol to study mitochondrial activity on whole blood of XCGD patients before and after transplantation, as well as on XCGD carriers. Here we present results of these analyses and of the restoration of mitochondrial activity in hyperinflamed X-linked Chronic Granulomatous Disease after hematopoietic stem cell transplantation. Moreover, we show a strong direct correlation between mitochondrial activity, chimerism, and DHR monitored before and after transplantation and in XCGD carriers. In conclusion, based on these findings, we suggest testing this new ready-to-use marker to better characterize patients before and after treatment and to investigate disease expression in carriers.

Hematopoietic Tumors in a Mouse Model of X-linked Chronic Granulomatous Disease after Lentiviral Vector-Mediated Gene Therapy.

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.

Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis.

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, a relevant 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, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) 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 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 HSPCs 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 bone marrow fibrosis.

T-cell defects in patients with ARPC1B germline mutations account for combined immunodeficiency.

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.

Multiparametric Whole Blood Dissection: A one-shot comprehensive picture of the human hematopoietic system.

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.

Transcriptomic analysis of BM-MSCs identified EGR1 as a transcription factor to fully exploit their therapeutic potential.

Bone marrow-mesenchymal stromal cells (BM-MSCs) are key components of the BM niche, where they regulate hematopoietic stem progenitor cell (HSPC) homeostasis by direct contact and secreting soluble factors. BM-MSCs also protect the BM niche from excessive inflammation by releasing anti-inflammatory factors and modulating immune cell activity. Thanks to these properties, BM-MSCs were successfully employed in pre-clinical HSPC transplantation models, increasing the rate of HSPC engraftment, accelerating the hematological reconstitution, and reducing the risk of graft failure. However, their clinical use requires extensive in vitro expansion, potentially altering their biological and functional properties. In this work, we analyzed the transcriptomic profile of human BM-MSCs sorted as CD45-, CD105+, CD73+, and CD90+ cells from the BM aspirates of heathy-donors and corresponding ex-vivo expanded BM-MSCs. We found the expression of immune and inflammatory genes downregulated upon cell culture and selected the transcription factor EGR1 to restore the MSC properties. We overexpressed EGR1 in BM-MSCs and performed in vitro tests to study the functional properties of EGR1-overexpressing BM-MSCs. We concluded that EGR1 increased the MSC response to inflammatory stimuli and immune cell control and potentiated the MSC hematopoietic supportive activity in co-culture assay, suggesting that the EGR1-based reprogramming may improve the BM-MSC clinical use.

Correction of osteopetrosis in the neonate oc/oc murine model after lentiviral vector gene therapy and non-genotoxic conditioning.

Introduction: Autosomal recessive osteopetrosis (ARO) is a rare genetic disease, characterized by increased bone density due to defective osteoclast function. Most of the cases are due to TCIRG1 gene mutation, leading to severe bone phenotype and death in the first years of life. The standard therapy is the hematopoietic stem cell transplantation (HSCT), but its success is limited by several constraints. Conversely, gene therapy (GT) could minimize the immune-mediated complications of allogeneic HSCT and offer a prompt treatment to these patients. Methods: The Tcirg1-defective oc/oc mouse model displays a short lifespan and high bone density, closely mirroring the human condition. In this work, we exploited the oc/oc neonate mice to optimize the critical steps for a successful therapy. Results: First, we showed that lentiviral vector GT can revert the osteopetrotic bone phenotype, allowing long-term survival and reducing extramedullary haematopoiesis. Then, we demonstrated that plerixafor-induced mobilization can further increase the high number of HSPCs circulating in peripheral blood, facilitating the collection of adequate numbers of cells for therapeutic purposes. Finally, pre-transplant non-genotoxic conditioning allowed the stable engraftment of HSPCs, albeit at lower level than conventional total body irradiation, and led to long-term survival and correction of bone phenotype, in the absence of acute toxicity. Conclusion: These results will pave the way to the implementation of an effective GT protocol, reducing the transplant-related complication risks in the very young and severely affected ARO patients.

Exonic knockout and knockin gene editing in hematopoietic stem and progenitor cells rescues RAG1 immunodeficiency.

Recombination activating genes (RAGs) are tightly regulated during lymphoid differentiation, and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem and progenitor cell (HSPC) transplantation is the treatment of choice but is limited by donor availability and toxicity. To overcome these issues, we developed gene editing strategies targeting a corrective sequence into the human RAG1 gene by homology-directed repair (HDR) and validated them by tailored two-dimensional, three-dimensional, and in vivo xenotransplant platforms to assess rescue of expression and function. Whereas integration into intron 1 of RAG1 achieved suboptimal correction, in-frame insertion into exon 2 drove physiologic human RAG1 expression and activity, allowing disruption of the dominant-negative effects of unrepaired hypomorphic alleles. Enhanced HDR-mediated gene editing enabled the correction of human RAG1 in HSPCs from patients with hypomorphic RAG1 mutations to overcome T and B cell differentiation blocks. Gene correction efficiency exceeded the minimal proportion of functional HSPCs required to rescue immunodeficiency in Rag1-/- mice, supporting the clinical translation of HSPC gene editing for the treatment of RAG1 deficiency.

Hematopoietic reconstitution dynamics of mobilized- and bone marrow-derived human hematopoietic stem cells after gene therapy.

Mobilized peripheral blood is increasingly used instead of bone marrow as a source of autologous hematopoietic stem/progenitor cells for ex vivo gene therapy. Here, we present an unplanned exploratory analysis evaluating the hematopoietic reconstitution kinetics, engraftment and clonality in 13 pediatric Wiskott-Aldrich syndrome patients treated with autologous lentiviral-vector transduced hematopoietic stem/progenitor cells derived from mobilized peripheral blood (n = 7), bone marrow (n = 5) or the combination of the two sources (n = 1). 8 out of 13 gene therapy patients were enrolled in an open-label, non-randomized, phase 1/2 clinical study (NCT01515462) and the remaining 5 patients were treated under expanded access programs. Although mobilized peripheral blood- and bone marrow- hematopoietic stem/progenitor cells display similar capability of being gene-corrected, maintaining the engineered grafts up to 3 years after gene therapy, mobilized peripheral blood-gene therapy group shows faster neutrophil and platelet recovery, higher number of engrafted clones and increased gene correction in the myeloid lineage which correlate with higher amount of primitive and myeloid progenitors contained in hematopoietic stem/progenitor cells derived from mobilized peripheral blood. In vitro differentiation and transplantation studies in mice confirm that primitive hematopoietic stem/progenitor cells from both sources have comparable engraftment and multilineage differentiation potential. Altogether, our analyses reveal that the differential behavior after gene therapy of hematopoietic stem/progenitor cells derived from either bone marrow or mobilized peripheral blood is mainly due to the distinct cell composition rather than functional differences of the infused cell products, providing new frames of references for clinical interpretation of hematopoietic stem/progenitor cell transplantation outcome.

Constitutive IL-1RA production by modified immune cells protects against IL-1-mediated inflammatory disorders.

Dysregulation of the interleukin-1 (IL-1) pathway leads to immune diseases that can result in chronic tissue and organ inflammation. Although IL-1 blockade has shown promise in ameliorating these symptoms and improving patients' quality of life, there is an urgent need for more effective, long-lasting treatments. We developed a lentivirus (LV)-mediated gene transfer strategy using transplanted autologous hematopoietic stem/progenitor cells (HSPCs) as a source of IL-1 receptor antagonist (IL-1RA) for systemic delivery to tissues and organs. Transplantation of mouse and human HSPCs transduced with an IL-1RA-encoding LV ensured stable IL-1RA production while maintaining the clonogenic and differentiation capacities of HSPCs in vivo. We examined the efficacy of cell-mediated IL-1RA delivery in three models of IL-1-dependent inflammation, for which treatment hindered neutrophil recruitment in an inducible model of gout, prevented systemic and multi-tissue inflammation in a genetic model of cryopyrin-associated periodic syndromes, and reduced disease severity in an experimental autoimmune encephalomyelitis model of multiple sclerosis. Our findings demonstrate HSPC-mediated IL-1RA delivery as a potential therapeutic modality that can be exploited to suppress tissue and organ inflammation in diverse immune-related diseases involving IL-1-driven inflammation.

Oncogene-induced senescence in hematopoietic progenitors features myeloid restricted hematopoiesis, chronic inflammation and histiocytosis.

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.

Lentiviral correction of enzymatic activity restrains macrophage inflammation in adenosine deaminase 2 deficiency.

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.

Bone marrow stromal cells from ß-thalassemia patients have impaired hematopoietic supportive capacity.

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. ß-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.

Lentiviral haemopoietic stem/progenitor cell gene therapy for treatment of Wiskott-Aldrich syndrome: interim results of a non-randomised, open-label, phase 1/2 clinical study.

BACKGROUND: Wiskott-Aldrich syndrome is a rare, life-threatening, X-linked primary immunodeficiency characterised by microthrombocytopenia, infections, eczema, autoimmunity, and malignant disease. Lentiviral vector-mediated haemopoietic stem/progenitor cell (HSPC) gene therapy is a potentially curative treatment that represents an alternative to allogeneic HSPC transplantation. Here, we report safety and efficacy data from an interim analysis of patients with severe Wiskott-Aldrich syndrome who received lentiviral vector-derived gene therapy. METHODS: We did a non-randomised, open-label, phase 1/2 clinical study in paediatric patients with severe Wiskott-Aldrich syndrome, defined by either WAS gene mutation or absent Wiskott-Aldrich syndrome protein (WASP) expression or a Zhu clinical score of 3 or higher. We included patients who had no HLA-identical sibling donor available or, for children younger than 5 years of age, no suitable 10/10 matched unrelated donor or 6/6 unrelated cord blood donor. After treatment with rituximab and a reduced-intensity conditioning regimen of busulfan and fludarabine, patients received one intravenous infusion of autologous CD34+ cells genetically modified with a lentiviral vector encoding for human WAS cDNA. The primary safety endpoints were safety of the conditioning regimen and safety of lentiviral gene transfer into HSPCs. The primary efficacy endpoints were overall survival, sustained engraftment of genetically corrected HSPCs, expression of vector-derived WASP, improved T-cell function, antigen-specific responses to vaccinations, and improved platelet count and mean platelet volume normalisation. This interim analysis was done when the first six patients treated had completed at least 3 years of follow-up. The planned analyses are presented for the intention-to-treat population. This trial is registered with ClinicalTrials.gov (number NCT01515462) and EudraCT (number 2009-017346-32). FINDINGS: Between April 20, 2010, and Feb 26, 2015, nine patients (all male) were enrolled of whom one was excluded after screening; the age range of the eight treated children was 1·1-12·4 years. At the time of the interim analysis (data cutoff April 29, 2016), median follow-up was 3·6 years (range 0·5-5·6). Overall survival was 100%. Engraftment of genetically corrected HSPCs was successful and sustained in all patients. The fraction of WASP-positive lymphocytes increased from a median of 3·9% (range 1·8-35·6) before gene therapy to 66·7% (55·7-98·6) at 12 months after gene therapy, whereas WASP-positive platelets increased from 19·1% (range 4·1-31·0) to 76·6% (53·1-98·4). Improvement of immune function was shown by normalisation of in-vitro T-cell function and successful discontinuation of immunoglobulin supplementation in seven patients with follow-up longer than 1 year, followed by positive antigen-specific response to vaccination. Severe infections fell from 2·38 (95% CI 1·44-3·72) per patient-year of observation (PYO) in the year before gene therapy to 0·31 (0·04-1·11) per PYO in the second year after gene therapy and 0·17 (0·00-0·93) per PYO in the third year after gene therapy. Before gene therapy, platelet counts were lower than 20 × 109 per L in seven of eight patients. At the last follow-up visit, the platelet count had increased to 20-50 × 109 per L in one patient, 50-100 × 109 per L in five patients, and more than 100 × 109 per L in two patients, which resulted in independence from platelet transfusions and absence of severe bleeding events. 27 serious adverse events in six patients occurred after gene therapy, 23 (85%) of which were infectious (pyrexia [five events in three patients], device-related infections, including one case of sepsis [four events in three patients], and gastroenteritis, including one case due to rotavirus [three events in two patients]); these occurred mainly in the first 6 months of follow-up. No adverse reactions to the investigational drug product and no abnormal clonal proliferation or leukaemia were reported after gene therapy. INTERPRETATION: Data from this study show that gene therapy provides a valuable treatment option for patients with severe Wiskott-Aldrich syndrome, particularly for those who do not have a suitable HSPC donor available. FUNDING: Italian Telethon Foundation, GlaxoSmithKline, and Orchard Therapeutics.

A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function.

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.