scholarly journals Incremental Innovation of Ex Vivo Hematopoietic Stem Cell Engineering to Expand Clinical Gene Therapy Applications

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4707-4707
Author(s):  
Erika Zonari ◽  
Giacomo Desantis ◽  
Carolina Petrillo ◽  
Oriana Meo ◽  
Samantha Scaramuzza ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Genetically Engineered ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Hematopoietic Reconstitution ◽  
Cd34 Cells ◽  
Clinical Gene Therapy ◽  
Ex Vivo Culture

Abstract Transplantation of genetically engineered, autologous hematopoietic stem and progenitor cells (HSPC) is becoming a promising alternative to allogeneic stem cell transplantation for curing genetic diseases, avoiding the risks of graft versus host disease and prolonged immunosuppression. Most clinical gene therapy protocols are based on CD34+ HSPC engineered during >2 days of ex vivo culture. By xenotransplanting mobilized peripheral blood (mPB) CD34+ HSPC, which were lentivirally (LV) marked with different fluorescent proteins according to CD38/CD90 expression levels allowing quantitative assessment of the contribution of CD38/CD90 subpopulations to hematopoietic reconstitution (n=48 NSG mice, 3 experiments), we identified 2 distinct waves of reconstitution: (1) short term repopulation (up to 2 months) mostly driven by CD34+CD38intCD90+/- cells and (2) long-term repopulation driven by CD34+CD38-CD90+ (70%) and CD34+CD38-CD90- cells (30%). Notably, an intermediate wave extending from 2 to 4 months driven by CD34+CD38low cells was selectively eliminated by prolonged ex vivo culture and could be rescued when culture time was reduced to 1 day. We therefore developed a novel LV transduction protocol able to provide curative levels of gene transfer during a single day of ex vivo culture. Stimulating CD34+ cells or CD34+CD38- cells with Prostaglandin E2 (PGE2) increased gene transfer with VSVg-pseudotyped LVs by 1.5-2 fold acting on early steps of transduction, an effect that was further potentiated by the late-acting compound Cyclosporin A. Using large-scale vector preparations for gene therapy of mucopolysaccharidosis type 1, chronic granulomatous disease or beta-thalassemia, we show by in vitro and xenotransplantation assays that a 1-day PGE2 protocol achieved similar transduction efficiencies into BM or MPB HSPC from healthy donors and patients as our 62h benchmark protocol. PGE2 treatment did not result in toxicity or skewed multi-lineage differentiation. However, shortening ex vivo culture increased engraftment levels in the NSG mouse model. To entirely avoid culturing progenitor cells, we explored the feasibility to limit ex vivo manipulation to HSC-enriched CD34+CD38- cells that may be co-transplanted with unmanipulated CD34+ progenitor cells devoid of long-term engraftment potential. This could further improve hematopoietic reconstitution, increase safety by reducing the LV integration load infused into the patient and downscale ex vivo manipulation making the process more efficient and economically sustainable. To this end, we optimized a sequential bead-based, GMP-compatible selection procedure to separate mPB into a CD34+CD38- stem and CD38+ progenitor cell fraction. We reached high purity (87+/-6.6% CD34+) and recovery of CD34+CD38- cells (37.3+/-8.7%), making their isolation clinically viable. Bead-selected CD34+CD38- cells showed higher engraftment potential than equivalent numbers of FACS-sorted cells. Co-infusion of unmanipulated (culture-sensitive) CD38+ supporter cells with genetically-engineered CD34+CD38- cells into NSG mice resulted in rapid engraftment followed by near-complete replacement of untransduced short-term repopulating progenitors by gene-marked HSPC deriving from CD34+CD38- cells after the 3rd month post-transplant. Finally, we explored ex vivo expansion of mPB CD34+CD38- cells with arylhydrocarbon receptor antagonists and/or pyrimido-indole-derivatives. These cells expanded 3-10 fold in a 7-14 d time-window, far less than seen for total CD34+ cells, thereby facilitating culture handling and reducing cost. Unlike CD34+ cells, expanded mPB CD34+CD38- cells largely maintained their SCID-repopulating potential providing proof-of-concept for the expansion of gene-modified HSC. This clinically applicable platform will improve the efficacy, safety and sustainability of ex vivo gene addition and open up new opportunities in the field of gene editing. Disclosures Ciceri: MolMed SpA: Consultancy.

scholarly journals Notch-Mediated Expansion of Human Hematopoietic Stem and Progenitor Cells By Culture Under Hypoxia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 28-29
Author(s):  
Daisuke Araki ◽  
Stefan Cordes ◽  
Fayaz Seifuddin ◽  
Luigi J. Alvarado ◽  
Mehdi Pirooznia ◽  
...  
Keyword(s):  
Er Stress ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Human Adult ◽  
Hypoxic Conditions ◽  
Cd34 Cells ◽  
Human Hscs ◽  
Ex Vivo Culture ◽  
Cells Cultured

Notch activation in human CD34+ hematopoietic stem/progenitor cells (HSPCs) by treatment with Delta1 ligand has enabled clinically relevant ex vivo expansion of short-term HSPCs. However, sustained engraftment of the expanded cells was not observed after transplantation, suggesting ineffective expansion of hematopoietic stem cells with long-term repopulating activity (LTR-HSCs). Recent studies have highlighted how increased proliferative demand in culture can trigger endoplasmic reticulum (ER) stress and impair HSC function. Here, we investigated whether ex vivo culture of HSPCs under hypoxia might limit cellular ER stress and thus offer a simple approach to preserve functional HSCs under high proliferative conditions, such as those promoted in culture with Delta1. Human adult mobilized CD34+ cells were cultured for 21 days under normoxia (21% O2) or hypoxia (2% O2) in vessels coated with optimized concentrations of Delta1. We observed enhanced progenitor cell activity within the CD34+ cell population treated with Delta1 in hypoxia, but the benefits provided by low-oxygen cultures were most notable in the primitive HSC compartment. At optimal coating densities of Delta1, the frequency of LTR-HSCs measured by limiting dilution analysis 16 weeks after transplantation into NSG mice was 4.9- and 4.2-fold higher in hypoxic cultures (1 in 1,586 CD34+ cells) compared with uncultured cells (1 in 7,706) and the normoxia group (1 in 5,090), respectively. Conversely, we observed no difference in expression of the homing CXCR4 receptor between cells cultured under normoxic and hypoxic conditions, indicating that hypoxia increased the absolute numbers of LTR-HSCs but not their homing potential after transplantation. To corroborate these findings molecularly, we performed transcriptomic analyses and found significant upregulation of a distinct HSC gene expression signature in cells cultured with Delta1 in hypoxia (Fig. A). Collectively, these data show that hypoxia supports a superior ex vivo expansion of human HSCs with LTR activity compared with normoxia at optimized densities of Delta1. To clarify how hypoxia improved Notch-mediated expansion of LTR-HSCs, we performed scRNA-seq of CD34+ cells treated with Delta1 under normoxic or hypoxic conditions. We identified 6 distinct clusters (clusters 0 to 5) in dimension-reduction (UMAP) analysis, with a comparable distribution of cells per cluster between normoxic and hypoxic cultures. Most clusters could be computationally assigned to a defined hematopoietic subpopulation, including progenitor cells (clusters 0 to 4) and a single transcriptionally defined HSC population (cluster 5). To assess the relative impact of normoxia and hypoxia on the HSC compartment, we performed gene set enrichment analysis (GSEA) of cells within HSC cluster 5 from each culture condition. A total of 32 genes were differentially expressed, and pathways indicative of cellular ER stress (unfolded protein response [UPR], heat shock protein [HSP] and chaperone) were significantly downregulated in hypoxia-treated cells relative to normoxic cultures (Fig. B). When examining expression of cluster 5 top differentially expressed genes across all cell clusters, we observed a more prominent upregulation of these genes within transcriptionally defined HSCs exposed to normoxia relative to more mature progenitors (Fig. C, red plots). Hypoxia lessened the cellular stress response in both progenitors and HSCs, but the mitigation was more apparent in the HSC population (Fig. C, grey plots), and decreased apoptosis was observed only within the HSC-enriched cluster 5 (Fig. D). These findings are consistent with several reports indicating that HSCs are more vulnerable to strong ER stress than downstream progenitors due to their lower protein folding capacity. In conclusion, we provide evidence that ex vivo culture of human adult CD34+ cells under hypoxic conditions enables a superior Delta1-mediated expansion of hematopoietic cells with LTR activity compared with normoxic cultures. Our data suggest a two-pronged mechanism by which optimal ectopic activation of Notch signaling in human HSCs promotes their self-renewal, and culture under hypoxia mitigates ER stress triggered by the increased proliferative demand, resulting in enhanced survival of expanding HSCs. This clinically feasible approach may be useful to improve outcomes of cellular therapeutics. Disclosures No relevant conflicts of interest to declare.

Ex Vivo Expansion of Retrovirally-Transduced Primate CD34+ Cells Results in Preferential Engraftment and Persistence of Clones with MDS1/EVI1 Insertion Sites.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 203-203
Author(s):  
Theo Gomes ◽  
Stephanie Sellers ◽  
Robert E. Donahue ◽  
Rima Adler ◽  
Andre La Rochelle ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Insertional Mutagenesis ◽  
Ex Vivo ◽  
Safety Issue ◽  
Retroviral Vectors ◽  
Ex Vivo Expansion ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Clinical Gene Therapy

Abstract There is increasing evidence that insertional activation of proto-oncogenes by retroviral vectors is a significant safety issue that must be addressed before clinical gene therapy, particularly targeting hematopoietic stem and progenitor cells, can be further developed. The risk of insertional mutagenesis for replication-incompetent retroviral vectors has been assumed to be low until the occurence of T cell leukemias in children treated with HSC-directed gene therapy for X-SCID, and recent evidence that retroviral integration is more common in the promoter region of transcriptionally-active genes. The occurence of “common integration sites” in a particular gene also suggests a non-random insertion pattern, and/or immortalization or other change in the behavior of a clone harboring an insertion in these particular genes. We have previously reported a highly non-random occurence of 14 unique vector integrations in the first two introns of the MDS1/EVI1 proto-oncogene out of a total of 702 identified from myeloid cells of 9 rhesus macaques at least 6 months post-transplantion of retrovirally-transduced CD34+ cells.(Calmels et al, 2005). This same gene locus was found frequently activated by insertions in murine bone marrow cells immortalized in long-term in vitro culture after transduction with retroviral vectors.(Du et al Blood, 2005) To begin to investigate the factors contributing to this worrisome finding, particularly given the very recent report of a marked over-representation of MDS1/EVI1 insertions in a human clinical gene therapy trial for chronic granulomatous disease, we asked whether continued ex vivo expansion of transduced CD34+ cells prior to transplantation would further select for clones with insertions in MDS1/EVI1 or other proto-oncogenes. Rhesus CD34+ cells were transduced with the G1Na standard retroviral vector, identical to that used in the prior studies, using our standard 96 hour transduction protocol in the presence of Retronectin and SCF, FLT3L and thrombopoietin. At the end of transduction, all cells were continued in culture for an additional 7 days under the same culture conditions, and then reinfused into the donor animal following 1200 rads TBI. At 1 month post-transplant there were no CIS and no MDS1/EVI1 insertions identified. However, at 6 months post-transplantation 5 out of 27 (19%) of the unique insertions identified in granulocytes were within the first two introns of MDS1/EVI1, very significantly higher than the 2% of MDS1/EVI1 insertions (14 of 702) identified in animals that were transplanted with cells not subjected to additional ex vivo expansion.(p<.0001) One MDS1/EVI1 clone constituted 14% of overall sequences identified, and the 5 clones constituted 37% of total sequences identified. This strongly suggests that the over-representation of this locus in engrafting cells is due to a potent immortalizing signal provided by activation of the MDS1/EVI1 gene products by the stonger retroviral promoter/enhancer, and that the need for extended ex vivo culture of target cells may select for insertion events activating this locus. It also suggests that strategies involving prolonged ex vivo expansion or selection of transduced cells could increase the risk of gene therapy utilizing integrating vectors targeting primitive hematopoietic cells.

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 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 Bone marrow homing and engraftment of human hematopoietic stem and progenitor cells is mediated by a polarized membrane domain

Blood ◽  
2012 ◽  
Vol 119 (8) ◽  
pp. 1848-1855 ◽  
Author(s):  
Andre Larochelle ◽  
Jennifer M. Gillette ◽  
Ronan Desmond ◽  
Brian Ichwan ◽  
Amy Cantilena ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Clinical Importance ◽  
Mechanistic Explanation ◽  
Membrane Domain ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Ex Vivo Culture

AbstractManipulation of hematopoietic stem/progenitor cells (HSPCs) ex vivo is of clinical importance for stem cell expansion and gene therapy applications. However, most cultured HSPCs are actively cycling, and show a homing and engraftment defect compared with the predominantly quiescent noncultured HSPCs. We previously showed that HSPCs make contact with osteoblasts in vitro via a polarized membrane domain enriched in adhesion molecules such as tetraspanins. Here we show that increased cell cycling during ex vivo culture of HSPCs resulted in disruption of this membrane domain, as evidenced by disruption of polarity of the tetraspanin CD82. Chemical disruption or antibody-mediated blocking of CD82 on noncultured HSPCs resulted in decreased stromal cell adhesion, homing, and engraftment in nonobese diabetic/severe combined immunodeficiency IL-2γnull (NSG) mice compared with HSPCs with an intact domain. Most leukemic blasts were actively cycling and correspondingly displayed a loss of domain polarity and decreased homing in NSG mice compared with normal HSPCs. We conclude that quiescent cells, unlike actively cycling cells, display a polarized membrane domain enriched in tetraspanins that mediates homing and engraftment, providing a mechanistic explanation for the homing/engraftment defect of cycling cells and a potential new therapeutic target to enhance engraftment.

scholarly journals Efficient Nanoparticle-Mediated Delivery of Gene Editing Reagents into Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2933-2933
Author(s):  
Rkia El Kharrag ◽  
Kurt Berckmueller ◽  
Margaret Cui ◽  
Ravishankar Madhu ◽  
Anai M Perez ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Quality Control ◽  
Progenitor Cells ◽  
Gene Editing ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Autologous hematopoietic stem cell (HSC) gene therapy has the potential to cure millions of patients suffering from hematological diseases and disorders. Recent HSCs gene therapy trials using CRISPR/Cas9 nucleases to treat sickle cell disease (SCD) have shown promising results paving the way for gene editing approaches for other diseases. However, current applications depend on expensive and rare GMP facilities for the manipulation of HSCs ex vivo. Consequently, this promising treatment option remains inaccessible to many patients especially in low- and middle-income settings. HSC-targeted in vivo delivery of gene therapy reagents could overcome this bottleneck and thereby enhance the portability and availability of gene therapy. Various kinds of nanoparticles (lipid, gold, polymer, etc.) are currently used to develop targeted ex vivo as well as in vivo gene therapy approaches. We have previously shown that poly (β-amino ester) (PBAE)-based nanoparticle (NP) formulations can be used to efficiently deliver mRNA into human T cells and umbilical cord blood-derived CD34 + hematopoietic stem and progenitor cells (HSPCs) (Moffet et al. 2017, Nature Communications). Here, we optimized our NP formulation to deliver mRNA into GCSF-mobilized adult human CD34 + HSPCs, a more clinically relevant and frequently used cell source for ex vivo and the primary target for in vivo gene therapy. Furthermore, we specifically focused on the evaluation of NP-mediated delivery of CRISPR/Cas9 gene editing reagents. The efficiency of our NP-mediated delivery of gene editing reagents was comprehensively tested in comparison to electroporation, the current experimental, pre-clinical as well as clinical standard for gene editing. Most important for the clinical translation of this technology, we defined quality control parameters for NPs, identified standards that can predict the editing efficiency, and established protocols to lyophilize and store formulated NPs for enhanced portability and future in vivo applications. Nanoformulations were loaded with Cas9 ribonucleoprotein (RNP) complexes to knock out CD33, an established strategy in our lab to protect HSCs from anti-CD33 targeted acute myeloid leukemia (AML) immunotherapy (Humbert et al. 2019, Leukemia). RNP-loaded NPs were evaluated for size and charge to correlate physiochemical properties with the outcome as well as establish quality control standards. NPs passing the QC were incubated with human GCSF-mobilized CD34 + hematopoietic stem and progenitor cells (HSPCs). In parallel, RNPs were delivered into CD34 + cells using our established EP protocol. NP- and EP-edited CD34 + cells were evaluated phenotypically by flow cytometry and functionally in colony-forming cell (CFC) assays as well as in NSG xenograft model. The optimal characteristics for RNP-loaded NPs were determined at 150-250 nm and 25-35 mV. Physiochemical assessment of RNP-loaded NP formations provided an upfront quality control of RNP components reliably detecting degraded components. Most importantly, NP charge directly correlated with the editing efficiency (Figure A). NPs achieved more than 85% CD33 knockout using 3-fold lower dose of CRISPR nucleases compared to EP. No impact on the erythromyeloid differentiation potential of gene-edited cells in CFC assays was observed. Finally, NP-modified CD34 + cells showed efficient and sustained gene editing in vivo with improved long-term multilineage engraftment potential in the peripheral blood (PB) and bone marrow stem cell compartment of NSG mice in comparison to EP-edited cells (Figure B). Here we show that PBAE-NPs enable efficient CRISPR/Cas9 gene editing of human GCSF-mobilized CD34 + cells without compromising the viability and long-term multilineage engraftment of human HSPCs in vivo. Most importantly, we defined physiochemical properties of PBAE-NPs that enable us to not only determine the integrity of our gene-editing agents but also predict the efficiency of editing in HSPCs. The requirement of 3-fold less reagents compared to EP, the ability to lyophilize quality-controlled and ready to administer gene therapy reagents, and the opportunity to engineer the surface of PBAE-NPs with HSC-targeting molecules (e.g. antibodies) could make this also a highly attractive and portable editing platform for in vivo HSC gene therapy. Figure 1 Figure 1. Disclosures Kiem: VOR Biopharma: Consultancy; Homology Medicines: Consultancy; Ensoma Inc.: Consultancy, Current holder of individual stocks in a privately-held company. Radtke: Ensoma Inc.: Consultancy; 47 Inc.: Consultancy.

scholarly journals Antagonizing PPARγ Expands Human Hematopoietic Stem and Progenitor Cells By Switching on FBP1-Repressed Glycolysis and Preventing Differentiation

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 709-709
Author(s):  
Bin Guo ◽  
Xinxin Huang ◽  
Hal E. Broxmeyer
Keyword(s):  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Metabolic Reprogramming ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Ppar Γ ◽  
Control Shrna ◽  
Cd34 Cells ◽  
Human Hscs

Abstract Allogeneic hematopoietic cell transplantation (HCT) is widely used as a life-saving treatment for malignant and non-malignant blood disorders. Hematopoietic stem cells (HSCs) are a major contributing cell population for a successful HCT. While cord blood (CB) is an acceptable source of HSCs for clinical HCTbecause of its many advantages including prompt availability, lower incidence of GvHD and virus infection, CB HCT is usually associated with slower time to engraftment especially in adult patients when compared with other cell sources; this is partly due to limiting numbers of HSCs in single cord units. In order to overcome this limitation, ex vivo expansion of CB HSCs has been evaluated in preclinical and clinical studies for improvement of the clinical efficacy of CB HCT. While a number of different ways have been evaluated to ex-vivo expand human HSCs, little is known about the mechanisms involved, and whether efficient expansion of CB HSCs could be achieved by metabolic reprogramming. In a compound screen for potential candidates which could promote ex vivo expansion of CB HSCs, we found that PPARγ antagonist GW9662 treatment significantly enhanced ex vivo expansion of CB phenotypic HSCs (~5 fold) and progenitor cells (HPCs) (~6.8 fold) in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and cytokines (SCF, FL, TPO) when compared with vehicle control. GW9662 significantly increased numbers of CB colony-forming unit (CFU) granulocyte/macrophage (GM) (~1.8 fold) and granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) (~3.2 fold) progenitors after 4 days ex vivo culture. To assess whether the ex vivo expanded CB HSCs enhanced by the PPARγ antagonist were functional in vivo, we performed both primary and secondary transplantation in immunocompromised NSG mice. Engraftment of CB CD34+ cells in primary recipients was significantly increased (~3 fold) both in bone marrow (BM) and peripheral blood (PB) by the cultured cells treated with GW9662. The percentages of both myeloid and lymphoid lineages were enhanced in BM of primary recipients transplanted with GW9662-treated CB CD34+ cells. We also transplanted CB CD34+ cells transfected with control shRNA or PPAR γ shRNA into NSG mice, and consistently found that both myeloid and lymphoid chimerism was enhanced in BM of recipients which were infused with PPAR γ shRNA transfected-CD34+ cells compared with control shRNA transfected-CD34+ cells. Long term reconstituting and self-renewing capability of GW9662-treated CB CD34+ cells with both enhanced myeloid and lymphoid chimerism, was confirmed in PB and BM in secondary recipients. Limiting dilution analysis was performed to calculate SCID-repopulating cells (SRC), a measure of the number of functional human HSCs. The SRC frequency of GW9662-cultured CB CD34+ cells was 4 fold greater than that of day 0 uncultured CD34+ cells, and 5 fold increased above that of vehicle-treated CD34+ cells with cytokines alone. To gain mechanistic insight into how PPARγ antagonism enhances expansion of human CB HSCs and HPCs, we performed RNA-seq analysis. Antagonizing PPARγ in CB CD34+ cells resulted in downregulation of a number of differentiation associated genes, including CD38, CD1d, HIC1, FAM20C, DUSP4, DHRS3 and ALDH1A2, which suggests that PPARγ antagonist may maintain stemness of CB CD34+ cells partly by preventing differentiation. Of interest, we found that FBP1, encoding fructose 1, 6-bisphosphatase, a negative regulator of glycolysis, was significantly down-regulated by GW9662, which was further confirmed by RT-PCR, western blot and flow cytometry analysis. GW9662 significantly enhanced glucose metabolism in CB HSCs and HPCs without compromising mitochondrial respiration. Enhanced expansion of CB HSCs by antagonizing PPARγ was totally suppressed by removal of glucose or by inhibition of glycolysis. Importantly, suppression of FBP1 greatly promoted glycolysis and ex vivo expansion of long-term repopulating CB HSCs (~3.2 fold). Overexpression of FBP1 significantly suppressed enhancedexpansion and engraftment of CB HSCs by PPARγ antagonist. Our study demonstrates that PPARγ antagonism drives ex vivo expansion of human CB HSCs and HPCs by switching on FBP1 repressed glucose metabolism and by preventing differentiation. This provides new insight into human HSC self-renewal, and suggests a novel and simple means by which metabolic reprogramming may improve the efficacy of CB HCT. Disclosures No relevant conflicts of interest to declare.

scholarly journals Lentiviral Vector-Mediated Gene Transfer Restores CD18 Expression in Long-Term Repopulating Hematopoietic Stem and Progenitor Cells Isolated from a Patient with Leukocyte Adhesion Deficiency Type 1

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1859-1859
Author(s):  
Richard H. Smith ◽  
Daisuke Araki ◽  
Andre Larochelle
Keyword(s):  
Gene Therapy ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Leukocyte Adhesion ◽  
Myeloid Cells ◽  
Cd11b Expression ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Ex Vivo Gene Therapy ◽  
Deficiency Type

Abstract Leukocyte adhesion deficiency type 1 (LAD-1) is an inherited primary immunodeficiency caused by loss-of-function mutation within the ITGB2 gene, which encodes the beta2 integrin subunit CD18. Individuals with LAD-1 experience significant loss of neutrophil-mediated innate cellular immune function, resulting in delayed wound healing, severe periodontitis, and life-long bouts of bacterial infection. LAD-1 is a prime candidate for lentiviral vector-mediated genetic intervention as i) it is an intractable, potentially life-threatening disease with limited treatment options, ii) it is amenable to current ex vivo gene therapy procedures, and iii) partial phenotypic correction would present a high likelihood of significant clinical benefit. Allogeneic stem cell transplant can be curative, but suffers from matched donor availability and the potential for graft-versus-host disease. Autologous ex vivo gene therapy may provide a viable alternative to allogeneic transplant in LAD-1 patients. We have evaluated the ability of a CD18-expressing lentiviral vector (LV-hCD18) to mediate ex vivo transduction of LAD-1 patient-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and subsequent long-term LAD-1 HSPC engraftment in immunodeficient NOD-scid IL2Rg null (NSG) mice. An open reading frame encoding human CD18 was placed under the transcriptional control of the MND promoter (a modified retroviral promoter associated with high levels of stable transgene expression) and packaged in VSV-G-pseudotyped lentiviral particles. After 1 day of pre-stimulation, LAD-1 HSPCs were transduced with LV-hCD18 (MOI = 10) in the presence or absence of transduction-enhancing adjuvants, poloxamer 407 (P407) and prostaglandin E2 (PGE 2), for 24 hours. Sublethally irradiated NSG mice (7 mice/group) were transplanted with either mock-transduced LAD-1 HSPCs, LAD-1 HSPCs transduced in the absence of adjuvants, or LAD-1 HSPCs transduced in the presence of P407/PGE 2. Bone marrow was harvested at ~5.5 months post-transplant for flow cytometric analyses of engraftment efficiency, transgene marking, and human blood cell lineage reconstitution. Bone marrow from mice that received mock-transduced LAD-1 HSPCs showed an average total of 6.45 ± 2.54% (mean ± SEM) CD45+ human cells. Mice that received LAD-1 HSPCs transduced in the absence of adjuvants showed 7.99 ± 1.82% CD45+ human cells, whereas mice transplanted with LAD-1 HSPCs transduced in the presence of adjuvants showed 7.33 ± 1.90% CD45+ cells. A Kruskal-Wallis statistical test indicated no significant difference in the level of human cell engraftment among the recipient groups (P=0.72). Consistent with the LAD-1 phenotype, human myeloid cells from mice that received mock-transduced LAD-1 HSPCs displayed only background levels of CD18 marking (0.13 ± 0.06% CD45+CD13+CD18+ cells). Mice that received LAD-1 HSPCs transduced in the absence of adjuvants showed 4.05 ± 0.40% CD18+ human myeloid cells (range 2.19% to 5.50%), whereas mice that received LAD-1 HSPCs transduced in the presence of P407/PGE 2 showed 9.56 ± 0.96% CD18+ human myeloid cells (range 4.63% to 13.10%), thus representing a &gt;2-fold increase in in vivo, vector-mediated transgene marking levels when adjuvant was used. Moreover, vector-mediated expression of CD18 rescued endogenous expression of a major CD18 heterodimerization partner in neutrophils, CD11b. In mock-transduced LAD-1 HSPC recipients, CD13+ human myeloid cells were devoid of cell surface CD11b expression (0.01 ± 0.01% CD45+CD13+CD11b+ cells). In contrast, CD13+ human myeloid cells in mice that received LAD-1 HSPCs transduced in the absence of adjuvant showed detectable levels of CD11b expression (2.62 ± 0.19% of CD18-expressing human myeloid cells), and CD11b levels were increased to 6.90 ± 0.98% in LAD-1 HSPCs transduced in the presence of P407/PGE 2. Multilineage engraftment, as evidenced by the presence of CD3+ T cells and CD20+ B cells, was noted within all groups; however, human myeloid cells represented the most prominent human blood cell compartment observed. Colony-forming-unit assays of transduced cells and non-transduced control cells pre-transplant showed similar clonogenic output and colony diversity. In sum, successful transduction, engraftment, transgene marking, CD11b rescue, and multilineage reconstitution supports further development of lentiviral vector-mediated gene therapy for LAD-1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Engraftment and Retroviral Marking of CD34+ and CD34+CD38− Human Hematopoietic Progenitors Assessed in Immune-Deficient Mice

Blood ◽  
1998 ◽  
Vol 91 (4) ◽  
pp. 1243-1255 ◽  
Author(s):  
Mo A. Dao ◽  
Ami J. Shah ◽  
Gay M. Crooks ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Low Levels ◽  
Ex Vivo Culture ◽  
Daunting Challenge

Abstract Retroviral-mediated transduction of human hematopoietic stem cells to provide a lifelong supply of corrected progeny remains the most daunting challenge to the success of human gene therapy. The paucity of assays to examine transduction of pluripotent human stem cells hampers progress toward this goal. By using the beige/nude/xid (bnx)/hu immune-deficient mouse xenograft system, we compared the transduction and engraftment of human CD34+progenitors with that of a more primitive and quiescent subpopulation, the CD34+CD38− cells. Comparable extents of human engraftment and lineage development were obtained from 5 × 105 CD34+ cells and 2,000 CD34+CD38− cells. Retroviral marking of long-lived progenitors from the CD34+ populations was readily accomplished, but CD34+CD38− cells capable of reconstituting bnx mice were resistant to transduction. Extending the duration of transduction from 3 to 7 days resulted in low levels of transduction of CD34+CD38− cells. Flt3 ligand was required during the 7-day ex vivo culture to maintain the ability of the cells to sustain long-term engraftment and hematopoiesis in the mice.

Adhesion to Fibronectin Maintains Regenerative Capacity During Ex Vivo Culture and Transduction of Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4612-4621 ◽  
Author(s):  
M.A. Dao ◽  
K. Hashino ◽  
I. Kato ◽  
J.A. Nolta
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Retroviral Vectors ◽  
Current Data ◽  
Regenerative Capacity ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Ex Vivo Culture

Abstract Recent reports have indicated that there is poor engraftment from hematopoietic stem cells (HSC) that have traversed cell cycle ex vivo. However, inducing cells to cycle in culture is critical to the fields of ex vivo stem cell expansion and retroviral-mediated gene therapy. Through the use of a xenograft model, the current data shows that human hematopoietic stem and progenitor cells can traverse M phase ex vivo, integrate retroviral vectors, engraft, and sustain long-term hematopoiesis only if they have had the opportunity to engage their integrin receptors to fibronectin during the culture period. If cultured in suspension under the same conditions, transduction is undetectable and the long-term multilineage regenerative capacity of the primitive cells is severely diminished.

Sign in / Sign up

Export Citation Format

Share Document