scholarly journals First-in-Human Mitochondrial Augmentation of Hematopoietic Stem Cells in Pearson Syndrome

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1024-1024 ◽  
Author(s):  
Elad Jacoby ◽  
Moriya Blumkin ◽  
Yair Anikster ◽  
Nira Varda-Bloom ◽  
Julia Pansheen ◽  
...  
Keyword(s):  
Quality Of Life ◽  
Cellular Therapy ◽  
Ex Vivo ◽  
O2 Consumption ◽  
Atp Content ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Background: Pearson Syndrome (PS) is an ultra-rare disease caused by de-novo mitochondrial DNA (mtDNA) deletions. Patients present at infancy with sideroblastic anemia and later develop a multisystem metabolic disorder, leading to death in early or late childhood. No disease-modifying treatments are available for PS. Ex-vivo enrichment of functional mitochondria into various cells has been previously demonstrated, as has inter-cellular mitochondrial transfer. In preclinical models of mitochondrial and lysosomal disorders, hematopoietic stem and progenitor cells (HSCs) have been shown capable of carrying and transferring normal organelles into diseased tissues, thereby altering disease phenotype. Here, we show enrichment of PS-derived HSCs with wild-type mitochondria, a process termed mitochondrial augmentation. We further report on three patients with PS treated with autologous HSCs following ex-vivo mitochondrial augmentation. Methods: Diagnosis of PS was confirmed by MLPA and deletion-specific dPCR. Colony formation assays were performed on PS patient-derived HSCs, prior to and after mitochondrial augmentation. HSC mobilization was performed with GCSF alone (n=1) or with plerixafor (n=2) prior to leukapheresis. Autologous CD34+ cells were positively-selected using a CliniMACS system, followed by ex-vivo mitochondrial augmentation of the cells with maternal cryopreserved mitochondria carrying normal mtDNA as confirmed by MLPA. Enriched cells were intravenously infused without conditioning. Level of heteroplasmy (relative normal to deleted mtDNA) was determined by deletion-specific dPCR of DNA from peripheral blood. Patients were followed for a period of up to 1 year including clinical and metabolic evaluations. Adverse events were reported as per CTCAE v4.03. Cellular mitochondrial function was studied on peripheral blood mononuclear cells (PBMCs) by ATP content, O2 consumption and flow cytometry for TMRE (tetramethylrhodamine ethyl ester) and MTG (mitotracker green). Results: Three patients were treated with production and safety data available, and in two patients efficacy data is available. PS-patient derived HSCs have a diminished capacity to form colonies in vitro (median, 360 colonies per 5x104 cells vs. 1090 in healthy donors). HSC colony forming capacity increased by an average of 30% after mitochondrial augmentation. Target cell dose (4x106 CD34+ cells/kg) was not reached despite two leukapheresis procedures in patients 1 and 2, who received 1.1 and 1.8 million CD34+ cells/kg recipient, respectively. Patient 3 received 2.8 million cells/kg following a single apheresis. Mitochondrial enrichment in the products was 156%, 162% and 114% for patients 1, 2 and 3. To date, the only treatment-related adverse events noted were leukapheresis related, including anemia, hypocalcemia and alkalosis. In two patients with more than 3 months follow-up, we observed in vivo mitochondrial enrichment starting 3-4 months after cellular therapy, and throughout the follow-up period (Figure). Metabolic function of PBMCs showed improvement at 5 months post-treatment in lymphocyte ATP content, O2 consumption and TMRE:MTG ratio, indicating improved mitochondrial respiratory capacity. Improvement in mitochondrial heteroplasmy and function was in line with clinical findings. Following cell therapy, no events of metabolic crisis occurred, along with normalization of a pre-treatment negative base excess in patient 1 and ongoing improvement in baseline lactate levels in patient 2. Aerobic ability and fine motor functions were superior compared to baseline in both patients. Importantly, quality of life, as measured by the International Pediatric Mitochondrial Disease Score (IPMDS), was greatly improved after treatment. Conclusion: We report a first in human study with a novel form of cellular therapy, mitochondrial augmentation, in which we enrich HSCs with organelles encoding non-mutated version of the mtDNA sequence. We show the ability of mitochondrial augmentation to improve in vitro PS-derived HSC function, and improvement in metabolic determinants, aerobic capacity and quality of life of two patients treated. Together, these preliminary clinical data suggest that mitochondrial augmentation therapy is safe, and may alter the clinical course for patients with mitochondrial deletions/mutations including PS. Figure Figure. Disclosures Jacoby: Novartis Israel: Consultancy. Blumkin:Minovia Therapeutics: Employment. Sher:Minovia Therapeutics: Employment. Yivgi Ohana:Minovia Therapeutics: Employment. Toren:Novartis Israel: Consultancy.

Long-Term Follow-up of Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Severe Alpastic Anemia After Cyclophosphamide Plus Antithymocyte Globulin Conditioning Regimen,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4090-4090
Author(s):  
Johanna Konopacki ◽  
Raphael Porcher ◽  
Marie Robin ◽  
Sabine Bieri ◽  
Jean Michel Cayuela ◽  
...  
Keyword(s):  
Quality Of Life ◽  
Chronic Gvhd ◽  
Conditioning Regimen ◽  
Hematopoietic Stem ◽  
Gvhd Prophylaxis ◽  
Increased Risk ◽  
Long Term Follow Up

Abstract Abstract 4090 Background: Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) from an HLA- identical sibling is the treatment of choice for young patients with acquired severe aplastic anemia (SAA). Due to increased rates of secondary solid cancer in patients with SAA who received an irradiation-based conditioning regimen, we decided 2 decades ago to use the association of Cyclophosphamide (CY) and Antithymocyte globulin (ATG). We report here the long-term follow-up of patients who underwent HSCT from an HLA-identical related donor after this conditioning regimen. Patients and Methods: 61 consecutive patients with SAA who received a first transplantation from June 1991 to February 2010 in our center were included. Patients with Fanconi anemia or other congenital bone marrow failure were excluded. The conditioning regimen consisted in CY (200mg/Kg) and ATG (2.5 mg/kg/day × 5 days). The donors were HLA-identical siblings in 60 cases and family HLA-matched in 1 case. Graft-versus -host disease (GvHD) prophylaxis associated cyclosporine and methotrexate (days 1, 3, 6 and 11). Long-term clinical outcome, immune recovery and quality of life were assessed. Results: The median age was 21 years [range: 4–43], 41 being adults. Median duration of the disease before HSCT was 93 days. Most of the patients had idiopathic aplastic anemia (n=49, 80%). Median time from diagnosis to HSCT was 3 months (range, 1 to 140). All but 2 patients received bone marrow as source of stem cells and all but 2 engrafted (primary graft failure= 3.4%) with a neutrophils count > 0.5 G/L and a platelets count >20 G/L after a median of 23 (range, 19 to 43) and 21 days (range, 10 to 177), respectively. In patients who had achieved neutrophil recovery, no secondary graft failure was observed. Cumulative incidence (CI) of acute grade II-IV GvHD was 23% (95%CI, 13 to 34) and 18 patients developed chronic GvHD (CI: 32%, 95% CI, 20 to 46). In multivariate analysis, a higher number of infused CD3 cells was associated with an increased risk of developing chronic GvHD (p=0.017). With a median follow-up of 73 months (8 to 233), the estimated 6-year overall survival was 87% (95%CI, 78 to 97). At 72 months, the CI of secondary malignancies was 9%, 10 patients developed avascular necrosis (21% CI), 12 patients were diagnosed with endocrine dysfunctions (19% CI) and 5 presented cardiovascular complications (CI of 10%). The CI of bacterial, fungal and viral infections were 25% (95% CI, 15 to 36), 8% (95% CI, 3 to 17) and 61% (95% CI, 46 to 73) at 72 months, respectively. At 2 years post HSCT, the immune reconstitution was normal for CD3, CD8 T-cells, B-cell and NK-cell but still incomplete for CD4 T-cells. A FACT-BMT questionnaire of quality of life (QOL) was sent to all survivors (n= 53) of who 26 accepted to respond to the questionnaire. There was no evidence for a change in QOL perception with time after transplantation. Our data were compared with those obtained from HSCT recipients from a Swiss transplant center (n=125 patients), mainly transplanted for hematological malignancies. The perception of QOL in patients who were transplanted for SAA was similar to the group of patients who were transplanted for other reason than SAA. Conclusions: Our results confirm that HSCT from HLA-identical sibling donors after CY-ATG conditioning regimen is a curative treatment for patients with SAA, with an excellent long-term outcome. We found an increased risk of chronic GvHD associated with the number of infused CD3 cells. Furthermore, we also found non negligible late complications as well as a similar quality of life with patients transplanted for hematological malignancies. Improving long-term health conditions must thus be a priority field for research, exploring the use of new conditioning regimen as well as new GvHD prophylaxis to improve the quality of life post HSCT of such patients. Disclosures: Peffault de Latour: Alexion: Consultancy, Research Funding.

Ex Vivo Generation of CD34+ Cells From CD34− Hematopoietic Cells

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4053-4059 ◽  
Author(s):  
Yoshihiko Nakamura ◽  
Kiyoshi Ando ◽  
Jamel Chargui ◽  
Hiroshi Kawada ◽  
Tadayuki Sato ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cell Population ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Calf Serum ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Cd34 Cells

Abstract The human Lin−CD34− cell population contains a newly defined class of hematopoietic stem cells that reconstitute hematopoiesis in xenogeneic transplantation systems. We therefore developed a culture condition in which these cells were maintained and then acquired CD34 expression and the ability to produce colony-forming cells (CFC) and SCID-repopulating cells (SRCs). A murine bone marrow stromal cell line, HESS-5, supports the survival and proliferation of Lin−CD34− cells in the presence of fetal calf serum and human cytokines thrombopoietin, Flk-2/Flt-3 ligand, stem cell factor, granulocyte colony-stimulating factor, interleukin-3, and interleukin-6. Although Lin−CD34− cells do not initially form any hematopoietic colonies in methylcellulose, they do acquire the colony-forming ability during 7 days of culture, which coincides with their conversion to a CD34+ phenotype. From 2.2% to 12.1% of the cells became positive for CD34 after culture. The long-term multilineage repopulating ability of these cultured cells was also confirmed by transplantation into irradiated NOD/SCID mice. These results represent the first in vitro demonstration of the precursor of CD34+ cells in the human CD34− cell population. Furthermore, the in vitro system we reported here is expected to open the way to the precise characterization and ex vivo manipulation of Lin−CD34− hematopoietic stem cells.

Genetic and Clinical Spectrum of Osteopetrosis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1087-1087
Author(s):  
Ansgar S Schulz ◽  
Despina Moshous ◽  
Klaus-Michael Debatin ◽  
Anna Villa
Keyword(s):  
Quality Of Life ◽  
Genetic Background ◽  
Clinical Presentation ◽  
Hematopoietic Stem ◽  
Long Term Outcome ◽  
Significant Difference ◽  
Unrelated Donors

Abstract Abstract 1087 Poster Board I-109 Background Osteopetrosis (OP) is a multi-systemic inborn disorder characterized by heterogeneous clinical presentations on the basis of a variety of mutated genes. We performed a retrospective analysis in patients with OP from 28 European centers on behalf of the ESID and the EBMT. Main objectives of this analysis were survival and quality of life with respect to genetic background, initial clinical presentation, and treatment modality. Methods A set of clinical parameters, genetic findings, transplant information and follow up data were collected using specific questionnaires. Questionnaires were sent at several time points to European centers treating patients with OP and the collected data were stored in a central OP database. DNA sequencing of candidate genes involved in human OP was done predominantly at laboratories in Milan, Paris and Ulm. Results Data of 173 patients and of 134 hematopoietic stem cell transplantations (HSCT) were obtained during a time frame between 1983 and 2008. In the whole cohort, mutations in the following genes were detected: 47% biallelic TCIRG1, 9% biallelic CLCN7, 6% monoallelic CLCN7, 5% OSTM1, 2% RANK, 1% RANKL; 30% of patients have no mutations in these genes or could not be tested completely. In 23% of patients HSCT was not considered because of severe neurological problems or other complications mostly in the context of OSTM1 or CLCN7 mutations, or because of less severe phenotypes mainly due to monoallelic CLCN7 mutations (autosomal dominant OP, ADO). Follow up data of 103 transplanted patients were available. With respect to donor type, the probability of survival at 2 years was 88% for matched family donors, 80% for matched unrelated donors, 68% for HLA-haploidentical donors and 54% for mismatched unrelated donors. There was no significant difference in survival of patients with different gene mutations but a trend to a worse long term outcome in CLCN7 patients. Most notably, several major sequelae were present in the majority of surviving patients. Visual impairment or dwarfism were found in about 2/3 of children showing no significant influence of the affected genes. About 10% of patients are suffering from significant persistent neurological problems after HSCT, which were found in patients with TCIRG and CLCN7 mutations. The quality of life were judged as normal by the parents in about 2/3 of surviving patients. Conclusion HSCT in infantile OP results in acceptable survival rates even after HLA-nonidentical transplants. However, outcome is influenced by a set of specific disease related problems and long term sequelae. The heterogeneity of OP necessitates an individualised therapeutic strategy respecting the genetic background, the clinical presentation and the type of the available donor. Disclosures No relevant conflicts of interest to declare.

Screen for Small Molecules Capable of Expanding Human Hematopoietic Stem Cell Ex Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1919-1919
Author(s):  
Iman Hatem Fares ◽  
Jalila Chagraoui ◽  
Jana Krosl ◽  
Denis-Claude Roy ◽  
Sandra Cohen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 1919 Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC. Disclosures: No relevant conflicts of interest to declare.

Initial Results from the Northstar Study (HGB-204): A Phase 1/2 Study of Gene Therapy for β-Thalassemia Major Via Transplantation of Autologous Hematopoietic Stem Cells Transduced Ex Vivo with a Lentiviral βΑ-T87Q -Globin Vector (LentiGlobin BB305 Drug Product)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 549-549 ◽  
Author(s):  
Alexis A. Thompson ◽  
John E Rasko ◽  
Suradej Hongeng ◽  
Janet L. Kwiatkowski ◽  
Gary Schiller ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Adverse Events ◽  
Research Funding ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Drug Product ◽  
Thalassemia Major ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Background: Hematopoietic stem cell (HSC) gene therapy has the potential to induce globin production and mitigate the need for blood transfusions in β-thalassemia major. Promising early results for 2 subjects with β0/βE -thalassemia major in the ongoing HGB-205 study suggested that transplantation with autologous CD34+ cells transduced with a replication-defective, self-inactivating LentiGlobin BB305 lentiviral vector containing an engineered β-globin gene (βA-T87Q) can be safe and yield robust production of βA-T87Qglobin resulting in rapid transfusion independence. The Northstar study (HGB-204), which uses the same lentivirus vector and analogous study design as study HGB-205, is multi-center and multi-national, and centralizes drug product manufacturing. Herein, we provide the initial data on subjects enrolled and treated in this study. Subjects and Methods: Transfusion-dependent subjects with β-thalassemia major undergo HSC collection via mobilized peripheral blood apheresis and CD34+ cells are selected. Estimation of the mean ex-vivo vector copy number (VCN) is obtained by quantitative PCR performed on pooled colony-forming progenitors. Subjects undergo myeloablation with intravenous busulfan, followed by infusion of transduced CD34+ cells. Subjects are monitored for hematologic engraftment, βA-T87Q -globin expression (by high performance liquid chromatography) and transfusion requirements. Integration site analysis (ISA, by linear amplification-mediated PCR and high-throughput sequencing on nucleated cells) and replication-competent lentivirus (RCL) assays are performed for safety monitoring. Results: As of 31 July 2014, 3 subjects have undergone HSC collection and ex-vivo LentiGlobin BB305 gene transfer. One subject (Subject 1102) has undergone myeloablation and drug product infusion. Outcomes data are shown in Table 1. The initial safety profile is consistent with myeloablation, without serious adverse events or gene therapy-related adverse events. This subject has increasing production of βA-T87Q-globin: the proportion of βA-T87Qglobin was 1.5%, 10.9% and 19.5% of total Hb at 1, 2 and 3 months post-infusion, respectively. This subject received pRBCs on Day +14 following drug product infusion and required no further transfusions until a single unit of pRBC was transfused on Day +96 for a Hb of 8.6 g/dL and fatigue. Two additional subjects have undergone drug product manufacture and are awaiting transplantation. Safety data related to ISA and RCL assays are pending. Abstract 549. Table 1 Preliminary results of dosing parameters and transplantation outcomes Subject Age (years) and Gender Genotype BB305 Drug Product Day of Neutrophil Engraftment Drug Product- related Adverse Events βA-T87Q-Hb at last follow-up visit /Total Hb (g/dL) VCN CD34+ cell dose (x106 per kg) 1102 18 F β0/βE 1.0/1.1a 6.5 Day +17 None 1.77/8.6 1104 21 F β0/βE 0.7/0.7a 5.4 P P P 1106 20 F β0/β0 1.5 12.3 P P P As of 31 July 2014; P, pending a If more than one drug product were manufactured, the VCN of each drug product lot is presented. Conclusion: The first subject treated on the Northstar study has safely undergone drug product infusion with autologous HSCs transduced with LentiGlobin BB305 lentiviral vector and is producing steadily increasing amounts of βA-T87Q-globin. Additional follow-up of this subject plus data on additional subjects who undergo drug product infusion will be presented at the meeting. Ex-vivo gene transfer of βA-T87Q-globin to autologous HSCs is a promising approach for the treatment of patients with β-thalassemia major. Disclosures Thompson: ApoPharma: Consultancy; Novartis: Consultancy, Research Funding; Amgen: Research Funding; Glaxo Smith Kline: Research Funding; Mast: Research Funding; Eli Lilly: Research Funding. Kwiatkowski:Shire Pharmaceuticals and Sideris Pharmaceuticals: Consultancy. Schiller:Sunesis, Amgen, Pfizer, Bristol Myers Squibb: Research Funding. Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Petrusich:bluebird bio, Inc.: Employment, Equity Ownership. Soni:bluebird bio, Inc.: Employment. Walters:Via Cord and AllCells, Inc.: Medical Director Other.

Hypoxia Potentiates Notch-Induced Expansion of Human Hematopoietic Stem/Progenitor Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2640-2640
Author(s):  
Jianfei Fu ◽  
Heather D. Huntsman ◽  
Ayla Cash ◽  
Patali S. Cheruku ◽  
Richard H. Smith ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Research Funding ◽  
Ex Vivo ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Cell Counts ◽  
Cd34 Cells ◽  
Number Of Cells

Abstract Activation of Notch signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with Notch ligand Delta1 has enabled a clinically relevant ex vivo expansion of short-term HSPCs. In vitro studies have also revealed a role of low O2 tension in HSPC regulation. A molecular link has been demonstrated in several stem/progenitor cell populations between Notch and hypoxia pathways but their interaction has not been investigated in human HSPCs. G-CSF mobilized human CD34+ cells from 4 healthy subjects were cultured in the presence of cytokines (SCF, FLT3L and TPO) in hypoxia (1.5-2% O2) or normoxia (21% O2) in vessels coated with fibronectin alone or combined with increasing concentrations of the immobilized ligand Delta1 (2.5, 5, 10 and 20 µg/mL). After 21 days in culture, cells were counted and characterized using CFU assays, flow cytometry for lineage (Glycophorin A+, CD13+, CD20+, CD3+ and CD41+ cells) and HSC (CD34+ CD38- CD45RA- CD90+ CD49f+ Rholow) phenotypes, and transplantation in immunodeficient (NSG) mice. In normoxia, the total number of cells increased 118-fold compared to baseline in the absence of Delta1 with limited residual CD34+ cells (1.5 ± 0.7%), extensive differentiation toward the myeloid lineage (96.3 ± 0.3% CD13+ cells) and minimal engraftment potential in NSG mice (0.2 ± 0.2% human CD45+ cells). With increasing concentrations of Delta1 in normoxia, consistent with the hypothesis that Delta1 delays differentiation, the total number of cells increased less (41-, 25-, 11- and 7-fold relative to baseline, respectively) CD34+ cells expanded more (4-, 4-, 3- and 2-fold relative to baseline, respectively), and CFU numbers increased more (8-, 7-, 4- and 3-fold relative to baseline, respectively) than without Delta1. However, phenotypically defined HSCs were undetectable or markedly decreased at the lowest Delta1 concentrations used (2.5 and 5 µg/mL) and their numbers were maintained or only minimally increased at the highest Delta-1 concentrations tested (10 and 20 µg/mL) relative to uncultured CD34+ cells. Accordingly, only cells cultured with 10 and 20 µg/mL Delta1 resulted in levels of engraftment in NSG mice (5.5 ± 5.4% and 5.4 ± 0.9% human CD45+ cells, respectively) comparable to uncultured cells (7.0 ± 0.1% human CD45+ cells). In hypoxia, total cell counts increased less than in normoxia both without (8-fold relative to baseline) and with increasing concentrations of Delta1 (11-, 11-, 9-, 9-fold relative to baseline, respectively) due to diminished myeloid differentiation. Total CD34+ cells decreased 1.7-fold in hypoxia in the absence of Delta1, but expanded modestly in the presence of Delta1 (3-, 3-, 2- and 2-fold, respectively). CFU numbers followed a similar trend. However, in hypoxic cultures with 2.5, 5 and 10 µg/mL Delta1, phenotypically defined HSCs increased 2.5-, 6.6- and 1.3-fold, respectively, compared to uncultured cells. Importantly, hypoxia combined with 2.5, 5 and 10 µg/mL Delta1 concentrations resulted in increased human cell engraftment in NSG mice (21.2 ± 4.4%, 29.3 ± 11% and 11.8 ± 5.4% human CD45+ cells, respectively) compared to uncultured cells (7.0 ± 0.1% human CD45+ cells). When 20 µg/mL Delta1 was used in hypoxia, engraftment potential in NSG mice was decreased (1.1 ± 0.6% human CD45+ cells). We next performed limiting dilution analysis to measure the frequencies of long-term repopulating HSCs (LT-HSCs) within the CD34+ cell compartment at baseline and after 21 days in hypoxic or normoxic cultures supplemented with the optimized concentrations of Delta1 (10 µg/mL in normoxia and 5 µg/mL in hypoxia). LT-HSCs in uncultured CD34+ cells were measured at the expected frequency (1 in 7,706; 95% CI of 3,446 to 17,232). When analyzed at 3 months post-transplantation, a limited (1.5-fold) increase in LT-HSC frequency (1 in 5,090; 95% CI 2.456 to 10,550) was obtained from Delta1 normoxic cultures compared to uncultured cells. In contrast, the frequency of LT-HSCs (1 in 1,586; 95% CI 680 to 3,701) was 4.9-fold higher in hypoxic Delta1 cultures compared to uncultured cells, and 4.2-fold higher than in normoxic Delta1 cultures. Similarly, absolute numbers of LT-HSCs per 100,000 Day 0 equivalent CD34+ cells increased from 13 (baseline) to 216 (normoxia) and 694 (hypoxia). Our data indicate that hypoxia potentiates Notch-induced expansion of human HSPCs and may be of benefit in stem cell transplantation and gene therapy applications. Disclosures Cheruku: Novartis: Research Funding. Larochelle:Novartis: Research Funding.

scholarly journals Mesenchymal Stem Cell-Derived Microvesicles Support Ex Vivo Expansion of Cord Blood-Derived CD34+Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Hui Xie ◽  
Li Sun ◽  
Liming Zhang ◽  
Teng Liu ◽  
Li Chen ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Promising Therapeutic Approach ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Mesenchymal stem cells (MSCs) are known to support the characteristic properties of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow hematopoietic microenvironment. MSCs are used in coculture systems as a feeder layer for the ex vivo expansion of umbilical cord blood (CB) to increase the relatively low number of HSPCs in CB. Findings increasingly suggest that MSC-derived microvesicles (MSC-MVs) play an important role in the biological functions of their parent cells. We speculate that MSC-MVs may recapitulate the hematopoiesis-supporting effects of their parent cells. In the current study, we found MSC-MVs containing microRNAs that are involved in the regulation of hematopoiesis. We also demonstrated that MSC-MVs could improve the expansion of CB-derived mononuclear cells and CD34+cells and generate a greater number of primitive progenitor cells in vitro. Additionally, when MSC-MVs were added to the CB-MSC coculture system, they could improve the hematopoiesis-supporting effects of MSCs. These findings highlight the role of MSC-MVs in the ex vivo expansion of CB, which may offer a promising therapeutic approach in CB transplantation.

scholarly journals Ex Vivo Expansion with E478 Increases the Rate of CRISPR-Mediated Homology Directed Repair of the Beta-Globin Gene in Human Hematopoietic Stem Cells By >10-Fold and Improves In Vivo Engraftment By >120-Fold

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4634-4634
Author(s):  
Kevin A. Goncalves ◽  
Megan D. Hoban ◽  
Sharon L. Hyzy ◽  
Katia S. George ◽  
Anthony E. Boitano ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Globin Gene ◽  
Fold Increase ◽  
Ex Vivo Expansion ◽  
Gene Correction ◽  
Employment Equity ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Equity Ownership

Background . Site-specific gene correction of hematopoietic stem cells (HSCs) via homology directed repair (HDR) has the potential to precisely repair defective genes and provide life-long cures for a variety of blood-based diseases. It is possible to obtain high levels of HDR during in vitro HSC culture, but these cells fail to robustly engraft in vivo, suggesting that the procedure of HDR compromises HSC function or that true HSCs are not undergoing HDR. Cells need to be actively cycling in order to undergo HDR, but conditions that allow HSC replication in vitro without compromising HSC number and function remain elusive. Thus, most HDR protocols minimize time in culture, potentially limiting HDR rates and cell yield. We recently reported that ex vivo expansion of HSCs with an aryl hydrocarbon receptor (AHR) antagonist is a clinically validated method to expand HSCs. The AHR antagonist-expanded CD34+ cell therapy, MGTA-456, results in rapid and durable recovery in patients with hematologic malignancies and inherited metabolic diseases (Wagner et al Cell Stem Cell 2016; Orchard et al AAN 2019). To apply this technology to gene-modified HSCs, we developed a novel AHR antagonist, E478, which expands NSG-engrafting cells 10-fold compared to uncultured primary human mobilized peripheral blood (mPB) CD34+cells in limit dilution studies. We previously showed that expansion with E478 results in up to 10-fold higher engraftment of lentiviral vector (LVV)-transduced cells and CRISPR/Cas9 knockout cells (Hoban et al ASGCT 2019). Here, we demonstrate that ex vivo expansion of mPB CD34+ cells with E478 results in >10-fold increase in rate of HDR and >120-fold increase in NSG engraftment of HDR+ cells compared to conventional approaches. Results . To determine whether more active cycling would lead to higher rates of HDR, we cultured cells for 1, 2, 3, and 4 days prior to electroporation with CRISPR gRNA targeting the beta-globin gene and transduction with a GFP-containing adeno-associated virus (AAV) donor template. Cell cycle analysis revealed that 33±1.8% of cells enriched for HSCs (CD34+CD90+ cells) remain quiescent after 2 days in culture, whereas 0.92±0.06% of CD34+CD90+ cells were quiescent after 3 and 4 days in culture (n=2 mPB donors). We then assessed HDR rates and HSC number after 1, 2, 3, and 4 days of additional culture. Compared to a conventional HDR protocol utilizing a 2-day pre-stimulation period followed by 1 day of culture after electroporation (herein called a 2+1 culture), we observed up to 8-fold increase in HDR with longer pre-stimulation periods, but this was accompanied with differentiation of CD34+CD90+ cells and loss of engraftment in NSG mice (79% decrease, p<0.001). We next evaluated whether E478 could increase the dose of HSCs and maintain high HDR rates. We cultured mPB CD34+ cells with E478 for a 4 day pre-stimulation, performed HDR, and continued the expansion for 4 days with E478 (herein called 4+4 culture). With the 4+4 protocol, we observed a 6-fold increase in the rate of HDR in vitro and a 134-fold increase in the number of CD34+CD90+ cells with E478 relative to 2+1 conditions with DMSO vehicle (n=2, p<0.01). Transplant of these cells into sublethally-irradiated NSG mice resulted in a 4-fold higher rate of engraftment (Figure A, p<0.01, n=8 mice), 12-fold higher rates of HDR (Figure B, p<0.001) and >120-fold increase in the number of HDR+ NSG-engrafting cells relative to 2+1 cultures (Figure C, p<0.001). Further, a 2+1 culture with E478 led to an 8-fold increase in number of HDR+ NSG-engrafting cells (p<0.001) relative to standard 2+1 approaches without a small molecule. Multi-lineage engraftment was observed in all groups. Studies using E478 with bone marrow from patients with sickle cell disease are in progress and will be presented. Conclusions. We demonstrate that ex vivo HSC expansion with E478 enables higher rates of HDR and a high dose of HDR+ HSCs, leading to >120-fold increase in the engraftment of HDR+ HSCs compared to conventional 2+1 approaches. Culture with E478 is a promising approach to realize the full potential of targeted gene correction in HSCs for a variety of genetic diseases. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hoban:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. George:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Targeted Gene Modification In Hematopoietic Stem Cells: A Potential Treatment For Thalassemia and Sickle Cell Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 434-434
Author(s):  
Andreas Reik ◽  
Kai-Hsin Chang ◽  
Sandra Stehling-Sun ◽  
Yuanyue Zhou ◽  
Gary K Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Target Gene ◽  
Ex Vivo ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Beta-thalassemia (β-thal) and sickle cell disease (SCD) are monogenic diseases caused by mutations in the adult β-globin gene. A bone marrow transplant (BMT) is the only curative treatment, but its application is limited since (i) HLA-matched donors can be found for <20% of cases, and (ii) the allogeneic nature of the transplant involves the significant risk of graft vs host disease (GvHD). Elevated levels of fetal γ-globin proteins observed in a subset of individuals carrying β-thal and SCD mutations ameliorate the clinical picture or prevent the development of disease complications. Thus, strategies for the selective and persistent upregulation of γ-globin represent an attractive therapeutic approach. Recent insights into the regulation of γ-globin transcription by a network of transcription factors and regulatory elements both inside and outside the β-globin locus have revealed a set of new molecular targets, the modulation of which is expected to elevate γ-globin levels for potential therapeutic intervention. To this end, we and others have established that designed zinc finger nucleases (ZFNs) transiently introduced into stem cells ex vivo provide a safe and efficient way to permanently ablate the expression of a specific target gene in hematopoietic stem cells (HSC) by introduction of mutations following target site cleavage and error-prone DNA repair. Here we report the development and comparison of different ZFNs that target various regulators of γ-globin gene transcription in human HSCs: Bcl11a, Klf1, and specific positions in the γ-globin promoters that result in hereditary persistence of fetal hemoglobin (HPFH). In all cases these target sites / transcription factors have previously been identified as crucial repressors of γ-globin expression in humans, as well as by in vitro and in vivo experiments using human erythroid cells and mouse models. ZFN pairs with very high genome editing activity in CD34+ HSCs were identified for all targeted sites (>75% of alleles modified). In vitro differentiation of these ZFN-treated CD34+ HSCs into erythroid cells resulted in potent elevation of γ-globin mRNA and protein levels without significant effects on erythroid development. Importantly, a similar and specific elevation of γ-globin levels was observed with RBC progeny of genome-edited CD34+ cells obtained from SCD and β-thal patients. Notably, in the latter case a normalization of the β-like to α-globin ratio to ∼1.0 was observed in RBCs obtained from genome-edited CD34s from two individuals with β-thalassemia major. To deploy this strategy in a clinical setting, we developed protocols that yielded comparably high levels of target gene editing in mobilized adult CD34+ cells at large scale (>108 cells) using a clinical-grade electroporation device to deliver mRNA encoding the ZFN pair. Analysis of modification at the most likely off-target sites based on ZFN binding properties, combined with the maintenance of target genome editing observed throughout erythroid differentiation (and in isolated erythroid colonies) demonstrated that the ZFNs were both highly specific and well-tolerated when deployed at clinical scale. Finally, to assess the stemness of the genome-edited CD34+ HSCs we performed transplantation experiments in immunodeficient mice which revealed long term engraftment of the modified cells (>16 weeks, ∼25% human chimerism in mouse bone marrow) with maintenance of differentiation in vivo. Moreover, ex vivo erythroid differentiation of human precursor cells isolated from the bone marrow of transplanted animals confirmed the expected elevation of γ-globin. Taken together, these data suggest that a therapeutic level of γ-globin elevation can be obtained by the selective disruption, at the genome level, of specific regulators of the fetal to adult globin developmental switch. The ability to perform this modification at scale, with full retention of HSC engraftment and differentiation in vivo, provides a foundation for advancing this approach to a clinical trial for the hemoglobinopathies. Disclosures: Reik: Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Lee:Sangamo BioSciences: Employment. Truong:Sangamo BioSciences: Employment. Wood:Sangamo BioSciences: Employment. Zhang:Sangamo BioSciences: Employment. Luong:Sangamo BioSciences: Employment. Chan:Sangamo BioSciences: Employment. Liu:Sangamo BioSciences: Employment. Miller:Sangamo BioSciences: Employment. Paschon:Sangamo BioSciences: Employment. Guschin:Sangamo BioSciences: Employment. Zhang:Sangamo BioSciences: Employment. Giedlin:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Urnov:Sangamo BioSciences: Employment.

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