Hematopoietic Stem Cell Mobilization for Gene Therapy: The Combination of G-CSF+Plerixafor in Patients with Beta-Thalassemia Major Provides High Yields of CD34+ Cells with Primitive Signatures

Hematopoietic stem cell engineering is a promising therapy to cure b-thalassemia, in particular for patients who lack a suitable BM donor for allogeneic transplantation. Since the engrafted gene-corrected stem cells will not have any selective advantage over the unmodified ones, the effectiveness of the therapy in this setting largely depends on the infusion of high numbers of gene-modified cells and on the conditioning regimen. The quality of the infused cells is also crucial for the clinical outcome and the duration of the therapeutic effect.

HSPCs mobilization, particularly when G-CSF and plerixafor are used in combination, has been proved to be the optimal approach to harvest a large number of CD34+cells in patients with hematological malignancies and in healthy volunteers. However adult heavily-transfused thalassemia patients have intrinsic characteristics that may adversely affect both the safety and the efficacy of mobilization.

We conducted a clinical trial to investigate the safety and effectiveness of mobilizing HSPCs with G-CSF+plerixafor in adult patients affected by β-thalassemia major with the aim to reach a cell dose of ≥8x10⁶ CD34+cells/Kg. We studied the kinetic of CD34+cells during mobilization and performed a comprehensive characterization of their molecular and functional properties.

All patients completed the mobilization according to the protocol (G-CSF 10 μg/kg/day for four days, followed by plerixafor 240 μg/kg in the evening on day 4) and no serious adverse events occurred. Leukapheresis was done 10-12 hours after plerixafor (on day 5). Three of the four patients reached the target cell dose or more in single-apheresis collections, even one patient where a significant dose reduction of G-CSF was halved due to early hyperleukocytosis. For one patients the number of cells collected in the first apheresis was slightly below the established target and therefore, according to the protocol, she was subjected to a second apheresis on day 6, after an additional dose of plerixafor. The total yield from the combined apheresis in this patient was 13.0 CD34+cells /Kg. CD34+ cell yields per single apheresis in our patients were comparable to those in healthy donors (12 pts) mobilized in our hospital with G-CSF alone.

A significant increase in the mean peripheral blood CD34+ cells (12.1± 8.2 fold), was unanimously observed after plerixafor addiction. The frequencies of the more primitive CD34+cell subtypes (CD34+CD38- and CD34+CD38-133+) as well as the clonogenic capacity tested in short term in vitro assay were found significantly increased too.

Comprehensive microarray analysis of genes expressed in the CD34+ cells purified from the same patient upon mobilization with G-CSF alone (G/CD34+cell) and with G-CSF+plerixafor (G+pl/CD34+cell) highlighted a different HSCs repertoire.

According to the mechanism of plerixafor mobilization, CXCR-4 gene expression was found 5-fold higher in G+pl/CD34+cells. CXCR-4 gene is known to be expressed on the surface of more primitive CD34+ HSCs with long-term repopulating potential and plays a central role in the regulation of adhesion of them to native niche in the BM.

A substantial number of genes with previously shown implication in mechanisms of homing and engraftment (CXCR4, CD82, DPP4, ROBO4), or genes linked to stress resistance (CXCL4, SOD2, IL8, PPBP) as well as several chemokines genes involved in cell mobility (CXCL2, CXC3, CXCR2) were also found to be up-regulated in G+pl/CD34+cells.

Overall, the yields, the primitive signatures of CD34+cells indicate the G-CSF+plerixafor mobilized peripheral blood as optimal graft that should favor HSPCs engraftment after transplantation. This findings has therapeutic implications not only for b-thalassemia but also for other hematopoietic stem cell gene therapy applications. This work was funded by the F and P Cutino Foundation - Project RiMedRi CUP G73F1200015000.