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

Somatic Mutation Tracking in Hematopoietic Stem Cell Gene Therapy Reveals Absence of Clonal Hematopoiesis

In hematopoietic stem cell Gene Therapy (GT), patients’ Hematopoietic Stem and Progenitor Cells (HSPCs) are genetically corrected ex vivo and reinfused to reconstitute the entire hematopoietic system and provide therapeutic benefit. During this process, HSPCs are subjected to tremendous pressures to sustain high levels of proliferation until the hematopoietic reconstitution is complete. So far it is unknown if this likely stressful condition may result in the accumulation of somatic mutations which may trigger decay of the hematopoietic functions and increased risk of oncogenesis. The impact of prolonged and heightened HSPCs proliferation rates on cellular fitness and safety remains an open question in GT. Indeed, HSPCs are not comprehensively geno-protected from DNA damage accumulation in HSPCs during aging and/or in specific disease conditions as it has been observed in sickle cell disease and Fanconi Anemia.Here we performed an analysis of somatic mutations in exons of 40 genes involved in clonal hematopoiesis and myeloid cancer in 23 GT patients treated for metachromatic leukodystrophy (MLD, 15 30 years old). We used genomic DNA from HSPCs cells prior infusion and peripheral blood mononuclear cells harvested at 2 years after GT and at the last available time point (2.5-7.5 years after GT). Sequencing reads correctly aligned on the targeted exon panel resulted in an average coverage of 4,400 and 4,300 reads/base in β-Thal and MLD patients respectively.The average mutation rate in the adult β-Thal patients was >2 fold higher than the rate measured in the pediatric patients (11.3 ± 11 vs 5.6 ± 3.5). Moreover, the average somatic mutation rates in adult and pediatric β-Thal patients were both significantly higher than in MLD patients (1.6 ± 0.72, p-value=0.0136 vs adult β-Thal, p-value=0.012 vs pediatric β-Thal, by Kruskal-Wallis test). None of the mutations were pathological or likely pathological. Most somatic mutations (85 out of 96) exhibited a Variant Allele Frequency of less than 2%. The average number of mutations in both the clinical trials remained consistent across all time points, showing no statistically significant variations. Considering that the sequenced genomic interval corresponds to 76,715 bps and that we analyzed a total of 8,100 equivalent genomes per patient, the resulting mutation rate in β-Thal patients was 1.21x10-8 mutations/bp, while MLD patients resulted in a mutation rate of 2.6x10-9 mutations/bp. Five mutations (4 in β-Thal and 1 in MLD) were found at more than one time point, but none showed a progressive increase in abundance, suggesting that these mutations did not confer a selective advantage to the mutated cell clones.Our work revealed that no somatic mutations known to drive clonal hematopoiesis or myeloid cancer, nor accumulation of somatic mutations, were found in our GT patients, indicating that the GT treatment was neutral in these conditions. However, the underlying disease in β-Thal patients resulted in a significantly higher mutation burden than MLD patients, a finding worth of deeper analysis and that supports long-term follow-up assessments of the clonal composition of the hematopoietic system in GT patients

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

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

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

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

Retroviral Integration Process in the Human Genome: Is It Really Non-Random? A New Statistical Approach

Retroviral vectors are widely used in gene therapy to introduce therapeutic genes into patients' cells, since, once delivered to the nucleus, the genes of interest are stably inserted (integrated) into the target cell genome. There is now compelling evidence that integration of retroviral vectors follows non-random patterns in mammalian genome, with a preference for active genes and regulatory regions. In particular, Moloney Leukemia Virus (MLV)–derived vectors show a tendency to integrate in the proximity of the transcription start site (TSS) of genes, occasionally resulting in the deregulation of gene expression and, where proto-oncogenes are targeted, in tumor initiation. This has drawn the attention of the scientific community to the molecular determinants of the retroviral integration process as well as to statistical methods to evaluate the genome-wide distribution of integration sites. In recent approaches, the observed distribution of MLV integration distances (IDs) from the TSS of the nearest gene is assumed to be non-random by empirical comparison with a random distribution generated by computational simulation procedures. To provide a statistical procedure to test the randomness of the retroviral insertion pattern, we propose a probability model (Beta distribution) based on IDs between two consecutive genes. We apply the procedure to a set of 595 unique MLV insertion sites retrieved from human hematopoietic stem/progenitor cells. The statistical goodness of fit test shows the suitability of this distribution to the observed data. Our statistical analysis confirms the preference of MLV-based vectors to integrate in promoter-proximal regions

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