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Hematopoietic stem and progenitor cell (HSPC)-based gene therapy (GT) requires the collection of a large number of cells. While bone marrow (BM) is the most common source of HSPCs in pediatric donors, the collection of autologous peripheral blood stem and progenitor cells (PBSCs) is an attractive alternative for GT. We present safety and efficacy data of a 10-year cohort of 45 pediatric patients that underwent PBSC collection for backup and/or purification of CD34+ cells for ex vivo gene transfer. Median age was 3.7 years and median weight 15.8 kg. After mobilization with lenograstim/plerixafor (n=41) or lenograstim alone (n=4), and 1-3 cycles of leukapheresis, median collection was 37 x106 CD34+ cells/kg. The procedures were well tolerated. Patients that collected ≥7 and ≥13 x106 CD34+ cells/kg in the first cycle had pre-apheresis circulating counts of at ≥42 and ≥86 CD34+ cells/μL respectively. Weight-adjusted CD34+ cell yield was positively correlated with peripheral CD34+ cell counts, and influenced by female gender, disease and drug dosage. All patients received a GT product above the minimum target, ranging from 4 to 30.9 x106 CD34+ cells/kg. Pediatric PBSC collection compares well to BM harvest in terms of CD34+ cell yields for the purpose of GT, with a favorable safety profile.

Emapalumab, a fully human anti-IFNγ monoclonal antibody, has been approved in the US as second-line treatment of primary hemophagocytic lymphohistiocytosis (HLH) patients and has shown promise in patients with graft failure (GF) requiring a second allogeneic hematopoietic stem cell transplantation (HSCT). The blockade of IFNγ activity may increase the risk of severe infections, including fatal mycobacteriosis. We report a case of secondary HLH-related GF in the context of HLA-haploidentical HSCT successfully treated with emapalumab in the presence of concomitant life-threatening infections, including disseminated tuberculosis (TB). A 4 years old girl with Adenosine Deaminase-Severe Combined Immunodeficiency complicated by disseminated TB came to our attention for ex-vivo hematopoietic stem cell-gene therapy. After engraftment failure of gene corrected cells, she received two HLA-haploidentical T-cell depleted HSCT from the father, both failed due to GF related to concomitant multiple infections and secondary HLH. Emapalumab administration allowed to control HLH, as well as to prevent GF after a third haplo-HSCT from the mother. Remarkably, all infections improved with antimicrobial medications and disseminated TB did not show any reactivation. This seminal case supports emapalumab use for treatment of secondary HLH and prevention of GF in patients undergoing haplo-HSCT even in the presence of multiple infections, including TB.

Introduction Autologous conditioned serum (ACS) is a novel blood product developed for intra-articular injection as a novel therapy for Osteoarthritis (OA). This study is a retrospective evaluation of 28 cases (25 Knee-OA and 3 hip-OA) treated with ACS between November 2013 and February 2016. Materials and methods ACS was prepared according to standards in an accredited Cell Manipulation Lab, and applied by an expert clinician (2 ml injection once weekly over 4 weeks). At any injection visit (Timepoints 1–4), and after a follow-up of 1 (Timepoint 5) and 6 months (Timepoint 6), patients were asked to describe the intensity of their pain with the VAS (visual analog scale) psychometric scale, and the objective parameter ROM (Range Of Motion) was recorded in case of injection in the knee. Results Pain (VAS) reduced in all cases since the first injection up to Timepoint 5. A significant improvement was observed in VAS between Timepoint 1 and 6 (primary objective), with a median VAS decrease of 60 mm (range 20–100, p A significant difference was also recorded in ROM between Timepoint 1 and 6 (secondary objective), with a median increase of 25° (range 5–40, p Ten out of 14 patients (71%) who were undergoing a chronic therapy to relieve pain were able to interrupt it. No serious adverse events were recorded. Conclusions Treatment with ACS produced a rapid decline in pain, accompanied by a large improvement in ROM. These results suggest that ACS is a valid option for the treatment of OA.

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 × 106/kg, with an optimal target at 5–10 × 106/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 × 106/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.

For decades, bone marrow (BM) has been the preferred source of hematopoietic stem and progenitor cells (HSPCs) for transplants following myeloablative conditioning. At present, mobilized peripheral blood stem cells are commonly used for transplantation, particularly in the autologous setting.[1][1

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. Disclosures Ciceri: MolMed SpA: Consultancy.