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.

IntroductionGene transfer into hematopoietic stem/progenitor cells (HSCs) by ␥-retroviral vectors (RVs) has been demonstrated as an effective treatment for inherited blood disorders, including X-linked severe combined immunodeficiency (SCID-X1), 1,2 adenosine deaminasedeficient SCID (ADA-SCID), 3 and X-linked chronic granulomatous disease (X-CGD). 4 However, after the occurrence of leukemic proliferation in 5 of 19 SCID-X1 patients 5,6 and of clonal expansion in 2 CGD patients, 7 the safety of the use of retroviral vectors turned out to be a primary concern. Because the viral RNA genome is reverse transcribed into a DNA copy that becomes integrated into the host cell genome, RV may induce cell transformation by insertional mutagenesis leading to proto-oncogene trans-activation, in combination with transgene-and disease-specific cofactors.The genomic features driving the preferential integration of RVs have been described in several large-scale survey of mapping studies in mice, nonhuman primates, and humans enrolled in clinical trials. [8][9][10][11][12][13][14] These studies have shown that RVs integrate nonrandomly in the host genome favoring transcription start sites (TSSs) and expressed genes, which makes productive interactions between the vector and host transcriptional machinery more probable. As a result of this bias, integrations that alter the expression of flanking genes involved in cell cycle, transcriptional activity, and signal transduction might influence the biologic fate of the affected cell clone, thereby conferring a growth or survival advantage. 4,15,16 However, to gain relevant information on the influence of vector integrations on transcriptional activity of nearby genes, studies should be carried out in relevant primary cells at the level of a clonal population containing defined insertion sites. Previous work conducted in the context of gene therapy with retrovirally transduced peripheral blood (PB) T cells revealed that RV insertion induced overexpression of a proximal gene at a frequency of almost 20%, although without measurable consequences on the biology and function of infused cells. 17 We showed previously that promoters and transcriptionally active genes are preferential sites of RV integration also in bone marrow-derived, transduced CD34 ϩ progenitor cells, and in their progeny isolated ex vivo from ADA-SCID patients. We also found that, in an ADA-deficient context, selective growth advantage of ADA ϩ lymphocytes is associated with a significant bias toward integrations in genomic regions, allowing a sustained transgene expression. Nevertheless, infusion of ADA-transduced HSCs is safe long-term and does not result in in vivo clonal expansion or malignant transformation up to 8 years after treatment. 11,18 Submitted February 4, 2009; accepted July 13, 2009. Prepublished online as Blood First Edition paper, August 3, 2009; DOI 10.1182 DOI 10. /blood-2009 *B.C. and E.M. contributed equally to this study.An Inside Blood analysis of this article appears at the front of this issue.The ...

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.

Acute lymphoblastic leukemia (ALL) in second complete remission is one of the most common indications for allogeneic hematopoietic stem cell transplantation (HSCT) in pediatric patients. We compared the outcome after HCST of adolescents, aged 14 to 18 years, with that of children (ie, patients < 14 years of age). Enrolled in the study were 395 patients given the allograft between January 1990 and December 2007; both children (334) and adolescents (61) were transplanted in the same pediatric institutions. All patients received a myeloablative regimen that included total body irradiation in the majority of them. The donor was an HLAidentical sibling for 199 patients and an unrelated volunteer in the remaining 196 patients. Children and adolescents had a comparable cumulative incidence of transplantation-related mortality, disease recurrence, and of both acute and chronic graft-versus-host disease. The 10-year probability of overall survival and event-free survival for the whole cohort of patients were 57% (95% confidence interval, 52%-62%) and 54% (95% confidence interval, 49%-59%), respectively, with no difference between children and adolescents. This study documents that adolescents with ALL in second complete remission given HSCT in pediatric centers have an outcome that does not differ from that of patients younger than 14 years of age.