Collection of an adequate amount of autologous haematopoietic stem progenitor cells (HSPC) is required for ex vivo manipulation and successful engraftment for certain inherited disorders. Fifty-seven ...paediatric patients (age 0.5-11.4 years) underwent a bone marrow harvest for the purpose of HSPC gene therapy (GT), including adenosine deaminase-severe combined immunodeficiency (ADA-SCID), Wiskott-Aldrich syndrome (WAS) and metachromatic leukodystrophy (MLD) patients. Total nucleated cells and the percentage and absolute counts of CD34+ cells were calculated at defined steps of the procedure (harvest, CD34+ cell purification, transduction with the gene transfer vector and infusion of the medicinal product). A minimum CD34+ cell dose for infusion was 2 × 10
/kg, with an optimal target at 5-10 × 10
/kg. Median volume of bone marrow harvested was 34.2 ml/kg (range 14.2-56.6). The number of CD34+ cells collected correlated inversely with weight and age in all patients and particularly in the MLD children group. All patients reached the minimum target dose for infusion: median dose of CD34+ cells/kg infused was 10.3 × 10
/kg (3.7-25.9), with no difference among the three groups. Bone marrow harvest of volumes > 30 ml/kg in infants and children with ADA-SCID, WAS and MLD is well tolerated and allows obtaining an adequate dose of a medicinal product for HSPC-GT.
Adenosine deaminase-deficient severe combined immunodeficiency disease (ADA-SCID) is a primary immune deficiency characterized by mutations in the ADA gene resulting in accumulation of toxic ...compounds affecting multiple districts. Hematopoietic stem cell transplantation (HSCT) from a matched donor and hematopoietic stem cell gene therapy are the preferred options for definitive treatment. Enzyme replacement therapy (ERT) is used to manage the disease in the short term, while a decreased efficacy is reported in the medium-long term. To date, eight cases of lymphomas have been described in ADA-SCID patients. Here we report the first case of plasmablastic lymphoma occurring in a young adult with ADA-SCID on long-term ERT, which turned out to be Epstein-Barr virus associated. The patient previously received infusions of genetically modified T cells. A cumulative analysis of the eight published cases of lymphoma from 1992 to date, and the case here described, reveals a high mortality (89%). The most common form is diffuse large B-cell lymphoma, which predominantly occurs in extra nodal sites. Seven cases occurred in patients on ERT and two after haploidentical HSCT. The significant incidence of immunodeficiency-associated lymphoproliferative disorders and poor survival of patients developing this complication highlight the priority in finding a prompt curative treatment for ADA-SCID.
...corticosteroids were never administered to the patient, neither before nor during pioglitazone treatment. ...the patient did not develop any adverse effects related to pioglitazone administration, ...confirming the safety of this therapeutic approach. ...Fernandez-Boyanapalli et al6 suggest that the mechanism responsible for peroxisome proliferator-activated receptor gamma agonist effect in mice with CGD is related to increased ROS production by mitochondria in a subpopulation of neutrophils, monocytes, and inflammatory macrophages.
X-linked adrenoleukodystrophy (X-ALD) is the most common inherited peroxisomal disorder caused by variants in the
gene. The main phenotypes observed in men with X-ALD are primary adrenal ...insufficiency, adrenomyeloneuropathy, and cerebral ALD (cALD). Cerebral ALD consists of a demyelinating progressive cerebral white matter (WM) disease associated with rapid clinical decline and is fatal if left untreated. Hematopoietic stem cell transplantation is the standard treatment for cALD as it stabilizes WM degeneration when performed early in the disease. For this reason, early diagnosis is crucial, and several countries have already implemented their newborn screening programs (NBS) with the assessment of C26:0-lysophosphatidylcholine (C26:0-LPC) values as screening for X-ALD.
In June 2021, an Italian group in Lombardy launched a pilot study for the implementation of X-ALD in the Italian NBS program. A three-tiered approach was adopted, and it involved quantifying the values of C26:0-LPC and other metabolites in dried blood spots with FIA-MS/MS first, followed by the more specific ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) technique and, finally, the genetic confirmation
focused NGS.
Genetically confirmed patients are set to undergo a follow-up protocol and are periodically evaluated to promptly start a specific treatment if and when the first signs of brain damage appear, as suggested by international guidelines. A specific disease monitoring protocol has been created based on literature data and personal direct experience.
The primary aim of this study was to develop a model able to improve the early diagnosis and subsequent follow-up and timely treatment of X-ALD.
The study was approved by the local ethics committee. The research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.
β‐Thalassemia is a common monogenic disorder due to mutations in the β‐globin gene and gene therapy, based on autologous transplantation of genetically corrected haematopoietic stem cells (HSCs), ...holds the promise to treat patients lacking a compatible bone marrow (BM) donor. We recently showed correction of murine β‐thalassemia by gene transfer in HSCs with the GLOBE lentiviral vector (LV), expressing a transcriptionally regulated human β‐globin gene. Here, we report successful correction of thalassemia major in human cells, by studying a large cohort of pediatric patients of diverse ethnic origin, carriers of different mutations and all candidates to BM transplantation. Extensive characterization of BM‐derived CD34+ cells before and following gene transfer shows the achievement of high frequency of transduction, restoration of haemoglobin A synthesis, rescue from apoptosis and correction of ineffective erythropoiesis. The procedure does not significantly affect the differentiating potential and the relative proportion of haematopoietic progenitors. Analysis of vector integrations shows preferential targeting of transcriptionally active regions, without bias for cancer‐related genes. Overall, these results provide a solid rationale for a future clinical translation.
See accompanying article: http://dx.doi.org/10.1002/emmm.201000086
Over the past decades outcomes of clinical hematopoietic stem cell transplants have established a clear relationship between the sources of hematopoietic stem cells (HSCs) infused and their ...differential homing and engraftment properties. For a long time, bone marrow (BM) harvest has been the preferred source of hematopoietic stem and progenitor cells (HSPCs) for hematopoietic reconstitution following myeloablative conditioning regimen. At present, mobilized peripheral blood (PB) is commonly used for hematopoietic cells transplantation in both adults and children, particularly in the autologous setting, and it has progressively replaced BM as the source of HSCs. So far, the intrinsic molecular features of human primitive HSCs from different sources have not been investigated in comparative studies to unravel their variable reconstitution potential.
Diverse strategies are currently used to disengage HSCs from the niche, promoting egress from BM to PB. Traditionally the growth factor granulocyte-colony stimulating factor (G-CSF) represents the gold standard agent to mobilize HSPCs for transplantation. Nevertheless, many other compounds have been tested to this regard. One of the most successful mobilizing agents is Plerixafor (AMD3100, Mozobil™), a bicyclam molecule that selectively and reversibly antagonizes the binding of stromal cell derived factor-1 (SDF-1), located on the surface of BM stromal cells and osteoclasts, to chemokine CXC-receptor-4 (CXCR4), located on the surface of HSPCs, with the subsequent mobilization in the PB. This drug, which was shown in preclinical combination studies with G-CSF to enhance mobilization compared to G-CSF alone, is currently approved by FDA and EMA "in combination with G-CSF to enhance the mobilization of HSCs into the peripheral blood for collection and autologous transplantation of patients affected by lymphoma or multiple myeloma whose cells mobilize poorly"
We investigated functional and molecular hallmarks of human HSCs from different sources, i.e. BM and PB following mobilization by G-CSF and/or Plerixafor. We show that Plerixafor alone mobilizes preferentially long-term hematopoietic stem cells (LT-HSCs), defined as CD34+ CD38/low CD90+ CD45RA- CD49f+ cells and primitive populations of HSCs. These cells are able to provide stable long-term hematopoietic engraftment in NOD/SCID/IL2rγnull (NSG) mice, resulting in enriched scid-repopulating cell frequency, in comparison to other sources. The quiescence status of these cells correlates with the enriched scid-repopulating cell frequency. Noteworthy, the combined use of G-CSF and Plerixafor mobilizes a CD34+ population enriched in immature cells and with a lower engraftment capacity respect to cells mobilized by Plerixafor alone.
Since the signaling provided by the interaction of SDF-1 with CXCR4, plays an essential role in maintaining HSC quiescence and regulating homing, we analyzed the CXCR4 expression. Interestingly, this analysis reveals that the proportion of CXCR4+ primitive cells was lower when using G-CSF combined to Plerixafor in respect to Plerixafor alone. These data indicate that the combination of the two mobilizing agents induce a higher amount of circulating CD34+ cells but containing a lower proportion of cells capable of homing to BM in NSG mice. . As a result, at a defined dose of transplanted CD34+ cells, less SRCs are observed when G-CSF is added to Plerixafor. Indeed, it is expected to observe also a rapid rescue of hematopoiesis in myeloablated subjects conferred by high amount of short-term progenitors.
Insights into the transcriptional program reveal the molecular machinery underlying stemness features of cells derived from different sources, defining their specific functional properties. Noteworthy, CD34+ cells exposed to Plerixafor but still resident in the BM acquire an intermediate signature between steady-state and circulating cells, suggesting an effect of this agent on HSC function.
From preliminary data, genes of Prostaglandin signaling are up-regulated in HSCs mobilized by Plerixafor, suggesting a role of this pathway.
These data uncover unique HSCs properties shaped by their origin and illuminate the choice of different transplantation strategies accordingly to the clinical need.
No relevant conflicts of interest to declare.
Over the past decades outcomes of clinical hematopoietic stem cell transplants have established a clear relationship between the sources of hematopoietic stem cells (HSCs) infused and their ...differential homing and engraftment properties. For a long time, bone marrow (BM) harvest has been the preferred source of hematopoietic stem and progenitor cells (HSPCs) for hematopoietic reconstitution following myeloablative conditioning regimen. At present, mobilized peripheral blood (PB) is commonly used for hematopoietic cells transplantation in both adults and children, particularly in the autologous setting, and it has progressively replaced BM as the source of HSCs.HSCs are maintained in their niche by binding to cellular determinants through adhesion molecules and diverse strategies are currently used to promote their egress from BM to PB. Traditionally, the growth factor granulocyte-colony stimulating factor (G-CSF) represents the gold standard agent to mobilize HSPCs for transplantation. Nevertheless, other compounds have been recently tested.
One of the most successful mobilizing agents is Plerixafor (AMD3100, Mozobil™), a bicyclam molecule that selectively and reversibly antagonizes the binding of stromal cell derived factor-1 (SDF-1), located on the surface of BM stromal cells and osteoclasts, to chemokine CXC-receptor-4 (CXCR4), located on the surface of HSPCs, with the subsequent mobilization in the blood. The use of this drug is currently approved by FDA and EMA in combination with G-CSF, in patients affected by lymphoma or multiple myeloma whose cells mobilize poorly with G-CSF alone. Clinical trials demonstrated that Plerixafor alone safely and rapidly mobilizes HSCs also in healthy donors, beta-thalassemia patients and pediatric patients affected by malignancies. Previous characterization studies on non-human primates and human samples of Plerixafor mobilized cells in comparison to cells mobilized by G-CSF alone or in combination with Plerixafor showed a different expression profile, cell composition and engrafting potential in a xenotransplant model. From these studies remains unsolved whether Plerixafor, G-CSF, or their combination mobilizes different primitive HSC populations, defined both by multimarker immunophenotype and in vivo functional analysis.
In the present study we investigated by controlled comparative analysis the functional and molecular hallmarks of human HSCs collected from BM, G-CSF and/or Plerixafor mobilized peripheral blood. We show that Plerixafor alone mobilizes preferentially long-term hematopoietic stem cells (LT-HSCs), defined as CD34+CD38/lowCD90+CD45RA-CD49f+ cells and primitive populations of HSCs. These cells possess higher ability to home to hematopoietic niches and engraft in NOD/SCID/IL2rγnull (NSG) mice, resulting in enriched scid-repopulating cell frequency, in comparison to other sources. The higher content of CXCR4+ and CD49f+ cells correlates with this feature. Furthermore, global gene expression profiling highlights the superior in vivo reconstitution activity of Plerixafor mobilized cells. The "stemness" signature of cells dislodged from their niche by the drug is attenuated by the combined use with G-CSF, which emphasizes the gene expression profile induced by G-CSF treatment.
These data indicate that a qualitative advantage accounts for the superior performance of Plerixafor mobilized cells. These findings provide the rationale for using a suboptimal dose of more primitive HSCs when target cell number for transplantation is limited, or when G-CSF mobilization is too risky like in sickle cell anemia patients.
Moreover, CD34+ cells mobilized by Plerixafor alone or with the combination of G-CSF are efficiently transduced by a lentiviral vector encoding for human ß-globin gene (GLOBE LV) and are able to engraft and differentiate in vivo, supporting their use for gene therapy applications.
Ciceri:MolMed SpA: Consultancy.
β-Thalassemia is a severe inherited anemia caused by insufficient production of β-globin chains. Allogeneic hematopoietic stem cell (HSC) transplantation is currently the only cure, and is limited by ...donor availability and regimen-related toxicity and mortality. Gene therapy is a promising therapeutic tool for all thalassemic patients lacking a compatible donor and potentially provides transfusion independence in the absence of transplant-related complications, such as graft rejection and graft-versus-host disease. The issue of HSC procurement is critical in this setting because of the specific features of thalassemic syndromes, which include bone marrow (BM) expansion, ineffective erythropoiesis, and splenomegaly. Little is known about the efficiency of CD34(+) cell yield from steady-state BM harvests from thalassemic patients. We have collected data on safety and cell yield from 20 pediatric patients with β-thalassemia who underwent autologous BM harvest before allogeneic HSC transplantation, and from 49 age-matched sibling donors who also underwent BM harvest. The procedure was safe, as no significant adverse events occurred. In terms of cell yield, no difference was found between patients and normal donors in the number of CD34(+) cells and total nucleated cells harvested. Most importantly, no difference was found in the proportion of myeloid and erythroid progenitors, suggesting a similar repopulating capacity. On the basis of these results, we conclude that steady-state BM can be used as a safe and efficient source of HSC for gene therapy of β-thalassemia.
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