Hematopoietic Stem Cell Mobilization for Gene Therapy: Superior Mobilization by the Combination of Granulocyte–Colony Stimulating Factor Plus Plerixafor in Patients with β-Thalassemia Major

Abstract

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.

Customizing the genome as therapy for the β-hemoglobinopathies

“…Harvest of CD34 1 HSPCs from peripheral blood is preferred due its minimal invasiveness and higher yield of CD34 1 HSPCs following mobilization by granulocyte colony-stimulating factor (G-CSF). 170 Use of G-CSF as a mobilizing agent is generally well tolerated for healthy adults and cancer patients. However, there are significant risks of G-CSF administration for patients with b-globin disorders.…”

Plerixafor and G-CSF combination mobilizes hematopoietic stem and progenitors cells with a distinct transcriptional profile and a reduced in vivo homing capacity compared to plerixafor alone

“…Further studies will contribute to dissecting the molecular basis underlying the direct and indirect actions of single and combined treatment on the HSC compartment in humans. Antonello Spinelli, 5 Bernhard Gentner, 4 Matilde Zambelli, 6 Cristina Parisi, 6 Laura Bellio, 6 Elena Cassinerio, 7 Laura Zanaboni, 7 Maria Domenica Cappellini, 7 Fabio Ciceri, 4 Sarah Marktel 4 and Giuliana Ferrari …”

Plerixafor+G-CSF–mobilized CD34+ cells represent an optimal graft source for thalassemia gene therapy

“…9,10 The bulk of the purified CD34 1 cells was cryopreserved for future clinical-grade gene transfer, whereas small aliquots were frozen for the preclinical studies, described herein. CD34 1 cell aliquots with .85% purity, from 31 differently mobilized thalassemic patients (HU1G-CSF: n 5 7, G-CSF: n 5 7, Plerixafor: n 5 13 and Plerixafor1G-CSF: n 5 4; supplemental Table 1 available on the Blood Web site) were thawed, prestimulated overnight, and subjected to two 24-hour transductions (multiplicity of infection 25 3 2) with the researchgrade TNS9.3.55 b-globin lenti-vector, 11 currently being clinically tested (#NCT01639690).…”

“…7,8 We previously defined Plerixafor1granulocyte colonystimulating factor (G-CSF) as the optimal mobilization approach for thalassemia GT over other strategies (G-CSF, hydroxyurea [HU]1G-CSF, Plerixafor) in 2 clinical trials. 9,10 Because differently mobilized cells may possess distinct intrinsic characteristics, we compare here their potency regarding gene transfer and vector-encoded b-globin expression in vitro and in xenografts to define the optimal graft for thalassemia GT.…”

“…Similar manipulation for Plerixafor1G-CSF-mobilized cells might thus boost VCN, maintaining their high b-globin expression/VCN and their long-term repopulating potential. Overall, the high CD34 1 cell numbers obtained by Plerixafor1G-CSF mobilization through single collections, even in hard-to-mobilize thalassemic patients, 10 coupled with the superior short-term engraftment and increased b-globin production/VCN of Plerixafor1G-CSF cells, render them an optimal graft for thalassemia GT. Importantly, a given required expression level might be achieved by Plerixafor1G-CSFmobilized cells at a lower VCN and, subsequently, higher biosafety for clinical applications.…”