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

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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Integrated Single Cell Transcriptomics Defines an Engineered Niche Supporting Hematopoietic Stem Cell Development Ex Vivo

Blood ◽

10.1182/blood-2019-126109 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3699-3699

Author(s):

Brandon Hadland ◽

Barbara Varnum-Finney ◽

Stacey Dozono ◽

Tessa Dignum ◽

Cynthia Nourigat-Mckay ◽

...

Keyword(s):

Stem Cell ◽

Single Cell ◽

Research Funding ◽

Signal Pathways ◽

Self Renewal ◽

Hematopoietic Stem ◽

Single Cell Rna Sequencing ◽

Equity Ownership

During embryonic development, hematopoietic stem cells (HSC) arise from hemogenic endothelial cells (HEC) within arterial vessels such as the aorta of the AGM (aorta-gonad-mesonephros) region, in a process referred to as the endothelial to hematopoietic transition (EHT). Although numerous signal pathways have been implicated in EHT, the precise combination of niche-derived signals required to support the generation and self-renewal of functional, long-term engrafting HSC remains poorly defined. To elucidate the niche signals regulating HSC emergence, we used single cell RNA-sequencing to simultaneously analyze the global transcriptional profiles of HEC during their transition to HSC and the AGM-derived endothelial cell stroma (AGM-EC) that supports the generation and expansion of functional HSC. Trajectory analysis of single cell transcriptomes enabled reconstruction of EHT in pseudotime, revealing dynamics of gene expression, including genes encoding cell surface receptors and downstream pathways, during the process of HSC genesis and self-renewal in vivo and in vitro. Transcriptional profiles of niche AGM-EC enabled identification of corresponding ligands which serve to activate these receptors during HSC generation. We integrated this knowledge to engineer a stromal cell-free niche for generation of engrafting HSC from hemogenic precursors in vitro. Specifically, we defined serum-free conditions combining immobilized Notch1 and Notch2-specific antibodies to activate Notch receptors, recombinant VCAM1-Fc chimera or fibronectin fragment to bind VLA-4 integrin, recombinant interleukin-3, stem cell factor, thrombopoietin, and CXCL12 to activate their respective cytokine/chemokine receptors, and small molecule inhibition of TGF-β Receptor 1. We demonstrated that this engineered niche is sufficient to support the generation of functional HSC, as measured by long-term (24 week) multilineage engraftment after transplantation to immune-competent, lethally irradiated adult recipient mice, following culture of hemogenic precursors isolated from E9.5 to E10.5 murine embryos. The observed efficiency of generating long-term engrafting HSC, particularly from precursors derived from early embryonic stages before E10, was lower in engineered conditions compared with AGM-EC stroma, suggesting additional niche signal factors remain to be defined to optimally support HSC maturation and self-renewal in the engineered niche. Single cell RNA-sequencing of hematopoietic progeny generated following culture in the engineered niche demonstrated the formation of populations with transcriptional signatures of HSC, as well as multipotent and lineage-specific progenitors, comparable to those generated following co-culture with niche AGM-EC stroma. However, we observed relative overexpression of Notch target genes promoting early T-lymphoid fate in cells generated from the engineered niche compared to those from AGM-EC stroma. Incorporating stage-specific attenuation of Notch1 receptor activation with soluble Notch1 blocking antibody during culture was sufficient to limit markers of early T-cell precursors, suggesting that temporal titration of Notch signal activation could be used to further modulate HSC and T-lymphoid output in the engineered niche. Altogether, these studies enhance our understanding of the core signal pathways necessary for the embryonic development of functional HSC, with the potential to advance in vitro engineering of therapeutically relevant pluripotent stem cell-derived HSC in stromal cell-free culture. Disclosures Bernstein: Lyell Immunopharma: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; Nohla Therapeutics: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

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Plerixafor (Mozobil ®)Plus G-CSF Is Effective in Mobilizing Hematopoietic Stem Cells in Patients with Concurrent Thrombocytopenia Undergoing Autologous Hematopoietic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v114.22.3229.3229 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3229-3229 ◽

Cited By ~ 1

Author(s):

Ivana N Micallef ◽

Eric Jacobsen ◽

Paul Shaughnessy ◽

Sachin Marulkar ◽

Purvi Mody ◽

...

Keyword(s):

Stem Cell ◽

Platelet Count ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Platelet Counts ◽

Cd34 Cells ◽

Low Platelet Count ◽

Equity Ownership

Abstract Abstract 3229 Poster Board III-166 Introduction Low platelet count prior to mobilization is a significant predictive factor for mobilization failure in patients with non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD) undergoing autologous hematopoietic stem cell (HSC) transplantation (auto-HSCT; Hosing C, et al, Am J Hematol. 2009). The purpose of this study is to assess the efficacy of HSC mobilization with plerixafor plus G-CSF in patients with concomitant thrombocytopenia undergoing auto-HSCT. Methods Patients who had failed successful HSC collection with any mobilization regimen were remobilized with plerixafor plus G-CSF as part of a compassionate use program (CUP). Mobilization failure was defined as the inability to collect 2 ×106 CD34+ cells/kg or inability to achieve a peripheral blood count of ≥10 CD34+ cells/μl without having undergone apheresis. As part of the CUP, G-CSF (10μg/kg) was administered subcutaneously (SC) every morning for 4 days. Plerixafor (0.24 mg/kg SC) was administered in the evening on Day 4, approximately 11 hours prior to the initiation of apheresis the following day. On Day 5, G-CSF was administered and apheresis was initiated. Plerixafor, G-CSF and apheresis were repeated daily until patients collected the minimum of 2 × 106 CD34+ cells/kg for auto-HSCT. Patients in the CUP with available data on pre-mobilization platelet counts were included in this analysis. While patients with a platelet count <85 × 109/L were excluded from the CUP, some patients received waivers and were included in this analysis. Efficacy of remobilization with plerixafor + G-CSF was evaluated in patients with platelet counts ≤ 100 × 109/L or ≤ 150 × 109/L. Results Of the 833 patients in the plerixafor CUP database, pre-mobilization platelet counts were available for 219 patients (NHL=115, MM=66, HD=20 and other=18.). Of these, 92 patients (NHL=49, MM=25, HD=8 and other=10) had pre-mobilization platelet counts ≤ 150 × 109/L; the median platelet count was 115 × 109/L (range, 50-150). The median age was 60 years (range 20-76) and 60.4% of the patients were male. Fifty-nine patients (64.1%) collected ≥2 × 109 CD34+ cells/kg and 13 patients (14.1%) achieved ≥5 × 106 CD34+ cells/kg. The median CD34+ cell yield was 2.56 × 106 CD34+ cells/kg. The proportion of patients proceeding to transplant was 68.5%. The median time to neutrophil and platelet engraftment was 12 days and 22 days, respectively. Similar results were obtained when efficacy of plerixafor + G-CSF was evaluated in 29 patients with platelet counts ≤ 100 × 109/L (NHL=12, MM=10, HD=3 and other=4). The median platelet count in these patients was 83 × 109/L (range, 50-100). The median age was 59 years (range 23-73) and 60.4% of the patients were male. The minimal and optimal cell dose was achieved in 19(65.5%) and 3(10.3%) patients, respectively. The median CD34+ cell yield was 2.92 × 106 CD34+ cells/kg. The proportion of patients proceeding to transplant was 62.1%. The median time to neutrophil and platelet engraftment was 12 days and 23 days, respectively. Conclusions For patients mobilized with G-CSF alone or chemotherapy ±G-CSF, a low platelet count prior to mobilization is a significant predictor of mobilization failure. These data demonstrate that in patients with thrombocytopenia who have failed prior mobilization attempts, remobilization with plerixafor plus G-CSF allows ∼65% of the patients to collect the minimal cell dose to proceed to transplantation. Thus, in patients predicted or proven to be poor mobilizers, addition of plerixafor may increase stem cell yields. Future studies should investigate the efficacy of plerixafor + G-CSF in front line mobilization in patients with low platelet counts prior to mobilization. Disclosures Micallef: Genzyme Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding. Jacobsen:Genzyme Corporation: Research Funding. Shaughnessy:Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Marulkar:Genzyme Corporation: Employment, Equity Ownership. Mody:Genzyme Corporation: Employment, Equity Ownership. van Rhee:Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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A Short Pulse of Prostaglandin E2 (PGE2) Induces Long Term Chromatin Changes in Hematopoietic Stem Cells Leading to Increased Self-Renewal and Engraftment

Blood ◽

10.1182/blood.v126.23.246.246 ◽

2015 ◽

Vol 126 (23) ◽

pp. 246-246

Author(s):

Eva M Fast ◽

Ellen M Durand ◽

Audrey Sporrij ◽

Leslie Ojeaburu ◽

Rebecca Maher ◽

...

Keyword(s):

Gene Expression ◽

Open Chromatin ◽

Advisory Committees ◽

Self Renewal ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells ◽

Equity Ownership ◽

Induced Genes

Abstract Hematopoietic stem cells (HSCs) offer promising treatment options for many blood diseases. We have previously identified Prostaglandin E2 (PGE2), a small molecule that increased HSC numbers in the zebrafish embryo. In an adult mammalian transplantation setting a two hour treatment significantly enhanced HSC engraftment. Currently PGE2 is being tested in a phase 2 clinical trial to improve cord blood transplants. To better understand PGE2 effect on HSCs mouse multipotent progenitors (MPP), short term (ST) HSCs, and long term (LT) HSCs were isolated via FACS and given a two hour pulse of PGE2 followed by a competitive transplantation assay. Surprisingly, PGE2 treatment mainly affected ST-HSCs by dramatically prolonging their ability to contribute to peripheral blood. The effect of the two hour treatment persisted through secondary competitive transplants in which robust peripheral blood chimerism of ST-HSCs was evident even 1.5 years after having been exposed to the drug. To elucidate underlying molecular changes gene expression right after PGE2 treatment as well as in ST-HSCs after transplantation was assessed. PGE2 target genes were divided into two categories; "transiently induced" and "permanently induced" genes. Most of the transcripts upregulated two hour after PGE2 treatment were "transiently induced" meaning that they did not continue to be differentially expressed after transplantation. In contrast, a few transcripts including chemokines such as Cxcl2, Cxcl3, members of the Fos gene family as well as Nr4a1, 2 and 3 were both upregulated right after PGE2 treatment as well as in ST-HSCs after transplantation. We classified these genes as "permanently induced". ATAC (Assay for Transposase-Accessible Chromatin)-seq analysis of the transplanted PGE2 treated cells indicated that these "permanently induced" genes maintained a distinctly open chromatin profile in both promotor and enhancer regions, whereas the "transiently induced" genes did not. Gene expression in human CD34+ cells included a signature implying CREB as the main transcription factor responsible for the acute PGE2 response. Phospho-FACS in mouse ST-HSCs and Western-blot analysis in human CD34+ cells confirmed a significant increase in CREB phosphorylation after PGE2 stimulation. Chromatin immunoprecipitation (ChIP)-seq analysis of pCREB was able to identify specific genomic regions where pCREB is recruited to after PGE2 treatment. Compared to unstimulated CD34+ cells an increased binding of pCREB could be detected in promotor regions near transcription start sites. In addition over 90% of de-novo pCREB binding occurred in intergenic and intronic regions. To determine the activation state of these putative enhancers changes in the histone mark H3K27ac and open chromatin state (via ATAC-seq) were assessed after PGE2 treatment. The data suggest that PGE2-induced pCREB binding correlates with remodeling of chromatin already after two hours of drug treatment. Furthermore chromatin sites opened by PGE2 were significantly enriched for the CREB motif both in human CD34+ cells acutely after treatment as well as in mouse ST-HSCs 1.5 years after transplant. In summary this work shows that a two hour treatment with PGE2 is sufficient to confer long-term engraftment properties to ST-HSCs. PGE triggers a chromatin remodeling event through CREB that can permanently alter epigenetic state and gene expression of ST-HSCs. Understanding the self-renewal network induced by PGE2 will not only enrich current clinical applications targeted at increasing engraftable HSC numbers but also further basic understanding of HSC self-renewal. Disclosures Zon: FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

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

Blood ◽

10.1182/blood.v106.11.1709.1709 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1709-1709

Author(s):

Andre Larochelle ◽

Cynthia L. Perez ◽

Allen Krouse ◽

Mark Metzger ◽

Simon Fricker ◽

...

Keyword(s):

Stem Cell ◽

Single Dose ◽

Peripheral Blood ◽

Rhesus Macaques ◽

Myeloablative Conditioning ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Preparative Regimen ◽

Hsc Transplantation

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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ETO2 Controls Hematopoietic Stem Cell Expansion.

Blood ◽

10.1182/blood.v114.22.396.396 ◽

2009 ◽

Vol 114 (22) ◽

pp. 396-396

Author(s):

Stephane Barakat ◽

Julie Lambert ◽

Guy Sauvageau ◽

Trang Hoang

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Short Term ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 396 Hematopoietic stem cells that provide short term reconstitution (ST-HSCs) as well as hematopoietic progenitors expand from a small population of long term hematopoietic stem cells (LT-HSCs) that are mostly dormant cells. The mechanisms underlying this expansion remain to be clarified. SCL (stem cell leukemia), is a bHLH transcription factor that controls HSC quiescence and long term competence. Using a proteomics approach to identify components of the SCL complex in erythroid cells, we and others recently showed that the ETO2 co-repressor limits the activity of the SCL complex via direct interaction with the E2A transcription factor. ETO2/CBF2T3 is highly homologous to ETO/CBFA2T1 and both are translocation partners for AML1. We took several approaches to identify ETO2 function in HSCs. We initially found by Q-PCR that ETO2 is highly expressed in populations of cells enriched in short-term HSC (CD34+Flt3-Kit+Sca+Lin-) and lympho-myeloid progenitors (CD34+Flt3+Kit+Sca+Lin-) and at lower levels in LT-HSCs (CD34-Kit+Sca+Lin- or CD150+CD48-Kit+Sca+Lin-). Next, the role of ETO2 was studied by overexpression or downregulation combined with transplantation in mice. Ectopic ETO2 expression induces a 100 fold expansion of LT-HSCs in vivo in transplanted mice associated with differentiation blockade in all lineages, suggesting that ETO2 overexpression overcomes the mechanisms that limit HSC expansion in vivo. We are currently testing the role of the NHR1 domain of ETO2 in this expansion. Conversely, shRNAs directed against ETO2 knock down ET02 levels in Kit+Sca+Lin- cells, causing a ten-fold decrease in this population after transplantation, associated with reduced short-term reconstitution in mice. Finally, proliferation assays using Hoechst and CFSE indicate that ETO2 downregulation affects cell division (CFSE) and leads to an accumulation of Kit+Sca+Lin-cells in G0/G1 state (Hoescht). In conclusion, we show that ETO2 is highly expressed in ST-HSCs and lymphoid progenitors, and controls their expansion by regulating cell cycle entry at the G1-S checkpoint. In addition, ETO2 overexpression converts the self-renewal of maintenance into self-renewal of expansion in LT-HSCs. Disclosures: No relevant conflicts of interest to declare.

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Preliminary Phase 2 Results Demonstrate Engraftment with Minimal Neutropenia with MGTA-456, a CD34+ Expanded Cord Blood (CB) Product in Patients Transplanted for Inherited Metabolic Disorders (IMD)

Blood ◽

10.1182/blood-2018-99-115102 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3467-3467

Author(s):

Paul Orchard ◽

Glen D. Raffel ◽

Carolyn H Condon ◽

Catherine A Monaghan ◽

Demetra Vernet ◽

...

Keyword(s):

Stem Cell ◽

Research Funding ◽

Graft Failure ◽

Phase 2 ◽

Employment Equity ◽

Historical Cohort ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Equity Ownership ◽

Grade 3

Abstract Background: IMDs including mucopolysaccharidosis type IH (MPS1/Hurler Syndrome), metachromatic leukodystrophy (MLD), globoid cell leukodystrophy (GLD) and cerebral adrenoleukodystrophy (cALD) are progressive, fatal diseases affecting the central nervous system which are treatable through allogeneic hematopoietic stem cell transplantation (HSCT). CB, in the absence of a matched donor, is the preferred source of stem cells as it is rapidly available and allows greater flexibility in allele matching. As a result of low cell doses, CB transplants in IMD are associated with prolonged periods of neutropenia and reported graft failure rates in up to ~20% (Lum et al 2017 Bone Marrow Transplant 52:846-53; Mallhi et al 2017 BBMT 23:119-25). MGTA-456 is a first-in-class cell therapy produced from a single CB unit using an aryl hydrocarbon receptor antagonist in a 15-day expansion culture of CD34+ cells. In previous phase 1/2 studies, 24 adult and 3 pediatric patients with hematologic malignancies treated with myeloablative conditioning (MAC) and MGTA-456 demonstrated a median 324-fold expansion of CD34+ cells, all patients engrafted, and the time to neutrophil recovery was significantly reduced by a median of 9 days compared to historical controls (Wagner et al 2016 Cell Stem Cell 18:144-55; Wagner et al 2017 Blood 130 supp:662 abstr). Furthermore, higher CD34+ dose has been correlated with improved engraftment and outcomes in IMD transplant patients (Prasad et al 2008 Blood 112:2979-89). Based on these promising data, we postulate the increased CD34+ dose provided by MGTA-456 would reduce the length of neutropenia and risk of graft failure in IMD patients. Patients and Methods: A Phase 2, open-label trial (NCT03406962) initiated in Feb 2018 is enrolling up to ~12 patients <16 yo with a diagnosis of MPS1, cALD, MLD, and GLD lacking a non-carrier matched related donor. Eligible CB units were matched at ≥ 6 of 8 HLA loci (A, B, C and DRB1) using allele-based typing. The reduced toxicity MAC regimen consists of anti-thymocyte globulin (days -9 to -6) followed by fludarabine (40 mg/m2 days -5 to -2) and busulfan (total exposure 21,000 to 22,000 μM/min/L-1 days -5 to -2). Immunoprophylaxis consists of cyclosporin and methylprednisolone. Results: Four patients have been treated thus far (Table 1). The MGTA-456 process provided a marked expansion of CD34+ cells (median 482-fold, Figure 1) with a median infused CD34+ cell dose of 84 x 106 cells/kg and median total nucleated cell (TNC) dose from the expanded fraction of 19.6 x 107 (Table 1). TNC dosing was capped at 27.0 x 107 cells/kg per protocol. The only infusion-related reaction noted was grade 3 nausea in 1 patient. Two patients had no days of neutropenia and 2 patients had 1 and 4 days respectively (mean 1.25 days) in contrast to a mean of 7.8 days for a historical cohort of 27 IMD patients undergoing CB transplantation at the same institution with identical conditioning (Figure 2). Myeloid chimerism (CD33+/66+) achieved ≥98% donor by day +14 in all patients. For the 3 patients with data at time of reporting, days to discharge after transplant were 17, 12 and 18. No patients experienced acute or chronic GVHD. One patient developed autoimmune cytopenia (not related to MGTA-456) which is a known complication reported in 20-56% of IMD patients undergoing HSCT that resulted in death at day +143 (Page et al 2008 BBMT 14:1108-17; Khalil et al 2014 Sci World J 581657). Long-term disease specific outcome measures including enzymatic activity are being collected and will be reported over time. Conclusions: Preliminary results of transplantation in IMD patients with MGTA-456, containing highly expanded CD34+ cell doses, demonstrated early and robust engraftment in all patients with marked reduction in days of neutropenia (to 0-4 days or mean 1.25 days) in comparison to a historical cohort. MGTA-456 was well tolerated with one infusion-related event of grade 3 nausea. These data, in combination with the previous 27 hematologic malignancy patients treated, suggest that MGTA-456 substantially enhances the engraftment potential of CB. Based on these promising data, MGTA-456 has potential to improve transplant-related outcomes in patients undergoing HSCT and increasing the availability of well-matched CB units that may have been previously excluded due to inadequate CD34+ dose. Disclosures Orchard: Magenta Therapeutics: Research Funding. Raffel:Magenta Therapeutics: Employment, Equity Ownership. Condon:Magenta Therapeutics: Employment, Equity Ownership. Monaghan:Magenta Therapeutics: Employment, Equity Ownership. Vernet:Magenta Therapeutics: Employment. Sheirr:Magenta Therapeutics: Employment, Equity Ownership. Braun:Magenta Therapeutics: Research Funding. Shanley:Magenta Therapeutics: Research Funding. Lund:Magenta Therapeutics: Research Funding. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Davis:Magenta Therapeutics: Employment, Equity Ownership. Wagner:Novartis: Research Funding; Magenta Therapeutics: Consultancy, Research Funding.

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Age-Associated Myeloid Biased Hematopoiesis Depends on Relative Decrease of Short-Term Hematopoietic Stem Cell

Blood ◽

10.1182/blood-2019-122355 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2481-2481

Author(s):

Katsuyuki Nishi ◽

Taro Sakamaki ◽

Kevin Shuolong Kao ◽

Kay Sadaoka ◽

Momo Fujii ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Research Funding ◽

Bone Marrow Cells ◽

Self Renewal ◽

Myeloid Lineage ◽

Hematopoietic Stem ◽

Aged Mice ◽

Donor Cells

Aging in the human hematopoietic system is known to be associated with reduced self-renewal capacity, myeloid biased hematopoiesis, and increased incidence of hematological disorders. Amongst these changes, myeloid-biased hematopoiesis leads to decreased adaptive immune system function and increased propensity for myeloid malignancies in aged individuals. Recent reports have supported the concept that myeloid-biased alteration is most likely due to cell intrinsic alterations in the hematopoietic stem cell (HSC) compartment and a clonal shift toward myeloid-biased HSCs. Recently, we demonstrated that Hoxb5 specifically marks HSCs with long-term self-renewal capacity (LT-HSC) within the bone marrow of young mice. Additionally, we further demonstrated that around 80% of the cells included within the immunophenotypic HSC fraction (defined by the cell surface markers: Lineage-c-Kit+Sca-1+Flk2-CD150+CD34-/lo) (hereafter referred to as pHSCs) are not LT-HSCs (Chen JY et al., Nature 2016). In this study, using our LT-HSC-specific reporter mouse model, we sought to understand the mechanisms underlying the myeloid-biased alteration with age. First, to verify that Hoxb5 can be used as a LT-HSC-specific marker in aged mice, we utilized a transplantation assay in which 10 cells of Hoxb5+ or Hoxb5- pHSCs isolated from 2-year-aged mice were transplanted with supporting bone marrow cells into lethally irradiated mice. Any mice showing long-term (≧16week) granulocyte reconstitution (≧1% generation in peripheral blood) were considered as a positive for long-term hematopoiesis. Refer to this criteria, continuous hematopoiesis was observed only within the Hoxb5+pHSC recipients, indicating that Hoxb5 marks LT-HSCs within the pHSC compartment throughout the mouse lifespan. Interestingly, the lineage output derived from transplanted aged-Hoxb5+-HSCs was not skewed towards the myeloid lineage when compared to their young counterparts (the frequency of myeloid lineage contribution in donor cells: Aged vs Young = 42.6% vs 50.5%, n.s.). Our findings suggest that the balance between myeloid and lymphoid lineage output of the LT-HSC (Hoxb5+ pHSC) persists throughout life. To further confirm this hypothesis, we co-transplanted 10 cells of 2-year-aged Hoxb5+ pHSC with 10 cells of EGFP-overexpressed young Hoxb5+ pHSC along with supporting bone marrow cells into the same recipients. At 12 weeks posttransplant, peripheral blood (PB) analysis demonstrated that the frequency of myeloid output in both aged or young donor cells was nearly equivalent (Aged vs Young = 48.0% vs 43.2%, n.s.). According to previous reports, myeloid-related genes are known to be enriched in HSC compartment with age. However, given our previous findings demonstrating that ~80% of the pHSC compartment consists of non-LT-HSCs, we conducted RNA-seq analysis for bulk pHSCs (a mixture of Hoxb5+ and Hoxb5- pHSCs) and pure Hoxb5+ pHSCs isolated from young and 2-year-old mice respectively. Gene Set Enrichment Analysis showed that the previously defined geneset: preGM (granulocyte/macrophage progenitors)-specific genes (Pronk, C. J. et al., Cell Stem Cell 2007) are more highly expressed in aged pHSCs than in young pHSCs (NES = 1.25, q=0.067), whereas no significant gene enrichment of preGM-specific genes in aged Hoxb5+ pHSCs was observed compared to young Hoxb5+pHSCs (NES=-1.02, q=0.333). Given that the frequency of the Hoxb5- pHSC (which possesses only transient self-renewal and give rise to mainly lymphoid cell) in the pHSC compartment is decreased in aged mice (Aged vs Young = 43.3% vs 73.6%, p<0.0001), we hypothesized that the myeloid biased phenotypic change in the HSC compartment with age depends on a relative decrease of Hoxb5- pHSCs. To verify this, we transplanted young Hoxb5+ pHSCs and Hoxb5- pHSCs in a 5:5 ratio (the ratio between Hoxb5+ and Hoxb5- pHSCs in aged mice) or 2:8 ratio (the ratio in young mice). PB analysis at 16 weeks post-transplant demonstrated that recipient mice transplanted with Hoxb5+ pHSCs and Hoxb5- pHSCs in the 5:5 ratio have a greater myeloid lineage output than recipients of the 2:8 ratio (the frequency of myeloid lineage contribution in donor cells: 5:5 ratio vs 2:8 ratio = 31% vs 7%, p<0.05). In conclusion, the present study demonstrates that myeloid biased hematopoiesis in the aged setting results from the alteration of the ratio between Hoxb5+ and Hoxb5- pHSCs rather than cell-intrinsic change. Disclosures Takaori-Kondo: Ono: Research Funding; Takeda: Research Funding; Kyowa Kirin: Research Funding; Chugai: Research Funding; Janssen: Honoraria; Pfizer: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.

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Phase 2 Trials with Mgta-456, Single Cord Blood Units (CBU) Expanded with an Aryl Hydrocarbon Receptor (AHR) Antagonist, Demonstrate Uniform Engraftment and Rapid Hematopoietic Recovery in Patients Following Myeloablative or Non-Myeloablative Conditioning

Blood ◽

10.1182/blood.v130.suppl_1.662.662 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 662-662

Author(s):

John E. Wagner ◽

Claudio G Brunstein ◽

Todd E. DeFor ◽

Anthony E. Boitano ◽

David McKenna ◽

...

Keyword(s):

Cord Blood ◽

Research Funding ◽

Phase 2 ◽

Employment Equity ◽

Myeloablative Conditioning ◽

Historical Cohort ◽

Aryl Hydrocarbon ◽

Hematopoietic Recovery ◽

Cd34 Cells ◽

Equity Ownership

Abstract Background. The use of umbilical cord blood (UCB) in transplant has been limited by the low number of CD34+ cells, resulting in prolonged periods of cytopenia for patients and high risk of graft failure, thereby restricting its widespread application. MGTA-456 is the hematopoietic cell product after cord blood CD34+ cells are placed in expansion culture for 15 days with an aryl hydrocarbon receptor (AHR) antagonist in the presence of SCF, Flt-3L, IL-6 and TPO. In a prior Phase 1/2 safety study, 18 patients received MGTA-456, its accompanying CD34negfraction and a larger, unexpanded UCB unit. All patients engrafted at a median of 14.5 days (range, 7-23), significantly faster than similarly treated historical controls (p<0.01). Based on these results, two Phase 2 studies were initiated to evaluate the effectiveness of MGTA-456 as a stand-alone graft after myeloablative conditioning (MAC) or non-myeloablative conditioning (NMAC). Patients and Methods : Twenty patients with high-risk hematologic malignancy and a partially HLA-matched CBU were enrolled; 10 were treated with cyclophosphamide (CY) 120 mg/kg, fludarabine (FLU) 75 mg/m2 and total body irradiation (TBI) 1320 cGy (MAC) and 10 with CY 50 mg/kg, FLU 200 mg/m2 and TBI 200 cGy (NMAC). All patients received cyclosporine and mycophenolate mofetil post-transplant immunoprophylaxis. Expansion was low in 2 UCB units, therefore 18 patients received MGTA-456 and its CD34neg fraction. Results : Expansion culture yielded a median of 1,227 x 106 CD34+ cells (range, 201-8969) as compared to the input number of 4.2 x 106 (range, 1.4-16.3) after CD34 selection - a 324-fold (range, 42-1643) expansion of CD34+ cells. As transplant results vary by intensity of the conditioning, patient outcomes were compared to similarly treated historical cohorts between 2006 and 2015 (n=151 MAC; n=132 NMAC). For both groups, demographics were similar except for more recent year of transplant for recipients of MGTA-456. For recipients of MAC, MGTA-456 engrafted in all patients at a median of 14 days (range, 7-32) as compared to 89% engraftment at a median of 23 days (range, 19-31) in the control population (p<0.01, see Figure 1). Complete chimerism was rapid for both myeloid and T cells with no late graft failures; the longest follow-up was 5.6 years in recipients of MGTA-456. For recipients of NMAC, MGTA-456 also engrafted in all patients at a median of 7 days (range, 6-14) as compared to 94% engraftment at a median of 15 days (range, 7-22). In contrast to complete chimerism seen after MAC, chimerism is often mixed for the first month in both myeloid and T cells after NMAC. Compared to the historical cohort, recipients of MGTA-456 had more rapid chimerism after NMAC. CD34 cell dose correlates with speed of recovery but only in recipients with MAC; in recipients of NMAC, recovery is uniformly rapid regardless of CD34 cell dose. Additionally, immune recovery as measured by an absolute CD4 count >200/uL was achieved at day 60 (median) in recipients of MGTA-456 regardless of conditioning regimen. Results were also encouraging for other transplant outcomes. For recipients of MGTA-456 compared to the historical cohort after MAC, incidence of acute GVHD (aGVHD) grade 3-4 was 22% vs 24%; chronic GVHD (cGVHD), 11% vs 21%; transplant-related mortality (TRM), 11% vs 34%; and overall survival (OS), 67% vs 55%. After NMAC, results were similar between cohorts except for a higher risk of aGVHD in recipients of MGTA-456 (aGVHD 3-4, 43% vs 15%; cGVHD, 0% vs 19%; TRM, 22% vs 20%; and OS, 44% vs 49%). The increased rate of aGVHD in the NMAC cohort likely reflects non-compliance with prescribed GVHD immunoprophylaxis in 2 of 9 recipients. Conclusion : In these studies, MGTA-456 significantly accelerated hematopoietic recovery and abrogated the engraftment barrier typically associated with UCB transplantation. The marked expansion of CD34+ cells in MGTA-456 suggests that a significant number of patients will have an adequate single CBU and better HLA matched graft since a greater proportion of the cord blood inventory will be available irrespective of weight. Given these promising results, additional studies are being planned. Disclosures Wagner: Novartis: Research Funding; Magenta Therapeutics: Research Funding. Brunstein: Novartis: Research Funding; Magenta Therapeutics: Research Funding. Boitano: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties; Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. McKenna: Magenta Therapeutics: Research Funding; Novartis: Research Funding. Sanna: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Bleul: Hoffmann-La Roche AG: Employment. Cooke: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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YY1 Controls Hematopoietic Stem Cell Quiescence By Repressing Cohesin Expression

Blood ◽

10.1182/blood-2018-99-118679 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3831-3831

Author(s):

Zhanping Lu ◽

Anna L F. V. Assumpção ◽

Aaron D Viny ◽

Ross L. Levine ◽

Xuan Pan

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Research Funding ◽

Bone Marrow Cells ◽

Cell Metabolism ◽

Conditional Knockout ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Quiescence ◽

Equity Ownership

Abstract Hematopoietic stem cells (HSCs) are undifferentiated, self-renewing, pluripotent cells that have the capacity to differentiate into all mature lineage-specific cells in adult blood. Adult HSCs can remain in a quiescent state for a prolonged time, and quiescence is a fundamental characteristic of HSCs in adult bone marrow. Thus, the cell cycle must be precisely regulated. Yin Yang 1 (YY1) is a multifunctional transcription factor and Polycomb Group Protein (PcG) that is important for embryonic development, adult hematopoiesis, cell proliferation and maintaining higher-order chromosomal structure. We have generated YY1 conditional knockout mice (Yy1f/f Mx1-Cre) and showed that Yy1 deficient HSCs fail to self-renewal and had disrupted HSCs quiescence. Stem cell factor (SCF)/c-Kit signaling, a critical regulatory pathway in HSC development, is significantly downregulated in Yy1-/- HSCs. Interestingly, YY1 regulation of HSCs self-renewal and quiescence is independent on its PcG domain/function. Instead, YY1 occupied at Smc3 promoter area and repressed Smc3 expression. In Yy1-/-HSCs, Smc3 expression was upregulated. SMC3 is a core component of cohesin protein complex and plays critical roles in HSC self-renewal, myeloid differentiation and leukemogenesis. To further dissect the underlying mechanisms by which YY1 regulates SMC3 expression in HSCs, we have generated conditional knockout mice with YY1 homozygous deletion and SMC3 heterozygous deletion (Yy1f/f Smc3f/+Mx1-Cre). In Yy1-/- Smc3+/- bone marrow cells, SMC3 expression was normalized to the wild-type level. In adult bone marrow cells, YY1 physically interacted with cohesin complex proteins through its zinc finger domain. By analyzing the YY1, SMC1A and SMC3 ChIP-Seq database, our study showed that YY1 and cohesin co-occupied at promoter areas of genes that are critical for cell metabolism. Evidence from previous study showed that impaired metabolism, including increased reactive oxygen species (ROS) and decreased mitochondrial function, can cause defect in stem cell self-renewal and quiescence. In Yy1-/- Smc3+/-HSCs, cell quiescence was restored although HSC self-renewal was still impaired, indicates that YY1 and SMC3 may control HSC cell quiescence via regulating genes critical for cell metabolism. Our study identified YY1 as the first transcription factor that regulates expression of cohesin complex component SMC3. We are currently further dissecting underlying mechanisms and functional significances of metabolic pathways regulated by YY1-SMC3 axis in HSCs. Disclosures Levine: Imago: Equity Ownership; Isoplexis: Equity Ownership; Janssen: Consultancy, Honoraria; Gilead: Honoraria; Epizyme: Patents & Royalties; Loxo: Consultancy, Equity Ownership; C4 Therapeutics: Equity Ownership; Roche: Consultancy, Research Funding; Prelude: Research Funding; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Novartis: Consultancy.

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Safety and Clinical Benefit of Lentiviral Hematopoietic Stem Cell Gene Therapy for Wiskott-Aldrich Syndrome

Blood ◽

10.1182/blood.v126.23.259.259 ◽

2015 ◽

Vol 126 (23) ◽

pp. 259-259 ◽

Cited By ~ 4

Author(s):

Francesca Ferrua ◽

Maria Pia Cicalese ◽

Stefania Galimberti ◽

Samantha Scaramuzza ◽

Stefania Giannelli ◽

...

Keyword(s):

Clinical Trial ◽

Gene Therapy ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Clinical Benefit ◽

Research Funding ◽

Lentiviral Vector ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Wiskott-Aldrich Syndrome (WAS) is an X-linked primary immunodeficiency characterized by thrombocytopenia, recurrent infections, eczema, autoimmunity and increased susceptibility to malignancies. Allogeneic hematopoietic stem cell transplantation (HSCT) is a recognized curative treatment for WAS, but is still associated with transplant-related complications and long-term morbidity, particularly in the absence of fully matched donors. In April 2010, we initiated a phase I/II clinical trial with hematopoietic stem cell (HSC) gene therapy (GT) for WAS. The investigational medicinal product (IMP) consists of autologous CD34+ HSC engineered with a lentiviral vector (LV) driving the expression of WAS cDNA from an endogenous 1.6 kb human WAS promoter (LV-WAS), infused after a reduced intensity conditioning (RIC) based on anti-CD20 mAb, targeted busulfan and fludarabine. We previously reported early follow up (FU) results from the first 3 patients (Aiuti et al., Science 2013). Seven patients (Zhu score ≥3) have now been treated at a median age of 1.9 years (1.1 - 11.1). As of May 2015, all patients are alive with a median FU of 3.2 years (0.7 - 5.0). CD34+ cell source was bone marrow (BM) (n=5), mobilized peripheral blood (MPB) (n=1) or both (n=1). IMP dose ranged between 7.0 and 14.1 x106 CD34+/kg, containing on average 94.4 ± 3.5% transduced clonogenic progenitors and a mean vector copy number (VCN)/genome in bulk CD34+ cells of 2.7 ± 0.8. No adverse reactions were observed after IMP infusion and RIC was well tolerated. Median duration of severe neutropenia was 19 days; granulocyte-colony stimulating factor was administered to 1 patient. In the first 6 treated patients with FU >2 years, we observed robust and persistent engraftment of gene corrected cells. At the most recent FU, transduced BM progenitors ranged between 20.7 and 59.7%, and LV-transduced cells were detected in multiple lineages, including PB granulocytes (VCN 0.34 - 0.93) and lymphocytes (VCN 1.18 - 2.73). WAS protein expression, measured by flow-cytometry, was detected in the majority of PB platelets [mean ± standard deviation (SD), 71.4 ± 14.0%], monocytes (63.3 ± 18.5%) and lymphocytes (78.9 ± 14.9%). Lymphocyte subset counts were normal in most patients and proliferative response to anti-CD3 mAb was in the normal range in all 6 patients. After immune reconstitution, a marked reduction in the annualized estimated rate of severe infections was observed, as compared with baseline (figure 1A). The first 6 treated patients discontinued anti-infective prophylaxis and no longer require a protected environment. Four patients stopped immunoglobulin supplementation and 2 of them developed specific antibodies after vaccination. Eczema resolved in 4 patients and remains mild in 2. No clinical manifestations of autoimmunity were observed ≥1 year after GT in accordance with improved B-cell development and decreased autoantibody production. All patients became platelet transfusion independent at a median of 4 months after GT (range: 1.0 - 8.7). Mean platelet counts progressively increased after treatment (mean ± SD: before GT, 13.4 ± 7.8 x109/l; 24-30 month FU, 45.8 ± 22.0 x109/l; 36-42 month FU, 57.0 ± 18.7 x109/l). The frequency and the severity of bleeding events decreased after the 1st year of FU. No severe bleedings were recorded after treatment (figure 1B). Quality of life improved in all patients after GT. From the 2nd year of FU, the number of hospitalizations for infections decreased and no hospitalizations due to bleeding were observed after treatment. The seventh patient treated, who received MPB derived CD34+ cells only, showed the fastest platelet recovery with the highest level of transduced myeloid cell engraftment, and is clinically well. No Serious Adverse Events (SAE) related to the IMP were observed. The most frequent SAE were related to infections (85%), occuring mainly during the 1st year of FU. Importantly, no evidence of abnormal clonal proliferations emerged after GT and the LV integration profile show a polyclonal pattern, with no skewing for proto-oncogenes. In conclusion, this updated report in 7 WAS patients show that GT is well tolerated and leads to a sustained clinical benefit. The high level of gene transfer obtained with LV-WAS results in robust engraftment of transduced HSC, even when combined with RIC. Prolonged FU will provide additional information on the long-term safety and clinical efficacy of this treatment. Figure 1. Figure 1. Disclosures Villa: Fondazione Telethon: Research Funding. Dott:GlaxoSmithKline: Consultancy. van Rossem:GlaxoSmithKline: Employment. Naldini:Salk Institute: Patents & Royalties: Lentiviral vectors; San Raffaele Telethon Institute: Patents & Royalties: Lentiviral vector technology; GlaxoSmithKline: Other: GSK licensed gene therapies developed at my Institute and the Institute receives milestone payments; Sangamo Biosciences: Research Funding; Biogen: Research Funding; Genenta Sciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Aiuti:GlaxoSmithKline (GSK): Other: PI of clinical trial which is financially sponsored by GSK; Fondazione Telethon: Research Funding.

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