The safety and efficacy of hematopoietic stem cell (HSC) mobilization was investigated in adult splenectomized (SPL) and non-SPL patients with thalassemia major, in two clinical trials, using different mobilization modes: granulocyte-colony-stimulating factor (G-CSF)-alone, G-CSF following pretreatment with hydroxyurea (HU), plerixafor-alone. G-CSF-mobilization was both safe and effective in non-SPL patients. However, in SPL patients the procedure resulted in excessive response to G-CSF, expressed as early hyperleukocytosis necessitating significant dose reduction, and suboptimal CD34(+) cells yields. One-month HU-pretreatment prevented hyperleukocytosis and allowed successful CD34(+) cell collections when an optimal washout period was maintained, but it significantly prolonged the mobilization procedure. Plerixafor resulted in rapid and effective mobilization in both SPL and non-SPL patients and was well-tolerated. For gene therapy of thalassemia, G-CSF or Plerixafor could be used as mobilization agents in non-SPL patients whereas Plerixafor appears to be the mobilization agent of choice in SPL adult thalassemics in terms of safety and efficacy.

Successful stem cell gene therapy requires high numbers of genetically engineered hematopoietic stem cells collected using optimal mobilization strategies. Here we focus on stem cell mobilization strategies for thalassemia and present the results of a plerixafor-based mobilization trial with emphasis on the remobilization with granulocyte-colony stimulating factor (G-CSF)+plerixafor in those patients who had previously failed mobilization. Plerixafor rapidly mobilized CD34(+) cells without inducing hyperleukocytosis; however, 35% of patients failed to reach the target cell dose of ≥6×10(6) CD34(+) cells/kg. Four subjects who failed on either plerixafor or G-CSF were remobilized with G-CSF+plerixafor. The combination proved highly synergistic; the target cell dose was readily reached and the per-apheresis yield was significantly increased over initial mobilization, ultimately resulting in single-apheresis collections, despite a more than 50% reduction of the dose of G-CSF in splenectomized patients to avoid hyperleukocytosis. The total stem and progenitor cells mobilized in G-CSF+plerixafor patients were higher than in patients treated by plerixafor alone. Importantly, the G-CSF+plerixafor-mobilized cells displayed a primitive stem cell phenotype and higher clonogenic capacity over plerixafor-mobilized cells. G-CSF+plerixafor represents the optimal strategy when very high yields of stem cells or a single apheresis is required. The high yields and the favorable transplantation features render the G-CSF+plerixafor-mobilized cells the optimal CD34(+) cell source for stem cell gene therapy applications.

Key Points Effective gene correction and long-term engraftment of human thalassemic CD34+ cells mobilized with different strategies. Plerixafor+G-CSF–mobilized CD34+ cells produce higher β-globin/VCN and superior early engraftment over single agent-mobilized cells.

Stable gene marking and effective engraftment of gene-modified CD34 hematopoietic stem cells is a prerequisite for gene therapy success but may be challenged by the inevitable cryopreservation of the final product prior to extensive quality assurance testing. We investigated the β-globin gene transfer potency in fresh and cryopreserved CD34 cells from mobilized patients with β-thalassemia, as well as the qualitative impact of repeated freeze/thaw cycles on the functionality of cultured and unmanipulated CD34 cells in terms of engrafting capacity in a xenotransplantation model, under partial myeloablation. Cells transduced fresh or after one freeze-thaw cycle yielded similar clonogenic and gene transfer frequencies. Repeated cryopreservation cycles did not affect the transduction rates whereas either one or two freeze-thaw cycles of cultured-but not of unmanipulated-cells significantly reduced their clonogenicity. No differences in the engrafting potential of gene-corrected cells subjected to either none or up to two cryopreservation cycles, were encountered post xenotransplantation. Overall, we assessed the gene transfer efficiency, clonogenicity and engrafting capacity of cryopreserved CD34 cells and the impact of repeated freeze/thaw cycles in their performance. These observations may prove essential in the design of gene therapy trials, considerably facilitating their logistics.

BACKGROUND Hematopoietic stem cell mobilization and leukapheresis in adult beta-thalassemia patients have been recently optimized in the context of clinical trials for obtaining hematopoietic stem cells for thalassemia gene therapy. In some patients, however, CD34+ cell yield was poor despite successful mobilization and the modification of apheresis settings was mandatory for harvest rescue. STUDY DESIGN AND METHODS The data of twenty adult β-thalassemia patients who were enrolled in a clinical trial for optimizing mobilization strategies for stem cell gene therapy were analyzed. The aim of this post-hoc analysis was to assess how certain hematological and/or clinical parameters may correlate with low collection efficiency in the presence of adequate numbers of circulating stem cells, after pharmacological mobilization and standard leukapheresis procedures. RESULTS Four patients among 19 optimally mobilized with Plerixafor or remobilized with G-CSF+Plerixafor patients demonstrated disproportionally poor CD34+ cell harvest, with respect to their circulating CD34+ cells after mobilization. All four patients who had undergone splenectomy presented at baseline and before first apheresis with lymphocytosis resulting in a lymphocyte/neutrophil ratio well above 1 and marked reticulocytosis as compared to patients with optimal mobilization/CD34+ cell harvest. Such unexpected expansion of specific cell populations disrupted the normal cell layer separation and necessitated modification of the apheresis settings in order to rescue the harvests. CONCLUSIONS By close examination of certain hematological and/or clinical parameters prior to leukapheresis, patients who, in spite of adequate mobilization are at risk for poor CD34+ cell harvests, may be identified and harvest failure can be prevented by adjusting of the apheresis settings.