Wiskott-Aldrich Syndrome (WAS) is an inherited immunodeficiency caused by mutations in the gene encoding WASP, a protein regulating the cytoskeleton. Hematopoietic stem/progenitor cell (HSPC) transplants can be curative but, when matched donors are unavailable, infusion of autologous HSPCs modified ex vivo by gene therapy is an alternative approach. We used a lentiviral vector encoding functional WASP to genetically correct HSPCs from three WAS patients and re-infused the cells after reduced-intensity conditioning regimen. All three patients showed stable engraftment of WASP-expressing cells and improvements in platelet counts, immune functions, and clinical score. Vector integration analyses revealed highly polyclonal and multi-lineage haematopoiesis resulting from the gene corrected HSPCs. Lentiviral gene therapy did not induce selection of integrations near oncogenes and no aberrant clonal expansion was observed after 20–32 months. Although extended clinical observation is required to establish long-term safety, lentiviral gene therapy represents a promising treatment for WAS.

SummaryHepatic veno-occlusive disease (VOD) is a common complication of haematopoietic stem cell transplantation (HSCT), with reported incidences of 5-40% in children. Recently, defibrotide (DF) has been successfully used as prophylaxis and treatment of VOD. This study reports data on 63 human leucocyte antigen-matched HSCT performed in 57 children affected by beta thalassemia at very high risk for developing VOD (liver fibrosis, iron overload, hepatitis C virus infections, busulphan-based conditioning, methotraexate + ciclosporine). All patients received a busulphan-based conditioning regimen, either orally (four HSCT) or intravenously (59 HSCT). All patients received oral DF (40 mg/kg per day, final dose) as VOD prophylaxis from median day )9 to median day +29. In order to overcome the lack of oral paediatric formulations, a galenic formulation was administered. DF was well tolerated. Only one patient fulfilled Seattle Criteria for VOD diagnosis. This patient had discontinued DF 6 d prior to VOD onset, due to high risk of haemorrhage. We concluded that oral defibrotide prophylaxis and i.v. busulphan safely abated VOD incidence in high-risk patients who had undergone HSCT. A galenic preparation of oral DF also permits this treatment in low-weight patients. Costs of DF prophylaxis are acceptable considering the reduced incidence of VOD.

beta-thalassemia is a major health problem worldwide, and stem cell transplantation (SCT) is the only curative option. Oral Busulfan (Bu) based conditioning is widely used in this setting. Due to the variability of Bu systemic exposure, intravenous (i.v.) Bu has been proposed as a standard of care, with no need for drug monitoring and dose adjustment. Patients with beta-thalassemia from countries with limited resources might be at higher risk of erratic Bu metabolism because of liver dysfunction, severe iron overload, and specific ethnic/genetic features. We studied Bu pharmacokinetics in 53 children with advanced beta-thalassemia from Middle Eastern countries who underwent a total of 57 matched related donor SCTs. Forty-two percent of the children required dose adjustment because they did not achieve the therapeutic window after the first dose. With a Bu dose-adjustment policy, regimen-related toxicity was limited. At a median follow-up of 564 days, the probabilities of 2-year survival, current thalassemia-free survival, rejection, and treatment-related mortality were 96%, 88%, 21%, and 4%, respectively. Conditioning with i.v. Bu and dose adjustment is feasible and well tolerated, although recurrence of thalassemia remains an unsolved problem in children with advanced disease.

Mixed chimerism (MC) and secondary graft failure are frequent events following SCT for thalassemia. There is limited information regarding the outcome of donor lymphocyte infusion (DLI) to prevent rejection, mainly from case reports describing only successful cases. We describe a series of seven children affected by b-thalassemia treated with escalating doses of DLI for level 2-3 MC (donoro90%) following myeloablative SCT from a matched family donor. The infusions were safe and no acute or chronic GVHD were documented; five patients experienced neutropenia and thrombocytopenia resolving spontaneously. DLI was successful in converting to full donor chimerism two patients stratified in the low-risk class (Pesaro class II). Conversely, for five high-risk patients, DLI was not effective in preventing secondary graft failure. This limited series suggests that escalating doses of DLI are safe in thalassemia patients post myeloablative therapy but efficacy may be jeopardized by rapidly growing antidonor alloimmunity in high-risk patients. We suggest giving escalating doses of donor T cells to attempt a graft-versusthalassemia as soon as level 2-3 MC is detected.

Allogeneic BMT represents the only chance of cure for b-thalassemia. Occasionally, two affected individuals from the same family share a matched healthy sibling. Moreover, a high incidence of transplant rejection is still observed in Pesaro class III patients, requiring a second BMT procedure. In these settings, one option is to perform a second BM harvest from the same donor. Although BM harvest is a safe procedure in children, ethical issues concerning this invasive practice still arise. Here, we describe our series of seven pediatric, healthy donors, who donated BM more than once in favor of their b-thalassemic HLA-identical siblings between June 2005 and January 2008. Three donors donated BM twice to two affected siblings and four donors donated twice for the same sibling following graft rejection of the first BMT. All donors tolerated the procedures well and no relevant side effects occurred. There was no significant difference between the two harvests concerning cell yield and time to engraftment. Our experience shows that for pediatric donors, a second BM donation is safe and feasible and good cellularity can be obtained. We suggest that a second harvest of a pediatric donor can be performed when a strong indication for BMT exists.