Plerixafor enables safe, rapid, efficient mobilization of hematopoietic stem cells in sickle cell disease patients after exchange transfusion

Abstract

Sickle cell disease is characterized by chronic anemia and vaso-occlusive crises, which eventually lead to multi-organ damage and premature death. Hematopoietic stem cell transplantation is the only curative treatment but it is limited by toxicity and poor availability of HLA-compatible donors. A gene therapy approach based on the autologous transplantation of lentiviral-corrected hematopoietic stem and progenitor cells was shown to be efficacious in one patient. However, alterations of the bone marrow environment and properties of the red blood cells hamper the harvesting and immunoselection of patients' stem cells from bone marrow. The use of Filgrastim to mobilize large numbers of hematopoietic stem and progenitor cells into the circulation has been associated with severe adverse events in sickle cell patients. Thus, broader application of the gene therapy approach requires the development of alternative mobilization methods. We set up a phase I/II clinical trial whose primary objective was to assess the safety of a single injection of Plerixafor in sickle cell patients undergoing red blood cell exchange to decrease the hemoglobin S level to below 30%. The secondary objective was to measure the efficiency of mobilization and isolation of hematopoietic stem and progenitor cells. No adverse events were observed. Large numbers of CD34+ cells were mobilized extremely quickly. Importantly, the mobilized cells contained high numbers of hematopoietic stem cells, expressed high levels of stemness genes, and engrafted very efficiently in immunodeficient mice. Thus, Plerixafor can be safely used to mobilize hematopoietic stem cells in sickle cell patients; this finding opens up new avenues for treatment approaches based on gene addition and genome editing. Clinicaltrials.gov identifier: NCT02212535.

Copyright © 2018 Ferrata Storti Foundation.

Figures

Figure 1.

Plerixafor is highly efficient at mobilizing hematopoietic stem and progenitor cells from sickle cell disease patients. (A) Changes in white blood cell (WBC) and (B) CD34+ hematopoietic stem/progenitor cell (HSPC) counts over the 66 h following Plerixafor administration in SCD Pler 1 (red squares), SCD Pler 2 (blue circles) and SCD Pler 3 (green triangles). Arrows indicate time and duration of apheresis. (C) Number of hematopoietic stem cells (HSC, black bars) and multipotent progenitors (MPP, dotted bars) per 1,000 CD34+ cells in samples of various origins. HD: healthy donor, BM: bone marrow, SCD: sickle cell disease, Pler: Plerixafor, Filg: Filgrastim.

Figure 2.

Analysis of the transcriptomic profiles of hematopoietic stem and progenitor cells from different sources (A) Hierarchical clustering analysis of HD BM, SCD BM, SCD Plerixafor-mobilized (Pler), HD Plerixafor-mobilized and HD Filgrastim-mobilized (Filg) HSPC (cluster method: average; distance: correlation). The color of the sample name indicates the classification. (B) Gene ontology analysis of differentially expressed genes. The most enriched biological process categories are shown on the y-axis. The x-axis shows sample comparisons, as defined in Table 3. The orange and green color gradients correspond to the statistical significance of the enrichment [expressed as –log10 (qvalue)] in up- and downregulated genes, respectively. The first color bar at the top indicates comparisons between HSPC from different types of source (dark red) or the same type of source (light red). The second color bar at the top indicates comparisons between HSPC from different types of donor (dark blue) or the same type of donor (light blue). (C) Heat map of genes involved in HSC and progenitor biology. A proportion of the HSC markers were highly expressed in SCD Plerixafor-mobilized HSPC compared with the other samples. The row Z-score is plotted on a red-blue color scale, where red indicates high expression and blue indicates low expression. The color bar at the top indicates the sample classification. HD: healthy donor; BM: bone marrow; HSPC: hematopoietic stem and progenitor cells; HSC: hematopoietic stem cells; SCD: sickle cell disease; Pler: Plerixafor; Filg: Filgrastim.

Figure 3.

Plerixafor-mobilized CD34+ cells from sickle cell disease patients engraft to the same degree as Filgrastim-mobilized CD34+ cells from healthy donors in NSG mice. NSG mice were sacrificed 3 to 4 months after the injection of SCD (SCD Plerixafor, n=3) or HD (HD Filgrastim, n=2) CD34+ cells. (A) Bone marrow cells and (B) splenocytes were isolated, stained and analyzed by flow cytometry. The chimerism (defined as % human CD45+cells/total CD45+cells) and the numbers of human B lymphocytes (CD19+IgM+), granulocytes (CD11b+CD15+), and monocytes (CD11b+CD14+) were evaluated in each group of mice (red circles and red triangles SCD Pler1; blue circles and blue triangles SCD Pler2; green circles and green triangles SCD Pler3; the two HD Filg control are represented by gray squares/gray diamond and black squares/black diamonds, respectively). Each dot represents an individual mouse. HD: healthy donor; SCD: sickle cell disease; Pler: Plerixafor; Filg: Filgrastim.