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.

Key Points Safe mobilization of CD34+ cells in adults with β-thalassemia and effective transduction with a globin vector under cGMP conditions. Stable vector copy number and β-globin expression in BFU-Es derived from engrafted CD34+ HPCs 6 months post-transplant in NSG mice.

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.