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

Anticancer Gene Transfer for Cancer Gene Therapy

Gene therapy vectors are among the treatments currently used to treat malignant tumors. Gene therapy vectors use a specific therapeutic transgene that causes death in cancer cells. In early attempts at gene therapy, therapeutic transgenes were driven by non-specific vectors which induced toxicity to normal cells in addition to the cancer cells. Recently, novel cancer specific viral vectors have been developed that target cancer cells leaving normal cells unharmed. Here we review such cancer specific gene therapy systems currently used in the treatment of cancer and discuss the major challenges and future directions in this field

Dual-regulated lentiviral vector for gene therapy of X-linked chronic granulomatosis

Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34(+) HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development

Invasive characteristics of human prostatic epithelial cells: understanding the metastatic process

Prostate cancer has a predilection to metastasise to the bone marrow stroma (BMS) by an as yet uncharacterised mechanism. We have defined a series of coculture models of invasion, which simulate the blood/BMS boundary and allow the elucidation of the signalling and mechanics of trans-endothelial migration within the complex bone marrow environment. Confocal microscopy shows that prostate epithelial cells bind specifically to bone marrow endothelial-to-endothelial cell junctions and initiate endothelial cell retraction. Trans-endothelial migration proceeds via an epithelial cell pseudopodial process, with complete epithelial migration occurring after 232±43 min. Stromal-derived factor-1 (SDF-1)/CXCR4 signalling induced PC-3 to invade across a basement membrane although the level of invasion was 3.5-fold less than invasion towards BMS (P=0.0007) or bone marrow endothelial cells (P=0.004). Maximal SDF-1 signalling of invasion was completely inhibited by 10 μM of the SDF-1 inhibitor T140. However, 10 μM T140 only reduced invasion towards BMS and bone marrow endothelial cells by 59% (P=0.001) and 29% (P=0.011), respectively. This study highlights the need to examine the potential roles of signalling molecules and/or inhibitors, not just in single-cell models but in coculture models that mimic the complex environment of the bone marrow

Urogenital Abnormalities in Adenosine Deaminase Deficiency

BACKGROUND: Improved survival in ADA-SCID patients is revealing new aspects of the systemic disorder. Although increasing numbers of reports describe the systemic manifestations of adenosine deaminase deficiency, currently there are no studies in the literature evaluating genital development and pubertal progress in these patients. METHODS: We collected retrospective data on urogenital system and pubertal development of 86 ADA-SCID patients followed in the period 2000-2017 at the Great Ormond Street Hospital (UK) and 5 centers in Italy. In particular, we recorded clinical history and visits, and routine blood tests and ultrasound scans were performed as part of patients' follow-up. RESULTS AND DISCUSSION: We found a higher frequency of congenital and acquired undescended testes compared with healthy children (congenital, 22% in our sample, 0.5-4% described in healthy children; acquired, 16% in our sample, 1-3% in healthy children), mostly requiring orchidopexy. No urogenital abnormalities were noted in females. Spontaneous pubertal development occurred in the majority of female and male patients with a few cases of precocious or delayed puberty; no patient presented high FSH values. Neither ADA-SCID nor treatment performed (PEG-ADA, BMT, or GT) affected pubertal development or gonadic function. CONCLUSION: In summary, this report describes a high prevalence of cryptorchidism in a cohort of male ADA-SCID patients which could represent an additional systemic manifestation of ADA-SCID. Considering the impact urogenital and pubertal abnormalities can have on patients' quality of life, we feel it is essential to include urogenital evaluation in ADA-SCID patients to detect any abnormalities, initiate early treatment, and prevent long-term complications

Estimated Comparative Integration Hotspots Identify Different Behaviors of Retroviral Gene Transfer Vectors

Integration of retroviral vectors in the human genome follows non random patterns that favor insertional deregulation of gene expression and may cause risks of insertional mutagenesis when used in clinical gene therapy. Understanding how viral vectors integrate into the human genome is a key issue in predicting these risks. We provide a new statistical method to compare retroviral integration patterns. We identified the positions where vectors derived from the Human Immunodeficiency Virus (HIV) and the Moloney Murine Leukemia Virus (MLV) show different integration behaviors in human hematopoietic progenitor cells. Non-parametric density estimation was used to identify candidate comparative hotspots, which were then tested and ranked. We found 100 significative comparative hotspots, distributed throughout the chromosomes. HIV hotspots were wider and contained more genes than MLV ones. A Gene Ontology analysis of HIV targets showed enrichment of genes involved in antigen processing and presentation, reflecting the high HIV integration frequency observed at the MHC locus on chromosome 6. Four histone modifications/variants had a different mean density in comparative hotspots (H2AZ, H3K4me1, H3K4me3, H3K9me1), while gene expression within the comparative hotspots did not differ from background. These findings suggest the existence of epigenetic or nuclear three-dimensional topology contexts guiding retroviral integration to specific chromosome areas

Lentiviral gene therapy corrects platelet phenotype and function in patients with Wiskott-Aldrich syndrome

BACKGROUND: Thrombocytopenia is a serious issue for all patients with classical Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) because it causes severe and life-threatening bleeding. Lentiviral gene therapy (GT) for WAS has shown promising results in terms of immune reconstitution. However, despite the reduced severity and frequency of bleeding events, platelet counts remain low in GT-treated patients. OBJECTIVE: We carefully investigated platelet defects in terms of phenotype and function in untreated patients with WAS and assessed the effect of GT treatment on platelet dysfunction. METHODS: We analyzed a cohort of 20 patients with WAS/XLT, 15 of them receiving GT. Platelet phenotype and function were analyzed by using electron microscopy, flow cytometry, and an aggregation assay. Platelet protein composition was assessed before and after GT by means of proteomic profile analysis. RESULTS: We show that platelets from untreated patients with WAS have reduced size, abnormal ultrastructure, and a hyperactivated phenotype at steady state, whereas activation and aggregation responses to agonists are decreased. GT restores platelet size and function early after treatment and reduces the hyperactivated phenotype proportionally to WAS protein expression and length of follow-up. CONCLUSIONS: Our study highlights the coexistence of morphologic and multiple functional defects in platelets lacking WAS protein and demonstrates that GT normalizes the platelet proteomic profile with consequent restoration of platelet ultrastructure and phenotype, which might explain the observed reduction of bleeding episodes after GT. These results are instrumental also from the perspective of a future clinical trial in patients with XLT only presenting with microthrombocytopenia

Lymphomononuclear cells from multiple sclerosis patients spontaneously produce high levels of oncostatin M, tumor necrosis factors a

Proinflammatory cytokines are deemed to play a pivotal role in the pathogenesis of multiple sclerosis (MS). They provide signals for T-cell activatio

Clinical, immunological, and molecular features of typical and atypical severe combined immunodeficiency: Report of the italian primary immunodeficiency network

Severe combined immunodeficiencies (SCIDs) are a group of inborn errors of the immune system, usually associated with severe or life-threatening infections. Due to the variability of clinical phenotypes, the diagnostic complexity and the heterogeneity of the genetic basis, they are often difficult to recognize, leading to a significant diagnostic delay (DD). Aim of this study is to define presenting signs and natural history of SCID in a large cohort of patients, prior to hematopoietic stem cell or gene therapies. To this purpose, we conducted a 30-year retro-prospective multicenter study within the Italian Primary Immunodeficiency Network. One hundred eleven patients, diagnosed as typical or atypical SCID according to the European Society for Immune Deficiencies criteria, were included. Patients were subsequently classified based on the genetic alteration, pathogenic mechanism and immunological classification. A positive relationship between the age at onset and the DD was found. SCID patients with later onset were identified only in the last decade of observation. Syndromic SCIDs represented 28% of the cohort. Eight percent of the subjects were diagnosed in Intensive Care Units. Fifty-three percent had an atypical phenotype and most of them exhibited a discordant genotype-immunophenotype. Pre-treatment mortality was higher in atypical and syndromic patients. Our study broadens the knowledge of clinical and laboratory manifestations and genotype/phenotype correlation in patients with SCID and may facilitate the diagnosis of both typical and atypical forms of the disease in countries where newborn screening programs have not yet been implemented