High Dose Hematopoietic Stem Cell Transplantation Leads to Rapid Hematopoietic and Microglia Recovery and Disease Correction in a Mouse Model of Hurler Syndrome

Background . Allogeneic hematopoietic stem cell transplant (HSCT) is a promising approach to halt disease progression and prevent or ameliorate neurological symptoms arising from select inherited metabolic disorders (IMDs). Donor-derived cells, including microglia, limit disease progression post-HSCT via production of normal enzyme in a process called cross-correction. A standard cell dose used in HSCT is sub-optimal, resulting in delayed hematopoietic recovery and slower correction of central nervous system (CNS) defects (Lund et al BBMT 2019). To address these limitations, we developed MGTA-456, a cell therapy that contains large numbers of CD34+ cells and has led to accelerated neutrophil recovery and 100% engraftment post-HSCT in patients with malignant and non-malignant diseases (Wagner et al Blood 2017; Orchard et al AAN 2019). We previously showed that MGTA-456 leads to faster hematopoietic and microglia recovery in the brains of NSG mice (Goncalves et al AAN 2019); however, the impact of cell dose on disease outcomes and mechanism of cross-correction are unknown. Here, we show that faster and greater hematopoietic and microglia recovery leads to rapid and complete resolution of disease endpoints in a mouse model of mucopolysaccharidosis I (Hurler syndrome) and that, mechanistically, donor engraftment in the brain is required for disease cross-correction. Results . To determine whether cell dose impacts microglial engraftment, CD45.2 mice were conditioned with a clinically-relevant, myeloablative dose of busulfan and transplanted with increasing doses of CD45.1 bone marrow cells, beginning with 0.3x106 cells/mouse (2x106 cells/kg) based on allometric scaling to model high dose cell therapies. A dose-dependent increase in microglia was observed as early as 1 week post-HSCT, where 10x106 cells led to a 26-fold higher number of donor microglia compared to 0.3x106 cells (p<0.01), an effect that was sustained through 16 weeks post-HSCT (p<0.001). Despite high donor chimerism in the periphery at all cell doses (75-99%), only partial chimerism was observed in the brain. At 16 weeks, donor microglia represented only 2% of microglia after transplant of 0.3x106 cells but this was increased to 35% of total microglia in the brain following transplant of 10x106 cells. These data indicate that while busulfan can facilitate a low level of microglia engraftment, this effect can be enhanced by transplant of high cell doses. To evaluate the impact of cell dose on disease outcomes, we transplanted a low (0.3x106) or high (10x106) cell dose of wild-type bone marrow cells into busulfan-conditioned Idua-/- mice, a model of Hurler syndrome. At 1 month post-HSCT, peripheral donor myeloid chimerism was >75% and >99% for 0.3x106 and 10x106 cells, respectively. In the brain, transplant of 10x106 cells led to significantly higher donor microglial engraftment versus 0.3x106 cells (Figure A). Notably, high cell dose resulted in significantly higher levels of IDUA enzyme in the brain (Figure B), reduced levels of β-hexosaminidase and glycosaminoglycan (GAG) substrate, and normalization of behavioral outcomes, including rotarod performance, to wild type levels (Figure C). In peripheral tissues, transplant of 10x106 cells, but not 0.3x106 cells, led to a reduction of GAGs to wild type levels as early as 1 week post-HSCT (p<0.01). To determine if donor engraftment in the brain is required for cross-correction, we transplanted 10x106 cells into mice conditioned with a myeloablative dose of treosulfan, which is not sufficient to condition the brain for microglia engraftment. Treosulfan conditioning, followed by high dose HSCT, led to >99% donor myeloid chimerism in the periphery but neither increased microglial levels nor corrected CNS defects (Figures A-C), suggesting that donor engraftment in the brain is required for disease modification. Long-term outcomes and impact on skeletal phenotype in this model will also be presented. Conclusions . We demonstrate that high dose HSCT leads to robust microglia engraftment in the brain and improved disease endpoints. These data suggest that strategies to increase cell dose, such as MGTA-456, may accelerate resolution of neurologic disease in patients with IMDs. Similar approaches, possibly coupled with gene modification technologies, could be used to improve microglial function in other neurodegenerative diseases where defective microglia have been implicated. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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Understanding the Impact of Inflammation on Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v116.21.2629.2629 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2629-2629

Author(s):

Ying Zhao ◽

Flora Ling ◽

Hong-Cheng Wang ◽

Xiao-Hong Sun

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Chronic Inflammation ◽

Bone Marrow Cells ◽

Wild Type ◽

Hematopoietic Stem ◽

Inflammatory Conditions ◽

The Impact ◽

Lps Treatment ◽

Marrow Cells

Abstract Abstract 2629 The overall objectives of this study are to investigate the impact of inflammatory conditions on hematopoietic stem cell (HSC) maintenance and to elucidate the underlying mechanisms. HSCs are exposed to a variety of inflammatory conditions through life. How these conditions influence the integrity of HSCs is a fundamental issue of clinical importance but it is poorly understood. Equally unknown is the molecular regulation of HSC maintenance during inflammatory. In this context, our focus is on the role of basic helix-loop-helix (bHLH) proteins, which include transcription activators such as E2A proteins and their inhibitors including Id proteins. We and others have shown that these regulators are involved in normal hematopoiesis such as stem cell function and lineage specific differentiation. Recently, we have obtained evidence to suggest that signaling through Toll-like receptors (TLRs), which is closely linked to inflammation, causes down-regulation of E2A function by stimulating Id1 expression. Therefore, we hypothesize that inflammatory conditions causes down-regulation of E protein function, which disturbs the quiescence of long-term (LT)-HSC, leading to stem cell exhaustion over time. To test this hypothesis, we induced chronic inflammation in wild type and Id1-/- mice by daily injection of 1 mg of LPS, i.p. for 30 days. Peripheral blood was collected on days 15 and 30 and levels of a panel of inflammatory cytokines were assayed using a Luminex multiplex kit. On day 15, dramatic increases were found in the levels of IL-10, IL-6, KC and TNFα but not IFN-γ, IL12-p70 and IL-1β. Interestingly, levels of IL-6 and TNFα were significantly lower in Id1-/- mice compared to wild type mice. By day 30 of LPS treatment, levels of these cytokines returned to the levels in animals without LPS injection. These results suggest that this chronic LPS treatment indeed elicited an inflammatory response that included transient elevation of inflammatory cytokines. Whether secretion of these cytokines has any direct effects on HSCs remains to be determined. To measure HSC activity in these LPS-treated mice, we performed serial bone marrow transplant assays. Lin−Sca-1+c-kit+ (LSK) stem/progenitor cells were isolated from wild type or Id1-/- mice treated with or without LPS. These cells were transplanted into lethally irradiated CD45.1+ recipients along with equal numbers of YFP-expressing LSK as competitors. Six weeks later, cohorts of mice were sacrificed and bone marrow cells were collected. Pooled whole bone marrow cells within each cohort were injected into lethally irradiated secondary recipients. Secondary recipients were sacrificed 8 and 16 weeks post transplant. For assessment of primary and secondary engraftment, bone marrow cells were examined for expression of donor and lineage specific markers. Robust engraftment was observed in primary or secondary recipients. Donor derived cells were then gated for YFP− and YFP+ cells, which separate cells originated from tester and competitor LSK, respectively. While YFP− and YFP+ cells engrafted equivalently in primary recipients transplanted with cells treated with or without LPS, LPS treatment of wild type mice caused a great disparity in secondary recipients. In contrast, HSC in Id1-/- mice did not appear to be affected by the same treatment even though HSCs in Id1 deficient mice are normally lower in numbers and activities as we previously reported. These results suggest that chronic inflammation diminishes the LT-stem cell activity and this may involve the up-regulation of Id1 expression. To investigate the underlying mechanism, we performed label retaining assays to examine the quiescence of LT-HSCs. We found that BrdU-labeling in HSCs was 2-fold lower in mice treated with LPS compared to the untreated controls, suggesting that treatment with LPS promoted the cycling of HSCs, thus impairing their stem cell function. Taken together, our study illustrates that chronic inflammation has a detrimental effect on LT-stem cell activity. Although HSCs have an enormous capability to repopulate the bone marrow by compensatory proliferation, pro-longed inflammation could eventually lead to stem cell exhaustion and seriously compromise hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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MGTA-456, a First-in-Class Cell Therapy Produced from a Single Cord Blood Unit, Enables a Reduced Intensity Conditioning Regimen and Enhances Speed and Level of Human Microglia Engraftment in the Brains of NSG Mice

Blood ◽

10.1182/blood-2018-99-118258 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 115-115

Author(s):

Kevin A. Goncalves ◽

Shuping Li ◽

Melissa L. Brooks ◽

Sharon L. Hyzy ◽

Anthony E. Boitano ◽

...

Keyword(s):

Fold Increase ◽

High Dose ◽

Reduced Intensity Conditioning ◽

Employment Equity ◽

Nsg Mice ◽

Hematopoietic Recovery ◽

Cd34 Cells ◽

Equity Ownership ◽

The Brain ◽

Reduced Intensity

Abstract Background. Allogeneic bone marrow transplant (BMT) is a promising, curative approach for patients with inherited metabolic disorders (IMDs), a class of pediatric diseases characterized by a single enzyme deficiency. The goal of transplant is to provide cells that produce functional enzymes otherwise deficient in these patients, and thereby prevent or ameliorate neurological complications associated with selected IMDs. Donor-derived microglial cells are protective, limiting neurological disease progression. For IMD patients who do not have an HLA matched, non-carrier related donor, cord blood (CB) is the preferred HSPC source given its rapid availability and superior clinical outcomes compared to other graft sources. CB, however, is associated with delayed hematopoietic recovery and relatively poor engraftment due to the limited numbers of hematopoietic stem cells (HSCs) in a CB unit, delaying enzyme/protein reconstitution and cross-correction of non-hematopoietic cells. An aryl hydrocarbon receptor antagonist (AHRa)-based culture has been shown to expand CB CD34+ and CD34+CD90+ cells 330-fold and 100-fold, respectively, leading to rapid hematopoietic recovery after infusion of the clinical product, MGTA-456 (Wagner et al., Cell Stem Cell 2016 and Orchard et al., ASH 2018). As microglia are thought to be derived from HSCs, we hypothesized that MGTA-456 might lead to faster and greater microglial engraftment and potentially enable reduced intensity conditioning. Here, we assessed human hematopoietic and brain engraftment in NSG mice after transplant with MGTA-456 and showed that microglia engrafted faster with MGTA-456, less conditioning was needed, and that, mechanistically, these cells are derived from the CD34+CD90+ cell fraction. Methods. CB CD34+ cells were expanded in growth factor-supplemented media with or without an AHRa for 10 days. NSG mice were transplanted with unmanipulated CB CD34+ cells or the expanded product after 200 cGy total body irradiation or busulfan (BU) dosed at 20 or 40 mg/kg ip. Microglial engraftment was measured by flow cytometry of homogenized brains, quantitating the number of CD45+CD11b+Iba1+ cells, and by immunohistochemistry of brain sections. Results. Relative to naïve, unmanipulated CB CD34+ cells, transplant of MGTA-456 into sublethally irradiated mice led to an 8-fold increase in hematopoietic engraftment and a 10-fold increase in microglial engraftment in the brain (p<0.0001, n=15 mice), with histology consistent with engrafting microglia. As high dose BU enables enhanced microglia engraftment relative to irradiation by crossing the blood brain barrier and clearing host microglia (Capotondo et al., PNAS 2013), we evaluated the effectiveness of MGTA-456 after BU conditioning at 20 or 40 mg/kg. Transplant of MGTA-456 led to a 37-fold increase in engraftment relative to mice transplanted with unmanipulated CB CD34+ cells (p<0.001, n=8). Notably, transplant of MGTA-456 into mice conditioned with low-dose BU (20 mg/kg) led to a 15-fold increase in engraftment relative to high-dose BU (40 mg/kg)-conditioned animals transplanted with unmanipulated CB CD34+ cells (p<0.001, n=8). To evaluate speed of microglial engraftment, we evaluated brains weekly to 16 weeks after transplant. A 28-fold increase in microglial engraftment was demonstrated as early as 2 weeks post-transplant with MGTA-456 (p<0.0001, n=8). Number of engrafting hematopoietic cells in the periphery correlated with number of engrafting microglia in the brain (p<0.0001). Lastly, subpopulations of MGTA-456 were evaluated to determine the source of microglial engraftment. Only CD34+CD90+ cells, but not CD34+CD90- or CD34- cells, led to brain engraftment, consistent with the subpopulation of cells that result in hematopoietic engraftment following transplant of unexpanded cells (Radtke et al., Sci Trans Med 2017 and Goncalves et al., Blood 2017 130:659). Conclusions. These studies demonstrate that microglial engraftment is faster and greater in recipients of MGTA-456 even after lower dose BU conditioning, that microglial engraftment correlates with peripheral blood recovery, and that microglia cells are derived from CD34+CD90+ cells. These results suggest that lower dose BU may improve safety without jeopardizing efficacy in IMD recipients of MGTA-456. A Phase 2 clinical trial is ongoing to evaluate transplant of MGTA-456 in patients with select IMDs. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Li:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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Mutant ASXL1 Disrupts Paraspeckle Formation through Aberrant Interaction with Nono in Hematopoietic Cells

Blood ◽

10.1182/blood-2019-125472 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2514-2514

Author(s):

Yamamoto Keita ◽

Susumu Goyama ◽

Shuhei Asada ◽

Takeshi Fujino ◽

Tomofusa Fukuyama ◽

...

Keyword(s):

Bone Marrow ◽

Alternative Splicing ◽

Rna Splicing ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Wild Type ◽

Truncated Form ◽

Hematopoietic Stem ◽

Equity Ownership ◽

Marrow Cells

Paraspeckles are sub-nuclear structures found in the interchromatin space of mammalian cells. The core paraspeckle components include a lncRNA NEAT1 and members of the DBHS family of proteins: NONO, SFPQ, and PSPC1. Paraspeckles and their components play diverse roles in gene regulatory networks, including transcription, alternative RNA splicing, nuclear retention of RNA, and DNA repair. Although a previous study showed the presence of paraspeckles in hematopoietic stem and progenitor cells (HSPCs), their roles in normal and malignant hematopoiesis remain largely unknown. ASXL1 regulates gene expression through interactions with multiple epigenetic regulators. Somatic mutations in ASXL1 gene occur frequently in myeloid neoplasms. We previously generated a hematopoietic lineage-specific conditional knockin (KI) mouse of a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), and showed that ASXL1-MT inhibited repopulating capability of HSPCs. We performed deep RNA sequencing using HSPCs from ASXL1-MT-KI mice, and found aberrant alternative splicing in multiple genes involved in hematopoiesis. The altered splicing in ASXL1-MT-KI HSPCs included abnormal exon skipping or retention in Runx1, Traf6, Atm, and Dnmt3b. These findings, together with a previous report showing that ASXL1 mutations affect alternative splicing in U937 cells, strongly indicate the involvement of ASXL1 in RNA splicing machinery. Because a previous interactome analysis suggested the association between NONO and ASXL1, we hypothesized that ASXL1 may play a role in RNA maturation processes through interactions with paraspeckle proteins. To test this hypothesis, we examined physical and functional interactions between paraspeckle components and ASXL1. We found that both wild-type and mutant ASXL1 interact with NONO and SFPQ in 293T cells. Interestingly, protein and RNA immunoprecipitation (RIP) analyses revealed that coexpression of wild-type ASXL1, but not mutant ASXL1, enhanced interactions between NONO and histone H3 as well as NONO and NEAT1. These results suggest that ASXL1 acts as a scaffolding protein that assembles paraspeckle proteins and histones to promote transcription and RNA processing. Importantly, mutant ASXL1 loses this function. Next, we assessed subcellular localization of Nono in HSPCs from control and ASXL1-MT-KI mice. We observed predominant cytoplasmic expression of Nono in ASXL1-MT KI HSPCs, while Nono mainly localized in the nucleus in control cells (Figure 1). In addition, expression of NEAT1_2 isoform, which is essential for paraspeckle formation and maintenance, was substantially downregulated in ASXL1-MT-KI HSPCs. Consistent with these observations, RNA FISH against NEAT1 and immunofluorescence against NONO revealed disrupted paraspeckle formation in ASXL1-MT-KI HSPCs. These data suggest that ASXL1-MT promotes nuclear export of Nono, which results in disruption of paraspeckles in HSPCs. NONO has nuclear localization signal (NLS) at its C-terminus, and it was previously shown that a cytoplasmic C-truncated form of NONO induced senescence in fibroblasts. To assess the effect of forced expression of the cytoplasmic NONO in hematopoietic cells, we transduced vector or a NONO mutant lacking the NLS domain (NONO-ΔNLS) into c-Kit+ bone marrow cells, and transplanted these cells into recipient mice. NONO-ΔNLS induced overproduction of reactive oxygen species (ROS) and reduced engraftment of bone marrow progenitors as ASXL1-MT did. We then assessed the effect of Nono depletion in ASXL1-MT-KI HSPCs using CRISPR/Cas9 system. We crossed ASXL1-MT-KI mice with Rosa26-LSL-Cas9-KI mice, and c-Kit+ bone marrow cells derived from these mice were transduced with a non-targeting or Nono-targeting sgRNAs. This experiment revealed that Nono depletion reverted the impaired repopulation of ASXL1-MT-KI HSPCs after transplantation. Taken together, these data indicate that the cytoplasmic localization of Nono induced by ASXL1-MT has the negative impact on HSPC function. In summary, this study reveals a novel link between an epigenetic regulator ASXL1 and paraspeckle formation. The aberrant interaction between mutant ASXL1 and NONO results in NONO mislocalization, paraspeckle disruption and HSPC dysfunction. Our findings also suggest potentially important roles for paraspeckles to maintain normal hematopoiesis. Disclosures Ogawa: Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; Asahi Genomics: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Kan Research Laboratory, Inc.: Consultancy.

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Deletion of Puma protects hematopoietic stem cells and confers long-term survival in response to high-dose γ-irradiation

Blood ◽

10.1182/blood-2009-10-248278 ◽

2010 ◽

Vol 115 (17) ◽

pp. 3472-3480 ◽

Cited By ~ 84

Author(s):

Hui Yu ◽

Hongmei Shen ◽

Youzhong Yuan ◽

Richard XuFeng ◽

Xiaoxia Hu ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

High Dose ◽

Wild Type ◽

Γ Irradiation ◽

Term Survival ◽

Hematopoietic Stem ◽

Γ Radiation

Abstract Molecular paradigms underlying the death of hematopoietic stem cells (HSCs) induced by ionizing radiation are poorly defined. We have examined the role of Puma (p53 up-regulated mediator of apoptosis) in apoptosis of HSCs after radiation injury. In the absence of Puma, HSCs were highly resistant to γ-radiation in a cell autonomous manner. As a result, Puma-null mice or the wild-type mice reconstituted with Puma-null bone marrow cells were strikingly able to survive for a long term after high-dose γ-radiation that normally would pose 100% lethality on wild-type animals. Interestingly, there was no increase of malignancy in the exposed animals. Such profound beneficial effects of Puma deficiency were likely associated with better maintained quiescence and more efficient DNA repair in the stem cells. This study demonstrates that Puma is a unique mediator in radiation-induced death of HSCs. Puma may be a potential target for developing an effective treatment aimed to protect HSCs from lethal radiation.

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Conditioning with Fludarabine-Busulfan versus Busulfan-Cyclophosphamide Is Associated with Lower aGVHD and Higher Survival but More Extensive and Long Standing Bone Marrow Damage

BioMed Research International ◽

10.1155/2016/3071214 ◽

2016 ◽

Vol 2016 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Xin He ◽

YongBin Ye ◽

XiaoJun Xu ◽

Jing Wang ◽

YuXian Huang ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Conditioning Regimen ◽

Clinical Manifestations ◽

Major Complication ◽

Clinical Situation ◽

Hematopoietic Stem ◽

T Cell Infiltration ◽

Bone Marrow Damage ◽

The Impact

Acute graft-versus-host disease (aGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and a major cause of nonrelapse mortality after allo-HSCT. A conditioning regimen plays a pivotal role in the development of aGVHD. To provide a platform for studying aGVHD and evaluating the impact of different conditioning regimens, we established a murine aGVHD model that simulates the clinical situation and can be conditioned with Busulfan-Cyclophosphamide (Bu-Cy) and Fludarabine-Busulfan (Flu-Bu). In our study, BALB/c mice were conditioned with Bu-Cy or Flu-Bu and transplanted with 2×107 bone marrow cells and 2×107 splenocytes from either allogeneic (C57BL/6) or syngeneic (BALB/c) donors. The allogeneic recipients conditioned with Bu-Cy had shorter survivals (P<0.05), more severe clinical manifestations, and higher hepatic and intestinal pathology scores, associated with increased INF-γ expression and diminished IL-4 expression in serum, compared to allogeneic recipients conditioned with Flu-Bu. Moreover, higher donor-derived T-cell infiltration and severely impaired B-cell development were seen in the bone marrow of mice, exhibiting aGVHD and conditioned with Flu-Bu. Our study showed that the conditioning regimen with Bu-Cy resulted in more severe aGVHD while the Flu-Bu regimen was associated with more extensive and long standing bone marrow damage.

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Impact of Amotosalen/UVA Pathogen Inactivated Platelet Components on Outcomes after Allogeneic Hematopoetic Stem Cell Transplantation: A Single Center Analysis

Blood ◽

10.1182/blood-2019-122330 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1167-1167

Author(s):

Andreas S. Buser ◽

Laura Infanti ◽

Andreas Holbro ◽

Joerg Halter ◽

Sabine Gerull ◽

...

Keyword(s):

Stem Cell ◽

Cox Regression ◽

Univariate Analysis ◽

Conditioning Regimen ◽

Disease Status ◽

Risk Scores ◽

Employment Equity ◽

Stem Cell Source ◽

Equity Ownership ◽

The Impact

Background: Platelet component (PC) transfusion is required for allogeneic hematopoietic stem cell transplantation (HCT) recipients. Contamination with infectious pathogens (bacteria, viruses, or protozoa) and T-cells is a risk factor for transfusion-transmitted infection (TTI) and transfusion associated graft-versus-host disease (TA-GVHD). Pathogen inactivation (PI) treatment of PC with amotosalen-UVA (PI-PC, INTERCEPT Blood System, Cerus Corp) in platelet additive solution (PAS) without bacterial screening, gamma irradiation, CMV serology, and with 7-day storage has been the standard of care in Switzerland since 2011 to manage risk of TTI and TA-GVHD. PI-PC have replaced conventional PC (C-PC) prepared in PAS with gamma irradiation and 5 day storage. We previously reported platelet usage in two consecutive five year periods at the University Hospital of Basel. Mean PI-PC dose was higher (3.0 vs. 2.8 x 1011, p=0.001) and mean storage duration longer (4.2 vs. 3.4 days: p=0.001) than with C-PC. PC expiration wastage was reduced with 7-day PI-PC storage vs. 5-day storage (1.5% vs. 8.7%). For HCT recipients, days of PC support; PC use per patient; and RBC use per patient were similar, despite 24.3% lower corrected count increments (CCI) with PI-PC. Now, we report the impact of these observations on treatment related mortality (TRM) and overall survival (OS) 100 days after HCT. Patients and Methods: A two-period retrospective cohort study was conducted to evaluate PI-PC impact on outcomes of consecutive first allogeneic HCT recipients from January 2006 to December 2010 (Period 1, P1), when gamma-irradiated apheresis C-PC were used, and Period 2 (P2) from January 2011 to December 2017, when apheresis and whole blood-derived PI-PC were used. The review utilized 100-day OS and 100-day TRM to determine the impact of PI-PC on HCT outcomes. Descriptive statistics were used for continuous variables and log-rank analysis for survival outcomes. Univariate analysis was performed using Pearson χ2 statistics. Multivariate Cox regression modelling analyses included: PC period (P1, P2), donor match (HLA identical/twin, matched related, matched unrelated), disease state (early, intermediate, late), and conditioning regimen (reduced intensity, myeloablative) with TRM as the outcome. This was an IRB approved single-center analysis. Results: In P1 and P2, 256 and 557 consecutive first-time allogeneic HCT recipients were included, respectively. By univariate analysis, the distribution of European Group for Bone Marrow Transplantation (EBMT) risk scores (grouped 0-2, 3-4, 5-7) and mean patient age were higher during P2 (p = 0.001 and p <0.001, respectively). Primary disease status (p = 0.039); stem cell source (p <0.001); GVHD prophylaxis with ATG (p <0.001); total body irradiation (p <0.001); and conditioning regimen (p <0.001) were different between P1 and P2. Donor match (p=0.084) and disease status (p = 0.628) were similar in P1 and P2. TRM at day 100 post HCT was significantly less (31/557, 5.5%) for PI-PC recipients in P2 vs. C-PC recipients in P1 (37/256, 14.5%, p<0.001). Overall proportion of survivors at day 100 post HCT was significantly greater for PI-PC recipients (507/557, 91.0 %) compared to C-PC recipients (209/256, 81.6%, p <0.001). By multivariate Cox regression analysis, P2 with PI-PC component support was associated with improved TRM (p = 0.001; adjusted hazard ratio 0.433; 95% confidence interval: 0.262, 0.716). Donor match (p = 0.019), disease state (p = 0.022), and myeloablative conditioning (p = 0.034) were associated with significantly poorer TRM (Table). Stem cell source was not significant (p=0.157) in the model. Hemorrhage was reported as cause of death in 1/50 (2.0%) patients during P2 with PI-PC and 4/47 (8.5%) patients during P1 with C-PCs. Conclusions: Universal implementation of PI-PC in routine with extended storage to 7 days in P2 was associated with reduced TRM and better overall survival 100 days post HCT, despite transplantation of older patients with higher EBMT risk scores. Multivariate analysis revealed an adjusted hazard ratio of 0.433 (95% C.I. 0.262, 0.716) for TRM by 100 days, suggesting better outcomes in P2. This retrospective analysis at a single site indicated that PI-PC treated with amotosalen /UVA stored up to 7 days did not have a negative impact on TRM and OS in HCT recipients, and was an integral part of improving clinical outcomes at our institution. . Table. Disclosures Heim: Novartis: Research Funding. Irsch:Cerus Corporation: Employment, Equity Ownership. Lin:Cerus Corporation: Employment, Equity Ownership. Benjamin:Cerus Corporation: Employment, Equity Ownership. Corash:Cerus Corporation: Employment, Equity Ownership.

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Astarabine, a Novel Leukemia-Targeted Cytarabine Composition Allows, for the First Time, the Delivery of High Cytarabine Doses for Older or Unfit Patients with Acute Leukemia. Results of an Ongoing Phase I/IIa Study

Blood ◽

10.1182/blood.v128.22.1650.1650 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1650-1650

Author(s):

Tsila Zuckerman ◽

Stela Gengrinovitch ◽

Ruth Ben-Yakar ◽

Ron Hoffman ◽

Israel Henig ◽

...

Keyword(s):

Bone Marrow ◽

Acute Leukemia ◽

De Novo ◽

Intensive Therapy ◽

High Dose ◽

Newly Diagnosed ◽

Employment Equity ◽

Unfit Patients ◽

Equity Ownership ◽

High Doses

Abstract Introduction: Therapy of acute myeloid leukemia (AML) has not changed significantly during several decades. High-dose cytarabine, although used as the first-line treatment for AML since 1970s and as a second-line treatment for acute lymphoblastic leukemia (ALL), is associated with severe side effects, such as cerebellar toxicity and bone marrow suppression. Hence, while the incidence of AML increases with age, doses of cytarabine are significantly attenuated or the drug is entirely excluded from the regimen used in older adults due to its potential toxicities, particularly in individuals with hepatic or renal dysfunction. Astarabine is a new composition of cytarabine covalently bound to asparagine. It is designed to target cytarabine to leukemic blasts, thus avoiding extramedullary toxicity. Leukemic cells, which are dependent on an external source of amino acids in general and asparagine in particular, due to their high metabolic rate, have a relatively increased uptake of Astarabine. Inside the blasts, Astarabine is cleaved to cytarabine, enabling targeted killing and relative sparing of normal hematopoiesis. As such, Astarabine may serve as an ideal therapy for leukemia, particularly for delivering high doses of cytarabine to medically unfit or older adults who otherwise can be given supportive therapy only. The aim of this study was to evaluate the safety and optimal dose of Astarabine in refractory/relapsed or medically unfit patients with acute leukemia. Methods: This Phase I/IIa prospective open label study enrolled patients aged ≥18 years with relapsed/refractory or newly-diagnosed acute leukemia unfit for intensive therapy, as judged by the treating physician. The study was approved by the Rambam IRB (approval #0384-11). Patients were enrolled into 6 Astarabine escalating-dose cohorts, each composed of 3-6 patients. Treatment was administered as a 1-hour single daily infusion for 6 days. For cohorts 1-4, Astarabine doses for each infusion were 0.5g/m2, 1.5g/m2, 3g/m2 and 4.5g/m2. The doses were reduced by 50% for patients >50 years. Since dose limiting toxicity (DLT) was not reached in cohorts 1-4, the study was extended to include cohorts 5 and 6 with daily Astarabine doses of 4.5g/m2 and 6g/m2, respectively, with no dose reduction for patients >50 years old. Results: The outcome of 15 patients is reported herein. Six patients with a median age of 64 years (range 27-81) had refractory/relapsed AML, 9 patients with a median age of 80 years (range 70-90) were newly diagnosed (secondary AML - 6, de-novo AML - 2, de-novo ALL - 1) and unfit for intensive therapy. Astarabine treatment was well-tolerated. Two patients died (one from pneumonia and one from sudden death 2 weeks from end of treatment) before completing 30 days post-treatment and hence were excluded from the outcome analysis. Response to the treatment was observed in the bone marrow of 6 of the 7 newly-diagnosed patients for whom bone marrow analysis was available, 3 of whom had a continuous complete remission (CR) for 4 (ongoing), 8, and 10 months post-treatment, and 3 had a continuous partial remission (PR) for 3,7, and 7 (ongoing) months. The median overall survival (OS) of the patients with CR/PR is 7 months to date (table 1). No significant response was observed in the relapsed/refractory patients, with a median OS of 2.5 months. Twelve patients died from disease progression. Conclusions: Astarabine, a new composition of leukemia-targeted cytarabine, is safe and very well tolerated, even in patients over 80 years of age, resulting in response in 6 of 7 newly diagnosed patients with acute leukemia. To the best of our knowledge, this is the first report permitting high-dose of cytarabine, considered a cornerstone of leukemia therapy, to be given to a population of patients that heretofore did not have this option. Further dose escalation studies are currently ongoing at a cytarabine-equivalent dose of 4.5 and 6 g/m2/day. A phase II study is planned to confirm these encouraging results and define the use of Astarabine for patients otherwise unable to receive high doses of cytarabine. Disclosures Zuckerman: BioSight Ltd: Consultancy, Research Funding. Gengrinovitch:BioSight Ltd: Employment, Equity Ownership, Patents & Royalties: Inventor all of the patents. Ben-Yakar:BioSight Ltd: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Inventor of all patents.

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Abstract P331: Angiotensin-salt Hypertension Requires Ouabain-sensitive Na + Pumps

Hypertension ◽

10.1161/hyp.70.suppl_1.p331 ◽

2017 ◽

Vol 70 (suppl_1) ◽

Author(s):

Ling Chen ◽

Jerry B Lingrel ◽

John M Hamlyn ◽

Mordecai P Blaustein

Keyword(s):

Low Dose ◽

High Dose ◽

Ang Ii ◽

Wild Type ◽

Dietary Salt ◽

Endogenous Ligand ◽

Ouabain Binding ◽

Fab Fragments ◽

Salt Hypertension ◽

The Brain

Dietary salt is a major factor in the pathogenesis of essential hypertension (EH), but the underlying links are unresolved. Animal models indicate that angiotensin (Ang) II and high dietary salt (HS) are convergent signals that act via the brain to elevate blood pressure (BP). Low-dose sc Ang II+HS is a common model for EH. We tested the Na + pump ouabain binding site’s role in this model because it is crucial in some other hypertension models (e.g., ACTH and Nedd4-2-knockout +HS). Mice that express Na + pumps with a mutant, ouabain-resistant α2 catalytic subunit (α2 R/R ; cation transport is normal), and wild type (WT), ouabain sensitive controls (α2 S/S ) were studied. [80-90% of rodent artery myocyte Na + pumps are ouabain-resistant (α1 R/R ); only 10-20% are α2.] BP was measured by telemetry. First, 3 basal 24 hr BPs were recorded. Osmotic 4-week minipumps were then implanted sc in all mice to deliver vehicle (saline; Expt. #1,3), or 400 (Expt. #1,2) or 800 (Expt. #3) ng/kg/min Ang II; simultaneously, in Expt. #2, the diet was switched from 0.4% (standard) to 2% NaCl (HS). BPs were monitored every 3-4 days for up to 4 weeks. Also, in Expt. #2, on day 21, all mice received 2 ip injections, 4 hrs apart, of 10 mg/kg DigiFab, Fab fragments that immuno-neutralize ouabain, while BP was continuously monitored; on day 23, the mice received 2 ip injections of CroFab, anti-crotalus toxin (‘control’) Fab fragments. Results: 1. Basal mean BP (MBP) was 10±2 mm Hg higher in α2 R/R than in WT mice ( P <0.01; n =21 & 29; ANOVA). 2. In WT mice, 400 ng/kg/min sc Ang II and Ang II+HS raised MBP by 15±1 and 34±1 mm Hg, respectively ( P <0.01; n =7-8; ANOVA). 3. The MPB elevation in Ang II+HS α2 R/R (17±2 mm Hg) was only half that in WT mice ( P <0.01; n =7 each; ANOVA). 4. DigiFab rapidly (<1 hr) reduced MBP by 14±2 mm Hg in Ang II+HS hypertensive WT mice ( P <0.001; n =7; T-test), but not in α2 R/R mice ( P <0.01; n =7 each; ANOVA); CroFab did not lower MBP in either strain. 5. 800 ng/kg/min sc Ang II elevated systolic BP by 55±3 mm Hg in WT mice, but by only 37±3 mm Hg in α2 R/R mice ( P <0.05; n =3-5; ANOVA). Conclusions: Ouabain-sensitive α2 Na + pumps and their endogenous ligand are both required for full expression of low-dose Ang II-salt hypertension. Ouabain-sensitive α2 pumps apparently also contribute to high-dose Ang II-hypertension.

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Phase I trial of retroviral-mediated transfer of the human MDR1 gene as marrow chemoprotection in patients undergoing high-dose chemotherapy and autologous stem-cell transplantation.

Journal of Clinical Oncology ◽

10.1200/jco.1998.16.1.165 ◽

1998 ◽

Vol 16 (1) ◽

pp. 165-172 ◽

Cited By ~ 174

Author(s):

C Hesdorffer ◽

J Ayello ◽

M Ward ◽

A Kaubisch ◽

L Vahdat ◽

...

Keyword(s):

Bone Marrow ◽

Phase I ◽

Marrow Transplantation ◽

Bone Marrow Cells ◽

Multiple Drug Resistance ◽

Autologous Bone Marrow Transplantation ◽

High Dose Chemotherapy ◽

Multiple Drug ◽

High Dose ◽

Hematopoietic Stem

PURPOSE Normal bone marrow cells have little or no expression of the MDR p-glycoprotein product and, therefore, are particularly susceptible to killing by MDR-sensitive drugs, such as vinca alkaloids, anthracyclines, podophyllins, and paclitaxel and its congeners. Here we report the results of a phase I clinical trial that tested the safety and efficacy of transfer of the human multiple drug resistance (MDR1, MDR) gene into hematopoietic stem cells and progenitors in bone marrow as a means of providing resistance of these cells to the toxic effects of cancer chemotherapy. PATIENTS AND METHODS Up to one third of the harvested cells of patients who were undergoing autologous bone marrow transplantation as part of a high-dose chemotherapy treatment for advanced cancer were transduced with an MDR cDNA-containing retrovirus; these transduced cells were reinfused together with unmanipulated cells after chemotherapy. RESULTS High-level MDR transduction of erythroid burst-forming unit (BFU-E) and colony-forming unit-granulocyte macrophage (CFU-GM) derived from transduced CD34+ cells was shown posttransduction and prereinfusion. However, only two of the five patients showed evidence of MDR transduction of their marrow at a low level at 10 weeks and 3 weeks, respectively, posttransplantation. The cytokine-stimulated transduced cells may be out-competed in repopulation by unmanipulated normal cells that are reinfused concomitantly. The MDR retroviral supernatant that was used was shown to be free of replication-competent retrovirus (RCR) before use, and all tests of patients' samples posttransplantation were negative for RCR. In addition, no adverse events with respect to marrow engraftment or other problems related to marrow transplantation were encountered. CONCLUSION These results indicate the feasibility and safety of bone marrow gene therapy with a potentially therapeutic gene, the MDR gene.

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Regulatory Roles of Bone in Neurodegenerative Diseases

Frontiers in Aging Neuroscience ◽

10.3389/fnagi.2020.610581 ◽

2020 ◽

Vol 12 ◽

Author(s):

Zhengran Yu ◽

Zemin Ling ◽

Lin Lu ◽

Jin Zhao ◽

Xiang Chen ◽

...

Keyword(s):

Stem Cells ◽

Neurodegenerative Diseases ◽

The Elderly ◽

Lymphoid Organ ◽

The Body ◽

Hematopoietic Stem ◽

Brain Functions ◽

And Function ◽

The Impact ◽

The Brain

Osteoporosis and neurodegenerative diseases are two kinds of common disorders of the elderly, which often co-occur. Previous studies have shown the skeletal and central nervous systems are closely related to pathophysiology. As the main structural scaffold of the body, the bone is also a reservoir for stem cells, a primary lymphoid organ, and an important endocrine organ. It can interact with the brain through various bone-derived cells, mostly the mesenchymal and hematopoietic stem cells (HSCs). The bone marrow is also a place for generating immune cells, which could greatly influence brain functions. Finally, the proteins secreted by bones (osteokines) also play important roles in the growth and function of the brain. This article reviews the latest research studying the impact of bone-derived cells, bone-controlled immune system, and bone-secreted proteins on the brain, and evaluates how these factors are implicated in the progress of neurodegenerative diseases and their potential use in the diagnosis and treatment of these diseases.

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