Improved collection of hematopoietic stem cells and progenitors from Fanconi anemia patients for gene therapy purposes

Abstract

Difficulties in the collection of hematopoietic stem and progenitor cells (HSPCs) from Fanconi anemia (FA) patients have limited the gene therapy in this disease. We have investigated (ClinicalTrials.gov, NCT02931071) the safety and efficacy of filgrastim and plerixafor for mobilization of HSPCs and collection by leukapheresis in FA patients. Nine of eleven enrolled patients mobilized beyond the threshold level of 5 CD34⁺ cells/μL required to initiate apheresis. A median of 21.8 CD34⁺ cells/μL was reached at the peak of mobilization. Significantly, the oldest patients (15 and 16 years old) were the only ones who did not reach that threshold. A median of 4.27 million CD34⁺ cells/kg was collected in 2 or 3 aphereses. These numbers were markedly decreased to 1.1 million CD34⁺ cells/kg after immunoselection, probably because of weak expression of the CD34 antigen. However, these numbers were sufficient to facilitate the engraftment of corrected HSPCs in non-conditioned patients. No procedure-associated serious adverse events were observed. Mobilization of CD34⁺ cells correlated with younger age, higher leukocyte counts and hemoglobin values, lower mean corpuscular volume, and higher proportion of CD34⁺ cells in bone marrow (BM). All these values offer crucial information for the enrollment of FA patients for gene therapy protocols.

Keywords: AMD3100; Fanconi anemia; HSPC collection; Mozobil; filgrastim; gene therapy; lentiviral vector; leukapheresis; mobilization; plerixafor.

Graphical abstract

Figure 1

HSPC mobilization induced by filgrastim and plerixafor in patients with Fanconi anemia (A) Schematic representation of the CD34⁺ cell mobilization protocol based on the subcutaneous injection of filgrastim (twice daily; 12 μg/kg/12 h for up to 8 days) and plerixafor (240 μg/kg body weight/day; up to 4 doses). After each administration of plerixafor, CD34⁺ cell numbers were determined, and aphereses were initiated when numbers were higher than 5 CD34⁺ cells/μL PB. (B–D) kinetics of PB leukocytes, CD34⁺ cells, and CFCs, respectively, at different time points after administration of the mobilizing drugs. (E) increment of CD34⁺ cells and CFCs at the peak of mobilization compared to basal numbers determined at visit 0. Data corresponding to each of the treated patients are represented by a different color according to the ID indicated in the figure.

Figure 2

Analysis of the CD34⁺ cell content in apheresis and immunoselection products after HSPC mobilization with filgrastim and plerixafor (A) Number of CD34⁺ cells/kg collected in each of the apheresis procedures from the nine patients who fulfilled apheresis criteria. Total number of collected CD34⁺ cells/kg corresponding to each patient are also shown (gray bars). (B) CD34⁺ cell numbers after immunoselection. (C) CD34⁺ cell recovery after immunoselection. Dashed bars in (B) and (C) represent data corresponding to immunoselection processes modified to improve the CD34⁺ cell yields. Black lines in each panel represent median values and shadow areas the interval range of the median. Patients’ color codes are the same as in Figure 1.

Figure 3

Analysis of different HSPC populations in mobilized PB CD34⁺ cells corresponding to FA patients and healthy donors The figure shows the content of HSCs (A) and more differentiated progenitor cells (B and C) in mPB CD34⁺ cells from nine HDs and six FA patients. FA patients were pre-treated with filgrastim and plerixafor (color codes as in Figure 1). HDs were treated only with filgrastim or with plerixafor and filgrastim (marked with a star). ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001.

Figure 4

Relationship between basal PB and BM parameters determined at visit 0 and values of mPB CD34⁺ cells 5 days after initiation of the mobilization protocol. (A–F) the relationship between numbers of mPB CD34⁺ cells at day 5 of mobilization and age of the patients and also basal hematological (leukocyte counts, mean corpuscular values, hemoglobin levels) and BM (percentage and total CD34⁺ cells/μL) parameters determined on visit 0. The dotted line in last panel shows the efficient HSPC mobilization (≥10 CD34⁺ cells/μL PB) of every patient containing at least 30 CD34⁺ cells/μL BM at visit 0. Patients’ color IDs are the same as in Figure 1. ∗p<0.05, ∗∗p<0.01.