Mobilization as a Preparative Regimen for Hematopoietic Stem Cell (HSC) Transplantation in Rhesus Macaques.

Abstract The myeloablative conditioning regimens currently used for hematopoietic stem cell (HSC) transplantation are associated with significant morbidity and mortality. Alternative strategies to promote engraftment of infused HSCs with increased safety warrant investigation. In a murine model, we previously demonstrated that, in absence of irradiation, mobilization with AMD3100 (a CXCR4 antagonist) before marrow transplantation vacated microenvironmental niches and resulted in higher levels of engraftment of transplanted HSCs compared to controls (no AMD3100 treatment before transplantation) (Abkowitz JL et al., Blood (ASH Annual Meeting Abstracts)104 (11): 1187, 2004). In this study, we hypothesized that AMD3100 mobilization before transplantation could also promote HSC engraftment in a large animal model, eliminating the need for toxic myeloablative conditioning. Peripheral blood cells from two rhesus macaques were collected by apheresis 3 hours after administration of a single dose of AMD3100 1mg/Kg. CD34+ cells were enriched and transduced for four days in the presence of cytokines and fibronectin with non-expression Moloney murine leukemia virus-derived retroviral vectors (G1PLI) that carry a bacterial neomycin phosphotransferase resistance gene (neoR). The neoR-marked CD34+ cells were reinfused in the non-myeloablated animals, immediately after AMD3100 mobilization and apheresis repeated on the day of transplantation. NeoR-marking levels of approximately 0.1% were detected in both peripheral blood MNC and granulocytes at two months (animal 2RC102) and four months (animal RQ4791) after transplantation. Previous transplantation studies performed without prior myeloablative conditioning or mobilization preparative regimen resulted in no long-term in vivo gene marking. We mathematically confirmed that this observed level of gene marking is what can be expected when AMD3100 mobilization is used as a conditioning regimen. Previous studies have estimated the number of long-term repopulating HSCs at 6 per 105 CD34+ cells (Abkowitz JL et al, Blood96: 3399, 2000). In animal RQ4791, approximately 4.5X107 CD34+ cells, and therefore 2700 HSCs, were mobilized after AMD3100 administration. The total number of HSCs per animal is thought to be conserved in mammals and has been estimated at 11,000 to 22,000 (Abkowitz JL et al, Blood100: 2665, 2002). Hence, 12–24% of HSCs were mobilized after a single dose of AMD3100, consequently opening 12–24% of microenvironmental niches for engraftment. If 1% of engrafted HSCs are marked, 0.12–0.24% long-term marking levels are expected, correlating well with the observed marking level of 0.1%. These results imply that the number of available niches in large animals, as in murine models, regulates the number of HSCs that engraft. As importantly, mobilization with AMD3100 could provide a non-toxic preparative approach in large mammals, including humans, to improve HSC engraftment in transplantation for genetic and other nonmalignant disorders.

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High Integrity and Fidelity of Long-Term Cryopreserved Umbilical Cord Blood for Transplantation

Journal of Clinical Medicine ◽

10.3390/jcm10020293 ◽

2021 ◽

Vol 10 (2) ◽

pp. 293

Author(s):

Gee-Hye Kim ◽

Jihye Kwak ◽

Sung Hee Kim ◽

Hee Jung Kim ◽

Hye Kyung Hong ◽

...

Keyword(s):

Stem Cell ◽

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Clinical Applications ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Hsc Transplantation

Umbilical cord blood (UCB) is used as a source of donor cells for hematopoietic stem cell (HSC) transplantation. The success of transplantation is dependent on the quality of cord blood (CB) units for maximizing the chance of engraftment. Improved outcomes following transplantation are associated with certain factors of cryopreserved CB units: total volume and total nucleated cell (TNC) count, mononuclear cell (MNC) count, and CD34+ cell count. The role of the storage period of CB units in determining the viability and counts of cells is less clear and is related to the quality of cryopreserved CB units. Herein, we demonstrate the recovery of viable TNCs and CD34+ cells, as well as the MNC viability in 20-year-old cryopreserved CB units in a CB bank (MEDIPOST Co., Ltd., Seongnam-si, Gyeonggi-do, Korea). In addition, cell populations in CB units were evaluated for future clinical applications. The stable recovery rate of the viability of cryopreserved CB that had been stored for up to 20 years suggested the possibility of uses of the long-term cryopreservation of CB units. Similar relationships were observed in the recovery of TNCs and CD34+ cells in units of cryopreserved and fresh CB. The high-viability recovery of long-term cryopreserved CB suggests that successful hematopoietic stem cell (HSC) transplantation and other clinical applications, which are suitable for treating incurable diseases, may be performed regardless of long-term storage.

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High Dose Plerixafor Is Safe and Mobilizes Higher Numbers of CD34+ Cells Compared with Standard Dose Plerixafor

Blood ◽

10.1182/blood.v120.21.585.585 ◽

2012 ◽

Vol 120 (21) ◽

pp. 585-585

Author(s):

Jeremy M Pantin ◽

Xin Tian ◽

Matthew M. Hsieh ◽

Lisa Cook ◽

Theresa Donohue ◽

...

Keyword(s):

Stem Cell ◽

Single Dose ◽

Adverse Events ◽

Dose Level ◽

Peripheral Blood ◽

Standard Dose ◽

High Dose ◽

Hematopoietic Stem ◽

Cell Numbers ◽

Cd34 Cells

Abstract Abstract 585 Introduction Plerixafor is a bicyclam compound that inhibits the binding of stromal cell derived factor-1 (SDF-1) to its cognate receptor CXCR4. This results in therapid release of CD34+ cells into circulation, which can then be collected by apheresis. Plerixafor is FDA approved at the 240 μg/kg dose to be used in conjunction with G-CSF to mobilize autografts for transplantation. Allogeneic grafts can also be mobilized using single agent plerixafor without G-CSF, and following transplantation, result in sustained donor derived hematopoiesis. However, when the 240 μg/kg dose is used, 1/3 of donors fail to mobilize minimally acceptable doses of CD34+ cells. Recently, we demonstrated the safety of administration of a single dose of 480 μg/kg of subcutaneous (sc) plerixafor in humans. We subsequently conducted a randomized cross-over trial comparing CD34+ mobilization in healthy subjects mobilized with a single dose of sc plerixafor given at either a high dose (480 μg/kg) or a conventional dose (240 μg/kg). Methods Twenty normal healthy volunteers were randomized and received either a 240 or 480 μg/kg dose of sc plerixafor followed by at least a 2 week wash out period then were administered the other dose of plerixafor. Circulating numbers of leukocytes and CD34+ cells/μlwere measured at multiple time points for 24 hours following each plerixafor injection and the CD34+ AUC over 24 hours was calculated for each subject at each dose level. Peripheral blood colony forming unit (CFU) assays were performed at baseline and 6 hours after plerixafor dosing. Adverse events were graded using CTCAE version 3.A sample size of 20 subjects was determined to have over 90% power to detect an absolute CD34+ count difference of 10/μl using this crossover design and a two-sidedpaired t-test at the 0.05 level. Results Twenty-three subjects were enrolled and 20 completed administration of both doses. Peak circulating CD34+ cell numbers (median 31.5 vs 25, p=0.0009), circulating CD34+ cell numbers at 24hrs (median 15.5 vs 9, p<0.0001), and the CD34+ AUC over 24 hours (median 543 vs 411, p<0.0001) were all significantly higher following the administration of the 480 μg/kg plerixafor dose compared to the 240 μg/kg dose. The time to peak CD34+ was also slightly longer after the 480 μg/kg dose (median 10 vs 8 hrs, p=0.011). These differences were not related to the order of administration of the 2 different plerixafor doses. Although GM-CFUs from the peripheral blood at 6hrs following plerixafor were significantly higher compared to baseline levels at both plerixafordoses, there was no dose-effect relationship observed between drug dose and fold increase in GM-CFUs. The incidence and severity of AE's did not differ between lower and higher doses of plerixafor and no grade 3 or greater adverse events occurred at either dose level. Conclusion These preliminary data suggest high dose plerixafor can be administered safely and may mobilize more CD34+ cells than standard dose plerixafor. Furthermore, these data suggest mobilization following a single dose of plerixafor and a single apheresis procedure would result in graft collections containing higher CD34+ cell numbers when allogeneic stem cell donors are mobilized with high-dose plerixafor compared to standard-dose. Disclosures: Off Label Use: Plerixafor, a hematopoietic stem cell mobilizer, is indicated in combination with granulocyte-colony stimulating factor (G-CSF) to mobilize hematopoietic stem cells to the peripheral blood for collection and subsequent autologous transplantation in patients with non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM).

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Estrogens Enhance Human Hematopoietic Stem Cell Engraftment in a Xenogenic Transplantation Model

Blood ◽

10.1182/blood-2018-99-112104 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2042-2042

Author(s):

Sara Fañanas-Baquero ◽

Israel Orman ◽

Federico Becerra Aparicio ◽

Silvia Bermudez de Miguel ◽

Jordi Garcia Merino ◽

...

Keyword(s):

Stem Cell ◽

Research Funding ◽

Short Term ◽

Myeloablative Conditioning ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic Recovery ◽

Cd34 Cells ◽

Equity Ownership

Abstract Hematopoietic Stem Cells (HSCs) is a rare cell population that sits atop a hierarchy of progenitors that become progressively restricted to several or a single blood lineage. HSCs are capable of self-renewal and multipotent differentiation to all blood cell lineages. HSCs are crucial in the maintenance of lifelong production of all blood cells. HSCs are highly regulated to maintain homeostasis through a delicate balance between quiescence, self-renewal and differentiation. However, this balance is altered during the hematopoietic recovery after Hematopoietic Stem Cell Transplantation (HSCT). HSCT is routinely used to reconstitute hematopoiesis after myeloablation, being the most commonly-used cell therapy. HSCT efficacy and multilineage reconstitution can be limited by inadequate HSC number, poor homing, engraftment, or limited self-renewal. Recent evidence indicates that estrogens are involved in regulating the hematopoietic system homeostasis. Estrogens are the primary female sex hormones and are responsible for controlling many cellular processes including growth, differentiation and function of the reproductive system. However, estrogens have also been proposed to regulate HSCs. b-Estradiol (E2) was shown to promote the cell cycle of HSCs and multipotent progenitors (MPPs) and increase erythroid differentiation in females (1). On the other hand, tamoxifen reduces the number of MPPs and short-term HSCs but activates proliferation of long-term HSCs (2). The potential clinical application of estrogens in HSCT mainly derives from the possibility that these drugs may enhance the engraftment of transplanted HSCs, thus reducing side effects associated to myeloablative conditioning. Here, we show that a short-term treatment of immunodeficient mice transplanted with hCD34+ cells with estrogens such as E2 and estetrol (E4) improves human hematopoietic engraftment. Fifty-thousand cord blood CD34+ cells (CB-CD34+) were transplanted into sublethally irradiated immunodeficient NSG mice. Three days after transplantation, mice were treated for four days with daily subcutaneous doses of E2, E4 or vehicle. Human hematopoietic engraftment was evaluated in the BM of transplanted mice at four months later. E2 and E4 estrogens increased the proportion of hCD45+ cells 1.8-fold and 2.4-fold as compared to values determined in control mice, without modifying the proportion of myeloid and lymphoid lineages. Significantly, animals treated with either estrogen had significantly higher levels of human hematopoietic progenitors (hCD45+CD34+). To study the engraftment of long-term engraftment HSCs in transplanted mice, human CD45+ cells from primary recipients were sorted and transplanted in secondary NSG recipients. Three months after transplants, the proportion of human hematopoietic cells in secondary recipients was also higher when primary recipients were treated with E2 or E4 than in vehicle-treated animals. Improved engraftments associated to the administration of E2 or E4 estrogens were confirmed when very low doses of CB-CD34+ cells were transplanted (5x103 hCD34+/mouse) in recipients of either sex. Collectively, our data support a new application of estrogens to improve the hematopoietic recovery after HSCT. This application may have particular relevance to enhance the hematopoietic recovery after myeloablative conditioning and when limiting numbers of HSCs are available. Disclosures Bueren: Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Segovia:Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

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Intrabone Delivery of CD34+ Cells Using an Optimized Delivery System Does Not Enhance Engraftment in a Rhesus Macaque Model of Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood-2018-99-114730 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5677-5677

Author(s):

Kate Stringaris ◽

Robert F Hoyt ◽

Jan Davidson ◽

Jeremy Pantin ◽

John F. Tisdale ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Rhesus Macaque ◽

Hematopoietic Stem Cell ◽

Rhesus Macaques ◽

Hematopoietic Progenitor ◽

Myeloablative Conditioning ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Marrow Space

Abstract Introduction: Umbilical cord blood (UCB) grafts are the only option for a significant minority of patients who require hematopoietic stem cell transplantation (HSCT) but lack a suitable related or unrelated donor. While UCB can serve as a suitable 'off the shelf' graft for many patients, units with the best HLA match often contain low and sometimes insufficient numbers of CD34+ cells for use in transplantation, particularly for adult patients. Furthermore, UCB grafts contain lower CD34+ cells number compared to BM or PBSC grafts, which leads to longer engraftment times and a higher risk of graft failure. BM and PBSC grafts are usually injected intravenously, homing to the bone marrow after several hours in the circulation. During this time, CD34+ cells are lost in the lungs, liver and spleen with typically < 20% making it to the bone marrow.1 Investigators have sought to overcome the limitation of low cell dose in UCB grafts and loss of CD34+ cells in the circulation by injecting CD34+ cells directly into the bone marrow space. However, we have recently shown that conventional intrabone delivery methods used in investigational trials to transplant UCB, result in low-level retention of hematopoietic progenitor cells in the intrabone space. Recently, we developed an optimized intrabone (OIB) transplant method using computer controlled low pressure and low volume injection (controlled infusion rate <0.2ml/min, total volume <5ml) under CT guidance. Utilizing porcine and rhesus macaque models, we have shown that OIB delivery of CD34+ cells improves intrabone retention, preventing circulation of CD34+ cells through the lungs, which is observed with conventional non-optimized intrabone methods.2 Here we conducted experiments using an autologous myeloablative transplant model in rhesus macaques comparing engraftment of gene-marked CD34+ cells transplanted intravenously, with cells transplanted using OIB delivery. Methods: Rhesus macaques received GCSF and plerixafor mobilization prior to apheresis. Products were CD34+ selected using Miltenyi beads. CD34+ cells were split equally for transduction with lentiviral vectors encoding the reporters GFP and YFP. After myeloablative conditioning with 10Gy total body irradiation, half the graft was injected directly intra-bone using the OIB method, with the other half of the autograft simultaneously being injected intravenously via slow iv push. Peripheral blood samples were measured daily by flow cytometry to assess the proportion of engrafting cells deriving from each source. To address whether OIB transplantation could allow engraftment of low CD34+ cell numbers, as found in UCB units, the cell doses transplanted in one rhesus macaque recipient were reduced to only 0.5 x 106CD34+ cells/kg. Results: CD34+ cells injected intrabone utilizing the OIB method engrafted in all 3 animals. However, flow cytometric analysis gating on GFP vs YFP positive neutrophils showed CD34+ cells injected utilizing OIB delivery did not engraft quicker than IV transplanted cells. Sequential monitoring of neutrophils over 30 days showed the contribution to hematopoiesis of OIB delivered cells was significantly lower than the cells injected IV (Table 1.) Conclusions: We developed a novel intrabone delivery system that optimizes the retention of CD34+ cells into the bone marrow space. Although autologous CD34+ cells injected using this OIB transplant method were capable of engrafting in rhesus macaques that had undergone myeloablative conditioning, they did not engraft faster and contributed less to hematopoiesis than CD34+ cells simultaneously transplanted using conventional IV infusion. These data raise questions over whether intrabone delivery, even when using techniques to optimize intrabone retention, has utility in improving CD34+ cell engraftment. References: 1. Van der Loo JC et al. Marrow and spleen seeding efficiencies of all murine hematopoietic stem cell subsets are de- creased by preincubation with hematopoietic growth factors. Blood. 1995; 85:2598-2606 2. Pantin et. al. Optimization of intrabone delivery of hematopoietic progenitor cells in a swine model using cell radiolabeling with 89zirconium. American J Transplant. 2015 Mar;15(3):606-17. Disclosures Davidson: Macrogenics: Employment. Pantin:NIH: Patents & Royalties: a patent application for an intrabone delivery device. Dunbar:National Institute of Health: Research Funding.

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Long-Term, Clonal Tracking Comparing Autologous Transplantation of G-CSF/SCF-Primed Bone Marrow CD34+ Cells with G-CSF/SCF-Mobilized Peripheral Blood CD34+ Cells in Rhesus Macaques

Blood ◽

10.1182/blood-2018-99-117047 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4524-4524

Author(s):

Sanggu Kim ◽

Robert E. Donahue ◽

Aylin Bonifacino ◽

Mark Metzger ◽

Cynthia E. Dunbar ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Stem Cell ◽

Peripheral Blood ◽

Rhesus Macaques ◽

Autologous Transplantation ◽

Low Frequency ◽

Time Points ◽

Cd34 Cells

Abstract Introduction : Although autologous transplantation of peripheral blood stem cells (PBSC) - mobilized with a combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) - has been well characterized, the efficacy of G-CSF/SCF-primed bone marrow stem cell (BMSC) transplantation, however, remains unclear and controversial. In our previous lentiviral vector-mediated PBSC and BMSC transplantation study, we reported efficient and long-term hematopoietic reconstitution by PBSC but not by BMSC - the later being associated with the gradual decline of vector markings in two BMSC-transplanted animals, with a loss of marking occurring in most lineages by 26 or 32 weeks after transplant. Follow-up analysis indicated that low-level yet consistent repopulation by BMSC continued in these animals for a longer period. Here we have compared peripheral blood (PB) markings and vector integration sites (VIS) in PBSC- and BMSC-transplanted animals for up to 12 years and 6 years, respectively. Methods : Young adult rhesus macaques were treated with G-CSF (10 mg/kg of body weight/day) and SCF (200 mg/kg/day) four days before the cell harvest for transplant. Mobilized PB leukapheresis cell products from five rhesus macaques (95E132, 2RC003, RQ5427, RQ3570, and 96E035) were harvested using a CS3000 Plus blood cell separator. Bone marrow (BM) cells from two animals (95E131 and 96E041) were surgically harvested from their femurs and iliac crests under anesthesia. After harvest, PBSC and BMSC were isolated by Ficoll-Hypaque density centrifugation followed by immunoselection of CD34+ cells, and transduced with HIV-based self-inactivating lentiviral vectors expressing EGFP. Vector-marked cells were then autologously transplanted into the host after total body irradiation (10 Gy). No further priming treatment was provided after transplant. PB from the 5 PBSC- and 2 BMSC-transplanted animals were serially collected over time and cryo-preserved for PCR, flow cytometry, and VIS analyses. Results: Both PBSC- and BMSC-transplanted animals showed long-term repopulation for lymphocytes, monocytes, granulocytes, platelets, and red blood cells. PBSC animals showed an average EGFP marking that ranged from 0.32 % to 10.24 %. From these animals, a total of 141 to 4,858 VIS were recovered. We found that the total number of VIS in each animal was proportional to the average EGFP marking in the same animals, and that both of these in turn were linearly correlated with the number of EGFP+ CD34+ cells initially transplanted (1.4 x106 - 28.8 x106 per animal). BMSC-transplanted animals did not show any such patterns. In two BMSC animals, the average EGFP marking levels remained at 0.05 % and 0.10 % until the end point (5 and 6 years) despite the fact that a comparatively large number of EGFP+ CD34+ cells had been transplanted (5.2 x106 and 17.7x106)and a large number of VIS recovered (793 and 680 VIS) in these animals. Temporal VIS analysis of PBSC animals showed that different groups of a large number of PBSC clones repopulated sequentially and reached a point of maximum repopulation at different time points, with some gradually declining after this. BMSC animals also showed a wave-like sequential repopulation similar to the patterns seen in PBSC animals. Unlike PBSC, however, nearly all BMSC clones were detected at a low frequency and at a single time point, except a few larger ones that were detected at multiple time points in a rising and falling pattern. There was no notable difference between the genomic features of VIS in PBSC- and BMSC-repopulating cells. Conclusions : Our data suggest that both the BMSC and PBSC consist of highly heterogeneous stem/progenitor cells that can provide long-term polyclonal repopulation through wave-like, sequential repopulation. Unlike PBSC, however, BMSC transplant was inefficient in PB repopulation resulting in only barely detectable markings in PB. The BMSC clonal profiles reflected the clonal patterns seen in PBSC animals, aside from BMSC animals having primarily low-frequency clones. We have previously shown significant differences in immunophenotype and cell cycle status between PBSC and BMSC, where BMSC were significantly lower in Thy-1 expression and had a higher percentage of cells in the S+G2/M phase of the cell cycle than PBSC. These differences may account for the inefficient differentiation and proliferation capabilities of BMSC compared to PBSC shown in this study. Disclosures Dunbar: National Institute of Health: Research Funding.

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Purified T Depleted Peripheral Blood and Bone Marrow CD34 Transplantation from Haploidentical Mother to Child with Thalassemia.

Blood ◽

10.1182/blood.v104.11.5161.5161 ◽

2004 ◽

Vol 104 (11) ◽

pp. 5161-5161

Author(s):

Pietro Sodani ◽

Marco Andreani ◽

Paola Polchi ◽

Javid Gaziev ◽

Filippo Centis ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

T Cell ◽

Peripheral Blood ◽

Therapeutic Option ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Preparative Regimen ◽

Chain Synthesis

Abstract Approximately 60% of thalassemic patients can not apply to “gene terapy today” which the insertion of one allogenic HLA identical stem cell into the empty bone marrow as the vector of the normal gene for beta globin chain synthesis. We studied the use of the haploidentical mother as the donor of hematopoietic stem cells assuming that the immuno-tollerance estabilished during the pregnancy will help to bypass the HLA disparity and allow the hemopoietic allogeneic reconstitution in the thalassemic recipient of the transplant.We have employed a new preparative regimen for the transplant in nine thalassemic children aged 3 to 8 years ( median age 5 years ) using T cell depleted peripheral blood stem cell (PBSCTs) plus bone marrow (BM) stem cells.. All patients received hydroxyurea (OHU) 60 mg/kg and azathioprine 3 mg/kg from day −59 untill day−11, fludarabine (FLU) 30 mg/m 2 from day −17 to day −11, busulphan (BU) 14 mg/kg starting on day −10, and cyclophosphamide(CY) 200mg/kg, Thiotepa 10 mg/kg and ATG Sangstat 2.5 mg/kg, followed by a CD34 + t cell depleted (CliniMacs sistem), granulocyte colony stimulating factor (G-csf) mobilized PBSC from their HLA haploidentical mother. The purity of CD34+ cells after MACS sorting was 98–99%, the average number of transplanted CD34+ cells was 15, 4 x 10 6/kg and the average number of infused T lymphocytes from BM was 1,8 x 10 5/Kg.The patients received cyclosporin after transplant for graft versus host disease( GVHD) prophilaxis. Four patients rejected the transplant and are alive with thalassemia: one patient received a different dose of CD3 without cyclosporine after transplant, two patients received a lower dose of CD34+, in the fourth patient the donor has been the haploidentical father instead than the mother. One of the nine patients, after the failure of the transplant from the mother, received a second transplant using purified CD34+ cells from the father, using the same preparative regimen and achieved a complete hematopoietic reconstitution. Six patients are alive disease free with a median follow up of 19 months (range 7–30). None of the six patients showed AGVHR. This preliminary study suggest that the transplantation of megadose of haploidentical CD34+ cell from the mother is a realistic therapeutic option for those thalassemic patients whithout genotipically or phenotipically HLA identical donor.

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Hematopoietic Stem Cell Gene Therapy Trial with Lentiviral Vector in X-Linked Adrenoleukodystrophy

Blood ◽

10.1182/blood.v112.11.821.821 ◽

2008 ◽

Vol 112 (11) ◽

pp. 821-821 ◽

Cited By ~ 3

Author(s):

Marina Cavazzana-Calvo ◽

Nathalie Cartier ◽

Salima Hacein-Bey Abina ◽

Gabor Veres ◽

Manfred Schmidt ◽

...

Keyword(s):

Gene Therapy ◽

Bone Marrow ◽

Stem Cell ◽

Lentiviral Vector ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Post Transplant ◽

Cd34 Cells ◽

Hsc Transplantation

Abstract We report preliminary results in 3 children with cerebral X-linked adrenoleukodystrophy (ALD) who received in September 2006, January 2007 and June 2008 lentiviral vector transduced autologous hematopoietic stem cell (HSC). We have previously demonstrated that cerebral demyelination associated with cerebral ALD can be stopped or reversed within 12–18 months by allogeneic HSC transplantation. The long term beneficial effects of HCT transplantation in ALD are due to the progressive turn-over of brain macrophages (microglia) derived from bone-marrow cells. For the current HSC gene therapy procedure, we used mobilized peripheral blood CD34+ cells that were transduced ex vivo for 18 hours with a non-replicative HIV1-derived lentiviral vector (CG1711 hALD) at MOI25 and expressing the ALD cDNA under the control of the MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substituted) promoter, and in the presence of 4 human recombinant cytokines (Il- 3, Stem Cell Factor [SCF], Flt3-ligand and Megakaryocyte Growth and Differentiation Factor [MGDF]) and CH-296 retronectine. Transduced cells were frozen to perform the required (RCL) safety tests. After thawing and prior to reinjection, 50%, 30% and 40% of transduced CD34+ cells expressed the ALD protein with a mean of 0.7, 0.6 and 0.65 copies of integrated provirus per cell. Transduced CD34+ cells were infused to ALD patients after a conditioning regimen including full doses of cyclophosphamide and busulfan. Hematopoietic recovery occured at day 13–15 post-transplant and the procedure was uneventful. In patient P1 and P2, the percentage of lymphocytes and monocytes expressing the ALD protein declined from day 60 to 6 months after gene therapy (GT) and remained stable up to 16 months post-GT. In P1, 9 to 13% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein 16 months post-transplant. In P2 and at the same time-point after transplant, 10 to 18% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein. ALD protein was expressed in 18–20% of bone marrow CD34+ cells from patients P1 and P2, 12 months post-transplant. In patient P3, 20 to 23% of CD3+, CD14+ and CD15+ cells expressed ALD protein 2 months after transplant. Tests assessing vector-derived RCL and vector mobilization were negative up to the last followups in the 3 patients. Integration of the vector was polyclonal and studies of integration sites arein progress. At 16 months post-transplant, HSC gene therapy resulted in neurological effects comparable with allogeneic HSC transplantation in patient P1 and P2. These results support that: ex-vivo HSC gene therapy using HIV1-derived lentiviral vector is not associated with the emergence of RCL and vector mobilization; a high percentage of hematopoietic progenitors were transduced expressing ALD protein in long term; no early evidence of selective advantage of the transduced ALD cells nor clonal expansion were observed. (This clinical trial is sponsored by Institut National de la Santé et de la Recherche Médicale and was conducted in part under a R&D collaboration with Cell Genesys, Inc., South San Francisco, CA)

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A Novel Small Molecule CXCR4 Antagonist Potently Mobilizes Hematopoietic Stem Cells in Mice and Monkeys

10.21203/rs.3.rs-49226/v1 ◽

2020 ◽

Author(s):

Xiao Fang ◽

Xiong Fang ◽

Yujia Mao ◽

Aaron Ciechanover ◽

Yan Xu ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Small Molecule ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Stromal Microenvironment ◽

Stromal Cell Derived Factor ◽

Hsc Transplantation

Abstract Background Hematopoietic stem cell (HSC) transplantation is an effective treatment strategy for many types of diseases. Peripheral blood (PB) is the most commonly used source of bone marrow (BM)-derived stem cells for current HSC transplantation. However, PB usually contains very few HSCs under normal conditions, as these cells are normally retained within the BM. This retention depends on the interaction between the CXC chemokine receptor 4 (CXCR4) expressed on the HSCs and its natural chemokine ligand, stromal cell-derived factor (SDF)-1α (also named CXCL12) present in the BM stromal microenvironment. In clinical practice, blocking this interaction with a CXCR4 antagonist can induce the rapid mobilization of HSCs from the BM into the PB.Methods C3H/HEJ, DBA/2, CD45.1+, CD45.2+ mice and monkeys were employed in colony-forming unit (CFU) assays, flow cytometry assays, and competitive/non-competitive transplantation assays, to assess the short-term mobilization efficacy of HF51116 and the long-term repopulating (LTR) ability of HSCs. Kinetics of different blood cells and the concentration of HF51116 in PB were also explored by blood routine examinations and pharmacokinetic assays. Results In this paper, we report that a novel small molecule CXCR4 antagonist, HF51116, which was designed and synthesized by our laboratory, can rapidly and potently mobilize HSCs from BM to PB in mice and monkeys. HF51116 not only mobilized HSCs when used alone but also synergized with the mobilizing effects of granulocyte-colony stimulating factor (G-CSF) after co-administration. Following mobilization by HF51116 and G-CSF, the long-term repopulating (LTR) and self-renewing HSCs were sufficiently engrafted in primary and secondary lethally irradiated mice and were able to rescue and support long-term mouse survival. In monkeys, HF51116 exhibited strong HSC mobilization activity and quickly reached the highest in vivo blood drug concentration. Conclusions These results demonstrate that HF51116 is a new promising stem cell mobilizer which specifically targets CXCR4 and merits further preclinical and clinical studies.

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Lower Hematopoietic Progenitor Cell Counts and Yields at Subsequent Donations Is Influenced By a Shorter Inter-Donation Interval between the First and Subsequent Mobilizations

Blood ◽

10.1182/blood-2019-123492 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1962-1962

Author(s):

Sandhya R. Panch ◽

Brent R. Logan ◽

Jennifer A. Sees ◽

Bipin N. Savani ◽

Nirali N. Shah ◽

...

Keyword(s):

Stem Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Time Interval ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Peripheral Blood Cd34 ◽

Cell Counts ◽

Cd34 Cells

Introduction: Approximately 7% of unrelated hematopoietic stem cell (HSC) donors are asked to donate a subsequent time to the same or different recipient. In a recent large CIBMTR study of second time donors, Stroncek et al. incidentally found that second peripheral blood stem cell (PBSC) collections had lower total CD34+ cells, CD34+ cells per liter of whole blood processed, and CD34+ cells per kg donor weight. Based on smaller studies, the time between the two independent PBSC donations (inter-donation interval) as well as donor sex, race and baseline lymphocyte counts appear to influence CD34+ cell yields at subsequent donations. Our objective was to retrospectively evaluate factors contributory to CD34+ cell yields at subsequent PBSC donation amongst NMDP donors. Methods. The study population consisted of filgrastim (G-CSF) mobilized PBSC donors through the NMDP/CIBMTR between 2006 and 2017, with a subsequent donation of the same product. evaluated the impact of inter-donation interval, donor demographics (age, BMI, race, sex, G-CSF dose, year of procedure, need for central line) and changes in complete blood counts (CBC), on the CD34+ cell yields/liter (x106/L) of blood processed at second donation and pre-apheresis (Day 5) peripheral blood CD34+ cell counts/liter (x106/L) at second donation. Linear regression was used to model log cell yields as a function of donor and collection related variables, time between donations, and changes in baseline values from first to second donation. Stepwise model building, along with interactions among significant variables were assessed. The Pearson chi-square test or the Kruskal-Wallis test compared discrete variables or continuous variables, respectively. For multivariate analysis, a significance level of 0.01 was used due to the large number of variables considered. Results: Among 513 PBSC donors who subsequently donated a second PBSC product, clinically relevant decreases in values at the second donation were observed in pre-apheresis CD34+ cells (73.9 vs. 68.6; p=0.03), CD34+cells/L blood processed (32.2 vs. 30.1; p=0.06), and total final CD34+ cell count (x106) (608 vs. 556; p=0.02). Median time interval between first and second PBSC donations was 11.7 months (range: 0.3-128.1). Using the median pre-apheresis peripheral blood CD34+ cell counts from donation 1 as the cut-off for high versus low mobilizers, we found that individuals who were likely to be high or low mobilizers at first donation were also likely to be high or low mobilizers at second donation, respectively (Table 1). This was independent of the inter-donation interval. In multivariate analyses, those with an inter-donation interval of >12 months, demonstrated higher CD34+cells/L blood processed compared to donors donating within a year (mean ratio 1.15, p<0.0001). Change in donor BMI was also a predictor for PBSC yields. If donor BMI decreased at second donation, so did the CD34+cells/L blood processed (0.74, p <0.0001). An average G-CSF dose above 960mcg was also associated with an increase in CD34+cells/L blood processed compared to donors who received less than 960mcg (1.04, p=0.005). (Table 2A). Pre-apheresis peripheral blood CD34+ cells on Day 5 of second donation were also affected by the inter-donation interval, with higher cell counts associated with a longer time interval (>12 months) between donations (1.23, p<0.0001). Further, independent of the inter-donation interval, GCSF doses greater than 960mcg per day associated with higher pre-apheresis CD34+ cells at second donation (1.26, p<0.0001); as was a higher baseline WBC count (>6.9) (1.3, p<0.0001) (Table 2B). Conclusions: In this large retrospective study of second time unrelated PBSC donors, a longer inter-donation interval was confirmed to be associated with better PBSC mobilization and collection. Given hematopoietic stem cell cycling times of 9-12 months in humans, where possible, repeat donors may be chosen based on these intervals to optimize PBSC yields. Changes in BMI are also to be considered while recruiting repeat donors. Some of these parameters may be improved marginally by increasing G-CSF dose within permissible limits. In most instances, however, sub-optimal mobilizers at first donation appear to donate suboptimal numbers of HSC at their subsequent donation. Disclosures Pulsipher: CSL Behring: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Research Funding; Bellicum: Consultancy; Amgen: Other: Lecture; Jazz: Other: Education for employees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Medac: Honoraria. Shaw:Therakos: Other: Speaker Engagement.

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Mobilization as a preparative regimen for hematopoietic stem cell transplantation

Blood ◽

10.1182/blood-2005-09-3593 ◽

2006 ◽

Vol 107 (9) ◽

pp. 3764-3771 ◽

Cited By ~ 53

Author(s):

Jing Chen ◽

André Larochelle ◽

Simon Fricker ◽

Gary Bridger ◽

Cynthia E. Dunbar ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Donor Cell ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Cell Engraftment ◽

Preparative Regimen ◽

And Control ◽

Hsc Transplantation ◽

Marrow Cells

Current myeloablative conditioning regimens for hematopoietic stem cell (HSC) transplantation are associated with significant morbidity and mortality. Thus, alternative strategies to promote engraftment of infused HSCs with increased safety warrant investigation. Using parabiotic mice, we determined that, after mobilization with AMD3100 (a CXCR4 antagonist), HSCs exited from marrow, transited blood, and engrafted in open niches in partner marrow. We then hypothesized that mobilization before transplantation might vacate niches and improve HSC engraftment. When PeP3b mice were treated with AMD3100 at 2 hours before the transplantation of 4 × 107 marrow cells, donor cell engraftment was higher (4.6% ± 1.1%) than in control animals (no AMD3100; 1.0% ± 0.24%, P < .001). When mice received weekly injections of AMD3100 on 3 consecutive weeks and marrow cells were transplanted 2 hours after each mobilization, donor cell engraftment further increased (9.1% ± 1.7%, P = .001). In contrast, in similar experiments with Balb/cByJ mice that mobilize poorly, there was no difference between the donor cell engraftment of AMD3100-treated and control recipients. These results indicate that the number of available niches regulates the number of HSCs. In addition, mobilization with AMD3100 may provide a safer preparative approach for HSC transplantation in genetic and other nonmalignant disorders.

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