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Hematopoietic stem and progenitor cell (HSPC)-based gene therapy (GT) requires the collection of a large number of cells. While bone marrow (BM) is the most common source of HSPCs in pediatric donors, the collection of autologous peripheral blood stem and progenitor cells (PBSCs) is an attractive alternative for GT. We present safety and efficacy data of a 10-year cohort of 45 pediatric patients that underwent PBSC collection for backup and/or purification of CD34+ cells for ex vivo gene transfer. Median age was 3.7 years and median weight 15.8 kg. After mobilization with lenograstim/plerixafor (n=41) or lenograstim alone (n=4), and 1-3 cycles of leukapheresis, median collection was 37 x106 CD34+ cells/kg. The procedures were well tolerated. Patients that collected ≥7 and ≥13 x106 CD34+ cells/kg in the first cycle had pre-apheresis circulating counts of at ≥42 and ≥86 CD34+ cells/μL respectively. Weight-adjusted CD34+ cell yield was positively correlated with peripheral CD34+ cell counts, and influenced by female gender, disease and drug dosage. All patients received a GT product above the minimum target, ranging from 4 to 30.9 x106 CD34+ cells/kg. Pediatric PBSC collection compares well to BM harvest in terms of CD34+ cell yields for the purpose of GT, with a favorable safety profile.

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, an important 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 (BM), supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPC). To overcome the limit of BM harvest/HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex vivo expansion of HSPC coupled with lentiviral gene transfer. Circulating CD34(+). cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector 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 NOD scid gamma common chain (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 HSPC 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 BM fibrosis.

Mucopolysaccharidosis type I (MPS-I) is a severe genetic disease caused by a deficiency of the alpha-L-iduronidase (IDUA) enzyme. Ex vivo hematopoietic stem cell (HSC) gene therapy is a promising therapeutic approach for MPS-I, as demonstrated by preclinical studies performed in naive MPS-I mice. However, after enzyme replacement therapy (ERT), several MPS-I patients develop anti-IDUA immunity that may jeopardize ex vivo gene therapy efficacy. Here we treat MPS-I mice with an artificial immunization protocol to mimic the ERT effect in patients, and we demonstrate that IDUA-corrected HSC engraftment is impaired in pre-immunized animals by IDUA-specific CD8+ T cells spared by pre-transplant irradiation. Conversely, humoral anti-IDUA immunity does not impact on IDUA-corrected HSC engraftment. The inclusion of lympho-depleting agents in pre-transplant conditioning of pre-immunized hosts allowes rescue of IDUA-corrected HSC engraftment, which is proportional to CD8+ T cell eradication. Overall, these data demonstrate the relevance of pre-existing anti-transgene T cell immunity on ex vivo HSC gene therapy, and they suggest the application of tailored immune-depleting treatments, as well as a deeper immunological characterization of patients, to safeguard the therapeutic effects of ex vivo HSC gene therapy in immunocompetent hosts., MPS-I patients receiving enzyme replacement therapy develop an anti-IDUA immune response that may affect ex vivo gene therapy. Squeri et al. show, in the MPS-I murine model, that pre-existing IDUA-specific CD8+ T cell response impairs LV-transduced HSC engraftment and that the efficacy of ex vivo gene therapy is rescued by lympho-depleting drugs.

GBM with an unmethylated MGMT gene promoter is associated with very poor prognosis. A subset of tumor associated macrophages expressing the angiopoietin receptor Tie2 (TEMs) can be genetically modified for local & tumor restricted release of interferon-α2 (IFN). IFN has antitumor effects, inhibits angiogenesis & modulates the immune system. Temferon consists of autologous HSPCs transduced ex-vivo with an LVV encoding an IFN gene & expression control sequences for TEMs. TEM-GBM is an open-label, Phase I/IIa study (Part A: 3x3x3 dose escalation; Part B: n=12), & Temferon (single dose) is given to patients with first diagnosis of GBM & unmethylated MGMT promoter. Part A 3rd cohort is ongoing & completes dosing in September 2020. Eight patients completed screening; one patient died (disease progression) before Temferon was administered. Six patients received Temferon (3 women, 3 men, mean age 52.3 years). Cohort 1 received Temferon 0.5x106 cells/kg & Cohort 2, 1x106 cells/kg. Neutropenia & thrombocytopenia occurred as expected following conditioning & hematologic recovery (HR) occurred median D+13. Transduced PBMCs were identified by vector copy number (VCN) on myeloid cells at HR & at later timepoints. In general, a dose-ordered increase in VCN was observed (mean VCN D+30 CD14+ Cohort 1: 0.094, cohort 2: 0.125); 1 patient in each cohort had low VCNs. VCN remained detectable up to recent follow up visits (≤ D+180). No dose-limiting toxicities have been reported. Four SAEs occurred in 3 patients who received Temferon (pneumonia, pulmonary embolism, febrile neutropenia, fatigue) but these events were not attributed to Temferon, resolved, & may have been related to the conditioning regimen (carmustine & thiotepa). Disease progression has been confirmed in 3 patients who received Temferon. These preliminary results indicate feasibility of engrafting a pre-determined fraction of Temferon cells in the bone marrow of GBM patients without, so far, causing dose-limiting toxicity.

Eradicating the malignant stem cell is the ultimate challenge in the treatment of leukaemia. Leukaemic stem cells (LSC) hijack the normal haemopoietic niche, where they are mainly protected from cytotoxic drugs. The anti-leukaemic effect of L-asparaginase (ASNase) has been extensively investigated in acute lymphoblastic leukaemia, but only partially in acute myeloid leukaemia (AML). We explored the susceptibility of AML-LSC to ASNase as well as the role of the two major cell types that constitute the bone marrow (BM) microenvironment, i.e., mesenchymal stromal cells (MSC) and monocytes/macrophages. Whilst ASNase was effective on both CD34+ CD38+ and CD34+ CD38- LSC fractions, MSC and monocytes/macrophages partially counteracted the effect of the drug. Indeed, the production of cathepsin B, a lysosomal cysteine protease, by BM monocytic cells and by AML cells classified as French-American-British M5 is related to the inactivation of ASNase. Our work demonstrates that, while MSC and monocytes/macrophages may provide a protective niche for AML cells, ASNase has a cytotoxic effect on AML blasts and, importantly, LSC subpopulations. Thus, these features should be considered in the design of future clinical studies aimed at testing ASNase efficacy in AML patients.

Umbilical cord blood (UCB) is a promising source of stem cells to use in early haematopoietic stem cell transplantation (HSCT) approaches for several genetic diseases that can be diagnosed at birth. Mucopolysaccharidosis type I (MPS-I) is a progressive multi-system disorder caused by deficiency of lysosomal enzyme α-L-iduronidase, and patients treated with allogeneic HSCT at the onset have improved outcome, suggesting to administer such therapy as early as possible. Given that the best characterized MPS-I murine model is an immunocompetent mouse, we here developed a transplantation system based on murine UCB. With the final aim of testing the therapeutic efficacy of UCB in MPS-I mice transplanted at birth, we first defined the features of murine UCB cells and demonstrated that they are capable of multi-lineage haematopoietic repopulation of myeloablated adult mice similarly to bone marrow cells. We then assessed the effectiveness of murine UCB cells transplantation in busulfan-conditioned newborn MPS-I mice. Twenty weeks after treatment, iduronidase activity was increased in visceral organs of MPS-I animals, glycosaminoglycans storage was reduced, and skeletal phenotype was ameliorated. This study explores a potential therapy for MPS-I at a very early stage in life and represents a novel model to test UCB-based transplantation approaches for various diseases.

Transplantation of hematopoietic cells from a healthy individual (allogeneic hematopoietic cell transplantation (allo-HCT)) demonstrates that adoptive immunotherapy can cure blood cancers: still, post-transplantation relapses remain frequent. To explain their drivers, we analyzed the genomic and gene expression profiles of acute myeloid leukemia (AML) blasts purified from patients at serial time-points during their disease history. We identified a transcriptional signature specific for post-transplantation relapses and highly enriched in immune-related processes, including T cell costimulation and antigen presentation. In two independent patient cohorts we confirmed the deregulation of multiple costimulatory ligands on AML blasts at post-transplantation relapse (PD-L1, B7-H3, CD80, PVRL2), mirrored by concomitant changes in circulating donor T cells. Likewise, we documented the frequent loss of surface expression of HLA-DR, -DQ and -DP on leukemia cells, due to downregulation of the HLA class II regulator CIITA. We show that loss of HLA class II expression and upregulation of inhibitory checkpoint molecules represent alternative modalities to abolish AML recognition from donor-derived T cells, and can be counteracted by interferon-gamma or checkpoint blockade, respectively. Our results demonstrate that the deregulation of pathways involved in T cell-mediated allorecognition is a distinctive feature and driver of AML relapses after allo-HCT, which can be rapidly translated into personalized therapies.

2532 Background: Genetically modified cell-based therapies are relevant in immuno-oncology due to their potential for tumor specificity & potential durability. We developed a cell-based treatment, Temferon, relying on ex-vivo transduction of autologous HSPCs to express therapeutic payloads within the tumor microenvironment. Temferon targets IFNa to Tie-2 expressing macrophages (TEMs). Methods: TEM-GBM is an open-label, Phase I/IIa dose-escalation study evaluating safety & efficacy of Temferon in up to 21 newly diagnosed patients with glioblastoma & unmethylated MGMT promoter. Autologous HSPCs are transduced ex-vivo with a lentiviral vector encoding for IFNa. The transgene expression is confined to TEMs due to the Tie2 promoter & the post-transcriptional regulation by miRNA-126. Results: As of January 17 2021, 15 patients have been enrolled; 9 received Temferon (D+0) with follow-up of 61 – 559 days. There was rapid engraftment & hematological recovery after the conditioning regimen. Median neutrophil & platelet engraftment occurred at D+13 & D+12, respectively. Temferon-derived differentiated cells, as determined by the presence of vector genomes in the DNA of peripheral blood & bone marrow cells, were found within 14 days post treatment & persisted subsequently, albeit at lower levels (up to 18 months). We also detected very low concentrations of IFNa in the plasma (median 5pg/ml at D+30; baseline < LLOQ) & in the cerebrospinal fluid, suggesting tight regulation of transgene expression. Three deaths occurred: two at D+343 & +402 after Temferon administration due to disease progression, & one at D+60 due to complications following the conditioning regimen. Seven patients had progressive disease (PD; range D+27-239) as expected for this tumor type. SAEs include infections, venous thromboembolism, brain abscess, hemiparesis, GGT elevation & poor performance status compatible with autologous stem cell transplantation, concomitant medications & PD. Four patients underwent second surgery. These recurrent tumors had gene-marked cells present & increased expression of IFN-responsive gene signatures compared to diagnosis, indicative of local IFNa release by TEMs. In one patient a stable lesion (as defined by MRI) had a higher proportion of T cells & TEMs within the myeloid infiltrate & an increased IFN-response signature than in a progressing lesion. The T-cell immune repertoire changed with evidence for expansion of tumor-associated clones. Tumor microenvironment characterization by scRNA & TCR sequencing is ongoing. Conclusions: Our interim results show that Temferon is well tolerated by patients, with no dose limiting toxicities identified to date. The results provide initial evidence of Temferon potential to modulate the TME of GBM patients, as predicted by preclinical studies. Clinical trial information: NCT03866109.

MicroRNA (miRNA)-126 is a known regulator of hematopoietic stem cell quiescence. We engineered murine hematopoiesis to express miRNA-126 across all differentiation stages. Thirty percent of mice developed monoclonal B cell leukemia, which was prevented or regressed when a tetracycline-repressible miRNA-126 cassette was switched off. Regression was accompanied by upregulation of cell-cycle regulators and B cell differentiation genes, and downregulation of oncogenic signaling pathways. Expression of dominant-negative p53 delayed blast clearance upon miRNA-126 switch-off, highlighting the relevance of p53 inhibition in miRNA-126 addiction. Forced miRNA-126 expression in mouse and human progenitors reduced p53 transcriptional activity through regulation of multiple p53-related targets. miRNA-126 is highly expressed in a subset of human B-ALL, and antagonizing miRNA-126 in ALL xenograft models triggered apoptosis and reduced disease burden.

Innate immune factors may restrict hematopoietic stem cell (HSC) genetic engineering and contribute to broad individual variability in gene therapy outcomes. Here, we show that HSCs harbor an early, constitutively active innate immune block to lentiviral transduction that can be efficiently overcome by cyclosporine H (CsH). CsH potently enhances gene transfer and editing in human long-term repopulating HSCs by inhibiting interferon-induced transmembrane protein 3 (IFITM3), which potently restricts VSV glycoprotein-mediated vector entry. Importantly, individual variability in endogenous IFITM3 levels correlated with permissiveness of HSCs to lentiviral transduction, suggesting that CsH treatment will be useful for improving ex vivo gene therapy and standardizing HSC transduction across patients. Overall, our work unravels the involvement of innate pathogen recognition molecules in immune blocks to gene correction in primary human HSCs and highlights how these roadblocks can be overcome to develop innovative cell and gene therapies.

Mendelian susceptibility to mycobacterial disease is a rare primary immunodeficiency characterized by severe infections caused by weakly virulent mycobacteria. Biallelic null mutations in genes encoding interferon gamma receptor 1 or 2 (IFNGR1 or IFNGR2) result in a life-threatening disease phenotype in early childhood. Recombinant interferon γ (IFN-γ) therapy is inefficient, and hematopoietic stem cell transplantation has a poor prognosis. Thus, we developed a hematopoietic stem cell (HSC) gene therapy approach using lentiviral vectors that express Ifnγr1 either constitutively or myeloid specifically. Transduction of mouse Ifnγr1-/- HSCs led to stable IFNγR1 expression on macrophages, which rescued their cellular responses to IFN-γ. As a consequence, genetically corrected HSC-derived macrophages were able to suppress T-cell activation and showed restored antimycobacterial activity against Mycobacterium avium and Mycobacterium bovis Bacille Calmette-Guerin (BCG) in vitro. Transplantation of genetically corrected HSCs into Ifnγr1-/- mice before BCG infection prevented manifestations of severe BCG disease and maintained lung and spleen organ integrity, which was accompanied by a reduced mycobacterial burden in lung and spleen and a prolonged overall survival in animals that received a transplant. In summary, we demonstrate an HSC-based gene therapy approach for IFNγR1 deficiency, which protects mice from severe mycobacterial infections, thereby laying the foundation for a new therapeutic intervention in corresponding human patients.

Two recent papers (one by Román-Rodríguez et al., 2019 in this issue of Cell Stem Cell) highlight how the power of biological selection on hematopoietic stem cell fitness can facilitate gene therapies for Fanconi Anemia. A clinical trial using lentiviral gene replacement and a proof-of-concept targeted genome editing study show robust engraftment and expansion of gene-corrected cells at levels reaching therapeutic relevance.

A precise understanding of the role of miR-223 in human hematopoiesis and in the pathogenesis of acute myeloid leukemia (AML) is still lacking. By measuring miR-223 expression in blasts from 115 AML patients, we found significantly higher miR-223 levels in patients with favorable prognosis, whereas patients with low miR-223 expression levels were associated with worse outcome. Furthermore, miR-223 was hierarchically expressed in AML subpopulations, with lower expression in leukemic stem cell–containing fractions. Genetic depletion of miR-223 decreased the leukemia initiating cell (LIC) frequency in a myelomonocytic AML mouse model, but it was not mandatory for rapid-onset AML. To relate these observations to physiologic myeloid differentiation, we knocked down or ectopically expressed miR-223 in cord-blood CD34 + cells using lentiviral vectors. Although miR-223 knockdown delayed myeloerythroid precursor differentiation in vitro, it increased myeloid progenitors in vivo following serial xenotransplantation. Ectopic miR-223 expression increased erythropoiesis, T lymphopoiesis, and early B lymphopoiesis in vivo. These findings broaden the role of miR-223 as a regulator of the expansion/differentiation equilibrium in hematopoietic stem and progenitor cells where its impact is dose- and differentiation-stage-dependent. This also explains the complex yet minor role of miR-223 in AML, a heterogeneous disease with variable degree of myeloid differentiation.

For decades, bone marrow (BM) has been the preferred source of hematopoietic stem and progenitor cells (HSPCs) for transplants following myeloablative conditioning. At present, mobilized peripheral blood stem cells are commonly used for transplantation, particularly in the autologous setting.[1][1

Clinical application of lentiviral vector (LV)‐based hematopoietic stem and progenitor cells (HSPC) gene therapy is rapidly becoming a reality. Nevertheless, LV‐mediated signaling and its potential functional consequences on HSPC biology remain poorly understood. We unravel here a remarkably limited impact of LV on the HSPC transcriptional landscape. LV escaped innate immune sensing that instead led to robust IFN responses upon transduction with a gamma‐retroviral vector. However, reverse‐transcribed LV DNA did trigger p53 signaling, activated also by non‐integrating Adeno‐associated vector, ultimately leading to lower cell recovery ex vivo and engraftment in vivo . These effects were more pronounced in the short‐term repopulating cells while long‐term HSC frequencies remained unaffected. Blocking LV‐induced signaling partially rescued both apoptosis and engraftment, highlighting a novel strategy to further dampen the impact of ex vivo gene transfer on HSPC. Overall, our results shed light on viral vector sensing in HSPC and provide critical insight for the development of more stealth gene therapy strategies.

New neurons, originating from the subventricular zone, are continuously integrating into neuronal circuitry in the olfactory bulb (OB). Using a transgenic sensor mouse, we found that adult-born OB interneurons express microRNA-125 (miR-125), whereas the pre-existing developmentally generated OB interneurons represent a unique population of cells in the adult brain, without miR-125 activity. Stable inhibition of miR-125 in newborn OB neurons resulted in enhanced dendritic morphogenesis, as well as in increased synaptic activation in response to odour sensory stimuli. These data demonstrate that miR-125 controls functional synaptic integration of adult-born OB interneurons. Our results also suggest that absence of an otherwise broadly expressed miRNA is a novel mechanism with which to achieve neuronal subtype specification.

Current understanding of acute myeloid leukemia (AML) assumes a developmental hierarchy, in which a minor fraction of primitive and quiescent leukemia stem cells (LSC) sustain clonal propagation of disease. These therapy-resistant LSC may be the basis of relapse, as supported by data correlating the presence of LSC gene expression signatures at diagnosis with poor prognosis (Ng et al, Nature 2016). Novel approaches tracing LSC fates at single cell level before, during and after chemotherapy (CTX) are needed to confirm their biological relevance and derive new, LSC-focused diagnostic and therapeutic strategies. We and others have previously linked key LSC properties, such as quiescence and therapy resistance, to complex transcriptional regulation orchestrated by miR-126 (Lechman et al, Cancer Cell 2016). Furthermore, we provided proof of concept that LSCs could be prospectively isolated as miR-126(high) cells exploiting a lentiviral reporter vector capturing miR-126 bioactivity in live cells with single cell resolution. To extend these studies, we transduced primary blasts from n=3 AML patients (pts) carrying NPM1 and FLT3-ITD mutations with the miR-126 reporter, followed by xenografting (PDX). Blasts showed intra-tumor heterogeneity (ITH) in terms of miR-126 activity, with a minor fraction identified as miR-126(high). Limiting dilution secondary transplantation of FACS sorted miR-126(high) and -(low) blasts proved strong enrichment of repopulating activity within the miR-126(high) compartment in all 3 pts. On the contrary, no LSC enrichment could be verified in the CD34+CD38- fraction in 1 patient, suggesting that high miR-126 activity represents a more robust LSC identifier than commonly used surface markers. Next, we investigated the impact of daunorubicin and cytarabine CTX on miR-126-reporter+ blasts (n=3 AML) in PDX. Surprisingly, overall miR-126 activity diminished in post CTX residual AML compared to controls, compatible with a loss of blast quiescence. CTX accentuated ITH by uncovering a subset of blasts with very high levels of miR-126, distinct from the bulk population, which may correspond to residual quiescent LSC (Fig A). We then performed bulk RNA sequencing on miR-126(high) and miR-126(low) subsets from CTX and control PDX. While miR-126(low) blasts from both groups expressed markers of myeloid differentiation, miR-126(high) blasts were enriched for published hematopoietic stem cell hallmark signatures. Integrating differentially expressed genes between miR-126(high) and-(low) subsets at steady state and post CTX, we extrapolated a novel 8 gene signature associated with miR-126(high) blasts. Of note, patients from the AML TCGA PanCancer Atlas Cohort (n=161) harboring overexpression in one or more of these 8 genes had significantly decreased overall survival (10 vs 19 months, Logrank test p-value = 0.018). To further test whether our 8-gene miR-126(high) signature reveals ITH in patients, we performed single cell RNA sequencing (scRNAseq) of AML patient BM aspirates at diagnosis (n= 6). Blasts were identified based on the detection of mutated NPM1 transcripts in single cells. In 5 out of 7 patients expression of the miR-126 signature mapped to specific clusters of blasts identified by unsupervised shared nearest neighbor algorithm, confirming that it identifies ITH in patient samples. Interestingly, we detected miR-126 signature(high) blasts in pts with poor prognosis (n=4) and not in those with favorable outcome (n=2) (Fig B). To investigate ITH across longitudinal samples, we next performed scRNAseq of residual AML from a representative patient assessed early after CTX. In line with our PDX CTX model displaying increased miR-126 ITH, blasts on day14 of induction CTX segregated into 2 different clusters: cluster 1 containing LSC-like cells with miR-126(high) signature and similar transcriptional profile to the diagnosis counterpart; cluster 2, instead, was composed of actively cycling blasts. Residual blasts at day30, in addition to uniformly expressing the miR-126(high) signature, differed from diagnosis and day14 blasts by displaying cell cycle quiescence and induction of oxidative phosphorylation genes (Fig C). In summary, we have set up and applied PDX LSC modeling to clinically relevant patient samples to address AML intra-tumor heterogeneity and to pinpoint novel relevant transcriptomic features of LSC at diagnosis and after chemotherapy. Figure Disclosures Gentner: Genenta Science: Consultancy, Equity Ownership, Research Funding.

BACKGROUND Most patients with GBM & an unmethylated O-6-methylguanine-DNA methylase (MGMT) gene promoter, have a poor prognosis with approximately 20% of patients surviving to 2 years. Poor prognosis is likely related to factors including a highly immunosuppressive tumor microenvironment (TME). The TME in GBM is mainly composed of tumor associated macrophages (TAMs) & microglia. A subset of tumor-infiltrating macrophages characterized by expression of the angiopoietin receptor Tie2 (TEMs) have features of M2-TAMs, promote tumor angiogenesis & are infrequently found in normal organs. Tie2 is significantly upregulated upon homing to tumors. Gene therapy technology has allowed TEMs to be used as carriers for the local and tumor restricted release of interferon-α (IFN). IFN has antitumor effects, inhibits angiogenesis & modulates the immune system. Cell-based delivery of IFN into the TME by TEMs is expected to provide efficacy, taking advantage of pleiotropic anti-tumor effects & avoiding tolerability issues associated with systemic IFN treatment. We are currently conducting a Phase I/IIa clinical study in Milan (INCB & OSR) to evaluate this therapeutic approach (Temferon) in 21 patients with GBM & unmethylated MGMT promoter (EudraCT Number 2018-001404-11). Eligible patients are identified immediately after first surgical resection. After screening, harvesting of HSPCs occurs followed by 6 weeks of radiotherapy. Non-myeloablative conditioning consists of BCNU & thiotepa followed by administration of non-manipulated HSPCs and Temferon. RESULTS In Part A of the study, 3 cohorts of 3 patients will receive escalating doses of Temferon. After Part A, a single dose of Temferon will be studied in a further 12 patients (Part B). Criteria for study eligibility are the same for both Part A and Part B. Part A is ongoing with the first 2 patients recruited (June 2019) and Temferon is scheduled for administration. An update on the status and progress of the study will be provided.

Genetically targeted T cells promise to solve the feasibility and efficacy hurdles of adoptive T-cell therapy for cancer. Selecting a target expressed in multiple-tumor types and that is required for tumor growth would widen disease indications and prevent immune escape caused by the emergence of antigen-loss variants. The adhesive receptor CD44 is broadly expressed in hematologic and epithelial tumors, where it contributes to the cancer stem/initiating phenotype. In this study, silencing of its isoform variant 6 (CD44v6) prevented engraftment of human acute myeloid leukemia (AML) and multiple myeloma (MM) cells in immunocompromised mice. Accordingly, T cells targeted to CD44v6 by means of a chimeric antigen receptor containing a CD28 signaling domain mediated potent antitumor effects against primary AML and MM while sparing normal hematopoietic stem cells and CD44v6-expressing keratinocytes. Importantly, in vitro activation with CD3/CD28 beads and interleukin (IL)-7/IL-15 was required for antitumor efficacy in vivo. Finally, coexpressing a suicide gene enabled fast and efficient pharmacologic ablation of CD44v6-targeted T cells and complete rescue from hyperacute xenogeneic graft-versus-host disease modeling early and generalized toxicity. These results warrant the clinical investigation of suicidal CD44v6-targeted T cells in AML and MM. ""Genetically targeted T cells promise to solve the feasibility and efficacy hurdles of adoptive T-cell therapy for cancer. Selecting a target expressed in multiple-tumor types and that is required for tumor growth would widen disease indications and prevent immune escape caused by the emergence of antigen-loss variants. The adhesive receptor CD44 is broadly expressed in hematologic and epithelial tumors, where it contributes to the cancer stem\\\/initiating phenotype. In this study, silencing of its isoform variant 6 (CD44v6) prevented engraftment of human acute myeloid leukemia (AML) and multiple myeloma (MM) cells in immunocompromised mice. Accordingly, T cells targeted to CD44v6 by means of a chimeric antigen receptor containing a CD28 signaling domain mediated potent antitumor effects against primary AML and MM while sparing normal hematopoietic stem cells and CD44v6-expressing keratinocytes. Importantly, in vitro activation with CD3\\\/CD28 beads and interleukin (IL)-7\\\/IL-15 was required for antitumor efficacy in vivo. Finally, coexpressing a suicide gene enabled fast and efficient pharmacologic ablation of CD44v6-targeted T cells and complete rescue from hyperacute xenogeneic graft-versus-host disease modeling early and generalized toxicity. These results warrant the clinical investigation of suicidal CD44v6-targeted T cells in AML and MM.""

Lineage specification is thought to be largely regulated at the level of transcription, where lineage-specific transcription factors drive specific cell fates. MicroRNAs (miR), vital to many cell functions, act posttranscriptionally to decrease the expression of target mRNAs. MLL-AF4 acute lymphocytic leukemia exhibits both myeloid and B-cell surface markers, suggesting that the transformed cells are B-cell myeloid progenitor cells. Through gain- and loss-of-function experiments, we demonstrated that microRNA 126 (miR-126) drives B-cell myeloid biphenotypic leukemia differentiation toward B cells without changing expression of E2A immunoglobulin enhancer-binding factor E12/E47 (E2A), early B-cell factor 1 (EBF1), or paired box protein 5, which are critical transcription factors in B-lymphopoiesis. Similar induction of B-cell differentiation by miR-126 was observed in normal hematopoietic cells in vitro and in vivo in uncommitted murine c-Kit + Sca1 + Lineage − cells, with insulin regulatory subunit-1 acting as a target of miR-126. Importantly, in EBF1-deficient hematopoietic progenitor cells, which fail to differentiate into B cells, miR-126 significantly up-regulated B220, and induced the expression of B-cell genes, including recombination activating genes-1/2 and CD79a/b. These data suggest that miR-126 can at least partly rescue B-cell development independently of EBF1. These experiments show that miR-126 regulates myeloid vs. B-cell fate through an alternative machinery, establishing the critical role of miRNAs in the lineage specification of multipotent mammalian cells.

Summary Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks, making them attractive candidate HSC regulators. We report that miR-126, a miRNA expressed in HSC and early progenitors, plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion, resulting in expansion of mouse and human long-term repopulating HSC. Conversely, enforced miR-126 expression impaired cell-cycle entry, leading to progressively reduced hematopoietic contribution. In HSC/early progenitors, miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway, attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size, demonstrating the importance of miRNA in the control of HSC function., Graphical Abstract Highlights ► miR-126 is a novel regulator of the HSC quiescence/proliferation equilibrium ► Reduction in miR-126 induces an expansion of long-term HSC without exhaustion ► Constitutive miR-126 expression promotes HSC quiescence and progenitor proliferation ► miR-126 attenuates PI3K/AKT activation in response to cytokine stimulation, miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway to promote HSC quiescence and progenitor proliferation.

Hematopoietic stem and progenitor cell (HSPC) expansion remains an important unmet goal for ex vivo gene therapy based on gene addition and editing to compensate for the negative impact of the gene transfer procedure enabling faster engraftment and less complications. Additionally, ex vivo expansion of corrected cells may improve efficacy at more sustainable manufacturing costs by downscaling transduction. To date, our knowledge of precise mechanisms of action of expansion compounds is limited, and it remains unclear whether cord blood expansion protocols also maintain stemness of mobilized peripheral blood CD34+ cells (mPB), the preferred HSPC source for gene therapy. We performed serial (day 0,4,8) droplet-based single cell RNA sequencing (scRNAseq) on lentivirally transduced mPB expanded with UM171 to dissect cellular heterogeneity, monitor population dynamics over time and identify a transcriptional profile of primitive cells in culture. By associating published HSPC gene expression profiles to our scRNAseq dataset from uncultured mPB, we found that 45% of cells harbored a myelo-lymphoid signature. Smaller cell clusters expressed a shared erythroid (ERY) and megakaryocytic (MK) signature (20%), or a more primitive multipotent HSC-like signature (15%) characterized by enhanced JAK/STAT signaling and expression of HSC associated genes (AVP, HOPX, ID3). Unsupervised ordering of cells within pseudotime separated emerging MK/ERYpoiesis (FCER1A, HBD) from lympho-myelopoiesis (CD52, JUN), with intermediate states of more primitive progenitors located in between. After 4 days in culture, we noted a general increase in nuclear and mitochondrial gene transcription with activation of oxidative metabolism, paralleled by cell cycle activation, as expected from cytokine stimulation. By d8 of culture these changes leveled off but remained higher than uncultured cells. Of note, cells at d8 revealed an activation of cellular stress response pathways (e.g. TNFa, IFNg) hinting towards a compromised culture that may eventually exhaust HSC. Unsupervised clustering of cultured mPB highlighted a dramatic expansion (70-80%) of MK/ERY progenitor cells with high cycling activity with only 20-30% cells showing myelo-lymphoid transcriptional features. In line, pseudotime analysis highlighted a main ERY and MK trajectory separated from that of cells characterized by the expression of HSPC genes (HOPX, SPINK2) and of an emerging myeloid trajectory (MPO). To profile HSC in culture, we sorted and sequenced CD34+90+201+ cells from d4 expansion culture (3% of total cells), which we show to contain >70% of SCID repopulating potential. ScRNAseq revealed transcriptional similarity with the myelo-lymphoid progenitor cluster identified in the unsorted d4 culture. Unsupervised clustering of the CD34+90+201+ population revealed cell cycle dependent heterogeneity, identifying a highly quiescent cluster with expression of HSC-like signatures. This cluster was also characterized by relatively low gene expression, possibly reflecting a non-activated cell state consistent with primitive HSPC. Pseudotime analysis produced a four-branched minimum spanning tree, which retained a clear cell cycle and metabolic effect. Top variable genes included cell cycle, glycolytic, mitochondrial and ribosomal genes, identifying different metabolic modules along the branched trajectory. These results highlight that cell heterogeneity within a purified, HSC-enriched population is driven mainly by metabolic activation and cell cycle status. As a complementary approach, we purified LT-HSC from uncultured mPB (CD34+38-90+45RA-49f+), marked them with CFSE and expanded them in UM171 culture. LT-HSCs expanded on average 3.5 fold in 7 days, with the following distribution: 0 divisions: 3%; 1: 26%; 2: 47%; 3: 21%; 4: 3%. We performed scRNAseq on LT-HSC pre culture and after 7d separating a highly proliferative (≥2 divisions) and quiescent (0 - 1 division) fraction, allowing us to obtain unprecedented insight into the response of engrafting cells to ex vivo culture and set a framework to dissect self-renewal (HSC expansion), HSC maintenance and loss through differentiation as potential culture outcomes. Our combined functional/transcriptomic approach will define new HSC markers in culture and greatly facilitate side-by-side comparison of different expansion protocols towards rapid clinical translation. Disclosures No relevant conflicts of interest to declare.

MicroRNAs are essential regulators of normal and malignant hematopoiesis. miRNAs are relevant for gene therapy, since they can be exploited to fine-tune the expression profile of vector constructs or to alter viral tropism (GentnerN Chiriaco et al, 2014; Escobar et al, 2014) and described the function of miR-126 in HSC where it regulates the balance between quiescence and self-renewal (Lechman et al, 2012). We here report a novel role for miR-126 in the induction and maintenance of high-grade B cell malignancies. By ectopically expressing miR-126 in transplanted BM cells, we observed that up to 60% of mice (n=71) developed B cell malignancies. LV insertion site (IS) analysis revealed that all tumors were monoclonal. We then tracked back leukemic clone to different hematopoietic lineages prospectively purified from the mice 2-6 months before disease onset. IS sharing between normal lineages and leukemic clone suggests stem or multipotent progenitor cell as origin for most tumors. Importantly, we show that miR-126 is the direct cause of genesis and maintenance of leukemia, since leukemogenesis is abolished when miRNA expression is inhibited by doxycycline (doxy) using a tetracycline-repressible miR-126 cassette, and established symptomatic leukemia completely regresses when miR-126 is switched off by doxy through induction of apoptosis. Transcriptional profiling indicated that miR-126 regulates multiple genes in p53 pathway both in murine blasts and in normal human CD34+ cells. Previous work suggested expression of miR-126 in acute lymphoblastic leukemia (ALL) and germinal center lymphoma. To further establish the relevance of miR-126 in human disease, we measured miR-126 expression in blasts from 16 adult patients with ALL. miR-126 was highly expressed in most studied ALL cases (Phil+: n=11, Phil-: n=5), at similar levels as CD34+ cells. We then down-regulated miR-126 in primary blasts from human B-ALL patients (n=5), and we observed increased apoptosis and impaired engraftment in xenograft models after primary and secondary transplantation (miR-126/KD: n=32 mice; Ctrl: n=37 mice), demonstrating the relevance of miR-126 in human B-ALL. In conclusion, we present a novel spontaneous mouse model for high grade B cell malignancies which are addicted to miR-126 expression, provide insight into the dynamic process of leukemogenesis by clonal IS tracking and unveil key tumor signaling pathways controlled by miR-126. Down-regulation of miR-126 could be exploited as therapeutic strategy in ALL, since it would deplete leukemic cells while expanding normal HSC, two ways to restore normal hematopoieis.

Lentiviral (LV)-based hematopoietic stem and progenitor cell (HSPC) gene therapy is becoming a promising alternative to allogeneic stem cell transplantation for curing genetic diseases. To potentially improve the efficacy, safety and economic sustainability of HSPC transduction, we reasoned to genetically manipulate only the more potent CD34+CD38- HSPC, thereby improving HSPC maintenance in culture in the absence of differentiating cells and downscaling the cell therapy product by a factor of ten without compromising long-term engraftment. This approach would also decrease the total load of vector integration infused in the patients, thus improving its overall safety.First, we determined the engraftment kinetics of CD34+ mobilized peripheral blood (mPB) subpopulations over a 24wk xenotransplantation period. We sorted CD34+ mPB into 4 fractions with increasing expression levels of CD38 and marked each fraction with a specific fluorescent protein allowing to track the population of origin driving hematopoietic reconstitution. Differentially labeled fractions were mixed, and various combinations of CD38-, CD38int and CD38hi HSPC were injected into NSG mice (2 exp, n=30). Almost all long-term repopulating capacity (>90%) was contained within CD34+CD38- cells, and these cells took over hematopoiesis by 9wks. Instead, early reconstitution was mainly driven by CD34+CD38int progenitor cells.In the prospect of a clinical translation, we then modeled the co-administration of gene-modified CD34+CD38- mPB cells with uncultured CD34+CD38int/+ supporter cells aimed to drive fast hematopoietic recovery (3 exp, n=38). Repopulation by gene-modified CD34+CD38- cells was slower (15wks) and incomplete ( 5 wks and re-established long-term (24wks) gene marking up to 85%, thus allowing to benefit from prompt hematopoietic recovery driven by transiently repopulating CD38int/+ supporter cells.Last, we optimized LV transduction in the framework of an improved culture protocol. Exposing CD34+ or CD34+CD38- mPB cells to prostaglandin E2 (PGE2) increased transduction efficiency 1.5-2.5x, allowing to markedly reduce pre-stimulation and LV exposure times better preserving HSC functions. Importantly, the higher gene-transfer efficiency was maintained for up to 24 wks following xenotransplantation (n=33), suggesting that PGE2 facilitated LV transduction in long-term HSC.In summary, these results support the clinical development of novel HSPC gene therapy protocols based on the modification of highly purified HSC subsets, with the prospect to improve the efficacy, safety and feasibility of future ex vivo gene therapy studies.

Globoid cell leukodystrophy (GLD) is a demyelinating lysosomal storage disease due to the deficiency of the galactocerebrosidase (GALC) enzyme. The favorable outcome of hematopoietic stem and progenitor cell (HSPC)-based approaches in GLD and other similar diseases suggests HSPC gene therapy as a promising therapeutic option for patients. The path to clinical development of this strategy was hampered by a selective toxicity of the overexpressed GALC in the HSPC compartment. Here, we presented the optimization of a lentiviral vector (LV) in which miR-126 regulation was coupled to codon optimization of the human GALC cDNA to obtain a selective and enhanced enzymatic activity only upon transduced HSPCs differentiation. The safety of human GALC overexpression driven by this LV was extensively demonstrated in vitro and in vivo on human HSPCs from healthy donors. No perturbation in the content of proapoptotic sphingolipids, gene expression profile, and capability of engraftment and mutlilineage differentiation in chimeric mice was observed. The therapeutic potential of this LV was then assessed in a severe GLD murine model that benefited from transplantation of corrected HSPCs with longer survival and ameliorated phenotype as compared to untreated siblings. This construct has thus been selected as a candidate for clinical translation.

To investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance. Lechman et al. show that miR-126 targets the PI3K/AKT/MTOR signaling pathway to preserve quiescence, increase self-renewal, and promote chemotherapy resistance of acute myeloid leukemia stem cells (LSC). Reducing the miR-126 level impairs LSC maintenance in contrast to expanding normal hematopoietic stem cells.

Several microRNAs (miRNAs) that are either specifically enriched or highly expressed in neurons and glia have been described, but the identification of miRNAs modulating neural stem cell (NSC) biology remains elusive. In this study, we exploited high throughput miRNA expression profiling to identify candidate miRNAs enriched in NSC/early progenitors derived from the murine subventricular zone (SVZ). Then, we used lentiviral miRNA sensor vectors (LV.miRT) to monitor the activity of shortlisted miRNAs with cellular and temporal resolution during NSC differentiation, taking advantage of in vitro and in vivo models that recapitulate physiological neurogenesis and gliogenesis and using known neuronal- and glial-specific miRNAs as reference. The LV.miRT platform allowed us monitoring endogenous miRNA activity in low represented cell populations within a bulk culture or within the complexity of CNS tissue, with high sensitivity and specificity. In this way we validated and extended previous results on the neuronal-specific miR-124 and the astroglial-specific miR-23a. Importantly, we describe for the first time a cell type- and differentiation stage-specific modulation of miR-93 and miR-125b in SVZ-derived NSC cultures and in the SVZ neurogenic niche in vivo, suggesting key roles of these miRNAs in regulating NSC function.

A productive immune response requires transient upregulation of the microRNA miR-155 in hematopoietic cells mediating innate and adaptive immunity. In order to investigate miR-155 in the context of tumor-associated immune responses, we stably knocked down (KD) miR-155 in the myeloid compartment of MMTV-PyMT mice, a mouse model of spontaneous breast carcinogenesis that closely mimics tumor-host interactions seen in humans. Notably, miR-155/KD significantly accelerated tumor growth by impairing classic activation of tumor-associated macrophages (TAMs). This created an imbalance toward a protumoral microenvironment as evidenced by a lower proportion of CD11c(+) TAMs, reduced expression of activation markers, and the skewing of immune cells within the tumor toward an macrophage type 2/T helper 2 response. This study highlights the importance of tumor-infiltrating hematopoietic cells in constraining carcinogenesis and establishes an antitumoral function of a prototypical oncomiR.

Over the past decades outcomes of clinical hematopoietic stem cell transplants have established a clear relationship between the sources of hematopoietic stem cells (HSCs) infused and their differential homing and engraftment properties. For a long time, bone marrow (BM) harvest has been the preferred source of hematopoietic stem and progenitor cells (HSPCs) for hematopoietic reconstitution following myeloablative conditioning regimen. At present, mobilized peripheral blood (PB) is commonly used for hematopoietic cells transplantation in both adults and children, particularly in the autologous setting, and it has progressively replaced BM as the source of HSCs.HSCs are maintained in their niche by binding to cellular determinants through adhesion molecules and diverse strategies are currently used to promote their egress from BM to PB. Traditionally, the growth factor granulocyte-colony stimulating factor (G-CSF) represents the gold standard agent to mobilize HSPCs for transplantation. Nevertheless, other compounds have been recently tested. One of the most successful mobilizing agents is Plerixafor (AMD3100, Mozobil™), a bicyclam molecule that selectively and reversibly antagonizes the binding of stromal cell derived factor-1 (SDF-1), located on the surface of BM stromal cells and osteoclasts, to chemokine CXC-receptor-4 (CXCR4), located on the surface of HSPCs, with the subsequent mobilization in the blood. The use of this drug is currently approved by FDA and EMA in combination with G-CSF, in patients affected by lymphoma or multiple myeloma whose cells mobilize poorly with G-CSF alone. Clinical trials demonstrated that Plerixafor alone safely and rapidly mobilizes HSCs also in healthy donors, beta-thalassemia patients and pediatric patients affected by malignancies. Previous characterization studies on non-human primates and human samples of Plerixafor mobilized cells in comparison to cells mobilized by G-CSF alone or in combination with Plerixafor showed a different expression profile, cell composition and engrafting potential in a xenotransplant model. From these studies remains unsolved whether Plerixafor, G-CSF, or their combination mobilizes different primitive HSC populations, defined both by multimarker immunophenotype and in vivo functional analysis. In the present study we investigated by controlled comparative analysis the functional and molecular hallmarks of human HSCs collected from BM, G-CSF and/or Plerixafor mobilized peripheral blood. We show that Plerixafor alone mobilizes preferentially long-term hematopoietic stem cells (LT-HSCs), defined as CD34+CD38/lowCD90+CD45RA-CD49f+ cells and primitive populations of HSCs. These cells possess higher ability to home to hematopoietic niches and engraft in NOD/SCID/IL2rγnull (NSG) mice, resulting in enriched scid-repopulating cell frequency, in comparison to other sources. The higher content of CXCR4+ and CD49f+ cells correlates with this feature. Furthermore, global gene expression profiling highlights the superior in vivo reconstitution activity of Plerixafor mobilized cells. The "stemness" signature of cells dislodged from their niche by the drug is attenuated by the combined use with G-CSF, which emphasizes the gene expression profile induced by G-CSF treatment. These data indicate that a qualitative advantage accounts for the superior performance of Plerixafor mobilized cells. These findings provide the rationale for using a suboptimal dose of more primitive HSCs when target cell number for transplantation is limited, or when G-CSF mobilization is too risky like in sickle cell anemia patients. Moreover, CD34+ cells mobilized by Plerixafor alone or with the combination of G-CSF are efficiently transduced by a lentiviral vector encoding for human ß-globin gene (GLOBE LV) and are able to engraft and differentiate in vivo, supporting their use for gene therapy applications. Disclosures Ciceri: MolMed SpA: Consultancy.

MicroRNA-126 reinforces hematopoietic stem cell (HSC) quiescence by dampening PI3K-AKT signaling. We recently reported key functions of miR-126 in acute leukemia: in human AML, it was required to maintain leukemic stem cell (LSC) quiescence (Lechman et al, Cancer Cell 2016), while its ectopic expression in mouse HSC induced leukemia (75% B-ALL, 25% AML) that fully regressed when switching a tetracycline-repressible miR-126 cassette off (Nucera et al, Cancer Cell 2016). RNA sequencing showed that miR-126 targeted cell cycle, apoptosis and p53 response genes, prevented differentiation and sustained oncogenic/pro-survival pathways typically associated with stem and progenitor cells (Kit, Wnt, Thy1, Jak/Stat, Bcl2). We quantified miR-126 expression levels in a cohort of 45 newly diagnosed AML patients (n=38 de novo, of which 37% favorable, 34% int-1, 16% int-2, 13% adverse according to the ELN classification) presenting at the San Raffaele Hospital between 2010 and 2015 using a robust digital droplet PCR assay. In addition to core-binding factor (CBF) mutated AML, we found that the group of AML with chromosomal aneuploidy showed significantly elevated miR-126 levels, suggesting that this subgroup is characterized by high LSC frequencies and/or a specific need to suppress p53 responses, a hypothesis supported by the data obtained in our mouse model. To measure miR-126 at single cell resolution, we stably transduced primary AML blasts (n=5 diseases) with a lentiviral miR-126 reporter vector, xenotransplanted them into NSG mice and quantified miR-126 activity by FACS in the engrafted cells recovered from the mice's bone marrow. Across different genetic subgroups including CBF leukemia, cells with the highest miR-126 activity were enriched in the CD34+ or CD34+CD38- fractions, consistent with LSC. To facilitate prospective LSC isolation based on miRNA activity, we screened a series of combination miRNA reporters incorporating response elements for LSC-enriched and LSC-depleted miRNAs and have now identified an optimized construct that highlights easily sortable, distinct subpopulations that are currently undergoing functional validation. Applying this tool to AML with complex and monosomal karyotype, we are addressing whether high miR-126 expression refers to elevated LSC frequency or is a specific feature of this clinically relevant AML entity. We next applied the miR-126 reporter to n=15 primary, human B-ALL measuring miR-126 activity in the xenograft. Surprisingly, we identified well-separated blast subpopulations that differed in miR-126 activity within single diseases. Heterogeneity for miR-126 appeared to be a general feature of B-ALL as we detected 2-3 subpopulations in most of the diseases studied. We purified miR-126(high) and miR-126(low) B-ALL subpopulations from the primografts and verified up to 1log differences in miR-126 expression, while we detected equal levels of the BCR-ABL fusion transcript in all subpopulations from Philadelphia+ B-ALL confirming their neoplastic nature. When transplanting miR-126(high) and miR-126(low) subpopulations into secondary or tertiary recipients, post-sorting miR-126 levels were tightly maintained indicating that miR-126 levels were static rather than dynamically regulated in distinct B-ALL subpopulations, as expected from a subclonal architecture. RNAseq performed on miR-126(high) and miR-126(low) human B-ALL fractions evidenced a miR-126 signature reminiscent of the one obtained in the B-ALL mouse model and cord blood CD34+ cells, uncovering physiological miR-126 activity in human primary B-ALL. Taken together, these data support a broad, pathogenetically important role for miR-126 in human AML and B-ALL that goes beyond LSC and open up opportunities to better understand leukemia disease biology, dissect intratumoral heterogeneity and therapeutically target resistant and refractory disease, considering that miR-126 knockdown expands normal HSC while depleting the leukemia. Disclosures Ciceri: MolMed SpA: Consultancy.

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.

New neurons are continuously generated from neural stem cells with astrocyte properties, which reside in close proximity to the ventricle in the postnatal and adult brain. In this study we found that microRNA-124 (miR-124) dictates postnatal neurogenesis in the mouse subventricular zone. Using a transgenic reporter mouse we show that miR-124 expression is initiated in the rapid amplifying progenitors and remains expressed in the resulting neurons. When we stably inhibited miR-124 in vivo, neurogenesis was blocked, leading to the appearance of ectopic cells with astrocyte characteristics in the olfactory bulb. Conversely, when we overexpressed miR-124, neural stem cells were not maintained in the subventricular zone and neurogenesis was lost. In summary, our results demonstrate that miR-124 is a neuronal fate determinant in the subventricular zone.

Over the past decades outcomes of clinical hematopoietic stem cell transplants have established a clear relationship between the sources of hematopoietic stem cells (HSCs) infused and their differential homing and engraftment properties. For a long time, bone marrow (BM) harvest has been the preferred source of hematopoietic stem and progenitor cells (HSPCs) for hematopoietic reconstitution following myeloablative conditioning regimen. At present, mobilized peripheral blood (PB) is commonly used for hematopoietic cells transplantation in both adults and children, particularly in the autologous setting, and it has progressively replaced BM as the source of HSCs. So far, the intrinsic molecular features of human primitive HSCs from different sources have not been investigated in comparative studies to unravel their variable reconstitution potential. Diverse strategies are currently used to disengage HSCs from the niche, promoting egress from BM to PB. Traditionally the growth factor granulocyte-colony stimulating factor (G-CSF) represents the gold standard agent to mobilize HSPCs for transplantation. Nevertheless, many other compounds have been tested to this regard. One of the most successful mobilizing agents is Plerixafor (AMD3100, Mozobil™), a bicyclam molecule that selectively and reversibly antagonizes the binding of stromal cell derived factor-1 (SDF-1), located on the surface of BM stromal cells and osteoclasts, to chemokine CXC-receptor-4 (CXCR4), located on the surface of HSPCs, with the subsequent mobilization in the PB. This drug, which was shown in preclinical combination studies with G-CSF to enhance mobilization compared to G-CSF alone, is currently approved by FDA and EMA "in combination with G-CSF to enhance the mobilization of HSCs into the peripheral blood for collection and autologous transplantation of patients affected by lymphoma or multiple myeloma whose cells mobilize poorly" We investigated functional and molecular hallmarks of human HSCs from different sources, i.e. BM and PB following mobilization by G-CSF and/or Plerixafor. We show that Plerixafor alone mobilizes preferentially long-term hematopoietic stem cells (LT-HSCs), defined as CD34+ CD38/low CD90+ CD45RA- CD49f+ cells and primitive populations of HSCs. These cells are able to provide stable long-term hematopoietic engraftment in NOD/SCID/IL2rγnull (NSG) mice, resulting in enriched scid-repopulating cell frequency, in comparison to other sources. The quiescence status of these cells correlates with the enriched scid-repopulating cell frequency. Noteworthy, the combined use of G-CSF and Plerixafor mobilizes a CD34+ population enriched in immature cells and with a lower engraftment capacity respect to cells mobilized by Plerixafor alone. Since the signaling provided by the interaction of SDF-1 with CXCR4, plays an essential role in maintaining HSC quiescence and regulating homing, we analyzed the CXCR4 expression. Interestingly, this analysis reveals that the proportion of CXCR4+ primitive cells was lower when using G-CSF combined to Plerixafor in respect to Plerixafor alone. These data indicate that the combination of the two mobilizing agents induce a higher amount of circulating CD34+ cells but containing a lower proportion of cells capable of homing to BM in NSG mice. . As a result, at a defined dose of transplanted CD34+ cells, less SRCs are observed when G-CSF is added to Plerixafor. Indeed, it is expected to observe also a rapid rescue of hematopoiesis in myeloablated subjects conferred by high amount of short-term progenitors. Insights into the transcriptional program reveal the molecular machinery underlying stemness features of cells derived from different sources, defining their specific functional properties. Noteworthy, CD34+ cells exposed to Plerixafor but still resident in the BM acquire an intermediate signature between steady-state and circulating cells, suggesting an effect of this agent on HSC function. From preliminary data, genes of Prostaglandin signaling are up-regulated in HSCs mobilized by Plerixafor, suggesting a role of this pathway. These data uncover unique HSCs properties shaped by their origin and illuminate the choice of different transplantation strategies accordingly to the clinical need. Disclosures No relevant conflicts of interest to declare.

Methods to analyze the clonality of an adverse event in preclinical or clinical retroviral stem cell gene therapy protocols are needed. We analyzed the progeny of retrovirally transduced human peripheral blood progenitor cells (PBPCs) after transplantation and engraftment in immune-deficient mice. The integration site of the provirus serves as a unique tag of the individual transduced PBPC. A plasmid library of junctions between proviral and human genomic DNA was generated. We were able to detect individual transduced cell clones that amounted to 0.14%–0.0001% of chimeric bone marrow cells. This is the first report in which the contribution of individual marrow-repopulating cells to human hematopoiesis is directly quantified.

Synergistic deregulation of HOXA9 and the HOX-gene cofactor MEIS1 is a commonly observed phenomenon in acute myeloid leukemia (AML). The leukemogenic potential of aberrant Hoxa9 and Meis1 expression has been shown in several AML models. However, the molecular mechanisms behind Hoxa9- and Meis1-induced leukemogenesis are still not well understood. In order to identify functionally relevant Meis1-induced microRNAs (miRNA), we profiled the global miRNA expression using a Hoxa9-Meis1 murine AML progression model. This two-step model allowed us to quantify miRNAs at a pre-leukemic stage through the overexpression of the proto-oncogene Hoxa9 (Hoxa9/ctrl), as well as after full leukemic transformation through co-overexpression of Hoxa9 and Meis1 (Hoxa9/Meis1). The pre-leukemic stage is characterized by in vitro immortalization without in vivo engraftment, whereas the transplanted leukemic cells induce full-blown AML in vivo. MiR-155 turned out to be one of the most significant differentially expressed miRNA species and its upregulation was independently validated in Hoxa9/Meis1 cells by qRT-PCR. Subsequent analysis of various AML subtypes (CN-AML, t(11q23), t(8;21), t(15;17), n=38) showed significantly elevated levels of miR-155 in CN-AML with NPM1mut (n=10, p Therefore, to dissect the leukemic potential of miR-155 to program mbm, 5-FU-stimulated mbm cells were retrovirally transduced with miR-155, leading to significantly increased proliferation in vitro (p The leukemic potency of Hoxa9/miR-155 was further investigated in a murine transplantation model in vivo. Transplantation of mbm co-overexpressing Hoxa9/miR-155 led to significantly increased engraftment levels already after four weeks (wks) (57.8%±31.3, n=16) compared to Hoxa9/ctrl (11.7%±19.3%, p Considering the central role of the Hoxa9/Meis1 in both myeloid and lymphoid acute leukemias, we demonstrate for the first time the leukemogenic relevance of a miRNA within this transcriptional axis. Disclosures No relevant conflicts of interest to declare.

We have recently shown that miR-126 expression faithfully identifies the engrafting fraction of bone marrow (BM) and cord blood (Gentner et al, Sci Transl Med 2010), in which it regulates hematopoietic stem cell (HSC) pool size by modulating cell cycle progression (Lechman et al., Cell Stem Cell 2012). Antagonizing miR-126 expands HSC by enhancing PI3K/AKT signaling, without causing their exhaustion or transformation. By reconstituting mice with lentivirally transduced BM cells ectopically expressing miR-126, we noted differential effects in HSC and progenitors: while miR-126 overexpressing (126OE) HSC were more quiescent and outcompeted by HSC with physiologic miR-126 expression, progenitor subsets were increased in number and proliferated more upon 126OE. In particular, B lymphopoiesis was enhanced, with the appearance of 126OE-vector marked, CD45(low)CD19+Ig(-) B cell progenitors in the peripheral blood. This abnormal population was transiently observed in all 126OE (n=45), but in none of the control mice (n=30), and BM B cell precursors and progenitors from 126OE mice showed significantly reduced levels of apoptosis, as well as a differentiation block at the immature B cell stage. At least part of this effect was due to interference of miR-126 with p53 activation. Consequentially, 20-30% of 126OE mice (as compared to 0% of control mice) followed over 1 year developed vector-marked, high-grade B-cell neoplasms which resembled diffuse large B-cell lymphoma, lymphoblastic lymphoma or acute lymphoblastic leukemia (ALL). Strikingly, by using a tetracycline-regulated, conditional miR-126OE vector, we observed full regression of the expanded abnormal B cell population after switching miR-126 expression off by doxycycline administration, demonstrating that miR-126 is an oncogenic driver in hematologic malignancies. To establish the relevance of miR-126 in human disease, we studied primary samples from patients with acute leukemia. We measured miR-126 expression in blasts from 12 adult patients with acute lymphoblastic leukemia (ALL). miR-126 was highly expressed in all studied cases of ALL (Phil+: n=7, Phil-: n=5), often surpassing the levels found in normal HSC. Unlike in HSC, miR-126 expression was independent from expression of its host gene EGFL7, suggesting an ALL-specific regulation of the miR-126 locus. By exploiting a lentiviral miR-126 reporter vector, we quantified miR-126 activity with single cell resolution after xenotransplantation of miR-126 reporter vector transduced ALL blasts into NSG mice. In 7 out of 12 ALL cases, we identified distinct subpopulations exhibiting different levels of miR-126 activity, and miR-126 (hi) ALL cells were more frequently contained in the CD34+ cell fraction. Moreover, high miR-126 expression correlated with faster and higher ALL engraftment in NSG mice. In acute myeloid leukemia (AML), miR-126 is significantly enriched in leukemic stem cells (LSC) where it governs quiescence and chemotherapy resistance (see abstract by Lechman et al), and analogous studies in ALL are ongoing. To further substantiate the role of miR-126 in AML, we studied miR-126 activity in 11 primary AML cases (4 AML with CBF, core binding factor, mutations; 7 AML with normal karyotype) using the reporter vector technology and the NSG xenotransplantation model. Globally, we found highest miR-126 activity in CBF-mutated AML. However, there was substantial heterogeneity in subpopulations of each single disease. Significantly higher miR-126 levels were found in CD34+, and particularly in CD34+CD38- AML cells, a fraction that is enriched for LSC. To see whether there is a correlation between miR-126 expression and disease progression, we measured miR-126 levels in sorted blasts from paired diagnosis/relapse (n=6) and diagnosis/chemotherapy-refractory (n=8) patient samples. Strikingly, we found a significant up-regulation of miR-126 in the disease progression sample, suggesting a mechanistic role for miR-126 in AML persistence after chemotherapy. In summary, these data support a clinically relevant role of miR-126 in the pathogenesis, treatment response and progession of acute leukemias. Authors AG and SN contributed equally, and LN and BG contributed equally. Disclosures: No relevant conflicts of interest to declare.

Background Targeting the interactions between tumor cells and their microenvironment is an exciting new frontier in cancer therapy. The biology of acute myeloid leukemia (AML) and multiple myeloma (MM) is characterized by addiction to specific signals uniquely provided within the bone marrow (BM), where tumor cells preferentially home and locally thrive. The hyaluronate receptor CD44 was shown to be required for retroviral-induced leukemogeneis in syngeneic mouse models. Conversely, CD44 mAbs interfere with human leukemia initiation in immunocompromised mice by inhibiting leukemia stem cell homing to the bone marrow (BM). The therapeutic potential of CD44 mAbs is also under clinical investigation in humans. Much less is known on the role of the differently spliced CD44 variant isoforms. The expression of exon 6 (CD44v6) conveys additional properties to standard CD44, like binding to osteopontin and cooperation with different tyrosine kinase receptors (RTKs), like VEGF receptor type II and c-Met. Interestingly, CD44v6 is the most abundantly expressed CD44 isoform in both AML and MM, where it correlates with a bad prognosis. Since CD44v6 expression is much more tumor-restricted than CD44, targeting this isoform may have a better efficacy/toxicity profile than targeting the standard molecule. Aim To preclinically validate CD44v6 as a therapeutic target in AML and MM Results By FACS analysis and RT-qPCR, we established CD44v6 over expression in a relevant fraction of leukemic blasts from AML pts (15/25, 60%) with preference for the M4-5 FAB subtypes, and in the majority of malignant plasmacells from MM pts (13/15, 87%). CD44v6 was also over expressed on THP-1, Kasumi and U937 human AML cells, and on MM.1S, XG-6 and XG-7 MM cells. To address the specific role of CD44v6 in BM homing, we pretreated MM1.S cells with either a CD44 mAb (SFF-2) or a CD44v6 mAb (VFF-18) and infused them i.v. in NSG mice. Unexpectedly, while SFF-2 almost completely inhibited early (18hrs) homing to the BM compared with an irrelevant mAb, VFF-18 had no effect. To rule out confounding variables associated with specific mAb clones, we silenced CD44v6 expression in MM1.S cells by lentiviral-mediated shRNA transduction and confirmed no difference in BM homing compared with control LV-transduced cells. Longer follow-ups (4-6 weeks) however revealed that, despite unaltered rates of in vitro proliferation, CD44v6-silenced MM1.S cells were severely hampered in their tumorigenic capacity in vivo (P Conclusions These results clearly indicate that CD44v6 is dispensable for BM homing, but responsible for AML and MM addiction to microenvironmental signals. Combining CD44v6 targeting with cytotoxic chemotherapy might interfere with this vicious circle and result in higher and/or more durable response rates. Disclosures: No relevant conflicts of interest to declare.

miRNA expression is deregulated in human acute myeloid leukemia (AML), however the impact of altered post-transcriptional programs on the genesis and maintenance of leukemia stem cells (LSC) remains undefined. In order to elucidate the functional role of miRNA in LSC and identify relevant miRNA candidates, we performed global miRNA profiling on sorted cell subpopulations from 16 AML patient and 3 umbilical cord blood samples (Eppert et al, Nature Medicine 2011). Supervised analysis guided by the ability of each sub-population to initiate leukemic engraftment after xenotransplantation into immune-deficient mice generated a unique miRNA signature. miR-126, a miRNA that we previously demonstrated to have a conserved role in maintaining hematopoietic stem cell (HSC) quiescence (Lechman et al. Cell Stem Cell, 2012), was more highly expressed in LSC-enriched fractions and chosen for further validation. To confirm that miR-126 is a bona fide LSC determinant, we utilized a bidirectional lentiviral reporter vector specific for miR-126 (Gentner et al. Science Translational Medicine, 2010) to sort cells from AML patient samples based on miR-126 bioactivity, and demonstrated that all in vivo leukemia-initiating capacity was confined to cells with elevated miR-126 bioactivity. Lentiviral enforced expression of miR-126 in primary AML patient samples significantly increased LSC frequency (3.5-52.3 fold) as assessed by limiting dilution transplantation assays, while diminishing cell cycle entry, differentiation marker expression (CD14,CD15) and colony forming potential. Sponge-mediated knockdown of miR-126 expression resulted in the opposite effects. These findings suggest that high levels of miR-126 bioactivity support self-renewal/maintenance of primitive AML cells at the cost of aberrant differentiation. Moreover, by preserving LSC quiescence miR-126 promoted chemotherapy resistance, in part through suppression of CDK3, a gatekeeper of G0 to G1 cell cycle transit. Enforced expression of CDK3 partially rescued the functional consequences of supra-physiological levels of miR-126 bioactivity, rendering previously resistant LSC susceptible to killing by AraC/Daunorubicin combination chemotherapy. Our human LSC miRNA signature, optimized by regression analysis on a cytogenetically normal AML patient cohort, was prognostic for survival in a large independent AML patient cohort (Ley et. al N Engl. J Med, 2013) further validating the clinical significance of miRNA as stem cell determinants. Furthermore, miRNA-126 alone was prognostic for survival in two independent cohorts of AML patients with normal cytogenetics. These data demonstrate a mechanistic role for miR-126 in governing intrinsic LSC properties and establish miR-126 as a critical biomarker for clinical outcome. Disclosures: Wang: Trillium Therapeutics/Stem Cell Therapeutics: Research Funding.

Successful gene therapy of inherited blood diseases relies on transplantation and engraftment of autologous genetically engineered hematopoietic stem/progenitor cells (HSPCs) in myeloablated patients. Hematopoietic reconstitution and clinical benefit are related to cell dose, although single disease features might play a role favoring selection of relevant progenitor populations. Gene therapy trials in young pediatric patients are performed isolating CD34+ cells from bone marrow (BM), while in adults mobilized peripheral blood stem cells (PBSC) should represent the favorite target. In the context of gene therapy for thalassemia, the choice of HSPC source is crucial since intrinsic characteristics of patients (splenomegaly and thrombophilia) dictate caution in the use of G-CSF as mobilization agent and prompt investigation of new agents. Moreover, adult thalassemic patients may possibly have a decreased BM stem cell reservoir, due to the BM suppression in response to multiple transfusions. A phase II clinical protocol exploring the use of Plerixafor as a single mobilizing agent in adult patients affected by transfusion dependent beta-thalassemia (EudraCT 2011-000973-30) started in 2012 at our hospital. Plerixafor selectively and reversibly antagonizes the binding of SDF-1 to its receptor CXCR4 with subsequent egress of HSCs to the peripheral blood. The availability of a new source of HSPCs, potentially superior in terms of CD34+ cell yield, transduction efficiency and biological features to steady-state BM, would have a significant impact on the feasibility and efficacy of gene therapy. Four subjects were enrolled and treated by subcutaneously administration of Plerixafor at the single dose of 0.24 mg/kg followed by leukoapheresis. Mobilization of CD34+ cells occurred very rapidly with a peak between 7 to 9 hrs. Three out of four patients achieved the minimal target cell dose (2 x 106 cells/kg) and no severe adverse event occurred. To the aim of engineering Plerixafor-mobilized CD34+ cells for gene therapy, we performed a comprehensive characterization of their biological, molecular and functional properties. In vivo reconstitution potential and lympho-myeloid differentiation were tested following transplantation in NSG mice and compared to those of PBSCs mobilized by G-CSF. Percentages of engrafted human cells in NSG mice transplanted with Plerixafor -PBSCs were about 2- to 5-fold higher than those found in mice transplanted with G-CSF PBSCs. On the same line, the SRC frequency, obtained by pooled engraftment data, was significantly higher (1 SRC out of 47.875 CD34+ cells vs.1 SRC out of 141.203 CD34+ cells). The phenotypic analysis of the frequency of primitive hematopoietic sub-populations revealed that Plerixafor mobilizes preferentially HSPCs and LT-HSPCs, with a percentage of CD34+ CD38-/low CD90+ CD45RA- CD49f+ cells higher than that found in G-CSF PBSCs. This result mirrors the enhanced number of SRCs found in the CD34+ cell population mobilized by Plerixafor. In order to further define the molecular features of HSPCs from different sources, we are studying signalling networks in response to specific cytokines by phospho-proteins analysis and gene expression by microarrays analysis. Our studies are focused on self-renewal, homing, engraftment and multilineage differentiation processes and bioinformatic analysis will reveal the molecular machinery underlying 'stemness' properties of Plerixafor mobilized cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 30 The precise mechanisms controlling hematopoietic stem cell (HSC) quiescence, self renewal and differentiation remain incompletely understood, and HSC expansion remains a long sought after goal for cell- and gene therapy. Genetic manipulation of single transcription factors and signal transduction pathways has so far failed to provide sustained HSC expansion, often resulting in stem cell exhaustion or the emergence of malignancy. MicroRNAs (miRNAs) post-transcriptionally regulate multiple genes in a coordinated fashion, making them attractive targets to manipulate signaling networks and complex cellular functions. We have systematically manipulated miR-126, a miRNA enriched in the primitive hematopoietic compartment, in mouse and human HSC by stably knocking down its activity with a sponge lentiviral vector (126/KD LV) or forcing its overexpression by a 126/OE LV. Hematochimeric mice were generated by transplanting 126/OE LV- or 126/KD LV- transduced progenitors into congenic mice (murine HSC) or NSG mice (human HSC), and the effects of altered miR-126 levels on hematopoiesis were investigated in vivo. Steady state hematopoiesis was comparatively normal, with no alterations in complete blood cell counts except a minor skewing towards or against lymphopoiesis upon 126/OE or 126/KD, respectively. Strikingly, under stress conditions such as 5-fluorouracil exposure, secondary transplantation or the NSG xenograft environment, 126/OE resulted in a progressive loss of HSC, while 126/KD increased HSC numbers as measured by immunophenotype and functional output upon competitive transplantation into secondary recipients. Limiting dilution analysis confirmed a 3–4 fold expansion of mouse and human HSC upon miR-126/KD, and the competitive advantage of 126/KD HSC was maintained long-term including post tertiary transplantation. To study the mechanism by which miR-126 regulates HSC numbers, cell cycle analysis was performed on 126/KD- or 126/OE progenitors isolated from steady state bone marrow. Human CD34+CD38-CD90+ and murine Lineage-Kit+Sca+CD150+ HSC were shifted towards the S/G2/M or the G0 phase upon 126/KD or 126/OE, respectively, indicating that miR-126 antagonism increased cycling while excessive levels of miR-126 favoured HSC quiescence within the niche microenvironment. Interestingly, this effect was highly specific for HSC and the most primitive progenitors. An in vitro culture model of HSC-enriched cord blood cells was set up to study the molecular basis of miR-126 mediated control of HSC cycling. This model reproduced key aspects of the HSC phenotype observed in vivo, i.e. increased or decreased proliferation of primitive progenitors upon 126/KD or 126/OE, respectively. Transcriptome analysis indicated that miR-126 manipulation altered several cellular functions and pathways, most significantly the PI3K/AKT/GSK3β signalling axis. We confirmed PIK3R2 and CRKII as direct miR-126 targets in CB CD34+38- cells and show enhanced PI3K pathway activation as reflected by increased AKT and GSK3β phosphorylation upon 126/KD, while 126/OE caused opposite effects. Pharmacologic inhibition of the beta subunit of PI3K abrogated the 126/KD phenotype, indicating that miR-126 mainly acts through the PI3K pathway. In contrast to published studies showing HSC expansion upon hyperactivation of the PI3K pathway using various genetic approaches, miR-126/KD did not cause HSC exhaustion or leukemogenesis. On the contrary, evidence will be provided that 126/OE is oncogenic in hematopoietic cells. Thus, miR-126 represents a principle target for HSC expansion for cell and gene therapy applications. In summary, we propose that in vivo HSC pool size is influenced by endogenous levels of miR-126 through modulation of the HSC quiescence/proliferation equilibrium. Disclosures: No relevant conflicts of interest to declare.

Abstract 949 Background: Acute myeloid leukemia (AML) and multiple myeloma (MM) share common features, such as preferential homing to the bone marrow (BM) and the tendency to relapse after chemotherapy. These phenomena are possibly linked, as signals provided within the BM niche induce chemoresistance. Relapsing AML and MM can however be cured by allogeneic hematopoietic stem cell transplantation (HSCT), suggesting that allospecific T may specifically sterilize tumor cells that survive chemotherapy. Unfortunaley, the universal application of HSCT is severely limited by the occurrence of life-threatening GVHD and by the fact that leukemia often escapes the alloimmune pressure by losing specific HLAs, as we have recently demonstrated (Vago et al., NEJM 2009). The isoform variant 6 of the adhesion receptor CD44 (CD44v6) is widely expressed in AML and MM, and is crucially involved in BM homing. In phase I/II trials, targeting CD44v6 with mAb conjugated to chemotherapeutic agents showed substantial efficacy, but also toxicity due to background expression of CD44v6 on the skin. Thanks to the chimeric antigen receptor (CAR) technology it is now possible to efficiently redirect T cells against virtually any antigen for which there is availability of a specific mAb. Our experience with suicide gene therapy in the context of HSCT (Ciceri et al., Lancet Oncol 2009) clearly indicates that genetic switches are efficient tools for controlling the off-tumor toxicities of T cells. Aim: To develop a strategy for the safe targeting of CD44v6 through the co-expression of a suicide gene in CAR-modified T cells for AML and MM treatment Results: CD44v6 surface expression was observed in 11/14 (79%) AML cases. Co-culturing primary leukemic cells with BM-derived mesenchymal stromal cells (MSCs) ex vivo caused the significant up-regulation of CD44v6 expression (P Conclusions: CD44v6 CAR-redirected suicidal T cells have the potential to eradicate AML and MM and can be ablated at will. We are currently designing a phase I/II clinical trial where CD44v6 CAR-redirected suicidal T cells will be applied soon after autologous HSCT for sterilizing residual tumor, and routinely ablated thereafter not to interfere will full hematological reconstitution. The co-expression of a suicide gene will also allow to control any potential toxicity deriving from background expression of CD44v6 on healthy tissues. Disclosures: Bordignon: Molmed SpA: Employment.

Abstract 586 Gene therapy of inherited blood diseases requires harvest of hematopoietic stem cells (HSCs) from patients and autologous transplantation of genetically modified cells. In order to achieve correction of the disease, high number of HSCs and previous conditioning of the host bone marrow (BM) are necessary. In the clinical application of gene therapy for thalassemic patients the choice of the HSC source is a crucial issue. On one side, the minimal target dose poses a challenge for the use of steady state BM since reinfusion of high numbers of beta globin gene modified CD34+ cells is probably necessary to gain sufficient correction of the genetic defect in order to achieve transfusion independency; on the other side, the disease related features and complications of thalassemic patients (i.e. splenomegaly and thrombophilia) dictate caution in the use of G-CSF as mobilizing agent. In April 2011 a clinical protocol exploring the use of Plerixafor (AMD3100) as single agent was started (“Plerixafor mobilized stem cells as source for gene therapy of beta-thalassemia”, acronym AMD-THAL, EudraCT2011-000973-30). Aims of the trial were to explore the ability of Plerixafor in inducing safe and effective stem cells mobilization in adult patients affected by beta-thalassemia, to characterize stem/progenitor cells mobilized from the BM and peripheral blood of treated subjects and to achieve gene transfer efficiency of mobilized CD34+ cells at a level comparable to that obtained using steady state BM. Four patients (01, 02, 03 and 04) were enrolled and already mobilized to date (August 2012). All patients are affected by transfusion dependent beta-thalassemia and aged 28 (01), 41 (02), 39 (03), 33 (04). Two are splenectomized (02 and 03); all subjects are regularly iron chelated with adequate organ function. Administration of Plerixafor subcutaneously as single agent and at the single dose of 0.24 mg/kg resulted in mobilization of CD34+ cells/mcl with a peak of 78 cells at 9 hrs (01), 70 cells at 7 hrs (02) and 69 cells at 8 hrs (03); suboptimal mobilization was observed in patient 04 (peak 18 at 8 hrs). Patient 03 received a second dose at 0.40 mg/kg 24 hrs after the first dose and underwent a second leucoapheretic procedure. Harvest by leukoapharesis resulted in procurement of the following CD34+ cells/kg: 1.84 × 106 (01) and 4.43 × 106 (02) with a unique leukoapheretic procedure, and 3.57 × 106 (03) with two leukoapheresis. No apheresis was performed for patient 04 because the minimum target of 20 CD34+ cells/mcl in peripheral blood was not reached. CD34+ cells selection through Clinimacs Miltenyi resulted in the following yield: 1.2 × 106 CD34+ cells/Kg, 65% recovery (01), 2.66 × 106 CD34+ cells/Kg, 60% recovery (02), 1.78 × 106 CD34+ cells/Kg, 50% recovery (03). No severe adverse event occurred. Recorded side effects were: grade 3 hypotension related to the apheretic procedure (01), mild grade 1 facial disestesia (02 and 04) and hyperleukocytosis (02: WBC from 13.6 to 42.6 × 103/mcl). In addition, steady state and Plerixafor primed BM aspirates were performed to analyze any modification in CD34+ concentration in the BM following Plerixafor administration. In fact, Plerixafor administration resulted in enrichment of CD34+ cells concentration in the BM. Purified CD34+ cells from leukoapheresis of the 4 treated patients were analyzed for their biological and functional properties, subpopulations composition and expression profile. In vivo reconstitution potential and lymphomyeloid differentiation of CD34+ cells were tested following transplantation in NSG mice. Experiments are ongoing but preliminary results indicate that cells mobilized by Plerixafor have a primitive phenotype with a high reconstitution potential and are efficiently transduced with a lentiviral based vector, named GLOBE, encoding for the human beta-globin (Roselli et al., 2010), thus being a suitable source of target cells for gene therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 3125 Background: We have previously demonstrated that the genetic induction of a conditional suicidal phenotype in donor T cells allows for an operational dissociation of the GVL effect from GVHD after allogeneic hematopoietic stem cell transplantation (HSCT). Unfortunately, leukemia often escapes the immunological pressure of alloreactive donor T cells by losing “passenger” mismatched HLAs. Conversely, redirecting T cells against a non HLA-restricted antigen critically involved in the neoplastic phenotype may circumvent tumor escape due to the emergence of antigen-loss variants. The isoform variant 6 of CD44 is expressed by different epithelial and hematological cancers, and is possibly involved in tumor-cell survival and proliferation. Clinical experience with chemo-conjugated CD44v6-specific mAbs in epithelial tumors showed substantial efficacy, which was however limited by skin toxicity due to background expression of CD44v6 on keratinocytes Aim: By analogy with our experience in HSCT, we reasoned that CD44v6 targeting with suicide gene-modified T cells would provide a major therapeutic effect against hematological tumors, while granting a safety switch in case of toxicity. To this aim, we designed a novel CD44v6-specific chimeric antigen receptor (CAR) and developed a strategy for its co-expression with a suicide gene Results: CD44v6 expression by FACS was observed at high levels in 6/17 (37%) cases of acute myeloid leukemia (AML), but did not associate with enhanced leukemia initiation after infusion into NSG mice (83% vs 88%). In all cases, however, AML cells isolated from the bone marrow (BM) of engrafting mice were brightly positive for CD44v6, suggesting in vivo regulation by microenvironmental factors. In vitro, co-culturing primary AML cells with human BM-derived mesenchymal stromal cells (MSCs) caused a selective up-regulation of CD44v6 (P90% elimination: 18 hrs vs 112 hrs average, P Conclusions: We demonstrated that LV-mediated dual transgenesis of primary human T cells with a novel CD44v6-specific CAR and a suicide gene is feasible, results into a powerful antitumor effect against chemoresistant AML and MM cells, and enables effective T-cell ablation in case of toxicity. The premise that suicide gene-modified CAR-redirected T cells can widen the therapeutic index of CD44v6 targeting awaits clinical confirmation Disclosures: Bordignon: Molmed SpA: Employment. Bonini:MolMed: Consultancy.

Abstract 2631 Little is known about microRNA function in hematopoietic stem and progenitor cells (HSPC). Using a lentivector genetic reporter strategy to functionally detect miRNA activity in hematopoietic cells at single cell resolution, we identified several miRNAs which were specifically expressed in mouse and human HSC and early progenitors, defined according to cell surface phenotype and functional repopulation assays. One of these HSPC-specific miRNAs, miR-126, was further studied. We generated a stable miR-126 knockdown (kd) or forced its expression (“knock-in”, ki) in mouse HSPC using lentiviral vectors. Kd or ki cells were competitively transplanted with congenic, control vector-transduced cells, and hematopoietic chimerism was followed for >1 year in both primary and secondary recipients. miR-126 kd HSPC displayed enhanced myeloid and/or lymphoid contribution during the early phases of reconstitution, while they subsequently contributed similarly as the control cells. When this steady state bone marrow (BM) was transplanted into secondary recipients, we noted an even more pronounced over-contribution of miR-126 kd cells to hematopoiesis. In the long run, however, some secondary mice showed signs of exhaustion of miR-126 kd cells. These data suggest that miR-126 kd enhances hematopoiesis, likely at the stem/early progenitor level and in particular under stress conditions. On the other hand, forced expression of miR-126 (ki) resulted in an early competitive disadvantage in vivo, with progressively decreasing contribution to all hematopoietic lineages, paralleled by a nearly complete depletion of Kit+Sca+Lin- (KSL) miR-126 ki cells in the BM at 6 weeks after transplant. At 3 weeks post-transplant, when miR-126 ki KSL cells could still be detected, we found an increased proliferative index in these cells as judged by EdU incorporation in vivo, paralleled by a higher hematopoietic output respect to control cells at week 2–4 after transplant. These data suggest that miR-126 ki might favor HSC commitment at the cost of self-renewal. This phenotype was specific for miR-126 and not due to vector toxicity, as we demonstrate stable, long term overexpression of several control miRNAs in vivo. Moreover, miR-126 ki cells showed normal clonogenic activity in vitro. We then optimized a protocol to stably knock down miR-126 in human cord blood (huCB) HSPC, and validated this approach by demonstrating upregulation of previously described miR-126 targets including the beta subunit of phosphoinositide-3-kinase. Manipulation of miR-126 activity changed cell growth and differentiation of huCB, and we show altered activation of key signal transduction pathways upon miR-126 kd. Identification of additional miR-126 targets is ongoing using unbiased proteomic and transcriptomic approaches. In summary, these data suggest that a narrow range of miR-126 activity is required for robust and sustained HSC function, and that its manipulation may provide novel insights into stem cell biology. Disclosures: No relevant conflicts of interest to declare.

Abstract 4438 BACKGROUND: Graft Failure (GF) occurs in 5–27% of patients (pts) after allogeneic hamatopoietic stem cell transplant (HSCT) and is associated with high morbidity and mortality related to infections and hemorrhagic events. Graft function may be poor as result of graft rejection, primary disease relapse or Poor Graft Function (PGF). The incomplete recovery of blood counts is defined primary PGF and the decreasing blood counts after successful engraftment secondary PGF. Several factors may determine GF: disease risk and status, conditioning regimen, HSC source, HLA compatibility, T cell content, immunosuppression, GvHD, viral infections, drugs. GCSF and Rhu-EPO are readily available and effective in PGF but with no effects on platelets. Second transplantation from the same donor, with or without conditioning therapy, can boost the haematopoietic recovery in pts with GF. Unfortunately, both a second peripheral CD34+ mononuclear cells (MNC) mobilization and a marrow harvest in the operating room may be contraindicated early after the first donation as not safe for donors. Intrabone SCT can overcome the risk of graft failure even with a low number of CD34+ MNC, as it has been demonstrated in cord blood transplant. Here we investigate in three adult pts with GF a bone-to-bone boost (BBB) with a small marrow harvest from respective donors, unfit for a second conventional donation. AIM: to evaluate the feasibility of the BBB technique in 3 pts with graft failure. METHODS: pts were 2 males (57, 53 y) with PGF with a diagnosis of AML and CMML, respectively, and a female (44 y) with graft rejection and AML relapse. In the first two patients prolonged pancytopenia and hypoplastic marrow were documented, with diagnosis of primary PGF and secondary PGF, respectively, donor chimerism ranging from 80–100% (STR and HLA), without evidence of leukemia. In the third patient, after prolonged pancytopenia an AML relapse was documented with 89% blasts on bone marrow aspirate. In PGF patients no conditioning regimen was administered before the boost at day 30 and 72 after SCT, respectively. In the patient with AML relapse Melphalan 200 mg/mq was given 48 h before the infusion, at day 35 after SCT. The 3 donors were related, haploidentical. For the BBB procedure small quantities of bone marrow (< 200ml) were collected from the posterior iliac crest bilaterally of the donors, at the bedside, during deep sedation and analgesya. Shortly after the unmanipulated marrow harvested was infused in superior-posterior iliac crest mono- or bilaterally, depending on the volume, during deep sedation and analgesya. In pt 1 Mononucleated cell (MNC) dose was 0.9 × 10^8/Kg for a volume of 166 ml. In pt 2 MNC dose was 0.4 × 10^8/Kg for a volume of 88 ml. In pt 3 MNC dose was 0.3 × 10^8/Kg for a volume of 140 ml. RESULTS AND CONCLUSION: In this cases the BBB technique proved feasible and safe for both the donor and the patient. Patient 1 received a second PBSC boost, without conditioning, 3 months after the BBB, and he's now alive, in CR, 13 months after the first transplant. Patient 2 died 3 months after the first transplant for pneumonia and sepsis. Patient 3 is alive, in CR, 4 months after the first HSCT. This practice can give the chance of HSC boost to patients with GF without the need of a GCSF mobilization for donors, with a minimal invasive operation. This can give the option to overcome and resolve infectious and hemorrhagic complications, bridging patients to further therapies and procedures. The intra-bone SCT may be a facilitating tool for microenvironment reconstitution, seeding and subsequent differentiation and may as well have a tolerogenic effect, through the mesenchymal stromal cells infused with the harvest. Further studies are necessary to assess the efficacy of this procedure. Disclosures: No relevant conflicts of interest to declare.

Leukemia stem cells (LSCs) are a biologically distinct blast population positioned at the apex of the acute myeloid leukemia (AML) developmental hierarchy. A more complete understanding of the unique properties of LSCs is crucial for the identification of novel AML regulatory pathways and the subsequent development of innovative therapies that effectively target these cells in leukemia patients. However, most studies overlook the heterogeneity of AML and the existence of LSC, potentially masking important molecular pathways. MicroRNAs (miRNAs) are an emerging class of non-coding small RNAs that negatively regulate the expression of protein-encoding genes. Normal miRNA expression is tissue and developmental stage restricted, suggesting important roles in tissue specification and/or cell lineage determination. Several studies have already demonstrated that miRNA expression levels are dysregulated in AML. However, little is known of the contribution of miRNAs to the regulation of gene expression and maintenance of LSCs. In order to elucidate the role of miRNAs in the regulation and maintenance of the leukemic stem cell state and the leukemogenic process, primary AML patient samples were fractionated into sub-populations based on the expression of CD34 and CD38 cell surface expression and purified RNA was extracted for miRNA array profiling. Supervised analysis guided by the in vivo SCID leukemia initiating capacity of each sub-population identified a unique 2 miRNA signature associated with leukemia stem cell enriched fractions. Validation of top miRNA candidates was confirmed by standard RT-PCR. However, to confirm that our miRNA candidates were biologically active within the AML developmental hierarchy at a single cell level, we utilized bidirectional miRNA sensor lentivectors (Bd.LV). Bd.LVs co-express truncated NGFR and enhanced GFP (eGFP), and permit post-transcriptional regulation of eGFP when miRNA target sequences (miRT) are placed downstream. One week post-transduction of a unique primary AML patient sample (8227), which can maintain a phenotypic and functional hierarchy for over 200 days in serum-free in vitro culture conditions, multicolor flow cytometry analysis revealed that miR-126 strongly suppressed (17 fold eGFP repression vs. control vector) eGFP expression in CD34+CD38-cells, the sub-fraction of AML that is highly enriched in LSC, while miR-126 activity declined along a gradient of further “differentiation” of the cells. Knockdown of miR-126 activity in 8227 cells with antagomirs designed to target miR-126 resulted in an increase in eGFP expression within the CD34+CD38-population, confirming the specificity of the biosensor lentivector. Furthermore, this expression gradient of miR-126 was conserved in 2 primary patient AML samples after 10 weeks post-transplant into pre-conditioned NOD/SCID mice. These experiments demonstrate that mir-126, a miRNA candidate identified by miRNA array, is more abundantly expressed and biologically active within the leukemia stem cell compartment of the AML hierarchy. Moreover, these data demonstrate the utility of biosensor vector technology in discerning miRNA expression patterns at the single cell level and will be useful for the further refinement of LSC purification.

The balance between self-renewal and differentiation is crucial to ensure the homeostasis of the hematopoietic system, and is a hallmark of hematopoietic stem cells. However, the underlying molecular pathways, including the role of micro-RNA, are not completely understood. To assess the contribution of micro-RNA, we performed micro-RNA profiling of hematopoietic stem cells and their immediate downstream progeny multi-potent progenitors from wild-type control and Pbx1-conditional knockout mice, whose stem cells display a profound self-renewal defect. Unsupervised hierarchical cluster analysis separated stem cells from multi-potent progenitors, suggesting that micro-RNA might regulate the first transition step in the adult hematopoietic development. Notably, Pbx1-deficient and wild-type cells clustered separately, linking micro-RNAs to self-renewal impairment. Differential expression analysis of micro-RNA in the physiological stem cell-to-multi-potent progenitor transition and in Pbx1-deficient stem cells compared to control stem cells revealed miR-127-3p as the most differentially expressed. Furthermore, miR-127-3p was strongly stem cell-specific, being quickly down-regulated upon differentiation and not re-expressed further downstream in the bone marrow hematopoietic hierarchy. Inhibition of miR-127-3p function in Lineage-negative cells, achieved through a lentiviral-sponge vector, led to severe stem cell depletion, as assessed with serial transplantation assays. miR-127-3p-sponged stem cells displayed accelerated differentiation, which was uncoupled from proliferation, accounting for the observed stem cell reduction. miR-127-3p overexpression in Lineage-negative cells did not alter stem cell pool size, but gave rise to lymphopenia, likely due to lack of miR-127-3p physiological downregulation beyond the stem cell stage. Thus, tight regulation of miR-127-3p is crucial to preserve the self-renewing stem cell pool and homeostasis of the hematopoietic system.

Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34(+) HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development.

Globoid cell leukodystrophy (GLD; also known as Krabbe disease) is an invariably fatal lysosomal storage disorder caused by mutations in the galactocerebrosidase (GALC) gene. Hematopoietic stem cell (HSC)-based gene therapy is being explored for GLD; however, we found that forced GALC expression was toxic to HSCs and early progenitors, highlighting the need for improved regulation of vector expression. We used a genetic reporter strategy based on lentiviral vectors to detect microRNA activity in hematopoietic cells at single-cell resolution. We report that miR-126 and miR-130a were expressed in HSCs and early progenitors from both mice and humans, but not in differentiated progeny. Moreover, repopulating HSCs could be purified solely on the basis of miRNA expression, providing a new method relevant for human HSC isolation. By incorporating miR-126 target sequences into a GALC-expressing vector, we suppressed GALC expression in HSCs while maintaining robust expression in mature hematopoietic cells. This approach protected HSCs from GALC toxicity and allowed successful treatment of a mouse GLD model, providing a rationale to explore HSC-based gene therapy for GLD.