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Filgrastim+Plerixafor (especially in very poor mobilizers) has been observed by various groups (including ours); the rapid decrease in peripheral blood stem cells may cause collection to fail when apheresis is initiated according to conventional guidelines.34 We monitored the egress of CD34+ cells into the blood every hour after Plerixafor injection. The time course of CD34+ mobilization was remarkably similar in our three patients. Peak counts of over 80 CD34+/μL were achieved as early as 2 to 3 h after Plerixafor administration; this confirms that HSPC can be harvested immediately in SCD patients. The decrease in CD34+ cell count observed after 6 h might be the conse- quence of a short-term mobilization of HSC by Plerixafor and therefore to the reduced egress of CD34+ cells from BM combined with their return to the BM. Another possi- bility is that the drop in CD34+ cells is due to the leuka- pheresis procedure. Pantin et al. analyzed the kinetics of CD34+ counts in healthy donors treated with Plerixafor and not subjected to the apheresis procedure.35 In their work, CD34+ cell counts peaked at 6 h at lower values than the ones observed in our study and start to decrease 10 h after Plerixafor administration, with differences in kinetics and range of mobilization probably related to the clinical conditions of healthy donors versus SCD patients. Overall, the study by Pantin et al. suggests that leuka- pheresis per se does not cause a decrease in CD34+ cell counts. Additionally, the limited collection efficiency (≤30% of the circulating CD34+ cells) (Table 2) does not support the hypothesis that the drop is due to the leuka- pheresis procedure. Close monitoring of peripheral blood CD34+ cell counts is therefore a crucial point for efficient apheresis in SCD patients mobilized with Plerixafor.
The leukapheresis product contained significantly more HSC than the other stem cell sources used as controls, i.e. 8- to 10-fold more than in BM from healthy donors or SCD patients and in Filgrastim- or Plerixafor-mobilized cells from healthy donors. Accordingly, HSPC from the patients’ Plerixafor-mobilized samples showed elevated transcription of several HSC-associated genes. We do not have a formal explanation for this result; we can only hypothesize that sickling cycles damage the BM stroma and favor the mobilization of HSC.
Genes involved in inflammatory and immune responses were upregulated in Plerixafor-mobilized SCD samples. The inflammation-related characteristics of HSC and their environment constitute a major obstacle to both allogene- ic and autologous transplantation. Significant pathological changes in hematopoiesis have been described by Weisser et al.36 in a setting of murine and human chronic granulo- matous disease, an inherited disease characterized by chronic, sterile, granulomatous inflammation). In mice and in humans with chronic granulomatous disease, BM and Filgrastim-mobilized grafts contain a low proportion of HSC; in transplanted mice, this feature is associated with low reconstitution potential.36 Hence, we transplant- ed mobilized CD34+ cells from SCD patients into
immunodeficient NSG mice, in order to establish whether an inflammatory expression profile interfered with engraftment and self-renewal capacity. In fact, the cells engrafted as well as Filgrastim-mobilized samples in both primary and secondary transplantation, demonstrating that Plerixafor-mobilized SCD CD34+ cells contain true stem cells that are able to reconstitute human hematopoiesis as well as their Filgrastim-mobilized coun- terparts. Moreover, it is possible to effectively correct Plerixafor-mobilized SCD HSC by gene addition (manu- script in preparation). Taken as a whole, our results show that the inflammatory characteristics of HSC do not impair self-renewal and engraftment.
In conclusion, the present results show that CD34+ cells can be safely mobilized with Plerixafor in SCD patients under well-defined clinical conditions, including a 3- month interruption of hydroxyurea treatment, monthly transfusions and red blood cell exchanges. The proportion of true stem cells in the Plerixafor-mobilized CD34+ popu- lation was significantly higher than the proportion from any other source, although their egress must be monitored carefully. After harvesting under specific conditions, the cells can be successfully immunoselected. In the case that the optimal dose of CD34+ cells required for gene therapy (6-9x106/kg) is not reached after one apheresis and in the absence of adverse events, a second mobilization by Plerixafor, 24 h after the first one, will be considered. In our small cohort, the high numbers of cells enabled us to extend our gene-addition therapy project without having to perform several low-yield BM harvesting steps. Furthermore, the results removed the obstacle of collect- ing an appropriate graft for genome-editing purposes. Lastly, our study emphasizes the importance of consider- ing the specific characteristics of a diseased BM; finding a way to put the patient’s hematopoietic system into a steady-state condition may circumvent a lack of true stem cells in the harvested product or poor engraftment of genetically-modified cells.
Acknowledgments
The authors would like to thank Jean-Marc Luby for excellent, dedicated technical assistance, Valérie Jolaine, Michaela Semeraro for the logistics of the clinical trial, Michaela Semeraro for care of patients, and Christine Bole and Olivier Alibeu for the RNA sequencing. We also thank Frédéric Galacteros, Pablo Bartolucci and Susanne Matthes-Martin for revision of the clini- cal protocol.
Funding
This work was supported by state funding from the Agence Nationale de la Recherche as part of the Investissements d’Avenir program (ANR-10-IAHU-01 and ANR-16-CE18-0004), the French National Institute of Health and Medical Research (INSERM), and Assistance Publique-Hôpitaux de Paris (AP- HP). This work was also supported by a grant from the European Research Council (ERC 2015-AdG, GENEFORCURE).
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