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)

scholarly journals Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis

Haematologica ◽  
2020 ◽  
Vol 106 (1) ◽  
pp. 74-86 ◽  
Author(s):  
Valentina Capo ◽  
Sara Penna ◽  
Ivan Merelli ◽  
Matteo Barcella ◽  
Serena Scala ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Progenitor Cells ◽  
Cell Transplantation ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cell Source

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis.

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.

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

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 259-259 ◽  
Author(s):  
Francesca Ferrua ◽  
Maria Pia Cicalese ◽  
Stefania Galimberti ◽  
Samantha Scaramuzza ◽  
Stefania Giannelli ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Gene Therapy ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Clinical Benefit ◽  
Research Funding ◽  
Lentiviral Vector ◽  
Hematopoietic Stem ◽  
Cd34 Cells

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

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

2021 ◽  
Vol 10 (2) ◽  
pp. 293
Author(s):  
Gee-Hye Kim ◽  
Jihye Kwak ◽  
Sung Hee Kim ◽  
Hee Jung Kim ◽  
Hye Kyung Hong ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Clinical Applications ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Hsc Transplantation

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

Ex Vivo Expansion and Long-Term Hematopoietic Reconstitution Ability of Sorted CD34+CD59+ Cells from Patients with Paroxysmal Nocturnal Hemoglobinuria.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2324-2324
Author(s):  
Juan Xiao ◽  
Bing Han ◽  
Wanling Sun ◽  
Yuping Zhong ◽  
Yongji Wu
Keyword(s):  
Bone Marrow ◽  
Ex Vivo ◽  
Peripheral Blood Cell ◽  
Intravascular Hemolysis ◽  
Blood Cell Count ◽  
Normal Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder characterized by intravascular hemolysis, venous thrombosis, and bone marrow (BM) failure. Until now, allogeneic hematopoietic stem cell transplantation is still the only way to cure PNH. Eculizumab, although very promising, is not the eradication of the disease because of raising the possibility of severe intravascular hemolysis if therapy is interrupted. Here we enriched the residual bone marrow normal progenitor cells (marked by CD34+CD59+) from PNH patients, tried to find an effective way of expanding the progenitors cells used for autologous bone marrow transplantation (ABMT). Objective To expand CD34+CD59+ cells isolated from patients with PNH and observe the long-term hemaotopoietic reconstruction ability of the expanded cells both ex vivo and in vivo. Methods CD34+CD59+ cells from 13 patients with PNH and CD34+ cells from 11 normal controls were separated from the bone marrow monouclear cells first by immunomagnetic microbead and then by flow cytometry autoclone sorting. The selected cells were then cultivated under different conditions for two weeks to find out the optimal expansion factors. The long-term hematopoietic supporting ability of expanded CD34+CD59+ cells was evaluated by long-term culture in semi-solid medium in vitro and long-term engraftment in irradiated severe combined immunodeficiency(SCID) mice in vivo. Results The best combination of hematopoietic growth factors for ex vivo expansion was SCF+IL-3+IL-6+FL+Tpo+Epo, and the most suitable time for harvest was on day 7. Although the CD34+CD59+ PNH cells had impaired ex vivo increase compared with normal CD34+ cells (the biggest expansion was 23.49±3.52 fold in CD34+CD59+ PNH cells and 38.82±4.32 fold in CD34+ normal cells, P<0.01 ), they remained strong colony-forming capacity even after expansion ( no difference was noticed in CFCs or LTC-IC of PNH CD34+CD59+ cells before and after expansion, P>0.05). According to the above data, 11/13(84.3%) patients with PNH can get enough CD34+CD59+cells for ABMT after expansion. The survival rate and human CD45 expression in different organs was similar between the irradiated SCID mice transplanted with expanded CD34+CD59+ PNH cells and those with normal CD34+ cells (P>0.05). The peripheral blood cell count recovered on day 90 in mice transplanted with PNH cells, which was compatible with those transplanted with normal cells (P>0.05). On secondary transplantation, the peripheral blood cell count returned to almost normal on day 30 in mice transplanted with either PNH cells or normal cells. Lower CD45 percentage was found in secondary transplantation compared with primary transplantation but no difference between mice transplanted with different cells. Conclusion Isolated CD34+CD59+ cells from patients with PNH can be effectively expanded ex vivo and can support lasting hematopoiesis both ex vivo and in vivo. These data provide a new potential way of managing PNH with ABMT.

Functional Screen Identifies Novel Regulators of Murine Hematopoietic Stem Cell Engraftment

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4321-4321
Author(s):  
Miguel Ganuza Fernandez ◽  
Per Holmfeldt ◽  
Himangi Marathe ◽  
Trent Hall ◽  
Jennifer Pardieck ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Ex Vivo ◽  
Significant Loss ◽  
Hematopoietic Stem ◽  
Individual Gene ◽  
Post Transplant ◽  
Functional Screen

Abstract Introduction: Hematopoiesis involves the hierarchical generation of the major blood lineages from a common ancestor, the Hematopoietic Stem Cell (HSC). HSC also have the intrinsic ability to repopulate an ablated hematopoietic compartment when introduced into the periphery of a recipient. This has allowed Hematopoietic Stem Cell transplantation (HSCT) to be used as a cell therapy over the last 45 years, benefiting thousands of patients. Unfortunately many patients succumb to disease while waiting for an adequate donor. Others have to undergo unrelated donor transplants, putting themselves at a risk of developing graft-versus-host disease. Improving HSC engraftment could ameliorate transplant morbidity. Thus, understanding mechanisms regulating HSC engraftment is key. Results: We used our recently published gene expression profiles of developing HSC and other public databases to prioritize 58 genes as putative regulators of adult HSC function. We confirmed by qRT-PCR that 51/58 candidates were enriched for gene expression in Lineage-Sca-1+c-Kit+ (LSK) bone marrow cells relative to downstream progeny, suggesting a role in hematopoietic stem and progenitor cells (HSPC). To functionally assay a role for each gene of interest (GOI) in HSC engraftment, we designed and validated ≥2 independent shRNAs/GOI that effected a >75% knockdown in gene expression in LSK cells. LSK cells were lentivirally transduced with control or individual gene-specific shRNAs and transplanted into lethally irradiated recipients along with mock-transduced LSK competitor cells congenic at the CD45 allele. In contrast to previous functional screens, transplant was performed within 24-hours of LSK cell isolation, avoiding extensive ex vivo culture. This minimal manipulation allowed us to detect genes critical for efficient HSC engraftment. Peripheral blood chimerism was analyzed for at least 16 weeks post-transplant. The major bone marrow hematopoietic compartments were also analyzed. 17 of 48 genes tested were identified as necessary for optimal HSPC engraftment (i.e. knockdown induced a significant loss of repopulation) and the knockdown of three genes enhanced HSPC repopulation. shRNAs targeting each “Hit” were interrogated ex vivo for non-specific effects on LSK cell viability and expansion. A 2° screen was performed to validate the results of this primary screen. Here, CD45.2 LSK cells transduced with control or individual gene-specific shRNAs were sorted 48 hours post-transduction for mCherry+ cells and then transplanted into lethally irradiated mice with mock-transduced and mock-sorted CD45.1 congenic LSK cells. 18 “Hits” were confirmed to perturb HSC repopulating potential in this 2° screen, including three whose loss enhanced HSPC repopulation. The transcription factor, Foxa3, is one hit identified here as necessary for HSC repopulation. We further found that that Foxa3-/- bone marrow displays a significant loss of repopulating potential >16 weeks post-transplant, confirming the results of our screen. As Foxa3-/- long-term HSC also display reduced colony forming potential in vitro and fail to contribute to downstream progenitor compartments in transplant recipients, we propose that Foxa3 is a novel regulator of HSC differentiation post-transplant. Foxa3 has never before been implicated in hematopoiesis or HSPC biology. Conclusions: Our novel functional screen has revealed 15 genes required for optimal HSPC engraftment and three genes whose knockdown improved HSPC engraftment. We further validated Foxa3 as a novel regulator of HSC activity by demonstrating that Foxa3-/- HSC are also deficient in repopulating activity. We are currently investigating the molecular mechanism of Foxa3’s role in HSC and, given that Foxa genes are known transcriptional pioneering factors, pursuing the hypothesis that Foxa3 functions as a novel epigenetic regulator of HSC activation and differentiation. Each gene identified in our screen represents a window into the discovery of novel mechanisms regulating HSC biology and engraftment. Disclosures No relevant conflicts of interest to declare.

Oncostatin M Enhances Hematopoietic Stem Cell Homing and Engraftment

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 31-31
Author(s):  
Hannah K Rasmussen ◽  
Frankie Wong ◽  
Tiffany Tate ◽  
David T. Scadden ◽  
Jonathan Hoggatt
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Vehicle Control ◽  
Oncostatin M ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Group 2 ◽  
Hsc Transplantation ◽  
Novel Therapeutic

Abstract Hematopoietic stem cell (HSC) transplant is widely used to treat hematologic, metabolic, and malignant diseases. HSCs reside within a microenvironmental stem cell niche; the cellular makeup of which is complex, with contributions from endothelial, mesenchymal, and mature hematopoietic cells. Despite the longstanding use of HSC transplantation, there are remarkably few, if any, therapeutics that are available today that can be administered to the recipient of a transplant to enhance hematopoietic engraftment. We believe that revealing the molecular toolkit that naturally exists within the niche provides a rich source of potential novel therapies. Recently, three independent groups (Winkler et al., 2010; Christopher et al., 2011; Chow et al., 2011) have implicated macrophages as a key regulator in G-CSF-mediated mobilization of HSCs. These groups demonstrated that macrophages express the G-CSF receptor, and that depletion of macrophages leads to niche attenuation, reduced levels of stromal derived factor-1 (SDF-1), and mobilization of HSCs. This data implied that macrophages produce a positive supporting factor(s) in the niche to support HSC retention, though none of these prior reports were able to identify the key molecule(s). We now report that Oncostatin M (OSM) is a macrophage-produced protein that regulates HSC retention within the niche. Initial identification was achieved utilizing an in vitro co-culture screening system with macrophage-conditioned media and response of MS-5 stromal cells as measured by SDF-1 production. As previously demonstrated, macrophages enhanced SDF-1 production; however, when OSM production was reduced via shRNA knockdown, or the OSM receptor was knocked down in MS-5 cells or blocked with antibody, the enhanced SDF-1 production was abrogated. Intriguingly, this effect was specific to OSM, as changes in IL-6 or LIF signaling, both members of a similar gp130 signaling family along with OSM, did not have any effects. When mice were treated G-CSF, significant reductions in bone marrow SDF-1 levels and mobilization resulted. However, when mice were co-treated with OSM, levels of bone marrow SDF-1 remained the same as untreated mice (n=10 mice per group, 2 independent expts, P<0.01). Similarly, when macrophages were depleted in vivo via clodronate-loaded liposome treatment, significant HSC mobilization occurred, which was blocked with co-treatment of OSM (n=10 mice per group, 2 independent expts, P<0.01). These results now elucidate a previously unknown key regulatory mechanism governing G-CSF and macrophage mediated mobilization of HSCs. We hypothesized that this new biologic insight could be leveraged as a novel therapeutic strategy to enhance HSC homing and engraftment. To test this, we conducted a series of experiments in which we conditioned mice with myeloablative irradiation and treated with either vehicle control or recombinant OSM (0.5ug per injection) every 6 hours for 48 hours at which point mice received a transplant of bone marrow cells and were subsequently analyzed post-transplant. Mice pre-treated with OSM prior to transplantation exhibited a 2-fold increase in HSC homing compared to vehicle control groups (n=10 mice per group, 3 independent expts, P<0.05). Furthermore, these homed progenitors demonstrated remarkably enhanced hematopoietic expansion in OSM treated mice, as demonstrated by increased numbers of colony forming units (CFUs) from bone marrow assessed days 4, 7, 11, and 14 post-transplant (n=10 mice per group, P<0.01). Excitingly, using a limited cell number transplant, mimicking settings of single cord blood unit transplantation in adults or other settings of limited HSC number, we demonstrate enhanced hematopoietic engraftment and survival in OSM pre-treated mice, with 100 percent survival compared to 50 percent survival of mice treated with vehicle control (n=20 per group, P<0.001). Collectively, our results demonstrate how illumination of endogenous regulatory mechanisms within the hematopoietic niche can reveal molecular agents and pathways with potential to serve as new therapeutic agents in the clinic. Specifically, we identify OSM as the key regulatory molecule governing the G-CSF-macrophage mediated mobilization of HSCs, and describe for the first time a novel therapeutic approach using recombinant OSM as a therapeutic to meet a currently unmet clinical need for HSC transplantation. Disclosures Hoggatt: Harvard University: Patents & Royalties.

scholarly journals An Ex Vivo Bioengineered Human Liver Microenvironment to Support Hematopoietic Stem Cell Maintenance and Expansion

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-10
Author(s):  
Na Yoon Paik ◽  
Grace E. Brown ◽  
Lijian Shao ◽  
Kilian Sottoriva ◽  
James Hyun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Human Umbilical Cord ◽  
Hematopoietic Stem ◽  
Main Site

Over 17,000 people require bone marrow transplants annually, based on the US department of Health and Human Services (https://bloodcell.transplant.hrsa.gov). Despite its high therapeutic value in treatment of cancer and autoimmune disorders, transplant of hematopoietic stem cells (HSC) is limited by the lack of sufficient source material due primarily inadequate expansion of functional HSCs ex vivo. Hence, establishing a system to readily expand human umbilical cord blood or bone marrow HSCs in vitro would greatly support clinical efforts, and provide a readily available source of functional stem cells for transplantation. While the bone marrow is the main site of adult hematopoiesis, the fetal liver is the primary organ of hematopoiesis during embryonic development. The fetal liver is the main site of HSC expansion during hematopoietic development, furthermore the adult liver can also become a temporary extra-medullary site of hematopoiesis when the bone marrow is damaged. We have created a bioengineered micropatterned coculture (MPCC) system that consists of primary human hepatocytes (PHHs) islands surrounded and supported by 3T3-J2 mouse embryonic fibroblasts. Long-term establishment of stable PHH-MPCC allows us to culture and expand HSC in serum-free medium supplemented with pro-hematopoietic cytokines such as stem cell factor (SCF) and thrombopoietin (TPO). HSCs cultured on this PHH-MPCC microenvironment for two weeks expanded over 200-fold and formed tight clusters around the periphery of the PHH islands. These expanded cells also retained the expression of progenitor markers of Lin-, Sca1+, cKit+, as well as the long-term HSC phenotypic markers of CD48- and CD150+. In addition to the phenotypic analysis, the expanded cells were transplanted into lethally irradiated recipient mice to determine HSC functionality. The expanded cells from the PHH-MPCC microenvironment were able to provide multi-lineage reconstitution potential in primary and secondary transplants. With our bioengineered MPCC system, we further plan to scale up functional expansion of human HSC ex vivo and to better understand the mechanistic, cell-based niche factors that lead to maintenance and expansion HSC. Disclosures No relevant conflicts of interest to declare.

scholarly journals Reactivation of γ-globin in adult β-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing

Blood ◽  
2018 ◽  
Vol 131 (26) ◽  
pp. 2915-2928 ◽  
Author(s):  
Chang Li ◽  
Nikoletta Psatha ◽  
Pavel Sova ◽  
Sucheol Gil ◽  
Hongjie Wang ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Binding Site ◽  
Genome Editing ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Cell Genome ◽  
Hsc Transplantation

Key Points CRISPR/Cas9-mediated disruption of a BCL11A binding site in HSCs of β-YAC mice results in the reactivation of γ-globin in erythrocytes. Our approach for in vivo HSC genome editing that does not require HSC transplantation and myeloablation should simplify HSC gene therapy.

A Non-Human Primate Model for Lentivirus-Mediated Anti-HIV RNAi Strategies.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3103-3103
Author(s):  
Karen Beagles ◽  
Brian Beard ◽  
John Rossi ◽  
Jiing-Kuan Yee ◽  
Shiu-lok Hu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Cell Line ◽  
Lentiviral Vector ◽  
Large Animal ◽  
Hematopoietic Stem ◽  
Hiv Tat ◽  
Cd34 Cells ◽  
Anti Hiv ◽  
Human Primate

Abstract AIDS remains a significant health problem worldwide despite the advent of highly active antiretroviral therapy (HAART). Although substantial efforts have been made to develop a vaccine there is still no cure and alternative strategies are needed to treat HIV infection and to control its spread. Our goal is to evaluate lentiviral vectors that inhibit HIV replication by RNA interference (RNAi) in a non-human primate SHIV model to develop a hematopoietic stem cell (HSC) gene therapy for AIDS. SHIV89.6 P is a chimeric virus comprised of an SIV genome that contains the tat, rev and env genes of HIV and infects both T lymphocytes and macrophages. Infection of non-human primates with SHIV89.6P results in significant decreases in CD4+ T cells as early as 4 weeks post infection, and is currently the best large animal model available to test gene therapy strategies for AIDS. We present here data showing efficient transduction of M. nemestrina CD34+ cells with an HIV-based lentiviral vector and RNAi-mediated inhibition of SHIV89.6 P replication in a hybrid T/B lymphocyte cell line (CEMx174). Although others reported a block to transduction of M. mulatta CD34+ cells with an HIV-based lentiviral vector, we observed efficient transduction rates (» 50%) of M. nemestrina CD34+ cells, comparable to transduction rates observed in human CD34+ cells (» 60%). To determine effectiveness of anti tat/rev shRNA to inhibit SHIV89.6P in vitro, a human T cell/B cell hybrid cell line (CEMx174) was transduced with a lentiviral vector expressing a short-hairpin RNA (shRNA) targeted to both HIV tat and rev sequences that also contained either a GFP reporter gene or a MGMT(G156A) resistance gene at MOIs of 1.3 and 3 respectively. Polyclonal populations of CEMx174 cells transduced with the GFP and MGMT(G156A) vectors were challenged with a 2.15x103 TCID50 dose of SHIV 89.6P. One week post challenge, expression of both tat and rev transcripts was reduced 88% and 97% respectively in these cultures as measured by real-time PCR. In summary, we have shown efficient HIV-based lentiviral transduction of M. nemestrina cells and efficient inhibition of SHIV infection by shRNA against HIV tat and rev thus providing a useful model to test lentiviral-mediated anti-HIV RNAi stem cell gene therapy in vivo.

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