Recombinant scAAV2 Vector-Mediated Ex Vivo Transduction of Primary Human Hematopoietic Stem Cells from a β-Thalassemia Patient and Human β-Globin Gene Expression in a Murine Xenograft Model in Vivo

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5951-5951
Author(s):  
Liu-Jiang Song ◽  
Xin-Hua Zhang ◽  
Jun Zhu ◽  
Jue-Lian Wu ◽  
Xiao-Ling Yin ◽  
...  

Abstract Background: The most severe form of β-thalassemia, β°-thalassemia major, is characterized by the complete absence of normal β-globin chain, and is often lethal. Autologous transplantation of genetically-modified hematopoietic stem cells (HSCs) using lentiviral vectors have been used successfully to achieve clinical efficacy in one patient, although clonal expansion of a myeloid cell population also occurred in this patient which was associated with the activation of a cellular proto-oncogene, HMGA2. We reasoned that recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus (AAV), might offer a safer alternative. We have previously documented that although the conventional single-stranded (ss) AAV2 vectors mediated β-globin gene transfer and expression in primary human fetal liver cells and in human HSCs from patients with β-thalassemia patients in vitro, the level of transgene expression was sub-optimal. In the present study, we investigated whether double-stranded self-complementary (sc) AAV2 vectors could overcome this limitation. Methods: Human HSCs, obtained from a β-thalassemia homozygous patient, were mock-transduced or transduced with recombinant scAAV2-β-globin vectors at 5×104 vgs/cell, followed by i.v. injection into sub-lethally irradiated NOD/SCID mice (2.65 cGy total body irradiation), which were also pre-treated with 200 µg purified anti-IL2RB/CD122 monoclonal antibody. Recipient mice were sacrificed 12 weeks post-transplantation. Bone marrow cells from recipient mice were analyzed by BFU-E assays. Human β-globin gene expression in human erythroid progenitor cells from transplanted mice was evaluated by RT-PCR. Results: Pre-treatment of NOD/SCID mice with anti-CD122 antibody improves engraftment of human HSCs in bone marrow of receipt mice. Human β-actin (538-bp) and β-globin (272-bp) transcripts were detected by RT-PCR in bone marrow cells from all recipient mice, indicating that recombinant scAAV2-β-globin–transduced HSCs from a patient with β-thalassemia were successfully transduced and transplanted in these mice and that human β-globin gene was transcriptionally active 12 weeks post-transplantation. Conclusion: Our results indicate that human HSCs from β-thalassemia patients can be efficiently transduced by recombinant scAAV2-β-globin vectors followed by expression of normal human β-globin gene. These studies provide the proof-of-concept that scAAV2 vector-mediated gene transfer into human HSCs might be a potentially safer alternative approach for gene therapy of β-thalassemia. Disclosures No relevant conflicts of interest to declare.

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4715-4715
Author(s):  
Mengqun Tan ◽  
Zhenqing Liu ◽  
Juan Zhang ◽  
Zhiyan Li ◽  
Liujiang Song ◽  
...  

Abstract Abstract 4715 β -Thalassemia is one of the most common worldwide monogenic human diseases,caused by molecular defects in the human β -globin gene cluster leading to decrease or absence of β-globin. Loss of β -globin chains causes ineffective production of oxygen-carrying hemoglobin and therefore results in severe anemia. The treatment for β -Thalassemia major usually includes lifelong blood transfusions but chronic blood transfusion often causes iron overload, and accumulated iron produces tissue damage in multiple organs, so that iron chelating treatment is also needed. Bone marrow transplantation is another effective therapy, which can eliminate a patient's dependence on blood transfusions, however, it is difficult to find a matching donor for most patients; therefore it is only available for a minority of patients. Gene therapy is one potential novel therapy for treatment of inherited monogenic disorders. The long–term therapeutic strategy for this disease is to replace the defective β-globin gene via introduction of a functional gene into hematopoietic stem cells (HSCs). Adeno-associated virus type 2 (AAV), a nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. AAV can infect both dividing and non-dividing cells and wild AAV integrates preferentially at a specific site on human chromosome 19. In the absence of helper virus, recombinant AAV will stably integrate into the host cell genome, mediating long-term and stable expression of the transgene. In this study, we used a hybrid rAAV6/2 vector carrying the human β-globin gene to transduce HSCs from a β -Thalassemia patient, followed by transplantation into irradiated BALB/c nude mice. One month post-transplantation, Hb was prepared from peripheral blood and analyzed by Western Blot and HPLC respectively. RNA and DNA were isolated from bone marrow cells (BMCs) from recipient mice transplanted with mock-infected or hybrid rAAV–globin-infected cells and analyzed by RT-PCR and PCR respectively. The results showed: 1. Human β-actin and β-globin transcripts were detected by RT-PCR in BMCs from all recipient mice, indicating that human HSCs were successfully transplanted in these mice and that the human β-globin gene was transcriptionally active in the donor cells. 2. The level of human hemoglobin expressed in peripheral red blood cells of recipient mice as measured by HPLC (ratio of β/α) was increased to 0.3 from 0.05 of pre-transplantation levels. Expression of human β-globin was also confirmed in recipient mice by Western Blot; a 2–3-fold increase compared with that of controls. Our results indicate that human HSCs from a β-Thalassemia patient can be efficiently transduced by a hybrid rAAV6/2-β-globin vector followed by expression of normal human β-globin protein. This study provides a proof-of-concept that rAAV6/2-mediated gene transfer into human HSCs might be a potential approach for gene therapy of β-Thalassemia. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2788-2788
Author(s):  
Lilia Stepanova ◽  
Brian P. Sorrentino

Abstract Homeobox (Hox) transcription factors are important regulators of hematopoietic cell proliferation and differentiation. Of them, HoxB4 is of particular interest because overexpression promotes rapid expansion of mouse hematopoietic stem cells (HSCs) without causing neoplastic transformation. Despite the effects of HoxB4 overexpression on HSCs, mice that are homozygous for HoxB4 gene deletion have only subtle defects in HSCs and progenitor cells. We hypothesized that other paralogs of HoxB4 may also be capable of inducing HSC expansion could thereby compensate for loss of HoxB4 function. To test this hypothesis, we have studied the effects of retroviral overexpression of a HoxC4 gene in murine progenitors and HSCs. The murine HoxC4 cDNA was cloned and inserted into an MSCV vector that co-expresses an IRES-YFP reporter gene. We transduced murine bone marrow cells with a MSCV-HoxC4-YFP vector and compared the secondary replating efficiency of myeloid colonies (CFU-Cs) to that seen using either a MSCV-HoxB4-GFP or an MSCV-GFP vector. This assay tests for progenitor cell self-renewal which is increased using HoxB4-expressing vectors. Cells transduced with the MSCV-HoxC4-YFP vector formed 20–40 times more secondary CFU-Cs than with cells transduced with the MSCV-GFP control vector. This increase in CFU-C replating efficiency was equivalent to that seen with the MSCV-HoxB4-IRES-GFP vector. To test the in vivo effects of the MSCV-HoxC4-YFP vector on self-renewal of HSCs, we transplanted lethally irradiated mice with a mixture of cells; 20% transduced with the MSCV-HoxC4-YFP vector and 80 % mock-transduced. Peripheral blood analysis of the transplanted recipients up to 28 weeks post-transplantation showed that the percentage of cells transduced with the MSCV-HoxC4-YFP vector was 70–85% in both lymphoid and myeloid cells in the peripheral blood. A similar degree of chimerism was noted in concurrent controls using the MSCV-HoxB4-GFP vector. In contrast, the percentages of peripheral blood cells transduced with the MSCV-GFP vector was only 15–25%, paralleling the input ratios of transplanted cells. Secondary transplantation experiments showed stable levels of chimerism in both HoxC4 and HoxB4 groups, indicating that the expansion seen with the MSCV-HoxC4-YFP vector occurred at the HSC level. These results indicate that retroviral-mediated expression of HoxC4, like HoxB4, can cause significant expansion of HSCs in vivo. Because several other Hox genes can cause hematopoietic abnormalities and leukemia when expressed from a retroviral vector, we transplanted lethally irradiated mice with 4x106 cells that were transduced with the MSCV-HoxC4-YFP vector and monitored the animals for survival and complete blood counts. Now, at 33 weeks post transplantation, no tumor formation was observed in mice expressing either the HoxB4 or the HoxC4 vector, and peripheral blood counts have remained normal. Our results show that retroviral overexpression of HoxC4 can induce a significant expansion of the HSCs in vivo, and suggest that expression of HoxC4 may compensate for the loss of HoxB4 in knockout mice. We are currently analyzing the effects of HoxA4 and HoxD4 to determine if they share the same functional characteristics, and are also determining whether HoxB4 and HoxC4 are modulating the same downstream genes using microarray analysis of transduced murine bone marrow cells.


Cell ◽  
1998 ◽  
Vol 93 (6) ◽  
pp. 1055-1065 ◽  
Author(s):  
Hartmut Geiger ◽  
Stefanie Sick ◽  
Constanze Bonifer ◽  
Albrecht M Müller

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 1975-1980 ◽  
Author(s):  
DM Bodine ◽  
T Moritz ◽  
RE Donahue ◽  
BD Luskey ◽  
SW Kessler ◽  
...  

Retroviral mediated gene transfer into stem cells has been proposed as therapy for many inherited hematopoietic diseases. Deficiency of the enzyme adenosine deaminase (ADA) results in depletion of T lymphocytes, causing severe combined immunodeficiency syndrome (SCIDS). In this report, we describe retroviral mediated gene transfer of a murine ADA cDNA into Rhesus monkey hematopoietic stem cells. Immunoselected CD34+ bone marrow cells were exposed to medium containing the ADA retrovirus during culture on a stromal cell line engineered to express the transmembrane form of stem cell factor. After infusion of autologous, transduced cells into irradiated recipients, gene transfer was observed in all three monkeys. The ADA provirus was detected in 2% of circulating granulocytes and T cells from 100 days post-transplantation to longer than 1 year and in B cells from 250 days post-transplantation and beyond. Mouse ADA activity was detected in peripheral blood cells at approximately 3% the activity of monkey ADA. Thus, we have shown gene transfer into repopulating cells that contribute to all hematopoietic lineages with persistent gene expression. These data provide support for the use of stem cell targeted gene transfer for therapy of ADA deficiency.


Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 1975-1980 ◽  
Author(s):  
DM Bodine ◽  
T Moritz ◽  
RE Donahue ◽  
BD Luskey ◽  
SW Kessler ◽  
...  

Abstract Retroviral mediated gene transfer into stem cells has been proposed as therapy for many inherited hematopoietic diseases. Deficiency of the enzyme adenosine deaminase (ADA) results in depletion of T lymphocytes, causing severe combined immunodeficiency syndrome (SCIDS). In this report, we describe retroviral mediated gene transfer of a murine ADA cDNA into Rhesus monkey hematopoietic stem cells. Immunoselected CD34+ bone marrow cells were exposed to medium containing the ADA retrovirus during culture on a stromal cell line engineered to express the transmembrane form of stem cell factor. After infusion of autologous, transduced cells into irradiated recipients, gene transfer was observed in all three monkeys. The ADA provirus was detected in 2% of circulating granulocytes and T cells from 100 days post-transplantation to longer than 1 year and in B cells from 250 days post-transplantation and beyond. Mouse ADA activity was detected in peripheral blood cells at approximately 3% the activity of monkey ADA. Thus, we have shown gene transfer into repopulating cells that contribute to all hematopoietic lineages with persistent gene expression. These data provide support for the use of stem cell targeted gene transfer for therapy of ADA deficiency.


Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4694-4694
Author(s):  
Jana Michalova ◽  
Ludek Sefc ◽  
Filipp Savvulidi ◽  
Katarina Forgacova ◽  
Katerina Faltusova ◽  
...  

Abstract Abstract 4694 Background: Quiescent hematopoietic stem cells (HSCs) located in stem cell niches are characterized by a relative resistance to hypoxia. This study is focused primarily on maintainance of the repopulating ability of HSCs in structurally intact BM exposed to anoxia, lack of metabolic substrates and accumulation of metabolic waste products during a period of ischemia at three different temperatures. In the case of a warm ischemia at 37°C, changes in gene expression profile in the whole bone marrow has been also examined. Methods: Murine congenic model C57Bl/6 Ly5.1/Ly5.2 was used in the experiments. Normal mice or mice recovering from a bone marrow damage induced either by cyclophosphamide or a sublethal irradiation were sacrified. Their BM was maintained in intact femurs at 37°C for different time periods up to 6 hours. For normal bone marrow, exposure to ischemia at 20°C and 4°C was also used for up to 20 and 48 hours, respectively. Afterwards, bone marrow cells were harvested and cells corresponding to a half of the femur were transplanted to sublethally (6 Gy) irradiated recipients in a competitive repopulation assay. Resulting chimerism was examined up to 6 months after transplantation to test for STRCs and LTRCs (Short and Long Term Repopulating Cells). Subpopulations of erythropoietic (Ter119+), B-lymphopoietic (B220+), granulo- and monocytopoietic (Gr-1/Mac+), and LSK (Lin-Sca-1+c-Kit+) bone marrow cells were analyzed for dead cells and apoptosis. Total RNA was isolated from bone marrow exposed to warm ischemia ranging 0 to 4 hours and dynamics of changes in its gene expression profile was determined by Illumina MouseRef8 BeadChip. Results: Repopulating ability of ischemic BM was fully preserved for 2 hour of the warm (37°C) ischemia and for 6 hours and 8 hours of 20°C and 4°C ischemia, respectively. There was no difference between STRCs and LTRCs in survival. STRCs and LTRCs from the bone marrow collected 2 days or 5 days after a single dose of cyclophosphamide exposed to warm ischemia showed decreased repopulating ability in comparison with those of normal mice. STRCs significantly prevailed over LTRCs in bone marrow collected 20 days after a sublethal irradiation and showed increased sensitivity to warm ischemia. B220+ cells were the most sensitive cells of the bone marrow to warm ischemia, LSK and Ter119 cells being the most resistant ones. Gene expression profile in bone marrow exposed to warm ischemia changed progressively over time. Despite the highly unfavorable metabolic conditions, hypoxia and lack of energy, a set of overexpressed genes equaled in number the one inhibited. Conclusions: HSCs exposed to warm or cold ischemia maintain their repopulating ability for a considerable time. Bone marrow ischemia activates specific gene expression in paralel with supression of others. Supported by projects LC06044, MSM 0021620806 and the grant SVV-2010-254260507. Disclosures: No relevant conflicts of interest to declare.


2022 ◽  
Author(s):  
Merve Aksoz ◽  
Grigore-Aristide Gafencu ◽  
Bilyana Stoilova Stoilova ◽  
Mario Buono ◽  
Yiran Meng ◽  
...  

Hematopoietic stem cells (HSC) reconstitute multi-lineage human hematopoiesis after clinical bone marrow transplantation and are the cells-of-origin of hematological malignancies. Though HSC provide multi-lineage engraftment, individual murine HSCs are lineage-biased and contribute unequally to blood cell lineages. Now, by combining xenografting of molecularly barcoded adult human bone marrow (BM) HSCs and high-throughput single cell RNA sequencing we demonstrate that human individual BM HSCs are also functionally and transcriptionally lineage biased. Specifically, we identify platelet-biased and multi-lineage human HSCs. Quantitative comparison of transcriptomes from single HSCs from young, and aged, BM show that both the proportion of platelet-biased HSCs, and their level of transcriptional platelet priming, increases with age. Therefore, platelet-biased HSCs, as well as their increased prevalence and elevated transcriptional platelet priming during ageing, are conserved between human and murine hematopoiesis.


Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 939-948 ◽  
Author(s):  
Y Tomita ◽  
DH Sachs ◽  
M Sykes

Abstract We have investigated the requirement for whole body irradiation (WBI) to achieve engraftment of syngeneic pluripotent hematopoietic stem cells (HSCs). Recipient B6 (H-2b; Ly-5.2) mice received various doses of WBI (0 to 3.0 Gy) and were reconstituted with 1.5 x 10(7) T-cell-depleted (TCD) bone marrow cells (BMCs) from congenic Ly-5.1 donors. Using anti-Ly-5.1 and anti-Ly-5.2 monoclonal antibodies and flow cytometry, the origins of lymphoid and myeloid cells reconstituting the animals were observed over time. Chimerism was at least initially detectable in all groups. However, between 1.5 and 3 Gy WBI was the minimum irradiation dose required to permit induction of long-term (at least 30 weeks), multilineage mixed chimerism in 100% of recipient mice. In these mice, stable reconstitution with approximately 70% to 90% donor-type lymphocytes, granulocytes, and monocytes was observed, suggesting that pluripotent HSC engraftment was achieved. About 50% of animals conditioned with 1.5 Gy WBI showed evidence for donor pluripotent HSC engraftment. Although low levels of chimerism were detected in untreated and 0.5-Gy-irradiated recipients in the early post-BM transplantation (BMT) period, donor cells disappeared completely by 12 to 20 weeks post-BMT. BM colony assays and adoptive transfers into secondary lethally irradiated recipients confirmed the absence of donor progenitors and HSCs, respectively, in the marrow of animals originally conditioned with only 0.5 Gy WBI. These results suggest that syngeneic pluripotent HSCs cannot readily engraft unless host HSCs sustain a significant level of injury, as is induced by 1.5 to 3.0 Gy WBI. We also attempted to determine the duration of the permissive period for syngeneic marrow engraftment in animals conditioned with 3 Gy WBI. Stable multilineage chimerism was uniformly established in 3-Gy-irradiated Ly-5.2 mice only when Ly-5.1 BMC were injected within 7 days of irradiation, suggesting that repair of damaged host stem cells or loss of factors stimulating engraftment may prevent syngeneic marrow engraftment after day 7.


Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 74-83 ◽  
Author(s):  
SJ Szilvassy ◽  
S Cory

Abstract Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient “compromised” bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.


2020 ◽  
Vol 16 (3) ◽  
pp. 313-325
Author(s):  
Monika Cymer ◽  
Katarzyna Brzezniakiewicz-Janus ◽  
Kamila Bujko ◽  
Arjun Thapa ◽  
Janina Ratajczak ◽  
...  

Abstract An efficient harvest of hematopoietic stem/progenitor cells (HSPCs) after pharmacological mobilization from the bone marrow (BM) into peripheral blood (PB) and subsequent proper homing and engraftment of these cells are crucial for clinical outcomes from hematopoietic transplants. Since extracellular adenosine triphosphate (eATP) plays an important role in both processes as an activator of sterile inflammation in the bone marrow microenvironment, we focused on the role of Pannexin-1 channel in the secretion of ATP to trigger both egress of HSPCs out of BM into PB as well as in reverse process that is their homing to BM niches after transplantation into myeloablated recipient. We employed a specific blocking peptide against Pannexin-1 channel and noticed decreased mobilization efficiency of HSPCs as well as other types of BM-residing stem cells including mesenchymal stroma cells (MSCs), endothelial progenitors (EPCs), and very small embryonic-like stem cells (VSELs). To explain better a role of Pannexin-1, we report that eATP activated Nlrp3 inflammasome in Gr-1+ and CD11b+ cells enriched for granulocytes and monocytes. This led to release of danger-associated molecular pattern molecules (DAMPs) and mitochondrial DNA (miDNA) that activate complement cascade (ComC) required for optimal egress of HSPCs from BM. On the other hand, Pannexin-1 channel blockage in transplant recipient mice leads to a defect in homing and engraftment of HSPCs. Based on this, Pannexin-1 channel as a source of eATP plays an important role in HSPCs trafficking.


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