The development of HIV-derived vectors and the optimization of HSC transduction conditions has provided a significant contribution to the field of gene therapy for β-thalassemia, leading to the application of LVs expressing the human β-globin gene in preclinical murine models and in human thalassemic cells (Malik et al, 2005
; Sadelain et al, 2007
). The unique feature of β-globin LVs, carrying the transgene expressed by a combination of large regulatory elements derived from the globin locus, greatly affects the production of high-titer viral stocks and the transduction efficiency of target cells. Moreover, a delicate balance must be achieved to reconcile the needs for high efficiency of transduction of a considerable number of stem/progenitor cells, obtainable by cytokine-mediated stimulation and expansion and the maintenance of their biological features. Therefore, the characterization of transduced cells is crucial to predict a favourable outcome of the overall procedure. We characterized the BM-derived CD34+
cell population, which represents the preferential target of gene therapy clinical trials for genetic diseases involving children (Aiuti et al, 2009
; Gaspar et al, 2004
; Hacein-Bey-Abina et al, 2002
), by analyzing the phenotype, clonogenic capacity and changes induced by the transduction procedure. BM will represent the most suitable source of haematopoietic progenitors for autologous transplantation in pediatric thalassemic patients in future gene therapy trials. Indeed, BM harvest is a safe procedure in children (Buckner et al, 1984
), while cytokine-mobilization in thalassemic patients poses some safety concerns, due to the chronic hypercoagulable state (Taher et al, 2008
) and the condition of splenomegaly often associated with the disease. We found that the frequency of multipotent progenitors and lineage-committed precursors in thalassemic BM is comparable to that in normal samples, and is not affected by the transduction procedure. The phenotype analysis for expression of surface markers characterizing the multipotent, common lymphoid and myeloid progenitors reveals that short exposure to cytokines, present in the preactivation/transduction medium, favours the expansion of the myeloid subpopulation against the lymphoid one. Experiments in normal donor samples give comparable results and the change in the relative proportion of committed progenitors is not reflecting a significant alteration in the number and quality of early progenitors, scored by clonogenic assay. Considering that, in the setting of gene therapy, the haematopoietic reconstitution will occur in the absence of immunosuppression, a decreased number of lymphoid progenitors in the transplant is not likely to compromise the outcome. Indeed, no evidence of immunological failure was reported in gene therapy clinical trials for genetic diseases. Moreover, in the presence of stressed erythropoiesis and erythroid expansion, like in thalassemia major BM, it is relevant to test the proportion of erythroid committed progenitors. Differently from previous results (Mathias et al, 2000
), by analyzing a large number of samples we show normal expression of the erythroid differentiation markers and clonogenicity. Differences in patients' age and clinical status at the time of marrow harvest could explain these findings. Moreover, gene expression profiling of cytokine-treated and -untreated CD34+
cells suggests that the effect of the transduction is similar between patients and healthy donors. Nevertheless, the nature of specific genes that are differentially expressed upon cytokine treatment might suggest a specific reponse in thalassemic cells. Indeed, analysis of specific pathways relevant for HSC biology and activity is in progress. However, the best prediction for repopulating activity of human cells comes from transplantation of high number of CD34+
cells in immunodeficient mice. The source of cells for this kind of experiment would come only from back-up, harvested from patients before transplantation, and we are planning these experiments for the future.
Recently, we achieved long-term correction of thalassemia in the th3 murine model using the novel GLOBE vector (Miccio et al, 2008
). Following on from this successful result, the exploitation of the therapeutic efficacy of GLOBE was investigated in BM-derived CD34+
cells. We have shown correction of hallmark features of the β-thalassemia phenotype, such as synthesis of HbA and ineffective erythropoiesis. Analyses of erythroid cultures demonstrate that GLOBE is able to efficiently transduce CD34+
cells, providing physiological levels of HbA in the erythroid progeny with a relatively low number of integrants per cell, in line with our results in the murine model (Miccio et al, 2008
). The beneficial effect of transgene expression results in the increased proportion of mature erythroblasts in comparison to untransduced controls. Importantly, HPLC analysis of newly synthesized globin chains reveals that α/β chain ratio is in the range of that reported in literature by the analysis of β-thalassemia carriers (Giordano et al, 1999
), indicating that gene transfer with GLOBE provides transgene expression at therapeutic level. Successful results in patients' cells were previously achieved by transduction with a β-globin LV contained almost all LCR sites (HS 2, 3 and 4), in addition to the 3′ enhancer and the 1.2 kb cHS4 insulator (Puthenveetil et al, 2004
). However, as reported in recent studies, large insulator elements negatively affect vector titer and stability of viral particles, thus limiting the scale-up production for clinical application (Hanawa et al, 2009
; Urbinati et al, 2009
). Recent work on smaller sequences from cHS4 gave promising results in murine cells (Arumugam et al, 2009
). So far, efficacy data obtained with GLOBE in a large and heterogenous group of patients' samples strengthen the therapeutic potential of transcriptionally regulated globin LVs for future clinical application.
An important issue to address is the prediction of the safety of a gene therapy approach (Nienhuis et al, 2006
). In contrast to RVs, LVs appear to integrate in the host genome throughout the transcriptional unit without preference for TSSs or promoters (Bushman et al, 2005
), and are therefore associated to a lower risk of insertional activation of cellular genes by transcriptional mechanisms. Data from the first trial using LV in two patients affected by adrenoleukodystrophy are reassuring in terms of safety, with no evidence of expansion of specific transduced clones (Cartier et al, 2009
). Moreover, vectors carrying β-globin promoters and LCR elements restrict transgene expression to the differentiated progeny within a single lineage, thereby reducing the risk of activating oncogenes in haematopoietic stem and progenitor cells. Our integration site analysis shows that GLOBE has integration preferences virtually indistinguishable from those of any other LV. GLOBE integration sites were associated with transcriptionally active genes, evenly distributed among low to high expression categories. Integrations hot spots are less frequent compared to RVs, and cancer-related genes do not appear to be over-represented compared to controls. We found that most of the target genes are maintained transcriptionally active in the genome of differentiating erythroblasts at day 7 and day 14 (unpublished results). Overall, these findings suggest the presence of a favourable chromatin context for transgene expression around GLOBE integration sites and no specific risk for the GLOBE vector. Notably, it was recently published that the epigenetic changes in the transgene promoter can be modulated by the presence of insulator elements in the vector (Arumugam et al, 2009
), thus representing a tool for further improvement of transgene expression.
In conclusion, our results demonstrate the efficacy of a gene therapy approach for β-thalassemia by transduction of human progenitor cells with GLOBE vector and set the basis for a future clinical trial.