Bone Marrow (BM) Delivery of Genetically-Modified (gm) Adult CD34+ Hematopoietic Stem and Progenitor Cells (HSPC) Improves Homing and Engraftment of Short-Term Progenitors over Long-Term Repopulating Hematopoietic Stem Cells

Gene therapy/editing of CD34+ HSPC ex vivo, followed by their transplantation, can cure a variety of hematologic diseases. However, a substantial loss of HSPC occurs from collection to transplant. Losses occur during processing for HSPC enrichment, ex vivo genetic manipulation and culture, formulation, and testing prior to transplant. Further, HSPC are lost to peripheral organs during homing when delivered intravenously (IV), reducing the effective gm HSPC dose; a loss compounded by the lack of helper cells that aid in the homing and engraftment process which are removed during enrichment. Direct BM delivery of gm HSPC can overcome some of these limitations. This has been tried previously, with non-enriched whole cord blood (CB) and non-gm HSPC, with conflicting results. We hypothesized that BM delivery of a limited dose of gm adult HSPC would improve long-term repopulation over that of IV delivery by bypassing HSPC loss during homing. Using bioluminescent imaging, we determined that CB HSPC transduced with a luciferase lentiviral vector (LV) delivered by intra-femoral (IF) injection localized to the injected femur, validating our injection method. Next, we delivered mobilized peripheral blood (MPB) HSPC transduced with a GFP LV into irradiated NOD.LtSz-scid IL2rg -/- (NSG) mice via IV or IF injection in limiting dilution. Total human engraftment (hCD45+ cells), transduced human engraftment (hCD45+GFP+ cells), and multi-lineage engraftment were measured in the BM at 3- and 6-months post-transplant. HSPC gave rise to a bi-lineage (B-myeloid) graft at 3 months, suggesting hematopoietic progenitor cell (HPC) engraftment, and a multi-lineage graft (hCD33+, hCD19+, hCD3+, and hCD34+ cells) at 6 months, suggesting engraftment from a long-term repopulating cell or hematopoietic stem cell (HSC). At 3 months, IF delivery of HSPC resulted in significantly higher total and transduced human cell engraftment, measured in the non-injected femur (Table 1). The engraftment was bi-lineage. At 6 months, IF delivery of HSPC no longer significantly increased engraftment over IV delivery (Table 1). However, a multi-lineage graft was present, indicating full hematopoietic repopulation. There was no significant difference in the lineage output between either delivery method at 3 or 6 months. These data suggest that HPC homed and engrafted more efficiently than HSC, when delivered IF. Alternatively, IF delivery altered the BM microenvironment, allowing preferential homing of HPC. However, CD34- cells injected IF, to simulate pressure and passage of cells through the BM with IF delivery, followed by IV delivery of CD34+ cells (sham IF with IV HSPC delivery) resulted in similar homing patterns to CD34+ cells delivered IV (p=0.1, Figure 1A), suggesting that differences between IV and IF delivery were likely due to cell-intrinsic rather than cell-extrinsic differences between HPC and HSC. To study the mechanism of preferential engraftment of HPC over HSC with IF delivery, we analyzed expression of the major homing receptors CXCR4 and VLA-4 on HPC and HSC. CXCR4 (Figure 1B) and VLA-4 were both expressed at significantly higher levels on HPC than on HSC (CXCR4 p<0.01; VLA-4 p<0.05) and their expression increased with increasing culture time and with HSPC cycling. However, VLA-4 expression was significantly increased in GFP+ (MFI 65313 ± 4750) compared to GFP- (MFI 48969 ± 2099; p<0.01) HSPC. CXCR4 expression was similar in both GFP+ (MFI 4261 ± 189) and GFP- (MFI 5245 ± 1186) HSPC, mimicking the in vivo engraftment pattern of GFP+ and GFP- cells, suggesting that CXCR4 may be the molecule responsible for enhancing HPC homing and engraftment with BM delivery. An initial experiment shows that when we remove the high CXCR4 expressing CD34+38+ HPC and deliver HSC-enriched CD34+38- cells IV or IF, IF delivery results in higher long-term engraftment (additional experiments ongoing, Figure 1C, D). These data support the hypothesis that cell-intrinsic differences in the homing behavior of HSC and HPC is likely due to their differential expression of CXCR4. Studies underway on blockade of CXCR4 or VLA-4 on gm HPC and/or gm HSC followed by their IF or IV delivery will be presented. Overall, we show IV delivery of gm HSPC is comparable to BM delivery. However, as HSC-enriched cells become clinically available for genetic therapies, BM delivery of enriched gm HSC may result in superior engraftment. Disclosures Malik: Aruvant Sciences, Forma Therapeutics, Inc.: Consultancy; Aruvant Sciences, CSL Behring: Patents & Royalties.

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High Efficiency Recovery of Hematopoietic Progenitors from Peripheral Blood Harvests after Short- and Long-Term Cryopreservation.

Blood ◽

10.1182/blood.v106.11.5275.5275 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5275-5275

Author(s):

Ulrich Denz ◽

Dagmar Wider ◽

Antonia Mueller ◽

Monika Engelhardt

Keyword(s):

Suspension Culture ◽

Peripheral Blood ◽

High Efficiency ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Viable Cells ◽

Cd34 Cells ◽

Significant Difference ◽

Group A

Abstract Introduction: Transplantation of functional hematopoietic stem cells (HSC) using peripheral blood (PB), bone marrow (BM) or cord blood (CB) cells is widely used to treat malignant and nonmalignant disorders. Because long-term cryopreservation is performed for PB, BM and CB cells, and these are often used years after cell harvests, the implementation of a quality-assurance is a major requirement to ensure graft safety for clinical use. Methods: We assessed the efficiency of recovery of viable HSC from 37 patients (pts; n=20 NHL, n=6 Hodgkin, n=9 MM, n=2 AML) and 6 allogeneic-donors (AD) with stored PBSC samples. All pts had received an auto-PBSCT between 1992–2004. Stored PBSC samples used in this analysis had been cryopreserved for a median of 5.6 years (y; range: 1.3–12). We determined post-thawing recovery, cell viability, ex vivo expansion potential, CD34+ numbers, CFU growth in methylcellulose culture and LTC-ICs. Viable cells were determined by trypan blue and propidium iodide via FACS analysis, CFUs in 0.9% methylcellulose (supplemented with IMDM, 30% FCS and EPO, IL-3+GM-CSF) and LTC-IC as previously described. Pts and AD were analyzed as a total group and within 3 subgroups of: A) ‘long-term’ cryopreservation: n=21 PBSC harvests had a median cryopreservation of 9.5y (8–12), B) ‘short-term’ cryopreservation: n=16 harvests had a 2.9y (1.3–5.6) cryopreservation period, and C) n=6 pts showing delayed engraftment (EG) or early death after auto-PBSCT: the cryopreservation in these 6 pts was 2.7y (2.2–3.5). Cryopreservation results were correlated with clinical results and EG. Results: Hematopoietic EG in group A and B was prompt with WBC>1000/μl and platelets>20,000/μl on d10–11 post PBSC reinfusion. EG in group C was delayed albeit 4.3x106 CD34+ cells/kg bw (2.1–8.6) had been retransfused (WBC>1000/μl + platelets>20,000/μl: d+13 post PBSC infusion, non-platelet-EG >20,000/μl before death: n=5). Primary cause of death in group C was progressive disease in 3 and serious infections in 5 pts. Group A showed 74.3% viable cells post-thawing in PBSC grafts. Median number of CD34+ cells were 2.9%. Median numbers of CFU-C, BFU-E and GEMM were 36, 60 and 7, respectively. This was comparable with results in group B, showing 70% viable cells post-thawing, CD34+ cells of 4.2% and CFUs of 43, 75 and 6, respectively (p>0.05). Proliferative capacity was intact in both groups after 7 days of suspension culture, generating CFU-C, BFU-E and GEMM of 67, 29 and 1, respectively. In group C, viable cells were present in only 58% and median CFU-C, BFU-E and GEMM were 21, 5 and 0, respectively (p<0.05). After 7 days of suspension culture, total CFUs were 5 (<5% as compared to group A+B). Mean CFU-Cs before and after LTC-IC were 9 and 8 after LTC-IC culture in group C, whereas these were 18 and 16 in group A (p<0.05). Thus, the percentage of viable cells, CFUs and LTC-ICs was preserved after long-term cryopreservation (group A), showed no significant difference between group A+B, but were decreased in group C. Conclusions: We show that human PBSC can be stored for more than a decade without apparent loss of HSC activity and can be efficiently retrieved. These results reinforce that expiration dates cannot be set for safely stored cryopreserved HSC. Assessment of CD34+ cell numbers, clonogenic potential via methylcellulose and LTC-IC assays are clinically relevant, since they may correlate with clinical outcome. Thus, these hematopoietic assays are valuable to assess the quality of cryopreservation and possibly also outcome of PBSCT.

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

Blood ◽

10.1182/blood.v112.11.2324.2324 ◽

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.

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Down-Modulating p18Ink4c for Ex Vivo Expansion of Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v104.11.1687.1687 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1687-1687

Author(s):

Tao Cheng ◽

Hui Yu ◽

Donna Shields ◽

Youzhong Yuan ◽

Hongmei Shen

Keyword(s):

Ex Vivo ◽

The Self ◽

Transduction Efficiency ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells ◽

Human Hscs

Abstract Our recent study demonstrated that the cyclin-dependent kinase inhibitor (CKI) p18Ink4c (p18), also an INK4 family protein acting at early G1-phase, exerts its inhibitory role during the self-renewing division of murine hematopoietic stem cells (HSC) in vivo (Nature Cell Biology 2004). Down-modulating p18 may permit enhanced stem cell expansion in vitro, a hypothesis that is now being testing in our laboratory. To provide the proof-of-the concept, we first took advantage of the murine system by testing the in vivo reconstituting ability of cells that had been cultured under the Dexter culture condition for 19 weeks. 2–20x105 cells with non-adherent and adherent populations were transplanted into lethally irradiated hosts. 3 of 7 mice revealed long-term engraftment in the p18−/− transplanted group (0.5–33% engraftment levels) while there was no engraftment in the p18+/+ group (n=7). Moreover, a substantial level (38.6% on average) of long-term engraftments (7 months) in multilineage was achieved in secondary recipients transplanted with the p18−/− cells (n=3), demonstrating the self-renewal potential of the expanded HSCs after the extended period of long-term culture. These data strongly indicate that p18 absence is able to substantially mitigate the differentiating effect of the ex vivo culture conditions on HSCs and therefore offer a strong rationale for targeting p18 in human HSC expansion. P18 mRNA was detected by RT PCR in human CD34+ cells with a higher expression level in the more primitive subset: CD34+CD38−. To explore the possibility of targeting p18 for expanding human HSCs, we have employed the RNA interference (RNAi) technology in CD34+ cord blood cells. We screened a pool of small interfering RNA (siRNA) oligos and three of them were able to effectively reduce p18 expression by 60–80% in 48 hours as assessed by both RNA and protein analyses in human cells. Further, we tested both transient and permanent delivery methods for introducing the RNAi effect in the CD34+CD38− cells. To demonstrate whether the RNAi method would be sufficient to impact the outcome of cell division after a single or limited cell cycle(s), we chose the nucleofector technology and were able to achieve 48.30±11.66% of transduction efficiency with good viability (50.63±9.38%, n=3) in human CD34+ cells. After a single electroporation pulse, we were able to increase by 2-fold the CD34+CD38− cells associated with the same magnitude of increased colony forming activity under culture condition supplemented with SCF, TPO and Flt3. To observe the long-term effect of p18 downregulation in human HSCs, we constructed a p18 short hairpin (shRNA)-expressing lentiviral vector that was engineered to have the mouse U6 promoter upstream of a CMV-EGFP expression cassette. A transduction efficiency of 30–60% was achieved after overnight infection of the human CD34+ cells with the p18 shRNA or with control lentiviral vectors pseudotyped with the VSV-g envelope. 72–96 hours after the transduction, human p18 protein can be knocked down by the p18 siRNA lentivector at near 100% in the HeLa cell line as determined on the western blot, and at more than 50% in human primary CD34+ cells as determined by real time RT PCR. We are currently undertaking further study aimed at assessing the repopulating ability of the transduced human HSCs with lentivirus-mediated p18 shRNA in NOD/SCID mice. Together, these findings suggest that down-modulating p18 might be a feasible approach for manipulating human HSCs ex vivo.

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

Blood ◽

10.1182/blood.v112.11.821.821 ◽

2008 ◽

Vol 112 (11) ◽

pp. 821-821 ◽

Cited By ~ 3

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)

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A novel lentiviral vector targets gene transfer into human hematopoietic stem cells in marrow from patients with bone marrow failure syndrome and in vivo in humanized mice

Blood ◽

10.1182/blood-2011-04-346619 ◽

2012 ◽

Vol 119 (5) ◽

pp. 1139-1150 ◽

Cited By ~ 31

Author(s):

Cecilia Frecha ◽

Caroline Costa ◽

Didier Nègre ◽

Fouzia Amirache ◽

Didier Trono ◽

...

Keyword(s):

Gene Therapy ◽

Cord Blood ◽

Mononuclear Cells ◽

Lentiviral Vector ◽

Ex Vivo ◽

Bone Marrow Failure ◽

Humanized Mice ◽

Hematopoietic Stem ◽

Cd34 Cells

AbstractIn vivo lentiviral vector (LV)–mediated gene delivery would represent a great step forward in the field of gene therapy. Therefore, we have engineered a novel LV displaying SCF and a mutant cat endogenous retroviral glycoprotein, RDTR. These RDTR/SCF-LVs outperformed RDTR-LVs for transduction of human CD34+ cells (hCD34+). For in vivo gene therapy, these novel RDTR/SCF-displaying LVs can distinguish between the target hCD34+ cells of interest and nontarget cells. Indeed, they selectively targeted transduction to 30%-40% of the hCD34+ cells in cord blood mononuclear cells and in the unfractionated BM of healthy and Fanconi anemia donors, resulting in the correction of CD34+ cells in the patients. Moreover, RDTR/SCF-LVs targeted transduction to CD34+ cells with 95-fold selectivity compared with T cells in total cord blood. Remarkably, in vivo injection of the RDTR/SCF-LVs into the BM cavity of humanized mice resulted in the highly selective transduction of candidate hCD34+Lin− HSCs. In conclusion, this new LV will facilitate HSC-based gene therapy by directly targeting these primitive cells in BM aspirates or total cord blood. Most importantly, in the future, RDTR/SCF-LVs might completely obviate ex vivo handling and simplify gene therapy for many hematopoietic defects because of their applicability to direct in vivo inoculation.

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

Blood ◽

10.1182/blood-2021-150924 ◽

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.

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Antibody targeting KIT as pretransplantation conditioning in immunocompetent mice

Blood ◽

10.1182/blood-2010-07-295949 ◽

2010 ◽

Vol 116 (24) ◽

pp. 5419-5422 ◽

Cited By ~ 45

Author(s):

Xingkui Xue ◽

Nancy K. Pech ◽

W. Christopher Shelley ◽

Edward F. Srour ◽

Mervin C. Yoder ◽

...

Keyword(s):

Low Dose ◽

Lentiviral Vector ◽

Ex Vivo ◽

Selective Advantage ◽

Wild Type ◽

Gene Correction ◽

Hematopoietic Stem ◽

Immunocompetent Mice

Abstract Inherited hematologic defects that lack an in vivo selective advantage following gene correction may benefit from effective yet minimally toxic cytoreduction of endogenous hematopoietic stem cells (HSCs) prior to transplantation of gene-modified HSCs. We studied the efficacy of administering a novel sequential treatment of parenteral ACK2, an antibody that blocks KIT, followed by low-dose irradiation (LD-IR) for conditioning of wild-type and X-linked chronic granulomatous disease (X-CGD) mice. In wild-type mice, combining ACK2 and LD-IR profoundly decreased endogenous competitive long-term HSC repopulating activity, and permitted efficient and durable donor-derived HSC engraftment after congenic transplantation. ACK2 alone was ineffective. The combination of ACK2 and LD-IR was also effective conditioning in X-CGD mice for engraftment of X-CGD donor HSCs transduced ex vivo with a lentiviral vector. We conclude that combining ACK2 with LD-IR is a promising approach to effectively deplete endogenous HSCs and facilitate engraftment of transplanted donor HSCs.

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Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666200225091339 ◽

2020 ◽

Vol 15 ◽

Author(s):

Fatima Aerts-Kaya

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Stress Conditions ◽

Stress Factors ◽

Hematopoietic Stem ◽

Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

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

Blood ◽

10.1182/blood.v130.suppl_1.709.709 ◽

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.

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

Blood ◽

10.1182/blood.v91.4.1243 ◽

1998 ◽

Vol 91 (4) ◽

pp. 1243-1255 ◽

Cited By ~ 69

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.

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