Second-Generation Chimeric Antigen Receptors Successfully Modify Hematopoietic Stem Cells for Immunotherapy of B-Lineage Malignancies

Abstract Patients with refractory or recurrent B-lineage hematological malignancies have less than 50% of chance of cure, despite intensive therapy. Chimeric Antigen Receptors (CARs) successfully engineer antigen specificity in immune cells, with clinical trials currently being conducted using ex vivo expanded gene-modified mature T cells. Results from preclinical studies and clinical trials show that effector cells usually have transient in vivo persistence that could significantly limit clinical efficacy and allow tumor recurrence. Our main hypothesis is that modification of hematopoietic stem cells (HSCs) with CARs will lead to persistent in vivo production of target-specific immune cells in multiple lineages, enhancing graft-versus-tumor activity and development of immunological memory. Using CD19 as target, we have generated first-generation and CD28- and 4-1BB-containing-second-generation CAR lentiviral constructs for modification of human HSCs, for assessment in vitro and in vivo. Gene modification with anti-CD19 CAR of CD34+cells isolated from human umbilical cord blood (UCB) did not impair normal differentiation and proliferation, with fully functional CAR-expressing cell progeny. Transduction with lentiviral vectors consistently achieved 40-50% efficiency at the clinically relevant vector copy number of 1-2 copies/cell. While first- and second-generation CARs triggered antigen-dependent cytotoxicity by myeloid and T cells in a similar fashion, only second-generation constructs successfully activated NK cells for antigen-dependent elimination of cell targets. In vivo studies using humanized NSG engrafted with CAR-modified human UCB CD34+ cells demonstrated similar levels of engraftment of human cells as compared to non-modified UCB CD34+ cells, with CAR-expressing cells in multiple lineages (myeloid, NK, T) successfully engrafted into bone marrow, spleen, peripheral blood and thymus detectable by flow cytometry and qPCR, in stable levels up to 35 weeks of life, with gene modification with first- or second-generation anti-CD19 CARs. No animals engrafted with CAR-modified HSCs presented signs of autoimmunity or chronic inflammation. Cells presented ex vivo antigen-dependent cytotoxicity against cell targets. Mice successfully engrafted with CAR-modified HSCs harbored decreased CD19+populations, and only HSCs modified with second-generation CARs successfully led to tumor growth inhibition and survival advantage at tumor challenge. CAR-modified HSCs led to development of T cell effector memory and T cell central memory subsets, confirming the expectation of development of long-lasting phenotypes due to directed antigen specificity. Longer survival of mice with developing tumors was also significantly correlated to higher number of CAR-expressing cells infiltrating subcutaneous tumors. Our results demonstrate feasibility of CAR modification of human HSCs for cancer immunotherapy. This approach can be applied to different cancers just by adjusting the target specificity. Furthermore, it could be easily employed in the context of HSC transplantation to augment the anti-leukemic activity, with CAR-expressing myeloid and NK cells to ensure tumor-specific immunity until de novo production of T cells from CAR-modified HSCs. It also bears the possibility of decreased morbidity and mortality, being desirable for vulnerable populations such as children and elderly patients, and offers alternative treatment for patients with no available HLA-matched sources for bone marrow transplantation, benefiting ethnic minorities. Disclosures Larson: Millenium Pharmaceuticals, Inc.: Speakers Bureau.

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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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Adoptive Therapy with T-Cell Precursors for Immune Reconstitution After Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v114.22.3525.3525 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3525-3525

Author(s):

Emanuela Burchielli ◽

Antonella Tosti ◽

Loredana Ruggeri ◽

Katia Perruccio ◽

Claudia De Angelis ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Triple Negative ◽

Ex Vivo ◽

Double Positive ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Single Positive ◽

Human Hscs

Abstract Abstract 3525 Poster Board III-462 Recipients of allogeneic hematopoietic transplantation experience a slow reconstitution of donor-derived B and T cell number and function. This post-transplant period of immunodeficiency is associated with an increased risk of infection and malignant relapse. The developement of these complications notably correlates with the recovery of CD4+T cell subset. We proposed a strategy to enhances in vivo reconstitution by promoting donor-derived T cell development in the recipient's thymus. Recently Notch1-based ex-vivo system have been established to mature cord blood- or bone marrow-derived human HSCs into committed T-cell precursors. We used this system for the generation of T-cell precursors starting from G-CSF mobilized human HSCs. We cultured mobilized human CD34+ hematopoietic stem cells (HSCs) (2.5 × 105) in vitro on OP9 mouse stromal cells expressing the Notch 1 ligand Delta-like-1 (OP9-DL1) in the presence of rhFLT3-ligand (5ng/ml) and rhIL7 (5 ng/ml). After 6 weeks of co-culture we obtained a 3 log increase of human T-linage precursors of CD45RA+CD7high phenotype. Further co-colture (7-9 weeks) leed to the generation of CD4+ and CD8+ double-positive (DP) T cells and even mature CD4+ and CD8+ single positive (SP) ab-TCR lymphocytes. Experiments were designed in order to evaluate whether human CD45RA+CD7high T cell precursors could 1) engraft into NOD-SCID IL2 rg-/− mice 2) leed to in vivo expansion and maturation along T cell developmental pathway. Control mice were irradiated and transplanted with G-CSF-mobilized human CD34+ (dose 5×106 i.v.). 4 weeks after transplant more than 20% human CD45 positive cells engrafted in the bone marrow. Thymic engraftment occured at 8 weeks after transplant, with 80% human CD45 positive cells (thymic cellularity: 2.7×105 cells), mostly with T cell-immature phenotype of CD3-CD4-CD8 triple negative (95%) (TN) and CD4+CD8+double positive (5%) (DP). Co-transplant of CD45RA+CD7high T cell precursors (106 cells i.v.) along with CD34+HSC leed to an accelerated thymic engraftment (95% human CD45 positive cells; thymic cellularity 2.5 × 106 cells) already at 6 wks after transplant. Thymocytes were CD3-CD4-CD8 triple negative (51%) (TN) and CD4+CD8+double positive (DP) (42%) cells and at 8 weeks after transplant matured into CD3+CD4+ and CD3+CD8+ single positive (SP) T cells. Spectratyping analyses revealed a broad diversity of the T-cell receptor (TCR) repertoire. This occured in the complete absence of Graft versus Host Disease (GvHD) suggesting that adoptively transferred ex vivo-generated T-cell precursors developed into host-tolerant mature T cells. Ongonig experiment are needed to clarify the beneficial effect of adoptive immunotherapy with human T cell precursors on peripheral T cell reconstitution and control of infection in the humanized mouse system. We conclude that ex-vivo generation of human T-linage precursors is feasible from the G-CSF-mobilized HSCs and that can be succesfully tranfered in-vivo as a new strategie to enhance T-cell reconstitution after allogeneic HSCT with no risk of GvHD. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood-2020-141121 ◽

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.

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In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo

Nature Communications ◽

10.1038/s41467-020-19486-2 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

N. N. Parayath ◽

S. B. Stephan ◽

A. L. Koehne ◽

P. S. Nelson ◽

M. T. Stephan

Keyword(s):

T Cells ◽

Ex Vivo ◽

Human Leukemia ◽

Antigen Receptors ◽

Wide Range ◽

Engineered T Cells ◽

Circulating T Cells ◽

The Cost

AbstractEngineering chimeric antigen receptors (CAR) or T cell receptors (TCR) helps create disease-specific T cells for targeted therapy, but the cost and rigor associated with manufacturing engineered T cells ex vivo can be prohibitive, so programing T cells in vivo may be a viable alternative. Here we report an injectable nanocarrier that delivers in vitro-transcribed (IVT) CAR or TCR mRNA for transiently reprograming of circulating T cells to recognize disease-relevant antigens. In mouse models of human leukemia, prostate cancer and hepatitis B-induced hepatocellular carcinoma, repeated infusions of these polymer nanocarriers induce sufficient host T cells expressing tumor-specific CARs or virus-specific TCRs to cause disease regression at levels similar to bolus infusions of ex vivo engineered lymphocytes. Given their ease of manufacturing, distribution and administration, these nanocarriers, and the associated platforms, could become a therapeutic for a wide range of diseases.

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Naive and Ex Vivo Activated Human T Cells Generate Consistent Engraftment and Lethal Graft-Versus-Host Disease (GvHD) in NOD SCID β 2M Null Mice: A New Xenogeneic Model for GvHD.

Blood ◽

10.1182/blood.v106.11.3106.3106 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3106-3106

Author(s):

Bruno Nervi ◽

Michael P. Rettig ◽

Julie K. Ritchey ◽

Gerhard Bauer ◽

Jon Walker ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Ex Vivo ◽

Conditioning Regimen ◽

Activation Markers ◽

In Vivo Models ◽

Hematopoietic Stem ◽

Human T Cells ◽

The Impact

Abstract GvHD remains a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion. The human GvHD pathophysiology includes recipient tissue destruction and proinflammatory cytokine production associated with the conditioning regimen; donor T cells become allo-activated, proliferate, and mediate tissue injury in various organs, including the liver, skin, and gut. Modern therapeutic strategies to control GvHD while maintaining the beneficial graft-versus-leukemia effects require ex vivo T cell stimulation and expansion. Multiple studies have demonstrated that these ex vivo expanded T cells exhibit decreased survival and function in vivo, including reduced alloreactivity and GvHD potential. Unfortunately no in vivo models exist to consistently examine the impact of ex vivo manipulation of human T cells (HuT) on T cell function. Naive HuT were compared to HuT activated using CD3/28 beads (XcyteTMDynabeads) with 50 U/ml IL-2 for 4 days (Act). We initially evaluated the HuT engraftment and GvHD potential of naive and Act in RAG2γ null mice (n=22) conditioned with clodronate liposomes on day −1 and 350cGy on day 0, as previously described by others. We injected 107 and 1.5x107 naive or Act HuT intravenously (iv). All mice exhibited low HuT engraftment and no lethal GvHD. NOD SCIDβ 2M null mice (β 2M) were next conditioned with 250cGy on day −1 (n=34), or 300cGy on day 0 (n=21). 107 naive vs Act HuT were injected retroorbitaly (ro). Lower HuT doses or iv injection resulted in no expansion or GvHD. Engraftment of HuT in peripheral blood of recipient mice was evaluated weekly by FACS and euthanasia was performed if mice lost > 20% body weight. 60% of the mice conditioned with 250cGy that received naive HuT developed lethal GvHD, in comparison to 75% of mice that received 300cGy and nave HuT, and 100% of mice that received 300cGy and Act HuT. Table 1 250cGy 300cGy Naive (n=34) Naive (n=8) Activated (n=13) *p<0.02 PB engraftment (%HuT) 20%±15 33%±21 59%±19 Lethal GvHD 60% 75% 100% All mice receiving 300cGy had well preserved CD4/CD8 ratios (1–1.5). Tissue infiltration was greatest in mice that had received 300cGy and Act HuT (spleen, liver, lung, kidney: 50–70%). Of interest, serum levels of hu IFNγ dramatically increased over time in all mice who went on to develop lethal GvHD (day 3=270 ug/ml and day 15=36,000 ug/ml) compared to mice that did not develop lethal GvHD (day 10=40 ug/ml and day 17=1,020 ug/ml)(p<0.05). Interestingly, the up-regulation of the activation markers CD25 and CD30 in HuT, and IFNγ production predicted lethal GvHD in β 2M null mice. In summary, we developed a xenogeneic model of lethal GvHD where naive or ex vivo Act HuT injected ro in sublethaly irradiated β 2M not only engraft, expand in vivo, but also infiltrate and damage different mouse target organs. HuT are allo-activated against mouse antigens and damage the target tissues, sharing the major characteristics of human GvHD and causing the death of mice. This model will allow us to study the effects of specific ex vivo T cell manipulation including transduction, selection, expansion, and the depletion or addition of various T cells and other cellular subsets on the outcome of GvHD, to determine improved therapeutic interventions.

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Clonal Analyses of Retroviral Vector Integrations in ADA-SCID Patients Treated with Stem Cell Gene Therapy.

Blood ◽

10.1182/blood.v108.11.3249.3249 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3249-3249

Author(s):

Barbara Cassani ◽

Grazia Andolfi ◽

Massimiliano Mirolo ◽

Luca Biasco ◽

Alessandra Recchia ◽

...

Keyword(s):

Gene Therapy ◽

T Cells ◽

T Cell ◽

Gene Transfer ◽

Human Genome ◽

Ex Vivo ◽

Cd34 Cells

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.

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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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ATG5 and ATG7 Fulfill Essential and Non-Redundant Roles in Human Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v124.21.4316.4316 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4316-4316

Author(s):

Hendrik Folkerts ◽

Maria Catalina Gomez Puerto ◽

Albertus T.J. Wierenga ◽

Koen Schepers ◽

Jan Jacob Schuringa ◽

...

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Target Genes ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Human Hscs

Abstract Macroautophagy is a catabolic process by which intracellular contents are delivered to lysosomes for degradation. ATG5 and ATG7 play an essential role in this process. Recent studies have shown that mouse hematopoietic stem cells (HSCs) lacking ATG7 were unable to survive in vivo, however, the role of macroautophagy in proliferation and survival of human HSCs has not yet been defined. Here, we demonstrate that autophagy is functional in human hematopoietic stem/progenitor cells. Robust accumulation of the autophagy markers LC3 and p62 were observed in cord blood (CB)-derived CD34+ cells treated with bafilomycin-A1 (BAF) or hydroxychloroquine (HCQ), as defined by Western blotting. When these cells were subsequently differentiated towards the myeloid or erythroid lineage, a decreased accumulation of LC3 was observed. In addition, CB CD34+CD38- cells showed enhanced accumulation of cyto-ID (a marker for autophagic vesicles) compared to CD34+CD38+ progenitor cells upon BAF or HCQ treatment. In line with these results, also more mature CB CD33+ and CD14+ myeloid cells or CD71+CD235+ erythroid cells showed reduced levels of cyto-ID accumulation upon BAF or HCQ treatment. These findings indicate that human hematopoietic stem and progenitor cells (HSPCs) have a higher basal autophagy flux compared to more differentiated cells. To study the functional consequences of autophagy in human HSCs and their progeny, ATG5 and ATG7 were downregulated in CB-derived CD34+ cells, using a lentiviral shRNA approach which resulted in 80% and 70% reduced expression, respectively. Downmodulation of ATG5 or ATG7 in CB CD34+ cells resulted in a significant reduction of erythroid progenitor frequencies, as assessed by colony forming cell (CFC) assays (shATG5 2.2 fold, p<0.05 or shATG7 1.4 fold p<0.05). Additionally, a strong reduction in expansion was observed when transduced cells were cultured under myeloid (shATG5 17.9 fold, p<0.05 or shATG7 12.3 fold, p<0.05) or erythroid permissive conditions (shATG5 6.7 fold, p<0.05 or shATG7 1.7 fold, p<0.05), whereby differentiation was not affected. The phenotype upon knockdown of ATG5 or ATG7 could not be reversed by culturing the cells on a MS5 stromal layer. In addition to progenitor cells, HSCs were also affected since long term culture-initiating cell (LTC-IC) assays in limiting dilution revealed a 3-fold reduction in stem cell frequency after ATG5 and ATG7 knockdown. The inhibitory effects of shATG5 and shATG7 in cultured CD34+ cells were at least in part due to a decline in the percentage of cells in S phase and (shATG5 1.4 fold, p<0.01 and shATG7 1.3 fold, p<0.01) and an increase of Annexin V positive cells. The changes in cell cycle and apoptosis coincided with a marked increase in expression of the cell cycle-dependent kinase inhibitor p21, an increase in p53 levels, and an increase in proapoptotic downstream target genes BAX, PUMA and PHLDA3. Additionally, ROS levels were increased after ATG5 and ATG7 knockdown. The increased apoptosis in shATG5 and shATG7 transduced cells might be triggered by elevated ROS levels. Taken together, our data demonstrate that autophagy is an important survival mechanism for human HSCs and their progeny. Disclosures No relevant conflicts of interest to declare.

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Small Molecule Combination Enhances CD34+/CD38- Cell Proliferation Via Inhibition of Differentiation

Blood ◽

10.1182/blood.v128.22.658.658 ◽

2016 ◽

Vol 128 (22) ◽

pp. 658-658

Author(s):

Lan Wang ◽

Xin Guan ◽

Huihui Wang ◽

Bin Shen ◽

Yu Zhang ◽

...

Keyword(s):

Cell Proliferation ◽

Small Molecules ◽

Cell Expansion ◽

Target Genes ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Qpcr Analysis ◽

Cd34 Cells ◽

Cells Cultured

Abstract Hematopoietic stem cells (HSCs) have become increasingly attractive for the therapy of various hematological system disorders. The aim of this study is to identify approaches that promote the expansion of HSCs. We present here the identification of a combination of small molecules and cytokines that is effective in retaining high stemness of hematopoietic stem/progenitor cells while promoting cell proliferation by inhibiting differentiation. Firstly, five small-molecule candidates were screened for their individual effects on ex vivo expansion of human peripheral blood CD34+ cells in the presence of selected cytokines. The best compounds at their optimal concentrations were further analyzed in combination, to achieve maximum capacity for stimulating the CD34+CD38- cell expansion ex vivo. The extent of cell expansion and the immunophenotype of expanded cells were assessed through flow cytometry. Additional cell and molecular assays were performed to confirm that the expanded CD34± cells are functionally normal in vitro. Subsequently, the expanded cells were transplanted into sublethally irradiated NOD/SCID mice for the assessment ofhuman cell viability and engraftment potential in vivo. Furthermore, the expression of several genes in the cell proliferation and differentiation pathways was analyzed through qPCR during the process of CD34±cell expansion. Following multiple rounds of screening, an optimal formula (named as "SVC cocktail") was obtained, which consisted of four cytokines (stem cell factor, flt-3 ligand, thrombopoietin and interleukin-6) and three small molecules (Stem Regenin 1, valproic acid and CAY10433). CD34+ cells cultured with SVC cocktail had a purity of 76.2%±7.5% and reached expansion folds of 27.9±4.3 for CD34+/CD38- HSCs on day 7. In contrast, CD34+ cells cultured with the cytokines alone displayed a purity of 27.4%±6.3% and expansion folds of 15.5±2.2 for CD34+/CD38- cells. The groups with small molecules only (plus DMSO, the vehicle), or with basal medium only, showed no surviving cells on day 4. Furthermore, cell cycle analysis indicated that the SVC cocktail-induced CD34+/CD38- cells stayed in a more quiescent state (G0/G1: 75.2%±3.6%; S: 9.2%±2.4%). On the other hand, the cells cultured without the three small molecules had active DNA synthesis (G0/G1: 56.0%±2.0%; S: 31.8%±3.2%), implicating a trend of enhanced cell differentiation in the cytokine alone group. RT-qPCR analysis further demonstrated that the expression of HSC stemness markers CD90, CD133, CD117, ALDH1, Bmi1, HoxB4, GATA-2, Runx1, and CXCR4 were elevated in the SVC cocktail-induced CD34+ cells, but dramatically reduced or barely detectable in the cytokine alone group. In addition, CFU assays for the SVC cocktail group vs the cytokine alone group demonstrated BFU-E of 54.0±4.6 vs 11.7±1.5, CFU-GM of 71.0±2.7 vs 8.3±2.5, CFU-GEMM of 40.7±3.8 vs 5.0±2.0 and CFU-Mk of 6.7±1.5 vs 0.7±0.6, respectively. For the in vivo engraftment in mouse bone marrow, human CD45 rate in the SVC cocktail group was much higher than in the cytokine alone group (21.1%±2.7% vs 0.5%±0.1%); similar group differences were also found in the CD34+ and CD34+CD38- rate (7.7%±1.4% vs 1.6%±1.2% and 6.8%±2.2% vs 1.6%±0.1% respectively), all at 8 weeks post transplantation. Moreover, qPCR analysis of Notch and Wnt signaling pathways for cultured cells on day 7 showed that the expression of Notch target genes (related to high activation of HSC property) was enhanced in the SVC cocktail group compare to the cytokine group (HES5: 9.2±2.3 vs 3.6±1.4 in arbitrary units; HEY1: 6.3±1.9 vs 2.6±1.2; HES1: 3.2±1.3 vs 1.3±0.4; Notch1: 1.4±0.3 vs 1.2±0.3), whereas the expression of Wnt target genes (related to activation of HSC differentiation) was greater in the cytokine alone group than in the SVC cocktail group (CCND1: 10.1±4.3 vs 1.2±0.8; LEF1: 4.3±0.6 vs 2.9±0.2; PPAR D: 3.4±0.3 vs 1.5±0.1; FZD2: 1.8±0.2 vs 1.0±0.1). Taken together, our results show that the new SVC cocktail is able to retain the characteristics of HSCs remarkably well, by enhancing their expansion while inhibiting their differentiation. Mechanistically, it appears that the three small molecules can effectively inhibit the cytokines' pro-differentiation effects on CD34+CD38- cells without affecting the cytokines' ability to stimulate cell proliferation. Disclosures Wang: Biopharmagen Corp.: Employment. Ren:Biopharmagen Corp: Employment. Jiang:Biopharmagen Corp: Consultancy.

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