Engraftment Potential Research Articles

PS967 PRECLINICAL ACTIVITY OF THE NOVEL ORAL DUAL BET‐CBP/P300 INHIBITORS, NEO1132 AND NEO2734, IN ACUTE MYELOID LEUKEMIA

Background:For decades, combination chemotherapy has been the therapeutic strategy to treat acute myeloid leukemia (AML). Although this treatment induces complete remission in most patients, only 30–40% of patients survive five years after diagnosis. These low cure rates are mainly due to relapse caused by the survival of a small subpopulation of leukemia cells with stem cell features, named “leukemic stem cells” (LSCs). The bromodomain (BRD) extra‐terminal (BET) protein Brd4 is a regulator of c‐Myc and an important therapeutic target in AML. In vivo, inhibition of Brd4 eradicates AML progenitors and reduces AML burden in mice. While treatment with BET inhibitors induces clinical responses in AML, these are usually partial and transient. Cyclic AMP response element‐binding protein (CREB)‐binding protein (CBP) and E1A interacting protein of 300 kDa (EP300 or P300) are homologues BRD‐containing transcriptional co‐activators that regulate transcription through their acetyltransferase activity. We studied NEO2734 and NEO1132, orally‐bioavailable, first‐in‐class dual BET and CBP/P300 inhibitors in preclinical models of AML.Aims:To evaluate the efficiency of NEO2734 and NEO1132 in AML (stem/progenitor) cell elimination.Methods:We studied the effect of treatment with NEO2734, NEO1132 and the CBP/P300 inhibitor tool compound, CPI637, on AML blast differentiation, apoptosis, engraftment potential in a patient‐derived xenograft AML mouse model (NSG mice), and on the clonogenic capacity of long‐ and short‐term surviving AML cells ex vivo and in vivo.Results:To assess the biological activity of NEO2734, we treated a panel of AML cell lines with various concentrations of NEO2734, NEO1132 and CPI637. Nanomolar concentrations of NEO2734 inhibited cell proliferation with IC50 values ranging from 32 nM to 141 nM. Next, we showed that NEO2734 inhibited cell proliferation and induced apoptosis in primary AML CD45dim blasts across a variety of genotypes (example Figure 1a and 1b)(IC50 between 30–100 nM for 5 samples tested). The potency of NEO2734 to inhibit proliferation and to induce apoptosis in AML cell lines and primary AML blasts was higher than that of NEO1132 and much higher than CPI637 (Figure 1a and 1b). Moreover, we observed no increase in the myeloid differentiation marker CD11b in primary AML blasts after NEO2734 treatment.To evaluate whether NEO2734 efficiently reduces human AML burden in the bone marrow in vivo, patient cells were transplanted into mice and the mice were treated with NEO2734 or NEO1132 (10 mg/kg, orally daily 10 times). The percentage of leukemic engraftment was reduced in the treated mice as compared to the controls (Figure 2). NEO2734 treatment inhibited leukemic engraftment more efficiently than NEO1132. Finally, we investigated whether NEO2734 treatment could inhibit clonogenic capacity of leukemic progenitors and long‐term surviving cells from primary AML cases. In all cases studied, a significant reduction in number of colonies after 1 week and 4 weeks of NEO2734 treatment was observed. Importantly, NEO2734 also reduced survival of immunophenotypically defined LSCs, CD34+CD38− cells. The potency of NEO2734 to inhibit LSC and leukemic progenitor survival was higher than that of NEO1132 and much higher than that of CPI637.imageSummary/Conclusion:The dual BET‐CBP/P300 inhibitor NEO2734 efficiently eliminates AML blasts and LSCs/progenitors, suggesting that AML patients should be included in clinical studies of NEO2734.

PS1221 LONG‐TERM EFFICACY AND SAFETY OF INOTERSEN FOR HEREDITARY TRANSTHYRETIN AMYLOIDOSIS: NEURO‐TTR OPEN‐LABEL EXTENSION 2‐YEAR UPDATE

Background:Hereditary transthyretin amyloidosis (hATTR) is a rare protein misfolding disorder that causes progressive and debilitating polyneuropathy. A randomized, controlled phase 3 trial (NEURO‐TTR; NCT01737398) demonstrated efficacy and safety of inotersen in patients with hATTR polyneuropathy (Benson 2018 NEJM).Aims:To provide an update on the long‐term efficacy and safety of inotersen, an antisense oligonucleotide inhibitor of transthyretin protein production, in patients with hereditary transthyretin amyloidosis (hATTR) with polyneuropathy.Methods:Patients who completed NEURO‐TTR were eligible to enroll in an ongoing open‐label extension (OLE) study (NCT02175004). We report an update on the long‐term efficacy and safety of inotersen in patients with hATTR polyneuropathy after 24 months in the OLE. Assessments included modified Neuropathy Impairment Score +7 neurophysiologic tests composite score (mNIS+7), Norfolk Quality of Life–Diabetic Neuropathy questionnaire total score (Norfolk QoL‐DN), Short Form 36 Health Survey version 2 (SF‐36v2) physical component summary (PCS), and safety monitoring.Results:Of 139 patients who completed NEURO‐TTR, 135 (97.1%) enrolled in the OLE. As of 5/31/2018, some patients were ongoing but had not yet completed 24 months in the OLE, and longest inotersen exposure in both studies was 5.2 years. Patients who switched from placebo to inotersen in the OLE demonstrated slowing of neurologic disease progression by mNIS+7 and Norfolk QoL‐DN as early as 6 months after starting inotersen (mean change from OLE baseline to month 6/year 2: 6.22/5.08 for mNIS+7 and 0.54/2.26 for Norfolk QoL‐DN). Patients who received inotersen for 39 months (15 months NEURO‐TTR + 24 months OLE) continued to show benefit (mean change from OLE baseline to year 2: 11.18 for mNIS+7 and 5.22 for Norfolk QoL‐DN). Patients who continued inotersen also showed stabilization of health‐related quality of life measured by SF‐36v2 PCS. No evidence of increased risk for grade 4 thrombocytopenia or severe renal events has been observed with increased exposure duration; no new safety concerns have been identified.Summary/Conclusion:In the OLE, inotersen improved, halted, or slowed progression of hATTR polyneuropathy, with greater stabilization observed in patients who initiated inotersen earlier.

PS1220 EX‐VIVO HEMATOPOIETIC STEM CELL GENE THERAPY (GT) FOR MUCOPOLYSACCHARIDOSIS TYPE I HURLER (MPSIH): PRELIMINARY RESULTS FROM A PHASE I/II CLINICAL STUDY

Background:MPSIH is a lysosomal storage disease caused by α‐L‐iduronidase (IDUA) deficiency that is associated with progressive multisystem morbidity including neurodevelopmental deterioration and severe orthopedic manifestations, leading to death in early childhood. Current therapeutic strategies include enzyme replacement therapy and allogeneic hematopoietic stem cell transplantation (HSCT), which however show limitations in preventing neurodevelopmental and orthopedic complications. Transplantation of genetically‐engineered autologous hematopoietic stem and progenitor cells (HSPC) is an attractive treatment option for MPSIH thanks to the possibility to obtain supraphysiologic levels of the missing enzyme in the hematopoietic progeny, thus allowing for increased cross‐correction.Aims:We recently opened a phase I/II clinical trial (NCT03488394) in children affected by MPSIH aimed at evaluating safety, tolerability and efficacy of the transplantation of autologous, IDUA lentiviral (LV)‐transduced CD34+ cells in MPSIH patients undergoing myeloablative conditioning.Methods:The study foresees the enrollment of 6 patients who lack a non‐heterozygous HLA‐matched donor and display an IQ/DQ>70. Primary endpoints of safety include: overall survival, hematological engraftment and safety of drug product (DP) administration. Primary endpoint of efficacy is represented by IDUA activity in peripheral blood up to supraphysiologic levels at 1 year post‐gene therapy (GT). Treatment impact on nervous system, skeleton and other target organs is assessed by clinical and radiologic parameters, as well as by disease‐specific biomarkers.Results:By the submission deadline, two patients (24 and 14 months old) were treated and have a follow‐up of 7 and 1.5 months, respectively. Autologous HSPC were harvested after mobilization with G‐CSF and Plerixafor; the procedure was uneventful and resulted in cryopreserved DP of 24 and 14 million CD34+ cells/kg, respectively. Transduction efficiency in the drug products was above 80% using an optimized transduction protocol that preserved engraftment potential of ex vivo‐cultured HSPC in preclinical models. No adverse events related to DP infusion were recorded. Hematopoietic engraftment was fast in both patients; neutrophil engraftment occurred on day +17 and +22, respectively; platelet engraftment at day+15 with counts never falling below 30.000/ul in both patients. Importantly, both patients reached supraphysiologic levels of IDUA by day+14 that stabilized around 10 fold above the mean of the normal range, along with an in vivo vector copy number ranging from 2 to 4 in multiple hematopoietic lineages. At the 6 month follow‐up of patient 1, stable IDUA activity was detected in the cerebrospinal fluid, and urinary GAGs normalized. A third patient has been successfully mobilized resulting in a cryopreserved DP of 13 million CD34+ cells/kg and will be treated shortly. Other patients are currently under evaluation.Summary/Conclusion:Preliminary clinical results indicate that our optimized transduction protocol allows efficient HSPC transduction without compromising their engraftment potential. Treated patients showed rapid hematopoietic recovery, supraphysiologic IDUA levels in the peripheral blood and metabolic correction of the MPSI‐H enzyme defect in critical target tissues. Long term‐follow up and treatment of further patients are necessary to confirm that IDUA supraphysiologic levels result in improvement of neurological and orthopedic outcome as compared to allogeneic HSCT.

CRISPR/Cas9 applications in gene therapy for primary immunodeficiency diseases.

Primary immunodeficiency diseases (PIDs) encompass a range of diseases due to mutations in genes that are critical for immunity. Haploinsufficiency and gain-of-function mutations are more complex than simple loss-of-function mutations; in addition to increased susceptibility to infections, immune dysregulations like autoimmunity and hyperinflammation are common presentations. Hematopoietic stem cell (HSC) gene therapy, using integrating vectors, provides potential cure of disease, but genome-wide transgene insertions and the lack of physiological endogenous gene regulation may yet present problems, and not applicable in PIDs where immune regulation is paramount. Targeted genome editing addresses these concerns; we discuss some approaches of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system applicable for gene therapy in PIDs. Preclinical repair of gene mutations and insertion of complementary DNA restore endogenous gene regulation and they have shown very promising data for clinical application. However, ongoing studies to characterize off-target genotoxicity, careful donor designs to ensure physiological expression, and maneuvers to optimize engraftment potential are critical to ensure successful application of this next-gen targeted HSC gene therapy.

(Epi)genetic Modifications in Myogenic Stem Cells: From Novel Insights to Therapeutic Perspectives.

The skeletal muscle is considered to be an ideal target for stem cell therapy as it has an inherent regenerative capacity. Upon injury, the satellite cells, muscle stem cells that reside under the basal lamina of the myofibres, start to differentiate in order to reconstitute the myofibres while maintaining the initial stem cell pool. In recent years, it has become more and more evident that epigenetic mechanisms such as histon modifications, DNA methylations and microRNA modulations play a pivatol role in this differentiation process. By understanding the mechanisms behind myogenesis, researchers are able to use this knowledge to enhance the differentiation and engraftment potential of different muscle stem cells. Besides manipulation on an epigenetic level, recent advances in the field of genome-engineering allow site-specific modifications in the genome of these stem cells. Combining epigenetic control of the stem cell fate with the ability to site-specifically correct mutations or add genes for further cell control, can increase the use of stem cells as treatment of muscular dystrophies drastically. In this review, we will discuss the advances that have been made in genome-engineering and the epigenetic regulation of muscle stem cells and how this knowledge can help to get stem cell therapy to its full potential.

Withdrawal: Generation of hematopoietic cells from mouse pluripotent stem cells in a 3D culture system of self-assembling peptide hydrogel.

In vitro generation of HSCs from pluripotent stem cells (PSCs) can be regarded as novel therapeutic approaches for replacing bone marrow (BM) transplantation without immune rejection or graft versus host disease(GVHD). To date, many differentiation approaches have been evaluated in terms of directing PSCs toward different hematopoietic cell types, yet, low efficiency and no function restrict the further hematopoietic differentiation study, our research aim to develop a three dimention (3D) hematopoietic differentiation approach that serve as recapitulation of embryonic development in vitro to a degree of complexity not achievable in a two dimention (2D) culture system. We first found that mouse PSCs could be efficiently induced to hematopoietic differentiation with expression of hematopoietic makers such as c-kit, CD41 and CD45 within self-assembling peptide hydrogel. Colony-forming cells assay results suggested mPSCs could differentiated into multipotential progenitor cells and 3D induction system derived hematopoietic colonies owned potential of differentiating into lymphocyte cells. In addition, in vivo animal transplantation experiment showed that mPSCs(CD45.2) could embedded into NOD/SCID mice(CD45.1) with about 3% engraftment efficiency after 3 weeks transplantation. This study demonstrated that we developed the 3D induction approach that could efficiently promoted the hematopoietic differentiation of mPSCs in vitro and obtained the multipotential progenitors that possessed the short-term engraftment potential. This article is protected by copyright. All rights reserved.

Engraftment and proliferation potential of embryonic lung tissue cells in irradiated mice with emphysema

Recently, there has been increasing interest in stem cell transplantation therapy, to treat chronic respiratory diseases, using lung epithelial cells or alveolospheres derived from endogenous lung progenitor cells. However, optimal transplantation strategy of these cells has not been addressed. To gain insight into the optimization of stem cell transplantation therapy, we investigated whether lung cell engraftment potential differ among different developmental stages. After preconditioning with irradiation and elastase to induce lung damage, we infused embryonic day 15.5 (E15.5) CAG-EGFP whole lung cells, and confirmed the engraftment of epithelial cells, endothelial cells, and mesenchymal cells. The number of EGFP-positive epithelial cells increased from day 7 to 28 after infusion. Among epithelial cells derived from E13.5, E15.5, E18.5, P7, P14, and P56 mice, E15.5 cells demonstrated the most efficient engraftment. In vitro, E15.5 epithelial cells showed high proliferation potential. Transcriptome analyses of sorted epithelial cells from E13.5, E15.5, E18.5, P14, and P56 mice revealed that cell cycle and cell-cell adhesion genes were highly enriched in E15.5 epithelial cells. Our findings suggest that cell therapy for lung diseases might be most effective when epithelial cells with transcriptional traits similar to those of E15.5 epithelial cells are used.

Long-Term Engraftment of ESC-Derived B-1 Progenitor Cells Supports HSC-Independent Lymphopoiesis.

SummaryIt is generally considered that mouse embryonic stem cell (ESC) differentiation into blood cells in vitro recapitulates yolk sac (YS) hematopoiesis. As such, similar to YS-derived B-progenitors, we demonstrate here that ESC-derived B-progenitors differentiate into B-1 and marginal zone B cells, but not B-2 cells in immunodeficient mice after transplantation. ESC-derived B-1 cells were maintained in the recipients for more than 6 months, secreting natural IgM antibodies in vivo. Gene expression profiling displayed a close relationship between ESC- and YS-derived B-1 progenitors. Because there are no hematopoietic stem cells (HSCs) detectable in our ESC differentiation culture, successful long-term engraftment of ESC-derived functional B-1 cells supports the presence of HSC-independent B-1 cell development.

Abstract B205: Adoptive transfer of autologous T-cells, modified with a MART-1 specific TCR and cultured in IL-7/IL-15, for the treatment of metastatic melanoma patients

Abstract Currently a TCR gene therapy trial to treat stage IV cutaneous and uveal melanoma patients is being conducted at the NKI. The TCR used for T-cell modification is specific for the HLA-A*0201 restricted MART-126-35 epitope, which is expressed on the majority of melanoma cells. Unique to this trial is the use of the combination of anti-CD3/CD28 beads for T-cell activation plus IL-7/ IL-15 for subsequent culture and expansion, instead of the more commonly used strategy that utilizes the combination of anti-CD3 and IL-2. The aim of this alternative production strategy is to generate a “less differentiated“ T-cell product that may have a better engraftment potential and antitumor activity. Thus far, a total of 12 patients have been treated, of whom 5 were uveal melanoma patients. 1 patient treated with 5x109 cells died 9 days after infusion, among others due to a severe cytokine release syndrome. The other 11 patients were treated in 3 different cell doses: 5x107, 25x107and10x107 transduced CD4 and CD8 T-cells. A prolonged engraftment and higher % of gene modified cells in the blood compartment were observed in patients treated with the highest number of cells infused. Gene modified T-cells could be found back in the circulation for up to two months post infusion, even when as few as 5x107 transduced T-cells were infused. In the highest cell dose, transduced T-cells could be detected up to 10 months following infusion. On-target toxicity against MART-1 expressing melanocytes in the skin (rash and vitiligo) was observed in all patients. Most severe skin toxicity, but also uveitis and inner ear toxicity, were observed in patients treated in the highest dose cohort of 2.5x107 transduced T-cells. These patients also developed a cytokine release syndrome requiring tocilizumab (anti-IL6R) treatment. Unexpected toxicity observed at this highest dose level consisted of development of pancytopenia around 6-8 weeks post T-cell infusion. In one of these patients, T-cell clonality of the infused cell product and of the peripheral blood compartment at the time of pancytopenia was analyzed in order to investigate the mechanism behind this toxicity. The highest clonality was found in the endogenous unmodified CD4 population. TCR diversity and evenness of the CD8 modified and unmodified populations were similar, suggesting that the pancytopenia was not associated with specific expansion of a TCR modified T-cell clone. However, this delayed pancytopenia could be related to 1) the conditioning chemotherapy regimen (fludarabine and cyclophosphamide), 2) cytokine release like IL-6, or 3) expansion of an autoreactive clone derived either from the infused T-cell product or newly generated from the endogenous T-cell pool. Of the 12 patients treated thus far, 8 patients had stable disease (SD) and two showed a partial response (PR) (5.2 and 9.5 month). The type of response or the duration of response was not correlated to the amount of cells infused or the peak and duration of engraftment of the infused T-cells. On the basis of these preliminary data, we conclude that TCR-modified T-cells produced by this method have a high engraftment potential and show in vivo activity at low infused cell doses, thereby suggesting the potential value of this protocol for TCR or CAR gene therapy for the treatment of cancer. Citation Format: Raquel Gomez-Eerland, Maartje Rohaan, Joost van den Berg, Maaike van Zon, Renate de Boer, Noor Bakker, Bastiaan Nuijen, Ton N.M. Schumacher, John B.A.G. Haanen. Adoptive transfer of autologous T-cells, modified with a MART-1 specific TCR and cultured in IL-7/IL-15, for the treatment of metastatic melanoma patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B205.

Assessment of Short-Term Engraftment Potential of Ex Vivo Expanded Hematopoietic Stem Cells Using Normal Fetal Mouse in Utero Transplantation Model.

Objective:Ex vivo expansion is a promising strategy to overcome the low number of human umbilical cord blood hematopoietic stem cells (hUCB-HSCs). Although based on the obtained results in unnatural physiological condition of irradiated genetically immune-deficient mouse models, there has always been concern that the expanded cells have less engraftment potential. The purpose of this study was to investigate effect of common ex vivo expansion method on engraftment potential of hUCB-mononuclear cells (MNCs), using normal fetal mouse, as a model with more similarity to human physiological conditions.Materials and Methods:In this experimental study, briefly, isolated hUCB-MNCs were cultured in common expansion medium containing stem cell factor, Flt3 ligand and thrombopoietin. The unexpanded and expanded cells were transplanted to the fetal mice on gestational days of 11.5-13.5. After administration of human hematopoiesis growth factors (hHGFs), presence of human CD45+ cells, in the peripheral blood of recipients, was assessed at various time points after transplantation.Results:The expanded MNCs showed 32-fold increase in the expression of CD34+38- phenotype and about 3-fold higher clonogenic potential as compared to the uncultured cells. Four weeks after transplantation, 73% (19/26) of expanded-cell recipients and 35% (7/20) of unexpanded-cell recipients were found to be successfully engrafted with human CD45+ cells. The engraftment level of expanded MNCs was significantly (1.8-fold) higher than unexpanded cells. After hHGFs administration, the level was increased to 3.2, 3.8 and 2.6-fold at respectively 8, 12, and 16 weeks of post transplantation. The increased expression of CXCR4 protein in expanded MNCs is a likely explanation for the present findings.Conclusion:The presented data showed that expanded MNCs compared to unexpended cells are capable of more rapid and higher short-term engraftment in normal fetal mouse. It could also be suggested that in utero transplantation (IUT) of normal fetal mice could be an appropriate substitute for NOD/SCID mice in xenotransplantation studies.

Mouse Hematopoietic Stem Cell Modification and Labelling by Transduction and Tracking Posttransplantation.

The tracking of the hematopoietic potential of genetically manipulated fluorescent hematopoietic stem cells (HSC) in the bone marrow (BM) allows the assessment of regulatory processes involved in the re-establishment of hematopoiesis posttransplant. Herein, we describe the means to assess the consequence of expressing specific genes in HSC on their engraftment potential posttransplant.

Genome Editing Via NHEJ-Mediated Targeted Integration Retains Engraftment Potential of Edited Hematopoietic Stem Cells

The ability to edit the genome of long-term-engrafting hematopoietic stem cells (LT-HSCs) would provide a curative therapy for a wide range of hematological disorders. While mixed LT/ST-HSC (ST, short-term) pools edited via non-homologous end joining (NHEJ) yield engraftable transgenic cells, pools made transgenic via homology-dependent repair (HDR) do not. This observation is likely explained by the absence of DNA replication required for HDR in LT-HSCs. Quiescence renders LT-HSCs refractory to transgene insertion via HDR, with the transgene-positive population consisting overwhelmingly of actively dividing, short-term-engrafting HSCs. In contrast to HDR, NHEJ is a DNA repair mechanism active in non-dividing cells. We hypothesized that CRISPR/Cas9-mediated transgenesis of HSC pools via NHEJ-mediated targeted integration (NHEJ-TI) would allow modification of LT-HSCs and produce edited, long-term engrafting progeny.First, we investigated whether NHEJ-TI is a feasible method of genome editing in HSCs. Utilizing CRISPR/Cas9 to induce a DSB in the AAVS1 (PPP1R12C) locus and a self-complementary rAAV6 (scAAV6) to deliver a GFP expression cassette dependent on the endogenous promoter, we optimized RNP nucleofection, scAAV6 infection, and HSC culture conditions for efficient NHEJ-TI in HSCs. Flow cytometry for eGFP and in-out PCR was used to confirm transgene integration at frequencies ranging from 4-6%.Next, we investigated whether the NHEJ-TI edited pool of mixed LT/ST-HSCs contained engraftable, edited cells by injection into irradiated NOD/SCID/IL2Rg-/- (NSG) mice. We compared the engraftment potential of transgenic HSCs cultured for either 2 days (2D) or 2 hours (2H) prior to editing. While the overall editing efficiency was ~20% higher in the 2D condition, the near absence of cell prestimulation in the 2H condition resulted in increased levels of overall engraftment, doubling the total engraftment level of edited hematopoietic cells at 10 weeks post-injection. Importantly, the percentage of edited cells among the engrafted human hematopoietic cell population remained essentially unchanged relative to the starting efficiency (2D: 4.0%, 2H: 3.6%; 10 weeks post-injection). Analysis of bone marrow-resident CD34-positive cells at 16 weeks post-engraftment will reveal the fraction of edited LT-HSCs. Our results show that NHEJ-TI is a feasible method of inserting transgenes into HSCs whilst retaining their engraftment potential. To our knowledge, this is the first study to achieve successful NHEJ-mediated targeted integration in HSCs while retaining engraftment potential and (perhaps) transgenesis as well. DisclosuresUenishi:Casebia Therapeutics: Employment. Abe:Casebia Therapeutics: Employment. Mallari:Casebia Therapeutics: Employment. Scharenberg:Generation Bio: Equity Ownership; Casebia Therapeutics: Employment; Alpine Immune Sciences: Equity Ownership. Cost:Casebia Therapeutics: Employment.

Inhibition of Amino Acid Metabolism Selectively Targets Human Leukemia Stem Cells

Outcomes for AML patients remain poor because of the inability to fully eliminate leukemia stem cells (LSCs). We have previously shown that primary human LSCs reside in a unique metabolic condition characterized by a relatively low oxidative state (termed “ROS-low”) and increased levels of glutathione (Lagadinou et al. Cell Stem Cell, 2013). Cells in this condition are highly dependent on oxidative phosphorylation (OXPHOS) for survival, in striking contrast to many tumor cells which often rely on glycolysis, suggesting that LSCs are governed by distinct metabolic properties. Therefore, the goal of this project was to identify and target metabolic vulnerabilities of LSCs.To achieve this objective, we used mass spectroscopy to interrogate the metabolome of leukemia stem cells (LSCs) isolated from primary human AML specimens. We observed significant increases in the levels, uptake, and metabolism of amino acids in LSCs compared to bulk AML cells. These data suggest that LSCs may preferentially rely on amino acids for survival. To investigate this hypothesis, we cultured LSCs and bulk leukemia cells isolated from primary leukemia specimens in media lacking amino acids and measured cell viability and colony forming potential. We found that LSCs were uniquely sensitive to amino acid loss. In addition, LSCs formed significantly fewer colonies upon amino acid depletion compared to LSCs cultured in media containing amino acids. To confirm that amino acid depletion was targeting functionally-defined LSCs, we employed engraftment assays in immune incompetent mice. Culturing primary AML cells without amino acids for 24 hours resulted in significantly reduced levels of leukemia cell engraftment. Next, we interrogated whether amino acid depletion impaired normal HSC survival and function by culturing mobilized peripheral blood without amino acids and measuring the frequency of CD34+ cells, colony forming ability, and engraftment into immune deficient mice. HSC frequency, colony forming ability, and engraftment potential were not changed by amino acid depletion. Altogether, these data demonstrate the LSCs are selectively dependent on amino acids for survival.We next determined how amino acids modulate LSC biology by measuring the consequences of amino acid loss on LSC metabolism. We observed that amino acid depletion decreased OXPHOS specifically in LSCs and not in bulk leukemia cells. We have previously shown that BCL-2 inhibition decreases OXPHOS in LSCs (Lagadinou et al. Cell Stem Cell, 2013). Importantly, recent studies have shown that inhibition of BCL-2 using the BCL-2 inhibitor venetoclax in combination with azacitidine has resulted in superior outcomes for AML patients (Dinardo et al. Lancet Oncology, 2018). Furthermore, our preliminary data demonstrates that venetoclax with azacitidine targets LSCs in AML patients. Therefore, we hypothesized that venetoclax with azacitidine may be targeting LSCs by modulating OXPHOS via amino acid metabolism.To test this hypothesis, we isolated LSCs from AML patients undergoing treatment with venetoclax and azacitidine. LSC specimens obtained pre and 24 hours after initiation of therapy were analyzed for changes in OXPHOS, gene expression, and the metabolome. We observed that venetoclax with azacitidine treatment decreased OXPHOS and reduced amino acid levels. In addition, expression of amino acid transporters was down-regulated. Finally, we sought to determine if culturing LSCs in high levels of amino acids before venetoclax and azacitidine treatment could rescue LSC viability and OXHPOS. We found that culturing LSCs with increased levels of amino acids rescued LSCs survival and OXPHOS, demonstrating that venetoclax with azacitidine targets LSCs by decreasing amino acid levels.Taken together, our data indicate that LSCs are selectively reliant on amino acid metabolism to fuel OXPHOS. Furthermore, amino acid metabolism can be targeted in AML patients by venetoclax with azacitidine treatment. These studies are the first to characterize metabolic targeting of LSCs in AML patients. DisclosuresNemkov:Omix Technologies inc: Equity Ownership. Pollyea:Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Curis: Membership on an entity's Board of Directors or advisory committees.

A Novel iPSC Model Reveals a Role for RUNX1 in the Maintenance of AML Leukemia Stem Cells

Leukemia stem cells (LSCs) are believed to be a prominent source of relapse in acute myeloid leukemia (AML). Human AML LSCs are classically studied via xenotransplantation and defined as cells with (a) self-renewal ability, giving rise to leukemic engraftment that can be maintained over serial transplantation; and (b) ability to give rise to more differentiated progeny that are unable to engraft. LSCs are chemoresistant presumably because they have properties distinct from those of the bulk AML cells. A better understanding of the properties of LSCs can ultimately allow us to develop new therapies specifically targeting these cells to achieve lasting responses or even cures.We recently reported that induced pluripotent stem cells (iPSCs) generated from AML patients (AML-iPSCs) exhibit leukemic features upon hematopoietic differentiation, including extensive proliferation, maintenance of hematopoietic stem cell (HSC) markers, and serial engraftment of a lethal leukemia in immunodeficient mice (Kotini et al. Cell Stem Cell 2017). More recently, we found that the hematopoietic stem/progenitor cells (HSPCs) derived from AML-iPSCs exhibit phenotypic and functional heterogeneity. We first observed the presence of two cell populations with distinct growth characteristics and morphology: a population exhibiting adherent growth (adherent, A) and a population growing in suspension (S). The A cell population contained cells with immature morphology and HSC immunophenotype (CD34+/CD38-/CD90+/CD45RA-/ CD49f+), while the S fraction contained more differentiated cells (CD34-/low/CD38+/CD90-/CD45RA+). Experiments utilizing serial replating, single-cell plating of GFP-labeled cells and mathematical modeling revealed a hierarchical organization, whereby the A cells continuously give rise to the S cell fraction. Time-lapse imaging showed that A cells divide both symmetrically and asymmetrically with the majority (~60%) of cell divisions giving rise to two A cells and less frequently generating either one A and one S (~15%) or two S cells (~15%). Serial transplantation experiments in NSG mice revealed that the engraftment potential was largely contained within the A cell fraction. Cell cycle analysis showed that adherent cells contained a lower S and higher G0/G1 phase fraction than suspension cells. Thus, these AML-iPSC-derived hematopoietic cells exhibit hallmarks of an LSC model, namely phenotypic and functional heterogeneity and hierarchical organization, with the A fraction containing LSCs that serially transplant leukemia and give rise to more differentiated cells (S fraction) without engraftment potential.Transcriptome analyses showed that the A to S transition was predominantly mediated by gene upregulation with only 2 out of 418 differentially expressed genes being downregulated and revealed enrichment for HSC and LSC gene sets in the A fraction. Consistent with a general transcriptional upregulation characterizing the A to S transition, differentially accessible regions by ATAC-seq analysis were found predominantly in intragenic regions and enhancers in A cells, while they mostly localized in gene promoters in S cells. Time-course single cell-RNA-seq analyses and their integration with bulk RNA-seq and ATAC-seq data revealed cell clusters specific to the A cell fraction, which were enriched in HSC, LSC and hESC genes. These integrated analyses revealed candidate genes with a role in maintaining LSC properties. The transcription factor RUNX1 was particularly prominent, with RUNX1 motifs found highly accessible in A compared to S cells. Overexpression of RUNX1 in S cells conferred a more LSC-like immunophenotype. RUNX1 is a key transcriptional regulator of hematopoiesis. It has a well-characterized role during development of the hematopoietic system and is often mutated or translocated in AML. A role of RUNX1 in LSC maintenance was unanticipated in view of its known tumor-suppressor function. In further support for a role for RUNX1 in LSCs, we found RUNX1 expression to significantly correlate with survival in AML patient cohorts.In summary, we developed a new model that enables us to prospectively isolate large numbers of genetically clonal human AML LSCs and perform genome-wide integrative molecular studies, with which we obtained new insights into the biology of AML LSCs. DisclosuresNo relevant conflicts of interest to declare.

Relapsed Acute Myeloid Leukemia Is Less Sensitive to Venetoclax + Azacitidine Due to Leukemia Stem Cell Resistance Driven By Fatty Acid Metabolism and Can be Overcome By Pharmacologic Inhibition of CPT1

Effective targeting of the acute myeloid leukemia (AML) leukemia stem cell (LSC) population may allow for deep, durable remissions and curative potential. In older, newly diagnosed AML patients who are not candidates for induction, venetoclax + azacitidine (aza) targets specific metabolic vulnerabilities of LSCs, resulting in very promising clinical outcomes. In our single institution experience treating 45 previously untreated AML patients with venetoclax + aza both in the context of the multi-institutional study NCT02203773 (N=33) and with off-label use (N=12), 36/45 (80%) achieved a complete remission (CR) or CR with incomplete count recovery (CRi). In the relapsed/refractory (R/R) setting, the efficacy of venetoclax + aza has been reported to be significantly worse. In our single-institution off-label experience (N=7), only 1/7 (14%) R/R patients had a CR/CRi (p=0.005 compared to the untreated group). R/R and untreated patients had similar baseline characteristics, although more R/R patients had an antecedent hematological disorder (Table 1). Multivariate analysis showed cytogenetic risk and R/R disease as the sole predictors of response to venetoclax + aza (Table 2). In light of existing data regarding biological changes that occur in LSCs after treatment and subsequent relapse, we aimed to determine whether laboratory analysis of LSCs from patients treated with venetoclax + aza would show differential sensitivity to this therapy in the up-front vs R/R setting that could help to explain the different clinical activity.We have previously shown that LSCs from untreated patients are uniquely reliant on oxidative phosphorylation (OXPHOS), and that venetoclax + aza targets LSCs by decreasing OXPHOS. Therefore, we tested the hypothesis that inferior responses of R/R patients to venetoclax + aza are due to changes in OXPHOS regulation in relapsed LSCs. LSCs were defined as cells bearing relatively low levels of reactive oxygen species (ROS-low), an effective means of enriching primary human LSCs.We found that in contrast with untreated patients, venetoclax + aza does not decrease viability or OXPHOS in LSCs from R/R patients (Fig1). Furthermore, R/R LSCs had altered fatty acid metabolism that contributed to these OXPHOS differences, with increased flux of fatty acids into the TCA cycle (Fig 2). In addition, R/R samples compensated for the metabolic perturbations that occurred upon exposure to venetoclax + aza through upregulation of fatty acid uptake and metabolism into the TCA cycle (Fig 3).Fatty acid metabolism is controlled by multiple genes and pathways. Integral to its activity is the gene Carnitine Palmitolytransferase 1 (CPT1), due to its pivotal role in the beta-oxidation of long chain fatty acids. Investigation of the Cancer Genome Atlas AML dataset reveals higher expression of CPT1 leads to significantly worse overall survival, suggesting increased fatty acid metabolism may drive a more resistant LSC population in R/R AML patients. We also found elevated baseline levels of CPT1 in patients who progressed on venetoclax + aza compared to those that had long term remissions (not shown). Therefore we utilized the CPT1 inhibitor etomoxir to block fatty acid metabolism. We found addition of etomoxir to cultures of R/R LSCs rescued the ability of venetoclax + aza to decrease OXPHOS and re-sensitized R/R LSCs to venetoclax + aza (Fig 4). To prove that this novel regimen targets functionally-defined R/R LSCs we performed ex vivo treatment followed by xenotransplantation of R/R patient specimens, which showed that upon etomoxir addition, engraftment potential is significantly decreased over venetoclax + aza alone (not shown).Therefore we propose a novel mechanism for the increased resistance of R/R AML patients to venetoclax + aza involving altered energy metabolism. We find increased fatty acid metabolism in R/R patient specimens, and targeting this pathway using the CPT1 inhibitor etomoxir leads to sensitization to venetoclax + aza and rescued targeting of OXPHOS, allowing for LSC eradication. [Display omitted] DisclosuresPollyea:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Curis: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees.

Brief Ex Vivo Incubation with Fas Ligand Selectively Depletes Alloreactive T Cells and Antigen Presenting Cells from Stem Cell Grafts

Graft versus host disease (GvHD) proceeds to be the Achilles' heel of hematopoietic stem cell transplantation, with clinicians continue facing a classic conflict: too much GvHD and the patient is at risk for transplant-related mortality and decreased quality of life; too little GvHD and the patient is at increased risk of relapse of their malignant disease. T cells and antigen presenting cells (APCs) are major components of the hematopoietic G-CSF mobilized peripheral blood cells (PBCs) graft. While GvHD is T cell mediated, the APCs are required for the initiation and maintenance of the GvHD. To reduce the risk for GvHD, grafts are sometimes depleted of their T cells, however, while preventing GvHD, the critically important attributes of graft versus leukemia (GvL) effect and engraftment are reduced significantly. Novel strategies that aim to abrogate or ameliorate GvHD, while preserving engraftment and GvL are of great need.A short incubation (2hr) of G-CSF mobilized PBCs with multimeric Fas ligand (i.e. ApoGraft) selectively induces apoptosis in T cell subsets and APCs (Panels A and B), but not in CD34+ progenitor cells (data not shown). FasL treatment preferentially induces apoptosis in mature T cell subsets which express high levels of Fas (CD95), such as T stem cell memory (TSCM), T central memory (TCM), and T effector memory (TEM) cells, as well as the pro-inflammatory T cell subtypes TH1 and TH17 cells, while no apoptotic signal is detected in the non-expressing CD95 naïve T cells (Panel A). The expression of T cells and APCs activation markers; CD25 and HLA-DR, respectively, is significantly reduced following apoptotic challenge in vitro (Panel C), as well as in transplanted mice (data not shown). Furthermore, upon an activation stimulus with anti CD3/CD28 beads in vitro, ApoGraft derived T cells secrete lower levels of IFN-γ, than G-CSF mobilized PBCs derived T cells (Panel D).To gain deeper understanding of the kinetics of GvHD development in vivo, NSG mice were transplanted with ApoGraft or G-CSF mobilized PBCs. Homing, expansion and differentiation of human leukocytes subtypes within the mice bone marrow, spleen and blood, were monitored 3, 7 and 14 days post transplantation. Decreased levels of T and B cells infiltration and expansion were detected in the spleen (Panels E and F), suggesting reduced formation of allo-reactive T cell clones. Reduced proliferation of these cells was associated with lower levels of IFN-γ secreted to the plasma (Panel H) and was in correlation with reduced GvHD and prolonged survival of the ApoGraft transplanted mice (Panel G). Importantly, we have previously demonstrated both in-vitro and in-vivo that ApoGraft has similar GvL and stem cell engraftment capabilities, compared to control G-CSF mobilized PBCs (data not shown). In conclusion, in contrast to conventional T- cell depletion methods, ApoGraft, an ex-vivo FasL-treated graft, affects both the T-cells and APCs, leading to reduced GvHD, while maintaining GvL and engraftment potential (Panel I). ApoGraft is currently being evaluated in a Phase I/II clinical trial (NCT02828878) in subjects with hematologic malignancies undergoing matched related allo-HSCT. [Display omitted] DisclosuresLevy-Barazany:Cellect Biotherapeutics Ltd: Employment. Pinkas:Cellect Biotherapeutics Ltd: Employment. Rodionov:Cellect Biotherapeutics Ltd: Employment. Marelly:Cellect Biotherapeutics Ltd: Employment. Tzadok:Cellect Biotherapeutics Ltd: Employment. Bakimer:Cellect Biotherapeutics Ltd: Employment. Yarkoni:Cellect Biotherapeutics Ltd: Employment. Peled:Cellect Biotherapeutics Ltd: Consultancy. Zuckerman:Cellect Biotherapeutics Ltd: Consultancy.

Induced Pluripotent Stem Cells to Model Blood Diseases

Our group is developing induced pluripotent stem cell (iPSC) models of myeloid malignancies, including MDS, MPN and AML. We are generating iPSCs from the bone marrow or blood of patients, which can be maintained indefinitely as pluripotent cell lines and, upon in vitro differentiation along hematopoietic lineages, exhibit hallmark features of these diseases. By integrating mutational analyses with cell reprogramming we can derive iPSCs capturing dominant clones, subclones and normal cells from the same patient and thus have established a collection of iPSC lines representing distinct disease stages along the spectrum of myeloid transformation: predisposition syndromes/preleukemic cells/clonal hematopoiesis; low risk MDS; high risk MDS; and MDS/AML. In parallel, we are using the CRISPR/Cas9 system to introduce or correct mutations in normal or malignant iPSCs, respectively, in isogenic settings and sequential CRISPR gene editing to model mutational cooperation. We recently reported that iPSC lines derived from patients with AML re-establish upon differentiation a leukemic phenotype characterized by extensive proliferation of immature myeloid cells that serially transplant a lethal leukemia into NSG mice (Kotini et al. Cell Stem Cell 2017). Strikingly, we observed that the AML-iPSC-derived hematopoietic stem/progenitor cells (HSPCs) contain two morphologically and immunophenotypically distinct cell subpopulations: a cell fraction (adherent, A) exhibiting adherent growth and containing immature cells with an HSC immunophenotype (CD34+/CD38-/CD90+/CD45RA-/CD49f+); and a non-adherent fraction (suspension, S) of more differentiated cells. Fate-tracking experiments revealed a hierarchical organization, with the A cells renewing themselves and continuously giving rise to the S cells through symmetric and asymmetric divisions. The NSG engraftment potential was largely contained within the adherent cell fraction. Thus, AML-iPSCs exhibit the hallmarks of a leukemia stem cell (LSC) model, namely phenotypic and functional heterogeneity and hierarchical organization, with the A fraction containing LSCs that serially transplant leukemia and give rise to more differentiated cells (S fraction) without engraftment potential. LSCs are believed to be a prominent source of AML relapse, but their rarity and the unavailability of universal and specific immunophenotypic markers prohibits their prospective isolation and makes the study of their properties challenging. This new iPSC-based AML-LSC model enables us for the first time to prospectively obtain large numbers of genetically clonal human LSCs and perform genome-wide integrative molecular analyses and large-scale screening to identify key molecular mechanisms sustaining the properties of LSCs as potential new therapeutic targets. Using this model we characterized the effects of previously proposed compounds with LSC selectivity in self-renewal vs differentiation of LSCs. We also screened a small molecule library of 1280 compounds in the A and S cells in parallel to identify compounds with selectivity for the former as candidates for LSC-specific targeting. DisclosuresNo relevant conflicts of interest to declare.

Mgta-456 Contains Large Numbers of Expanded Cord Blood (CB) CD34+CD90+ Hematopoietic Stem Cells (HSC) Which Confer All Engraftment Activity and Correlate with Accelerated Neutrophil Recovery after Myeloablative Conditioning in Patients with Hematologic Malignancy

Background: Patient access to well-matched CB containing high doses of stem cells remains a challenge for successful transplants. Low numbers of CD34+ cells in CB has resulted in delayed neutrophil recovery and a risk of graft failure relative to other hematopoietic stem cell (HSC) sources. MGTA-456 is a cell therapy that consists of CD34+ cells expanded in a 15-day culture in the presence of an aryl hydrocarbon receptor antagonist (AHRa) and the CD34 depleted fraction obtained from the same CB unit. Thus far, 40 patients with hematological malignancy (n=36) and non-malignant diseases (n=4) have received MGTA-456 with a median follow-up of 2.5 years (range 0.1 to 5 years) and 75 days (20 to 143 days) respectively. All patients engrafted at a significantly faster rate as compared to similarly treated historical controls (p<0.01). The aim of the current study was to fully characterize the expanded CD34+ cell fraction of MGTA-456 phenotypically and functionally and identify the cell population that correlates with time to neutrophil recovery. We found that the expanded CD34+CD90+ population of MGTA-456 were the cells responsible for engraftment in NOD-scid IL2Rgammanull (NSG) mice. We hypothesized that the dose of CD34+CD90+ cells/kg would have the strongest correlation with time to neutrophil recovery.Methods: The expansion culture consisted of StemSpan SFEM supplemented with SCF, FLT- 3L, TPO and IL-6 (all at 50 ng/mL) and the AHRa without the addition of antibiotics. After a 10 or 15 day culture, the CD34 expanded product was characterized by cell surface markers (CD34, CD90, CD133, CD41, CD71, CD235a, CD3, CD4, CD8, CD14, CD15, CD16, CD11b, CD33, CD19, CD56, and CD10), as well as colony-forming unit (CFU) capacity and engraftment potential in NOD-scid IL2Rgammanull (NSG) mice in a subset of products.Results: The expanded CD34+ cell fraction of MGTA-456 consisted of CD34+CD133-CD90- cells (late progenitors), CD34+CD133+CD90- cells (early progenitors) and CD34+CD133+CD90+ (progenitors and stem cells) as shown in Figure 1. The CD34- cells within the expanded fraction contained erythroid (CD71+) and megakaryocyte progenitors (CD41+), CD33+, CD14+, CD15+, and CD11b+ myeloid cells and CD56+ cells. CD3+, CD8+, CD4+, CD16+, CD19+ as well as CD10+ cells were not present (<1%) in the CD34 expanded fraction of MGTA-456. To identify which of these cell populations contain the NSG engraftment activity, we sorted CD34-, CD34+CD90- and CD34+CD90+ cells from MGTA-456 and transplanted the cell fractions into NSG mice. These studies clearly demonstrated that all NSG engraftment activity resided in the CD34+CD90+ cell subpopulation (Figure 2). Based on this result, we correlated the dose of CD34-, CD34+CD90-, CD34+CD90+ cells, and CFU cells/kg infused with time to neutrophil recovery of patients treated with MGTA-456. The dose of TNC (r2=0.49, P<0.05), CD34+CD90- (r2=0.45, p<0.05), and CD34+CD90+ (r2=0.52, p<0.05) cells/kg all significantly correlated with time to neutrophil recovery. The dose of CD34+CD90+ cells/kg had the strongest correlation with time to neutrophil recovery consistent with the NSG engraftment result.Conclusion: In these studies, we demonstrate that the expanded CD34+ cell fraction of MGTA-456 contains large doses of CD34+CD90+ HSC and progenitors, which are critical for long term engraftment in NSG mice and correlated with rapid neutrophil recovery clinically. [Display omitted] DisclosuresBoitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Goncalves:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Wagner:Magenta Therapeutics: Consultancy, Research Funding; Novartis: Research Funding.

Engineering Resistance to CD33-Targeted Immunotherapy in Normal Hematopoiesis By CRISPR/Cas9-Deletion of CD33 Exon 2

Background:Improved survival with gemtuzumab ozogamicin (GO) in some people with acute myeloid leukemia has validated CD33 as immunotherapeutic target and sparked interested in developing new, highly potent CD33-directed therapeutics. As a limitation of this treatment strategy, CD33 expression on maturing and mature myeloid cells causes significant on-target, off-leukemia effects. Toxicity of CD33-targeted immunotherapy should be minimal in the presence of normal hematopoietic stem and progenitor cells (HSPCs) engineered to lack CD33 variants recognized by therapeutic antibodies. Indeed, very recent studies have shown that CRISPR/Cas9 with a single guide RNA (gRNA) designed to target the CD33coding region reduces display of CD33 and protects engineered cells from CD33 CAR T-cells. However, this approach resulted in low levels of CD33disruption in vivoand off-target activity. We therefore developed an approach in which the Cas9 protein is complexed with two synthetic gRNAs for precise excision of the intervening sequence (i.e. more controlled genome editing than what can be accomplished with a single gRNA). We directed these two gRNAs to intronic sequences for precise excision of exon 2, which encodes the V-set domain of CD33 that is recognized by all current CD33 therapeutics including GO. This approach eliminates exonic indels and protects engineered cells from CD33-targeted immunotherapy while maintaining expression of an exon 2-free variant of CD33 (CD33∆E2), which has previously been identified as natural isoform in human HSPCs.Methods: We used human myeloid ML-1 cells and human fetal liver CD34+ HSPCs for CRISPR/Cas9 editing, which was carried out by electroporation of purified Cas9 protein complexed with synthetic gRNAs. In vitro cytotoxicity assays were performed to test sensitivity to GO and the CD33/CD3 bispecific antibody AMG 330.For in vivoassessment of engineered HSPCs, NSG neonate mice were infused with 6.0x105human CD34+ cells, and peripheral blood analyzed biweekly for 14 weeks.Results:Delivery of Cas9/sgRNA ribonucleoproteins (RNPs) in ML-1 cells resulted in exon 2 deletion and time-dependent reduction in cell surface display of full-length CD33 (CD33FL). Pure populations of CD33∆E2cells were generated via single-cell cloning. These sublines lacked surface display of CD33FLbut, instead, expressed increased levels of the CD33∆E2transcript. CD33∆E2ML-1 cells were completely resistant to GO and AMG 330, whereas wild-type ML-1 cells were highly sensitive to these two drugs. Consistent with the findings in ML-1 cells, delivery of CRISPR/Cas9 RNPs to human fetal liver CD34+ HSPCs resulted in the expected CD33exon 2 deletion and decreased CD33FLsurface expression, while it did not impact the distribution of colony-forming cellsand only minimally reduced colony-forming potential. In NSG mouse xenotransplantation experiments, we found these CD33∆E2human CD34+ HSPCs to have comparable engraftment and multilineage differentiation potential relative to HSPCs expressing CD33FL(Fig. 1A). As expected, CD33FLexpression was substantially reduced in peripheral blood monocytes from CD33∆E2 HSPC-engrafted animals (Fig. 1B). In addition, robust engraftment of CD33∆E2 -engineered HSPCs in bone marrow was demonstrated by over 50% biallelic CD33exon 2 deletion at time of necropsy from colony-forming cells analysis (Fig. 1C). To determine if in vivodifferentiated myeloid CD33∆E2cells derived from infused human CD34+ HSPCs are indeed resistant to CD33-directed therapies, we treated mice with GO intravenously. GO administration reduced the number of circulating CD33FLCD14+ myeloid cells while leaving the number of CD33∆E2cells unaffected.Conclusion:These findings support a novel strategy in which CD33∆E2-engineeredHSPCs are used to widen the therapeutic window of CD33-directed immunotherapies. Such cells could be envisioned for people at risk for the need of CD33-immunotherapy, e.g. AML patients in remission (where CD33-immunotherapy could be used to prevent/treat relapse) or, pre-emptively, for people with genetic AML-predisposition syndromes (such that CD33-targeted immunotherapy could be given safely if AML occurred). With this potential, further development of CD33∆E2-engineered HSPCs toward clinical application is warranted. [Display omitted] DisclosuresWalter:Amphivena Therapeutics, Inc: Consultancy, Other: Clinical Trial Support, Research Funding; Aptevo Therapeutics, Inc: Consultancy, Other: Clinical Trial Support, Research Funding; Covagen AG: Consultancy, Other: Clinical Trial Support, Research Funding; Actinium Pharmaceuticals, Inc: Other: Clinical Trial support , Research Funding; Amgen Inc: Other: Clinical Trial Support, Research Funding; Boehringer Ingelheim Pharma GmbH & Co. KG: Consultancy; Pfizer, Inc: Consultancy; Seattle Genetics, Inc: Consultancy, Other: Clinical Trial Support, Research Funding. Kiem:Magenta: Consultancy; Homology Medicine: Consultancy; Rocket Pharmaceuticals: Consultancy.

Potential Long-Term Treatment of Hemophilia a By Early Postnatal Co-Transplantation of Cord Blood Derived Endothelial Colony-Forming Cells and Placental Mesenchymal Stem Cells

Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the Factor VIII (FVIII) gene leading to deficient blood coagulation. As a monogenic disorder, HA is an ideal target for gene or cell-based therapy. Both mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) have been used independently to correct the HA phenotype, but poor survival and insufficient retention of transplanted cells in the long term limit their application. In this study, we sought to determine whether co-transplantation of endothelial colony-forming cells (ECFCs) with placental mesenchymal stem cells (PMSCs) can achieve stable, long-term engraftment. To investigate stem cell engraftment and therapeutic potential, ECFCs and PMSCs were transduced with a B domain deleted factor VIII (BDD-FVIII) expressing lentiviral vector. To distinguish between PMSCs and ECFCs in the co-transplantation group, a Td-Tomato containing lentiviral vector was used to label PMSCs while a green fluorescent protein (GFP) labeling vector was used to label ECFCs. They were then transplanted intramuscularly either as ECFC only group, PMSC only group or co-transplantation of ECFCs and PMSCs group into neonatal (3 days) or adult (12 weeks) immune deficient NOD scid gamma (NSG) mice. Using an in vivo bioluminescence imaging system, we investigated cell engraftment for up to 26 weeks after transplantation. ECFC only and the co-transplantation groups achieved better engraftment than PMSCs only group in both neonatal and adult mice during the entire study period. The co-transplantation group showed higher engraftment rate than the ECFC only group at 16 weeks and 20 weeks post-transplantation. In addition, transplantation of ECFCs only or in combination with PMSCs in neonatal period achieved higher engraftment than transplantation in adult period. Immunohistochemical analyses were further employed to determine the fate and functionality of the engrafted cells. We found that engrafted ECFCs maintained endothelial phenotype and expressed high level of FVIII at 26 weeks post-transplantation. The presence of ECFCs in the co-transplantation group also promoted formation of functional vasculature after transplantation. To study the functional reversal of HA phenotype we co-transplanted ECFCs and PMSCs into F8 knock-out HA mice. The transplanted cells reduced the blood loss volume from 562.13±19.84µl of blood to 155.78±44.93µl in a tail clip assay in these HA mice. This work demonstrates that co-transplantation of ECFCs with PMSCs at the neonatal period is a promising strategy to achieve stable, long-term engraftment of transplanted cells, and thus holds great promise for the treatment of HA and other genetic diseases and tissue regeneration applications. DisclosuresNo relevant conflicts of interest to declare.