Gene therapy for sickle cell disease is limited by the yield of hematopoietic progenitor cells that can be harvested for transduction or gene editing. We therefore performed a phase I dose-escalation study of the hematopoietic progenitor cell mobilizing agent plerixafor to evaluate the efficacy and safety of standard dosing on peripheral blood CD34+ cell mobilization. Of 15 patients enrolled to date, only one was chronically transfused and ten were on hydroxyurea. Of eight patients who achieved a CD34+ cell concentration >30 cells/μL, six were on hydroxyurea. There was no clear dose response to increasing plerixafor dosage. There was a low rate of serious adverse events; two patients developed vaso-occlusive crises, at the doses of 80 μg/kg and 240 μg/kg. Hydroxyurea may have contributed to the limited CD34+ mobilization by affecting baseline peripheral blood CD34 counts, which correlated strongly with peak peripheral blood CD34 counts. Plerixafor administration did not induce significant increases in the fraction of activated neutrophils, monocytes, or platelets. However, increased neutrophils positive for activated β2 integrin and Mac-1 were associated with serious adverse events. In summary, plerixafor was well tolerated but did not achieve consistent CD34+ cell mobilization in this cohort of patients, most of whom were being actively treated with hydroxyurea and only one was chronically transfused. The study will continue with escalation of the dose of plerixafor and modification of hydroxyurea administration. Clinicaltrials.gov identifier: NCT02193191.

An incorrect version of sentence appared on May 2018 Issue, pages 773.Our target goal of mobilizing at least 30 CD34 + cells/μL was, however, reached in only 50% of patients given the plerixafor dose of 80 μg/kg, 33% of patients given 160 μg/kg, and 33% of patients given 240 μg/kg. The corrected version of sentence is published below.Our target goal of mobilizing at least 30 CD34 + cells/μL was, however, reached in only 50% of patients given the plerixafor dose of 80 μg/kg, 67% of patients given 160 μg/kg, and 67% of patients given 240 μg/kg. An incorrect version of sentence appared on May 2018 Issue, pages 777.Eight of 15 patients (53%) with SCD treated with plerixafor reached the peripheral blood CD34 cell target count of at least 30 CD34 + cells/μL, including three of six patients treated at a dose of 240 μg/kg. The corrected version of sentence is published below.Nine of 15 patients (60%) with SCD treated with plerixafor reached the peripheral blood CD34 cell target count of at least 30 CD34 + cells/μL, including four of six patients treated at a dose of 240 μg/kg.

Sickle RBCs (SS) represent a naturally existing host-cell resistance mechanism to hemoparasite infections. Here, we investigate the basis of this resistance using Babesia divergens grown in sickle and sickle trait cells. We found that oxygenation and its corresponding effect on RBC-sickling, frequency of fetal hemoglobin (HbF+) cells, the cellular redox environment and parasite proliferation dynamics, all play a role in supporting or inhibiting Babesia proliferation. To identify cellular determinants that supported infection, an image-flow-cytometric tool was developed which could identify sickled-cells and constituent hemoglobin. We showed that hypoxic conditions impaired parasite growth in both HbSS and HbAS cells. Further, cell-sickling was alleviated by oxygenation which in turn relieved the inhibition of parasite proliferation in SS cells under hypoxic conditions. Interestingly, our tool identified HbF+-sickle cells as host-cells of choice under both hypoxia and hyperoxia, which was confirmed using cord RBCs which contain high amounts of HbF. In contrast to AA and AS cells, uninfected HbSS cells showed a higher ROS-containing environment, which was further perturbed on infection. We investigated the parasite's response in hostile SS cells and found that it alters its sub-population structure, with 1N dominance under hypoxic conditions yielding to equivalent ratios of all parasite forms at hyperoxic conditions favorable for its growth. Multiple factors, including oxygenation and its impact on cell-shape, HbF positivity, redox status, and parasite pleiotropy allow Babesia propagation in sickle RBCs. Our studies provide a cellular and molecular basis of natural resistance to Babesia which will aid in defining novel therapies against human babesiosis.

Background: Increased baseline white blood cell count (WBC) has been associated with vaso-occlusive complications in sickle cell disease (SCD), raising concern for the use of mobilizing agents that increase WBC count for hematopoietic progenitor cell (HPC) collection for gene therapy. Indeed, G-CSF, which predominantly increases neutrophil counts, when administered to SCD patients can precipitate life-threatening vaso-occlusion. Such vaso-occlusion, however, does not always correlate with degree of white blood cell count elevation (Fitzhugh C et al, Cytotherapy 2009) and G-CSF is associated with increases in neutrophil activation and adhesion. This raises the question whether WBC might be a surrogate for other proposed contributors to vaso-occlusion, such as activation of white cells, platelets, and coagulation. We examined this issue in the context of a clinical study of HPC mobilization with a single dose of plerixafor alone in clinically stable SCD patients. Materials and Methods: 14 patients (13 SS, 1 SB0, median age 31 with range 21-46) have completed the study so far (6 patients at 80 ug/kg, 3 patients at 160 ug/kg, and 5 patients at 240 ug/kg). 10 patients were on hydroxyurea (HU) at a median dose of 25 mg/kg (range 16-28 mg/kg) with a mean HbF of 17% versus 7% for the non-HU treated patients. Only one patient (non-HU treated) had received recent red cell transfusion (for leg ulcers) and had HbA (54%) present. Whole blood was collected at means of 4 hr pre-dose and 12 hr post-dose for activation marker studies by flow cytometry (cell studies) or ELISA (coagulation studies) as listed in Table 1. Results: Increases in absolute total white cell, neutrophil, monocyte, and lymphocyte concentrations in whole blood with plerixafor treatment were all highly significant (Table 2, p < 0.002), without significant differences between HU- and non-HU treated patients or between dose levels. Twelve of 14 patients, however, did not experience any significant adverse events from plerixafor administration. Absolute neutrophil counts (ANC) were strongly correlated with absolute concentrations (but not percentages) of activated β2 integrin neutrophils (r= 1.0) and activated Mac-1 neutrophils (r= 0.8) but no other activation markers. We observed significant plerixafor-associated increases in activation markers (Figure 1) only for ANC subsets positive for activated β2 integrin (1.7± 0.44 fold, mean ± SD, p=0.002) and activated Mac-1 (1.7 ± 1.2 fold, p=0.05), which the correlations with ANC partly explain. The activated Mac-1 significance, however, disappeared with Bonferroni correction. There were no significant differences in activation markers between dose levels or between HU- and non-HU treated patients. There were moderate correlations between activated β2 integrin or activated Mac-1 and absolute reticulocyte count (r= 0.6 for both) and HbF (r= -0.5 for both) but not between these activation markers and LDH. Two patients had pain crises starting at 48 hr (80 ug/kg dose) and 81 hr (240 ug/kg dose) post-plerixafor administration; their pre and post-dose (12 hr) white counts are shown in Table 3. The fold-increase in activation markers for these 2 patients is summarized in Figure 2 (for each activation marker, color-coded arrows point to the two patients) Conclusion: As with G-CSF, elevation of WBC or their subsets with plerixafor is not necessarily associated with vaso-occlusion, as manifested by pain crises. In our study, however, ANC elevation correlated with absolute concentrations of neutrophils positive for activated β2 integrin and activated Mac-1, which were the only activation markers significantly increased with plerixafor. These activation markers in turn correlated with reticulocyte count and HbF, which influence SCD vaso-occlusive severity. Vaso-occlusion as manifested by pain crises, however, was not consistently associated with the highest fold increases in ANC or activation markers. Therefore it remains unclear which bioassays correlate with vaso-occlusion associated with plerixafor mobilization. Disclosures No relevant conflicts of interest to declare.