scholarly journals Fully Closed, Large-Scale, and Clinical Grade Cell Sorting of Hematopoietic Stem Cell (HSC)-Enriched CD90+ Cells for Transplantation and Gene Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3246-3246
Author(s):  
Stefan Radtke ◽  
Margaret Cui ◽  
Anai M Perez ◽  
Yan-Yi Chan ◽  
Stefanie Schmuck ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Transduction Efficiency ◽  
Target Cells ◽  
Gene Modification ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cell Fractions

Introduction: Hematopoietic stem cell (HSC) gene therapy/editing is a viable treatment option for various hematological diseases and disorders including hemoglobinopathies and HIV/AIDS. Most if not all currently available approaches target CD34-enriched cell fractions, a heterogeneous mix of mostly committed progenitor cells and only very few true HSCs with long-term multilineage engraftment potential. As a consequence, gene therapy/editing approaches are currently limited in their HSC targeting efficiency, very expensive consuming huge quantities of modifying reagents, and can lead to unwanted side-effects in non-target cells. We recently described a novel HSC-enriched CD34 subset (CD90+CD45RA-) that is exclusively responsible for rapid recovery onset, robust long-term multilineage engraftment, as well as entire reconstitution of the bone marrow stem cell compartment in the nonhuman primate (NHP) stem cell transplantation and gene therapy model (Radtke et al. 2017, STM). Most importantly, we demonstrate that this CD34 subset reduces the number of target cells, modifying reagents and costs by more than 10-fold without compromising the long-term efficiency of gene-modification in the NHP (Humbert and Radtke et al. 2019, STM). Here, we aimed to develop a clinical protocol to reliably purify and efficiently gene-modify human HSC-enriched CD90+ cell fractions. Methods: Large-scale enrichment of CD34+ cells from GCSF-mobilized leukapheresis products was initially performed on the Miltenyi CliniMACS Prodigy according to previously established protocols (Adair et al. 2017, Nat. Comm.). Yield, purity, quality, and feasibility of CD90 sorting was then comprehensively tested on two different commercially available cell sorting systems comparing the jet-in-air sorter FX500 from Sony and the cartridge-based closed-system sorter MACSQuant Tyto from Miltenyi Biotech with our clinically approved gold-standard CD34-mediated gene therapy approach. Sorted CD90+ and bulk CD34+ cells were transduced with a clinical-grade lentivirus encoding for GFP and the multilineage differentiation as well as engraftment potential tested using in vitro assays and the NSG mouse xenograft model, respectively. Results: Flow-cytometric sort-purification of CD90+ cells was similarly efficient in purity and yield using either the FX500 or Tyto (Figure A,B). Both approaches reliably reduced the overall target cell count by 10 to 15-fold without impacting the cells viability and in vitro colony-forming cell potential. Unexpectedly, the transduction efficiency of sort-purified CD90+ cells was significantly improved compared to bulk-transduced CD34+ cells and especially the CD34+CD90+ subset (Figure C). All cell fractions demonstrated robust mouse xenograft potential (Figure D). Most importantly, significantly higher levels of GFP+ expression in the peripheral blood, bone marrow, spleen and thymus were observed after transplantation of gene-modified CD90+ compared to bulk CD34+ cells in NSG mice (Figure E). Conclusion: Here, we show that sort-purification of our HSC-enriched CD34+CD90+ cell subset is technically feasible and highly reproducible in two different systems. Purification of human CD90+ cell fractions significantly increased the gene-modification efficiency of primitive human HSCs with multilineage mouse engraftment potential. These findings should have important implications for currently available as well as future HSC gene therapy and gene editing protocols. Isolation of an HSC-enriched phenotype will allow more targeted gene modification and thus likely reduce unwanted off target effects. Our approach further reduced the overall costs for gene modifying reagents, can be combined with a closed transduction system, increase the portability and ultimately make HSC gene therapy GMP-facility independent and affordable. Finally, this stem cell selection strategy may also allow efficient and effective depletion of donor T cells in the setting of allogeneic stem cell or organ transplantation. Figure: A) Purity and B) yield of CD90+ cells after sort-purification. C) Transduction efficiency of bulk-transduced CD34+CD90+ cells and sort-purified CD90+ cells. Frequency of D) human chimerism and E) GFP+ human CD45+ cells in the peripheral blood (PB), bone marrow, spleen and thymus after transplantation of gene-modified bulk CD34+ or sort-purified CD90+ cells. Figure Disclosures Kiem: CSL Behring: Consultancy; Rocket Pharma: Consultancy, Equity Ownership; Homology Medicines: Consultancy, Equity Ownership; Magenta Therapeutics: Consultancy.

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

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 821-821 ◽  
Author(s):  
Marina Cavazzana-Calvo ◽  
Nathalie Cartier ◽  
Salima Hacein-Bey Abina ◽  
Gabor Veres ◽  
Manfred Schmidt ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Cd34 Cells ◽  
Hsc Transplantation

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

Retroviral transduction efficiency of G-CSF+SCF–mobilized peripheral blood CD34+ cells is superior to G-CSF or G-CSF+Flt3-L–mobilized cells in nonhuman primates

Blood ◽  
2003 ◽  
Vol 101 (6) ◽  
pp. 2199-2205 ◽  
Author(s):  
Peiman Hematti ◽  
Stephanie E. Sellers ◽  
Brian A. Agricola ◽  
Mark E. Metzger ◽  
Robert E. Donahue ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Peripheral Blood ◽  
Nonhuman Primates ◽  
Transduction Efficiency ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Retroviral Transduction ◽  
Peripheral Blood Cd34 ◽  
Cd34 Cells ◽  
Clinical Gene Therapy

Gene transfer experiments in nonhuman primates have been shown to be predictive of success in human clinical gene therapy trials. In most nonhuman primate studies, hematopoietic stem cells (HSCs) collected from the peripheral blood or bone marrow after administration of granulocyte colony-stimulating factor (G-CSF) + stem cell factor (SCF) have been used as targets, but this cytokine combination is not generally available for clinical use, and the optimum target cell population has not been systematically studied. In our current study we tested the retroviral transduction efficiency of rhesus macaque peripheral blood CD34+ cells collected after administration of different cytokine mobilization regimens, directly comparing G-CSF+SCF versus G-CSF alone or G-CSF+Flt3-L in competitive repopulation assays. Vector supernatant was added daily for 96 hours in the presence of stimulatory cytokines. The transduction efficiency of HSCs as assessed by in vitro colony-forming assays was equivalent in all 5 animals tested, but the in vivo levels of mononuclear cell and granulocyte marking was higher at all time points derived from target CD34+ cells collected after G-CSF+SCF mobilization compared with target cells collected after G-CSF (n = 3) or G-CSF+Flt3-L (n = 2) mobilization. In 3 of the animals long-term marking levels of 5% to 25% were achieved, but originating only from the G-CSF+SCF–mobilized target cells. Transduction efficiency of HSCs collected by different mobilization regimens can vary significantly and is superior with G-CSF+SCF administration. The difference in transduction efficiency of HSCs collected from different sources should be considered whenever planning clinical gene therapy trials and should preferably be tested directly in comparative studies.

Long-Term Safety and Efficacy of Stem Cell Gene Therapy for ADA-SCID.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 200-200
Author(s):  
Alessandro Aiuti ◽  
Ulrike Benninghoff ◽  
Barbara Cassani ◽  
Federica Cattaneo ◽  
Luciano Callegaro ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Severe Combined Immunodeficiency ◽  
Hematopoietic Stem ◽  
Enzyme Replacement ◽  
Cell Functions ◽  
Cd34 Cells

Abstract Severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency is a fatal congenital disorder of the immune system associated with systemic toxicity due to accumulation of purine metabolites. We previously showed that retroviral-mediated ADA gene transfer into autologous hematopoietic stem/progenitor cells (HSC) allowed restoration of immune and metabolic functions. We have now enrolled eight ADA-SCID children (age: 7–67 months) in our phase I/II gene therapy trial in which HSC are combined with low intensity conditioning with busulfan (total dose 4 mg/Kg i.v.). Previous treatment included haploidentical bone marrow transplant (n=3) or long-term (>1 year) enzyme replacement therapy (PEG-ADA) (n=4) associated with insufficient immune reconstitution or severe autoimmunity. In the latter case, PEG-ADA was discontinued to favour the growth advantage for gene corrected cells. The patients received a median dose of 8.8x106/Kg bone marrow CD34+ cells (range 0.9–10.8), containing on average 26.2±9.6% transduced CFU-C. Five patients experienced ANC <0.5x109/L, which was extended beyond day +30 in two patients. With a median follow up of 3.1 years (range 0.4–5.9), no adverse events related to gene transfer have been observed. Long-term engraftment of transduced HSC was demonstrated by stable multilineage marking, persisting more than 5 years from gene therapy. The average proportion of transduced cells in the peripheral blood at one year post-gene therapy (n=6) was 5% for granulocytes, 95% for T cells, 56% for B cells and 62% for NK cells. Comparison of the insertion sites retrieved ex vivo from patients with those identified in pre-transplant transduced CD34+ cells showed no skewing in the profile of genome distributions or in the gene families hit by the vector, and no clonal expansion. In the six children with a follow-up >1 year after gene therapy, we observed a progressive increase in lymphocyte counts which was sustained over time (median at 1.5 years 1.6x109/L), polyclonal thymopoiesis and normalization of T-cell functions in vitro. Serum Ig levels improved and evidence of antigen-specific antibodies was obtained, leading to IVIG discontinuation in five patients. All the children are currently healthy and thriving, and none of them showed severe infections. Sustained ADA activity in lymphocytes and RBC resulted in a dramatic reduction of RBC purine toxic metabolites (dAXP<30 nmoles/ml in 5 patients) and amelioration of children’s growth and development. In summary, these data confirm that gene therapy is safe and efficacious in correcting both the immune and metabolic defect in ADA-SCID, with proven clinical benefit.

The Hematopoietic Stem Cell Compartment Is Different in Polycythemia Vera and Idiopathic Myelofibrosis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 259-259 ◽  
Author(s):  
Chloe James ◽  
Frederic Mazurier ◽  
Ronan Chaligne ◽  
Sabrina Dupont ◽  
Francois Delhommeau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
B Lymphocytes ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
V617f Mutation ◽  
Myeloid Progenitors

Abstract X-linked clonality studies showed that myeloproliferative disorders derive from an abnormal hematopoietic stem cell (HSC). This has been recently confirmed by studies showing that the JAK2 V617F mutation was present in multipotent cells from patients with Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primitive Myelofibrosis (PMF). How one unique mutation could give rise to three different diseases remains unexplained and we hypothesized that the HSC compartment may be different in PV and PMF. To investigate whether the V617F mutation occurs in HSCs in PV and PMF, and whether the HSC compartments are different in these 2 diseases, we performed simultaneously two types of experiments: Myeloid, B, and NK in vitro differentiation assays and Repopulation of immune-deficient NOD/SCID mice with human JAK2 V617F CD34+ cells followed by analysis of the frequency of JAK2 V617F clones. We first confirmed that the JAK2 V617F mutation is present in HSCs, enabling long-term (15 weeks) in vivo hematopoietic reconstitution, both in PV and PMF patients. Nevertheless, we found marked differences between PV and PMF samples. Indeed, the frequency of JAK2 V617F lympho-myeloid progenitors was much higher in MF (6 PMF and one post PV-MF) than in PV patients (n = 9) (87.2% +/− 31.3 vs 21% +/− 21.1 respectively). Similarly, most of the human myeloid progenitors present in mice transplanted with CD34+ cells from MF patients (n = 7) 15 weeks post-transplantation were JAK2 V617F. On the contrary, human myeloid progenitors were predominantly JAK2 WT after transplantation of PV CD34+ cells (9 patients). To determine if the mutation was present in HSC able to differentiate into B-lymphocytes in PV and MF, we sorted and genotyped the fraction of B-lymphocytes (CD45+CD19+) that differentiated in the bone marrow of mice. In 5 mice transplanted with PV CD34+ cells, the fraction of B-cells was always JAK2 WT whereas in 2 out of 3 mice transplanted with MF CD34+ cells, B-lymphocytes were JAK2 V617F. To determine if the mutation was present in HSC capable of very long-term reconstitution in PV and PMF, we looked for the presence of long-term culture-initiating cells (LTC-IC) among human CD34+ cells isolated from the bone marrow of NOD/SCID mice (2 PV, 3 MF) 15 weeks after transplantation. In 2 mice transplanted with PMF CD34+ cells, the majority of LTC-ICs (70/70 and 95/166) were JAK2 V617F. On the contrary, in 3 mice reconstituted with PV CD34+ cells, most of the LTC-ICs were JAK2 WT (23/23, 4/4 and 126/168) although we could find some LTC-ICs that were JAK2 V617F, demonstrating that in PV also, the JAK2 V617F mutation is present in Long Term-HSC. Taken together these results demonstrate that the JAK2 V617F mutation is present in a small subset of HSCs in PV patients, whereas in MF, the vast majority of HSCs is JAK2 V617F. This suggests that these two diseases are two stages of the same pathology and that in MF the JAK2 V617F HSCs have acquired a proliferative advantage on JAK2 WT HSCs and thus have invaded the hematopoietic system.

Improving Hematopoietic Stem Cell Based Gene Therapy By Targeting CD133+ Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4202-4202
Author(s):  
Benjamin Goebel ◽  
Christian Brendel ◽  
Daniela Abriss ◽  
Sabrina Kneissl ◽  
Martijn Brugman ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Gene Transfer ◽  
Cell Population ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Transfer Vectors

Abstract Introduction Generally, CD34+ cells are used for genetic modification in gene therapy trials. CD34+ cells consist of a heterogeneous cell population with mostly limited long-term repopulating capabilities, resulting in low long-term engraftment levels in particular in those diseases in which gene modified cells lack a proliferative advantage over non-modified cells. Therefore, modifications in gene transfer vectors and gene transfer strategies are required to improve long-term clinical benefit in gene therapy patients. One particular attractive approach to solve this problem is the improvement of HSC based gene transfer by specifically targeting cells with long-term engraftment capabilities. Material and Methods We constructed lentiviral gene transfer vectors (LV) specifically targeting CD133+ cells, a cell population with recognized long-term repopulating capabilities. Targeting is achieved by pseudotyping with engineered measles virus (MV) envelope proteins. The MV glycoprotein hemagglutinin, responsible for receptor recognition, is blinded for its native receptors and displays a single-chain antibody specific for CD133 (CD133-LV). These vectors were compared to VSV-pseudotyped lentiviral vectors in in vitro and in vivocompetitive repopulation assays using mobilized peripheral blood CD34+ cells. Results Superior transduction of isolated human hematopoietic stem cell populations (CD34+CD38- or CD34+CD133+ cells) compared to progenitor cell populations (CD34+CD38+ or CD34+CD133-) could be shown using the newly developed CD133-LV. Transduction of total CD34+ cells with CD133-LV vectors resulted in stable gene expression and gene marked cells expanded in vitro, while the number of VSV-G-LV transduced CD34+ cells declined over time. Competitive repopulation experiments in NSG mice showed a significantly improved engraftment of CD133-LV transduced HSCs. At ∼12 weeks post-transplantation gene marked hematopoiesis was dominated by the progeny of CD133-LV transduced cells in 42 out of 52 transplanted animals in the bone marrow and 39 out of 45 transplanted animals in the spleen, respectively. Consistent with this data we could show that stem cell content in the CD133-LV transduced population is about five times higher compared to the VSV-transduced population using a limiting dilution competitive repopulation assay (LDA-CRU). Experiments showing proof of principle for the application of this technology for the correction of Chronic Granulomatous Disease (XCGD) using patient derived CD34+ cells are currently ongoing. Discussion In conclusions this new strategy may be promising to achieve improved long-term engraftment in patients treated by gene therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Etoposide-mediated interleukin-8 secretion from bone marrow stromal cells induces hematopoietic stem cell mobilization

2020 ◽  
Author(s):  
Ka-Won Kang ◽  
Seung-Jin Lee ◽  
Ji Hye Kim ◽  
Byung-Hyun Lee ◽  
Seok Jin Kim ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Cd34 Cells

Abstract Background This study assessed the mechanism of hematopoietic stem cell (HSC) mobilization using etoposide with granulocyte-colony stimulating factor (G-CSF) and determined how it differed from that using cyclophosphamide with G-CSF or G-CSF alone.Methods The study analyzed data from 173 non-Hodgkin’s lymphoma patients who underwent autologous peripheral blood stem cell transplantation (auto-PBSCT), in vitro experiments using HSCs and bone marrow stromal cells (BMSCs), and in vivo mouse model studies.Results The etoposide with G-CSF mobilization group showed the highest yield of CD34+ cells and the lowest change in white blood cell counts during mobilization. Etoposide triggered interleukin (IL)-8 secretion from BMSCs and caused long-term BMSC toxicity, which were not observed with cyclophosphamide treatment. The expansion of CD34+ cells cultured in BMSC-conditioned medium containing IL-8 was more remarkable than that without IL-8. The expression of CXCR2, mTOR, and cMYC in HSCs was gradually enhanced at 1, 6, and 24 h after IL-8 stimulation. In animal studies, the etoposide with G-CSF mobilization group presented stronger expression of IL-8-related cytokines and MMP9 and scantier expression of SDF-1 in the bone marrow, compared to the other groups not treated with etoposide.Conclusion Collectively, the unique mechanism of etoposide with G-CSF-mediated mobilization is associated with the secretion of IL-8 from BMSCs, causing the enhanced proliferation and mobilization of HSCs in the bone marrow, which was not observed in the mobilization using cyclophosphamide with G-CSF or G-CSF alone. Moreover, the long-term toxicity of etoposide to BMSC emphasizes the need for further studies to develop more efficient and safe chemo-mobilization strategies.

scholarly journals Identification and Characterization of a Distinct, Evolutionarily Conserved HSC Phenotype Associated with and Predicting Multi-Lineage Engraftment

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1153-1153
Author(s):  
Stefan Radtke ◽  
Morgan A. Giese ◽  
Yan-Yi Chan ◽  
Zachary K. Norgaard ◽  
Jennifer E Adair ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Body Weight ◽  
Stem Cell ◽  
Blood Cell ◽  
Hierarchical Organization ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
Group I

Abstract Current autologous stem cell transplantation and gene therapy strategies are limited by the inability to identify a true hematopoietic stem cell (HSC) propulation that reliably predicts engraftment. The ability to enrich and target a specific HSC pool and predict engraftment levels of the gene-modified cells would be a major advance, reducing manufacturing costs and off-target effects. Here we describe a distinct HSC phenotype, conserved between humans and nonhuman primates (NHP) which overcomes this limitation. We used our NHP stem cell transplantation and gene therapy model to study the engraftment potential of phenotypically distinct hematopoietic stem and progenitor cell (HSPC) subpopulations. We were able to identify an exclusive HSPC subpopulation capable of multi-lineage engraftment (Figure 1A and 1B). This HSPC subpopulation (denoted "I") accounts for ~3-5% of the entire CD34+ cell population in primed bone marrow, reducing the number of cells targeted for cell and gene therapy approaches by 20 to 30-fold. For autologous transplants, only 300-400K HSPCs/kg body weight were required to achieve rapid neutrophil and plateletet recovery within 9-10 and 19-20 days, respectively. Stable 30-35% gene-marking was obeserved in all blood lineages including T cell, B cell, NK cells, granulocyte, monocytes/macrophages, erythrocytes and platelets. Complete reconstitution of the bone marrow compartment was achieved within <100 days, with 31-33% gene-marking in HSCs as well as downstream progenitors from all lineages. Most importantly, and in support of the above described engraftment studies, the use of this HSPC population I phenotype allowed us to establish a reliable flow cytometry based analysis to assess and predict engraftment after HSC transplantion. This method predicted the onset of neutrophil and platelet recovery (Figure 1C and 1D). This assay was also able to predict the onset of NHP hematopoietic recovery for transplanted HSCs from different stem cell sources including steady state (unprimed) bone marrow, as well as gene-modified/transduced and ex vivo expanded HSCs. From this data, we were able to define the minimum cell number of HSCs/kg body weight required for sustained multi-lineage long-term engraftment with recovery of all blood cell lineages in the NHP transplant setting as 110,000 cells/kg. Importantly, we demonstrated that this cell population and phenotype is conserved between NHP and human hematopoiesis (Radtke et al. 2016, submitted). In summary, we identified a distinct and evolutionarily conserved HSC phenotype that is associated with multi-lineage engraftment in nonhuman primates and also reliably predicted the engraftment kinetics after transplant. This will greatly facilitate evaluation and "potency" of autolgous stem cell products especially after stem cell expansion or gene therapy. In addition, it will allow for an improved enrichment of the target cells for gene therapy and gene editing protocols and thus likely improve efficacy and safety. Figure 1 HSCs are exclusively driving multi-lineage engraftment and allow prediction of neutrophil and platelet engraftment in the NHP transplant model. (A) Hierarchical organization of HSPCs in the NHP. HSPC of group I, II, and III were sort-purified and transduced with LV vectors expressing either green fluorescent protein (GFP; population I), mCherry (population II) or mCerulean (population III), respectively. (B) Long-term follow-up of gene modified white blood cell (WBC) levels in vivo after myeloablative transplantation of these cell populations. (C and D) Correlation of transplanted HSCs/kg body weight with (C) neutrophil and (D) platelet recovery. Animals demonstrating engraftment failure (red squares) were excluded from this correlation. Figure 1. HSCs are exclusively driving multi-lineage engraftment and allow prediction of neutrophil and platelet engraftment in the NHP transplant model. / (A) Hierarchical organization of HSPCs in the NHP. HSPC of group I, II, and III were sort-purified and transduced with LV vectors expressing either green fluorescent protein (GFP; population I), mCherry (population II) or mCerulean (population III), respectively. (B) Long-term follow-up of gene modified white blood cell (WBC) levels in vivo after myeloablative transplantation of these cell populations. (C and D) Correlation of transplanted HSCs/kg body weight with (C) neutrophil and (D) platelet recovery. Animals demonstrating engraftment failure (red squares) were excluded from this correlation. Disclosures Adair: Rocket Pharmaceuticals: Consultancy, Equity Ownership. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Research Funding.

scholarly journals Stromal support enhances cell-free retroviral vector transduction of human bone marrow long-term culture-initiating cells

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1393-1399 ◽  
Author(s):  
KA Moore ◽  
AB Deisseroth ◽  
CL Reading ◽  
DE Williams ◽  
JW Belmont
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Long Term Culture ◽  
Stromal Support

Gene transfer into hematopoietic stem cells by cell-free virions is a goal for gene therapy of hematolymphoid disorders. Because the hematopoietic microenvironment provided by the stroma is required for stem cell maintenance both in vivo and in vitro, we reasoned that cell- free transduction of bone marrow cells (BMC) may be aided by stromal support. We used two high-titer replication-defective retroviral vectors to differentially mark progenitor cells. The transducing vector was shown to be a specific DNA fragment by polymerase chain reaction of colony-forming cells derived from progenitors maintained in long-term culture (LTC). BMC were infected separately by cell-free virions with or without pre-established, irradiated, allogeneic stromal layers, and in the presence or absence of exogenous growth factors (GF). The GF assessed were interleukin-3 (IL-3) and IL-6 in combination, leukemia inhibitory factor (LIF), mast cell growth factor (MGF), and LIF and MGF in combination. In addition, we developed a competitive LTC system to directly assess the effect of infection conditions on the transduction of clonogenic progenitors as reflected by the presence of a predominate provirus after maintenance in the same microenvironment. The results show gene transfer into human LTC-initiating cells by cell-free retroviral vector and a beneficial effect of stromal support allowing a transduction efficiency of 64.6% in contrast to 15.8% without a supporting stromal layer. A high transduction rate was achieved independent of stimulation with exogenous GF. We propose that autologous marrow stromal support during the transduction period may have application in clinical gene therapy protocols.

Similarities and differences in aging and in malignant transformation of human hematopoietic stem cells

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 6555-6555
Author(s):  
H. Lannert ◽  
T. Able ◽  
T. Franz ◽  
R. Hofmann ◽  
A. Lenze ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Leukemia Cells ◽  
Cell Aging ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Genomics And Proteomics ◽  
Cell Fractions

6555 Background: The tracking of stem cell aging, differentiation and deterioration by gene expression profiling and proteome analysis allows the comparison of different stages. The overall aim of a proteomic study is characterization of the complex network of cell regulation. We focused our investigations on different subsets of highly enriched CD34+ stem cells from different human origins: fetal liver, cord blood, bone marrow (BM), and mobilized stem cells from peripheral blood (PBSC), as well as CD34+ leukemia cells, thus e.g. to identify pathways and new targets for leukemia therapy. Methods: Mononuclear cells were isolated by a standard Ficoll separation method from the different blood sources. An Auto-MACS (Miltenyi) and FACS Vantage SE cell sorter (Becton Dickinson) was used to highly enrich (>99%) CD34+ cells fractions. Sample preparation: Total RNA was isolated from sorted 1 × 10e6 cells by standard methods using RNA isolation kit (Qiagen). For gene expression analysis topic-defined PIQOR stem cell microarrays (936 genes) were performed. Proteomics started with the determination of protein concentrations, 2D-gel-electrophoresis were described in Proteome Works System (BioRad). Sypro ruby and/or coomasie stained gels were used for protein identification by Q-TOF analyses. The subcellular localization of the identified proteins were performed by fluorescence and confocal microscopy of all cell fractions. Results: 1. The microarray gene expression correlation shows many similarities between human healthy stem cells of different sources and ages, otherwise many differences: 125 upregulated genes (kinases: PAK1, ATM1, CDKN2A) and 32 downregulated genes (EBCTF, RAMP1) in malignant cells compared to healthy stem cells. 2. Proteomics analyses of the different cell fractions show a large overlap of the most dominant protein spots, >200 spots were identified by Q-TOF. For example stathmin (oncoprotein Op18) is expressed at very high levels in leukemia cells and in PBSCs but not in BM cells, additionally demonstrated by fluorescence microscopy. Conclusions: Combining genomics and proteomics assays, pathways e.g. Op18 for proliferation and migration of healthy (mobilized) CD34+ cells from bone marrow and malignant leukemia cells could be identified. No significant financial relationships to disclose.

scholarly journals Hematopoietic Stem Cell–Based Gene Therapy for Acquired Immunodeficiency Syndrome: Efficient Transduction and Expression of RevM10 in Myeloid Cells In Vivo and In Vitro

Blood ◽  
1997 ◽  
Vol 89 (7) ◽  
pp. 2283-2290 ◽  
Author(s):  
Lishan Su ◽  
Robert Lee ◽  
Mark Bonyhadi ◽  
Hajime Matsuzaki ◽  
Sean Forestell ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Gene Transduction ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Acquired Immunodeficiency ◽  
Immunodeficiency Syndrome ◽  
Hiv 1

Abstract Gene delivery via the hematopoietic stem cell (HSC) offers an attractive means to introduce antiviral genes into both T cells and macrophages for acquired immunodeficiency syndrome (AIDS) gene therapy. An amphotropic retroviral vector encoding a bicistronic gene coexpressing RevM10 and the murine CD8α′ chain (lyt2) was developed to transduce HSC/progenitor cells. After transduction of CD34+ cells isolated from human umbilical cord blood, the lyt2 molecule detected by flow cytometry was used to monitor the level of gene transduction and expression and to enrich RevM10-expressing cells by cell sorting without drug selection. Using this quantitative method, high levels of gene transduction and expression (around 20%) were achieved by high-speed centrifugation of CD34+ cells with the retroviral supernatant (spinoculation). After reconstitution of human bone marrow implanted in SCID mice (SCID-hu bone) with the transduced HSC/progenitor cells, a significant number of donor-derived CD14+ bone marrow cells were found to express the RevM10/lyt2 gene. Finally, replication of a macrophage-tropic human immunodeficiency virus–type 1 (HIV-1) isolate was greatly inhibited in the lyt2+/CD14+ cells differentiated from transduced CD34+ cells after the enrichment of lyt2+ population. Thus, the RevM10 gene did not appear to inhibit the differentiation of HSC/progneitor cells into monocytes/macrophages. The level of retrovirus-mediated RevM10 expression in monocytes/macrophages derived from transduced HSCs is sufficient to suppress HIV-1 replication.

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