Summary Hematopoietic stem cells (HSCs) are used in transplantation therapy to reconstitute the hematopoietic system. Human cord blood (hCB) transplantation has emerged as an attractive alternative treatment option when traditional HSC sources are unavailable, however, the absolute number of hCB HSCs transplanted is significantly lower than bone marrow or mobilized peripheral blood stem cells (MPBSCs). We previously demonstrated that dimethyl-prostaglandin E2 (dmPGE2) increased HSCs in vertebrate models. Here, we describe preclinical analyses of the therapeutic potential of dmPGE2-treatment using human and non-human primate HSCs. dmPGE2 significantly increased total human hematopoietic colony formation in vitro and enhanced engraftment of unfractionated and CD34+ hCB following xenotransplantation. In non-human primate autologous transplantation, dmPGE2-treated CD34+ MPBSCs showed stable multilineage engraftment over one year post-infusion. Together, our analyses indicated that dmPGE2 mediates conserved responses in HSCs from human and non-human primates, and provided sufficient preclinical information to support proceeding to an FDA-approved phase 1 clinical trial.
SUMMARY In mice, clonal tracking of hematopoietic stem cells has revealed variations in repopulation characteristics. However, it is unclear whether similar properties apply in primates. Here, we examined this issue through tracking of thousands of hematopoietic stem and progenitor cells (HSPCs) in rhesus macaques for up to 12 years. Approximately half of the clones analyzed contributed to long-term repopulation (over 3–10 years) and likely represent self-renewing hematopoietic stem cells (HSCs), while the remainder contributed primarily for the first year. The long-lived clones could be further subdivided into functional groups contributing primarily to myeloid, lymphoid or both lineages. Over time, the 4–10% of clones with robust dual lineage contribution predominated in repopulation capacity. HSPCs expressing a CCR5 shRNA transgene behaved similarly to controls. Our study therefore documents HSPC behavior in a clinically-relevant model over a long time frame, and provides a substantial system-level dataset that is a reference point for future work.
RNAi is a powerful method for suppressing gene expression that has tremendous potential for therapeutic applications. However, because endogenous RNAi plays a role in normal cellular functions, delivery and expression of siRNAs must be balanced with safety. Here we report successful stable expression in primates of siRNAs directed to chemokine (c-c motif) receptor 5 (CCR5) introduced through CD34؉ hematopoietic stem/progenitor cell transplant. After hematopoietic reconstitution, to date 14 months after transplant, we observe stably marked lymphocytes expressing siRNAs and consistent down-regulation of chemokine (c-c motif) receptor 5 expression. The marked cells are less susceptible to simian immunodeficiency virus infection ex vivo. These studies provide a successful demonstration that siRNAs can be used together with hematopoietic stem cell transplant to stably modulate gene expression in primates and potentially treat blood diseases such as HIV-1.short-hairpin RNA ͉ siRNA ͉ rhesus macaque ͉ gene therapy s iRNAs recognize cognate mRNAs and induce sequence specific RNA degradation through a highly conserved cellular mechanism (1). Because siRNAs have the potential for therapeutic application, a number of vector systems have been developed to express short-hairpin RNAs (shRNAs) to produce siRNAs within mammalian cells in tissue culture and in animal model systems (2-7). The results of these studies indicate that expression of siRNAs can potentially be used to effectively down-regulate gene expression in vivo for therapeutic purposes; however, it is important to control for the negative effects of expressing siRNAs in mammalian cells.
In this study, we used the rhesus macaque model to determine the impact that AMD3100 has on lymphocyte mobilization, both alone and in combination with G-CSF. Our results indicate that, unlike G-CSF, AMD3100 substantially mobilizes both B and T lymphocytes into the peripheral blood. This led to significant increases in the peripheral blood content of both effector and regulatory T-cell populations, which translated into greater accumulation of these cells in the resulting leukapheresis products. Notably, CD4 ؉ / CD25 high /CD127 low /FoxP3 ؉ Tregs were efficiently mobilized with AMD3100-containing regimens, with as much as a 4.0-fold enrichment in the leukapheresis product compared with G-CSF alone. CD8 ؉ T cells were mobilized to a greater extent than CD4 ؉ T cells, with accumulation of 3.7 ؎ 0.4-fold more total CD8؉ T cells and 6.2 ؎ 0.4-fold more CD8 ؉ effector memory T cells in the leukapheresis product compared with G-CSF alone.Given that effector memory T-cell subpopulations may mediate less GVHD compared with other effector T-cell populations and that Tregs are protective against GVHD, our results indicate that AMD3100 may mobilize a GVHD-protective T-cell repertoire, which would be of benefit in allogeneic hematopoietic stem cell transplantation. (Blood. 2011;118(25): 6580-6590) IntroductionThe widespread use of cytokine-mediated mobilization has had a major impact on hematopoietic stem cell transplantation (HSCT). For auto-HSCT, peripheral blood-derived stem cell (PBSC) transplantation is associated with more rapid hematopoietic reconstitution and better outcomes compared with bone marrow transplantation. [1][2][3][4][5] For allo-HSCT, the choice is more complex. A metaanalysis showed that PBSC transplants in adults resulted in more rapid hematopoietic reconstitution, decreased relapse, and increased disease-free survival compared with bone marrow transplantation 6 but did not lead to an overall survival advantage compared with bone marrow, except in patients with late-stage disease. 6 This was probably because of the higher T-cell content of PBSC grafts (10-to 50-fold more than bone marrow-derived allografts), [7][8][9] leading to a significantly greater risk of GVHD. 6 In pediatrics, this increased risk of GVHD and transplant-related mortality shifted the risk/benefit balance, favoring bone marrow over PBSCs. 10 These dichotomous results between pediatric and adult patients suggest that a narrow therapeutic window exists for infused lymphocytes.With the FDA approval of AMD3100 (Plerixafor or Mozobil), 11 mobilization can now occur by multiple regimens, including G-CSF alone, AMD3100 alone, or G-CSF plus AMD3100. Therefore, the risks and benefits of each of these mobilization strategies must be understood and compared with those associated with bone marrow transplantation. AMD3100 is US Food and Drug Administration (FDA)-approved for auto-HSCT, and the combination of G-CSF and AMD3100 was shown to be superior to G-CSF for stem cell mobilization. 12-14 Furthermore, there was accelerated lymphocyte recov...
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