Exploring biological and functional features of HSCs from different sources for gene therapy of β-thalassemia

Gene therapy for inherited diseases affecting the hematopoietic system requires transplantation and engraftment of a high number of autologous genetically engineered hematopoietic stem cells (HSCs). In the context of gene therapy for nonfatal diseases, as thalassemia, a favorable balance between the extent of conditioning of the host bone marrow (BM) and the adequate dose of transduced HSCs needs to be achieved. For adult thalassemic patients, the issue of HSC source is crucial as the minimal target dose poses a challenge for the use of steady-state BM. Peripheral blood (PB) mobilized CD34+ cells represent a valid alternative, but intrinsic characteristics of thalassemic patients (splenomegaly and thrombophilia) dictate caution in the choice of full dose G-CSF as mobilizing agent. The recently available mobilizing agent Plerixafor, which reversibly inhibits the stromal cell-derived factor1/CXC-motif receptor-4 (SDF-1–CXCR4) interaction within the BM microenvironment could represent an attractive alternative. We characterized HSCs mobilized from adult patients affected by transfusion dependent β-thalassemia by Plerixafor (Thal Plx PB) or by the combination of G-CSF + Plerixafor (Thal G+Plx PB), as collaborative study between Hematology-BMT Unit and TIGET, at San Raffaele Hospital in Milan. In addition, we had the unique opportunity to compare Plerixafor-mobilized PB cells with BM CD34+ cells pre- and post-Plerixafor treatment, derived from the same patient. CD34+ cells purified from patients’ leukoaphereses were analyzed for their biological and functional properties, subpopulations composition and expression profile. In vivo reconstitution potential and lymphomyeloid differentiation were tested following transplantation in NOD/SCID/IL2rγnull (NSG) mice. We performed comparative studies using thalassemic patient-derived samples and also BM and G-CSF-mobilized CD34+ cells from healthy donors, in order to unravel whether different sources might determine intrinsic molecular and functional features of HSCs. We showed that Plerixafor mobilizes high-quality HSCs able to provide stable long-term hematopoietic engraftment in NSG mice. Moreover, the quiescence status of these cells correlates with the enriched scid-repopulating cell frequency, in comparison to the other sources. The 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 and harvested from the BM acquire an intermediate signature between resident and circulating cells, suggesting an effect of this agent on HSC function. Mobilized CD34+ cells isolated from thalassemic samples were also efficiently transduced with the GLOBE lentiviral vector carrying the β-globin gene, and showed stable transgene expression. The availability of high numbers of Thal Plx PB and Thal G+Plx PB CD34+ cells allowed us to test in vitro different transduction protocols and also to evaluate the reconstitution potential of transduced cells in NSG mice. These experiments allowed to optimize culture conditions and to confirm the maintenance of engraftment and repopulation capacities of the mobilized cells also after in vitro manipulation. Our studies uncovered previously unknown unique HSC properties shaped by their origin and illuminate the choice of different transplantation strategies accordingly to the clinical need. The availability of new sources of HSCs, superior in terms of CD34+ cell yield, transduction efficiency and biological features to BM, indeed, would have a significant impact on the feasibility and efficacy of gene therapy.