• gene replacement therapy
• gene therapy
• hereditary disorders
• viral vectors

• Child
• Humans
• Genetic Therapy
• Genetic Vectors
• Hemophilia A/genetics
• Hemophilia A/therapy
• Transgenes

• haematopoietic stem cells
• lymphocytes

• Humans
• CRISPR-Cas Systems/genetics
• Hematopoietic Stem Cells/metabolism
• Gene Editing/methods
• Gene Expression Regulation
• Hematopoietic Stem Cell Transplantation
• DNA-Binding Proteins/metabolism
• Nuclear Proteins/metabolism

• EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)
• Israel Science Foundation (ISF)

• CRISPR-Cas9
• HSPCs
• MT: RNA/DNA Editing
• RAG2
• SCID
• gene regulation
• genome editing
• rAAV6

• MLV
• gene therapy
• insertional mutagenesis
• mesoangioblast model
• retargeted integration
• retroviral vectors
• safer viral vectors

• Gene Editing
• Genetic Therapy
• Humans
• Metabolic Diseases/genetics
• Metabolic Diseases/therapy
• Zinc Finger Nucleases/genetics

• DP2 HL152427 NHLBI NIH HHS
• R01 DK123049 NIDDK NIH HHS
• R01 HL151352 NHLBI NIH HHS

• GT
• HSCT
• WAS
• Wiskott–Aldrich syndrome
• X-linked neutropenia
• X-linked thrombocytopenia
• XLN
• XLT
• gene therapy
• hematopoietic stem cell transplantation
• lentiviral vectors
• reduced intensity conditioning

• clinical trials
• gene therapy
• hemoglobin
• hemophilia B
• neurodegenerative
• ocular

• gammaretroviral vectors
• gene therapy
• integrase
• protein structure perturbation
• retroviral integrations
• transcriptional start sites

The EuroFlow PID consortium developed a set of flow cytometry tests for evaluation of patients with suspicion of primary immunodeficiency (PID). In this technical report we evaluate the performance of the SCID-RTE tube that explores the presence of recent thymic emigrants (RTE) together with T-cell activation status and maturation stages and discuss its applicability in the context of the broader EuroFlow PID flow cytometry testing algorithm for diagnostic orientation of PID of the lymphoid system. We have analyzed peripheral blood cells of 26 patients diagnosed between birth and 2 years of age with a genetically defined primary immunodeficiency disorder: 15 severe combined immunodeficiency (SCID) patients had disease-causing mutations in RAG1 or RAG2 (n = 4, two of them presented with Omenn syndrome), IL2RG (n = 4, one of them with confirmed maternal engraftment), NHEJ1 (n = 1), CD3E (n = 1), ADA (n = 1), JAK3 (n = 3, two of them with maternal engraftment) and DCLRE1C (n = 1) and 11 other PID patients had diverse molecular defects [ZAP70 (n = 1), WAS (n = 2), PNP (n = 1), FOXP3 (n = 1), del22q11.2 (DiGeorge n = 4), CDC42 (n = 1) and FAS (n = 1)]. In addition, 44 healthy controls in the same age group were analyzed using the SCID-RTE tube in four EuroFlow laboratories using a standardized 8-color approach. RTE were defined as CD62L+CD45RO-HLA-DR-CD31+ and the activation status was assessed by the expression of HLA-DR+. Naïve CD8+ T-lymphocytes and naïve CD4+ T-lymphocytes were defined as CD62L+CD45RO-HLA-DR-. With the SCID-RTE tube, we identified patients with PID by low levels or absence of RTE in comparison to controls as well as low levels of naïve CD4+ and naïve CD8+ lymphocytes. These parameters yielded 100% sensitivity for SCID. All SCID patients had absence of RTE, including the patients with confirmed maternal engraftment or oligoclonally expanded T-cells characteristic for Omenn syndrome. Another dominant finding was the increased numbers of activated CD4+HLA-DR+ and CD8+HLA-DR+ lymphocytes. Therefore, the EuroFlow SCID-RTE tube together with the previously published PIDOT tube form a sensitive and complete cytometric diagnostic test suitable for patients suspected of severe PID (SCID or CID) as well as for children identified via newborn screening programs for SCID with low or absent T-cell receptor excision circles (TRECs).

• EuroFlow
• diagnosis
• flow cytometric immunophenotyping
• primary immunodeficiencies (PID)
• severe combined immune deficiency (SCID)
• standardization

• GaLV
• chromatography
• lentivirus vectors
• production
• purification
• tangential flow filtration

• SB100X transposase
• angiogenesis
• iris pigment epithelial cells (IPE)
• pigment epithelium derived factor (PEDF)
• retinal pigment epithelium (RPE)
• vascular endothelial growth factor (VEGF)

• gene therapy, gene editing
• hematopoietic stem cells
• inherited diseases

• Genetic Therapy/methods
• Humans
• Mucopolysaccharidoses/therapy

Chimeric antigen receptor (CAR) T cell therapy, together with checkpoint inhibition, has been celebrated as a breakthrough technology due to the substantial benefit observed in clinical trials with patients suffering from relapsed or refractory B‐cell malignancies. In this review, we provide a comprehensive overview of the clinical trials performed so far worldwide and analyze parameters such as targeted antigen and indication, CAR molecular design, CAR T cell manufacturing, anti‐tumor activities, and related toxicities. More than 200 CAR T cell clinical trials have been initiated so far, most of which aim to treat lymphoma or leukemia patients using CD19‐specific CARs. An increasing number of studies address solid tumors as well. Notably, not all clinical trials conducted so far have shown promising results. Indeed, in a few patients CAR T cell therapy resulted in severe adverse events with fatal outcome. Of note, less than 10% of the ongoing CAR T cell clinical trials are performed in Europe. Taking lead from our analysis, we discuss the problems and general hurdles preventing efficient clinical development of CAR T cells as well as opportunities, with a special focus on the European stage.

• ATMPs
• cancer
• immunotherapy
• regulatory issues
• toxicities

• Animals
• Bone Marrow Transplantation/methods
• Busulfan
• Cell Survival
• Cellular Microenvironment
• Disease Models, Animal
• Female
• Fetal Therapies/methods
• Genetic Therapy/methods
• Genetic Vectors/therapeutic use
• Hematopoietic Stem Cell Transplantation/methods
• Hepatocytes/transplantation
• Lentivirus/genetics
• Liver/embryology
• Male
• Mice
• Mice, Inbred C57BL
• Mice, Mutant Strains
• Transplantation Chimera
• Transplantation Conditioning
• Vidarabine/analogs & derivatives
• beta-Thalassemia/embryology
• beta-Thalassemia/genetics
• beta-Thalassemia/therapy

• MR/R015325/1 Medical Research Council
• MR/N026101/1 Medical Research Council
• MR/P026494/1 Medical Research Council
• MR/N019075/1 Medical Research Council
• G1000709 Medical Research Council

• Chimeric antigen receptor
• Genetic engineering
• Lentiviral vector
• Transduction of hematopoietic stem cells

• CNV mouse model
• VEGF
• anti-angiogenesis
• microRNA
• multigenic lentiviral vector
• ocular gene therapy

The immune system is a complex system able to recognize a wide variety of host agents, through different biological processes. For example, controlled changes in the redox state are able to start different pathways in immune cells and are involved in the killing of microbes. The generation and release of ROS in the form of an “oxidative burst” represent the pivotal mechanism by which phagocytic cells are able to destroy pathogens. On the other hand, impaired oxidative balance is also implicated in the pathogenesis of inflammatory complications, which may affect the function of many body systems. NADPH oxidase (NOX) plays a pivotal role in the production of ROS, and the defect of its different subunits leads to the development of chronic granulomatous disease (CGD). The defect of the different NOX subunits in CGD affects different organs. In this context, this review will be focused on the description of the effect of NOX2 deficiency in different body systems. Moreover, we will also focus our attention on the novel insight in the pathogenesis of immunodeficiency and inflammation-related manifestations and on the protective role of NOX2 deficiency against the development of atherosclerosis.

• LAMP
• PCR
• gene doping

The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD)—a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs). Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs.

• CGD
• CRISPR/Cas9
• NHEJ
• chronic granulomatous disease
• hematopoietic cells
• in situ gene correction
• non-homologous end joining

Our objective was to evaluate the life-long safety profile of gene therapy using retroviral (non-replicating) vectors (nRCR), or cell products in 127 subjects with hemophilia, human immunodeficiency virus (HIV), or cancer, previously treated with such gene therapy.

We assessed the occurrence of serious adverse events (SAEs), deaths and presence of replication competent retrovirus (RCR).

A total of 23 subjects remained until the data cut-off date of 31 July 2013 and provided safety information of up to 18 years. Of the 104 subjects who discontinued, the primary reason was loss to follow-up (47.2 %; n = 60). The follow-up period for the 60 subjects lost to follow-up was 7–10 years. A total of 41 subjects experienced at least one SAE, and 15 subjects died. We reviewed SAEs and cause of death (none related to the active therapy), but no evidence was found for safety signals related to new malignancy or neurologic, rheumatological, autoimmune, or hematologic disorder. RCR results were negative, indicating no evidence for in vivo vector persistence.

Despite the loss of follow-up, which is the limiting factor in this long-term safety trial, the findings from this long-term follow-up study are encouraging.

• Gene therapy
• Lysosomal storage diseases
• Transplantation

• Animals
• Enzyme Replacement Therapy
• Gene Transfer Techniques
• Genetic Therapy
• Genetic Vectors
• Hematopoietic Stem Cell Transplantation
• Humans
• Leukodystrophy, Metachromatic/genetics
• Leukodystrophy, Metachromatic/therapy
• Lysosomal Storage Diseases/genetics
• Lysosomal Storage Diseases/therapy
• Mucopolysaccharidosis I/genetics
• Mucopolysaccharidosis I/therapy
• Treatment Outcome
• Viruses

• Fondazione Telethon

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.

• flow-cytometry
• hematopoiesis
• hematopoietic stem cells
• immunodeficiency
• leukemia

Primary immunodeficiencies (PIDs) are a widely heterogeneous group of inherited defects of the immune system consisting of many clinical phenotypes with at least 300 underlying genetic deficits currently known. Patients with PIDs can present with, or develop during the course of their life, a susceptibility to recurrent and chronic infection along with autoimmune, allergic, inflammatory, and/or proliferative disorders, all potentially leading to end-organ damage. In recent years, a combination of basic and clinical research has greatly improved understanding of the underlying immunological and genetic defects in PIDs, leading to improved diagnosis, classification, and treatment approaches. In this review, we consider some of the key understandings that should direct diagnostic and treatment approaches in PID and offer insights into current and emerging management approaches and the lifelong care of patients from childhood through to adulthood.

• Primary immunodeficiency
• continuity of care
• diagnosis
• management
• reference centers/networks
• teenagers
• transfer clinic
• transition care
• young adults

Retroviral vectors have shown their curative potential in clinical trials correcting monogenetic disorders. However, therapeutic benefits were compromised due to vector-induced dysregulation of cellular genes and leukemia development in a subset of patients. Bromodomain and extraterminal domain (BET) proteins act as cellular cofactors that tether the murine leukemia virus (MLV) pre-integration complex to host chromatin via interaction with the MLV integrase (IN) and thereby define the typical gammaretroviral integration distribution. We engineered next-generation BET-independent (Bin) MLV vectors to retarget their integration to regions where they are less likely to dysregulate nearby genes. We mutated MLV IN to uncouple BET protein interaction and fused it with chromatin-binding peptides. The addition of the CBX1 chromodomain to MLV IN_W390A efficiently targeted integration away from gene regulatory elements. The retargeted vector produced at high titers and efficiently transduced CD34⁺ hematopoietic stem cells, while fewer colonies were detected in a serial colony-forming assay, a surrogate test for genotoxicity. Our findings underscore the potential of the engineered vectors to reduce the risk of insertional mutagenesis without compromising transduction efficiency. Ultimately, combined with other safety features in vector design, next-generation BinMLV vectors can improve the safety of gammaretroviral vectors for gene therapy.

• gammaretroviral vectors
• gene therapy
• targeted integration

The marketing approval of genetically engineered hematopoietic stem cells (HSCs) as the first-line therapy for the treatment of severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID) is a tribute to the substantial progress that has been made regarding HSC engineering in the past decade. Reproducible manufacturing of high-quality, clinical-grade, genetically engineered HSCs is the foundation for broadening the application of this technology. Herein, the current state-of-the-art manufacturing platforms to genetically engineer HSCs as well as the challenges pertaining to production standardization and product characterization are addressed in the context of primary immunodeficiency diseases (PIDs) and other monogenic disorders.

• ADA-SCID
• LMO2
• SCID-X
• WAS
• gamma-retroviral vectors
• hematopoietic stem cell transplantation
• hematopoietic stem cells
• lentiviral vectors
• manufacturing
• primary immunodeficiency disease

Lentiviral vectors have been successfully used for human skin cell gene transfer studies. Defining the selection of integration sites for retroviral vectors in the host genome is crucial in risk assessment analysis of gene therapy. However, genome-wide analyses of lentiviral integration sites in human keratinocytes, especially after prolonged growth, are poorly understood.

In this study, 874 unique lentiviral vector integration sites in human HaCaT keratinocytes after long-term culture were identified and analyzed with the online tool GTSG-QuickMap and SPSS software.

The data indicated that lentiviral vectors showed integration site preferences for genes and gene-rich regions.

This study will likely assist in determining the relative risks of the lentiviral vector system and in the design of a safe lentiviral vector system in the gene therapy of skin diseases.

• bone marrow transplantation
• gene transfer techniques
• lentivirus
• megakaryocytes
• platelets

• Animals
• Blood Platelets/metabolism
• Bone Marrow Transplantation
• Cell Differentiation
• Cell Lineage
• Genetic Vectors
• Glycoproteins/genetics
• Green Fluorescent Proteins/metabolism
• HEK293 Cells
• Hematopoietic Stem Cells/cytology
• Humans
• Lentivirus/genetics
• Megakaryocytes/metabolism
• Mice
• Mice, Inbred C57BL
• Microscopy, Fluorescence
• Platelet Factor 4/genetics
• Promoter Regions, Genetic

• Acyl Coenzyme A/genetics
• Animals
• Antigens, CD34/genetics
• Cholesterol/biosynthesis
• Cholesterol/genetics
• Genetic Therapy
• Genetic Vectors/genetics
• Genetic Vectors/therapeutic use
• HIV-1/genetics
• Hematopoietic Stem Cells/metabolism
• Humans
• Lentivirus/genetics
• Transduction, Genetic

• Delivery of vascular contrasting agents
• Dexamethasone
• Drug delivery by red blood cells
• Enzyme replacement therapy
• Erythrocytes
• Immunophilins
• Tacrolimus

• Green fluorescent protein
• HIV-1 Gag
• Protein delivery
• Protein engineering
• Transcription factor
• VLP production
• Virus-like particles

• CD34(−) negative fraction
• engraftment
• hematopoietic stem cell transplantation
• mesenchymal stromal cells

• AgoshRNA
• CCR5
• HIV-1
• Humanized mice
• Lentiviral vector
• RNAi
• shRNA-miR
• si/shRNA

• Acquired Immunodeficiency Syndrome/genetics
• Acquired Immunodeficiency Syndrome/prevention & control
• Acquired Immunodeficiency Syndrome/therapy
• Acquired Immunodeficiency Syndrome/virology
• Animals
• Clustered Regularly Interspaced Short Palindromic Repeats
• Gene Editing
• HIV Infections/genetics
• HIV Infections/prevention & control
• HIV Infections/therapy
• HIV Infections/virology
• HIV-1
• Humans
• RNA Interference
• RNA, Small Interfering/genetics
• RNAi Therapeutics/methods

• R21 AI108259 NIAID NIH HHS
• R21 HL116268 NHLBI NIH HHS

• CRISPR–Cas9
• DNA repair
• gene therapy
• genetic toxicology
• mutation
• precise genome editing

• Adenosine deaminase deficiency
• Chronic granulomatous disease
• Leukemia cell vaccine
• Severe combined immune deficiency
• Wiskott-Aldrich syndrome

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.

•

Hematopoietic reconstitution occurs in two distinct clonal waves

• Bone marrow transplantation
• Haploidentical stem cell transplantation
• Primary immune deficiency
• Reduced intensity conditioning