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Background: Severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID) is a rare disorder caused by ADA gene mutations, leading to lymphotoxic build-up of purine metabolites and profound immunodeficiency. Historically, enzyme replacement therapy (ERT) has been used as a bridge therapy until patients can receive an allogeneic hematopoietic stem cell transplantation (HSCT), ideally from a matched related donor (MRD) or, if none is identified, a non-matched and/or unrelated donor. We developed a self-inactivating lentiviral vector (LV), denoted EFS-ADA LV, encoding the human ADA cDNA sequence under the control of a shortened human elongation factor 1α gene promoter. A fresh or cryopreserved formulation of a drug product (OTL-101), composed of autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with EFS-ADA LV, was evaluated in 2 prospective, non-randomized Phase I/II clinical trials at 2 USA centers. We report on safety and efficacy of OTL-101 in 30 ADA-SCID pediatric gene therapy (GT) subjects treated from 2013-2017 with a median follow up (FU) of 24 months (mo; range 12-26 mo), compared to a historical cohort of 26 ADA-SCID patients treated with HSCT. Methods: UCLA Fresh Study (NCT01852071): Autologous CD34+ HSPCs were isolated from bone marrow and pre-stimulated with cytokines before transduction with EFS-ADA LV to yield OTL-101, which was infused as a fresh formulation in 20 subjects (9 male, 11 female; aged 4 mo-4.3 yrs). Single dose busulfan (4 mg/kg) was administered prior to infusion of OTL-101. Subjects were followed for 24 mo. UCLA Cryo Study (NCT02999984): 10 subjects (4 male, 6 female; aged 5-15 mo) received a cryopreserved formulation of OTL-101, which allowed for an extended shelf-life and full quality control prior to infusion. Busulfan was administered in 2 doses, the first at 3 mg/kg and the second adjusted to target a total area under the curve of 4,900 µM*min (20 ng/mL*hr). At the time of analysis, all subjects reached 12 mo FU (except 1 subject who was withdrawn from the study due to lack of engraftment); 7 subjects reached 18 mo of FU. Historical Control Group: 26 patients (aged 0.2 mo-9.8 yrs) were treated with allogeneic HSCT (MRDs n=12, non-MRDs n=14) at Great Ormond Street Hospital, UK (n=16) or Duke University Children's Hospital, USA (n=10) from 2000-2016. Results: Sustained engraftment of genetically modified HSPCs was observed in 29/30 GT subjects by 6-8 mo and persisted through FU in both studies, based on vector gene marking in granulocytes and CD3+ T cell reconstitution (Figure). Subjects who engrafted maintained long-term metabolic detoxification from deoxyadenosine nucleotides after stopping ERT approximately 1 mo post-GT. At last FU (median 24 mo; range 12-24 mo) in the GT group, overall survival (OS) was 30/30 (100%) and event-free survival (survival in the absence of ERT reinstitution or rescue allogeneic HSCT; EvFS) was 29/30 (97%). OS and EvFS were higher in the GT group at last FU compared with HSCT controls (with or without an MRD) at 2 years (Table). One of 30 OTL-101 subjects (3%) did not engraft and was restarted on ERT; the subject was withdrawn from the study at 5.9 mo and subsequently received a rescue HSCT, whereas 42% of HSCT patients required rescue HSCT, PEG-ADA ERT or died. Among the 20 OTL-101 subjects in the UCLA Fresh Study who reached 2 years FU, 18 (90%) stopped immunoglobin replacement therapy (IgRT), compared to 52% of HSCT patients. Preliminary results were observed in 5/7 (71%) OTL-101 subjects in the UCLA Cryo Study with more limited (18 mo) FU. Twelve OTL-101 subjects experienced one or more serious adverse events, most frequently infections and gastrointestinal events; only 1 of which was considered treatment-related (bacteremia due to product contamination). In the GT group, there were no events of autoimmunity with ≤24 mo FU. Due to the autologous nature of OTL-101, there was no incidence of graft vs host disease (GvHD); in contrast, 8 HSCT patients experienced GvHD events (5 acute, 3 chronic events), 1 of which resulted in death. Conclusions: Based on sustained gene correction and restoration of immune function in all subjects who engrafted, treatment of ADA-SCID with OTL-101 has a favorable benefit-risk profile. Key correlates of engraftment were consistent across the expanded cohort. Importantly, higher rates of OS and EvFS compared with HSCT (with or without an MRD) were observed. Disclosures Kohn: Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Inventor on IP licensed from UC Regents to Orchard Therapeutics. Future royalties may occur., Research Funding; NIH: Research Funding. Shaw:Orchard Therapeutics: Consultancy, Other: Personal fees and non-financial support; NIH: Research Funding. Carbonaro-Sarracino:NIH: Other: Salary while working on project at UCLA 2013-2016, Research Funding; Orchard Therapeutics: Consultancy, Employment. De Oliveira:National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London: Research Funding; CIRM: Research Funding; National Gene Vector Repository: Research Funding; NIAID, NHI: Research Funding; Medical Research Council: Research Funding. Terrazas:California Institute for Regenerative Medicine: Research Funding; Gene Therapy Resource Program, NHLBI/NIH: Research Funding. Hollis:Curative Therapeutics: Consultancy, Other: Personal fees. Trevisan:Orchard Therapeutics: Research Funding. Arduini:Orchard Therapeutics: Employment, Equity Ownership. Lynn:Orchard Therapeutics: Employment, Equity Ownership. Kudari:Orchard Therapeutics: Employment, Equity Ownership. Spezzi:Orchard Therapeutics: Employment, Equity Ownership. Buckley:Duke University: Research Funding. Booth:SOBI: Consultancy; GSK: Honoraria; NovImmune: Consultancy. Thrasher:Generation Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 4BIOCapital: Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Gaspar:Orchard Therapeutics: Employment, Equity Ownership, Patents & Royalties: Lentiviral vector for gene therapy of ADA-SCID.

We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)–deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34+ cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m2). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency.

Although the mechanisms of cross-talk that regulate the hematopoietic and epithelial compartments of the thymus are well established, the interactions of these compartments with the thymic endothelium have been largely ignored. Current understanding of the thymic vasculature is based on studies of adult thymus. We show that the neonatal period represents a unique phase of thymic growth and differentiation, marked by endothelium that is organized as primitive, dense networks of capillaries dependent on vascular endothelial growth factor (VEGF). VEGF dependence in neonates is mediated by significantly higher levels of both VEGF production and endothelial VEGF receptor 2 (VEGF-R2) expression than in the adult thymus. VEGF is expressed locally in the neonatal thymus by immature, CD4−CD8− “double negative” (DN) thymocytes and thymic epithelium. Relative to adult thymus, the neonatal thymus has greater thymocyte proliferation, and a predominance of immature thymocytes and cortical thymic epithelial cells (cTECs). Inhibition of VEGF signaling during the neonatal period results in rapid loss of the dense capillaries in the thymus and a marked reduction in the number of thymocytes. These data demonstrate that, during the early postnatal period, VEGF mediates cross-talk between the thymocyte and endothelial compartments of the thymus.

The identity and lineage potential of the cells that initiate thymopoiesis remain controversial. The goal of these studies was to determine, at a clonal level, the immunophenotype and differentiation pathways of the earliest progenitors in human thymus. Although the majority of human CD34+lin− thymocytes express high levels of CD7, closer analysis reveals that a continuum of CD7 expression exists, and 1% to 2% of progenitors are CD7−. CD34+lin− thymocytes were fractionated by CD7 expression and tested for lineage potential in B-lymphoid, T-lymphoid, and myeloid-erythroid conditions. Progressive restriction in lineage potential correlated with CD7 expression, that is, the CD7hi fraction produced T and NK cells but lacked B and myelo-erythroid potential, the CD7int (CD10+) fraction produced B, T, and NK cells, but lacked myelo-erythroid potential. The CD7− fraction produced all lymphoid and myelo-erythroid lineages and expressed HSC-associated genes. However, CD34+lin−CD7− thymocytes also expressed early T lymphoid genes Tdt, pTα, and IL-7Rα and lacked engraftment capacity, suggesting the signals that direct lymphoid commitment and corresponding loss of HSC function are rapidly initiated on arrival of HSC in the human thymus. Thus, differential levels of CD7 identify the progressive stages of lineage commitment in human thymus, initiated from a primitive CD7− lympho-myeloid thymic progenitor.

A patient with adenosine deaminase–deficient severe combined immune deficiency (ADA-SCID) was enrolled in a study of retroviral-mediated ADA gene transfer to bone marrow hematopoietic stem cells. After the discontinuation of ADA enzyme replacement, busulfan (75 mg/m2) was administered for bone marrow cytoreduction, followed by infusion of autologous, gene-modified CD34+ cells. The expected myelosuppression developed after busulfan but then persisted, necessitating the administration of untransduced autologous bone marrow back-up at day 40. Because of sustained pancytopenia and negligible gene marking, diagnostic bone marrow biopsy and aspirate were performed at day 88. Analyses revealed hypocellular marrow and, unexpectedly, evidence of trisomy 8 in 21.6% of cells. Trisomy 8 mosaicism (T8M) was subsequently diagnosed by retrospective analysis of a pretreatment marrow sample that might have caused the lack of hematopoietic reconstitution. The confounding effects of this preexisting marrow cytogenetic abnormality on the response to gene transfer highlights another challenge of gene therapy with the use of autologous hematopoietic stem cells.

Rodent bone marrow cells can contribute to liver. If these findings are applicable to humans, marrow stem cells could theoretically be harvested from a patient and used to repair his/her damaged liver. To explore this potential, CD34+ or highly purified CD34+CD38−CD7− human hematopoietic stem cells from umbilical cord blood and bone marrow were transplanted into immunodeficient mice. One month after transplantation, carbon tetrachloride (CCl4) was administered into the mice to induce liver damage and hepatocyte proliferation. Mice were analyzed in comparison with CCl4-injured mice that did not receive transplants and noninjured controls that received transplants with the same stem cell populations, one month after liver damage. Human-specific albumin mRNA and protein were expressed in the mouse liver and human albumin was detected in the serum of mice that had received CCl4 injury. Human alpha-fetoprotein was never expressed, but in some mice, human cytokeratin 19 was expressed, which may indicate bile duct development in addition to the albumin-secreting hepatocyte-like cells. Human albumin was not expressed in the starting stem cell populations in injured mice that did not receive transplants nor in noninjured mice that had received transplants of human stem cells. Human albumin expression was detected only in CCl4-treated mice that received transplants of human stem cells, and recovery was increased by administration of human hepatocyte growth factor 48 hours after the CCl4-mediated liver injury. Our studies provide evidence that human “hematopoietic” stem/progenitor cell populations have the capacity to respond to the injured liver microenvironment by inducing albumin expression.

Adoptive cell therapy using T cells engineered to express antigen-specific T cell receptors (TCR-T) or chimeric antigen receptors (CAR-T) offer targeted and potentially curative treatments for malignancy. Current approaches rely on the genetic modification and expansion of mature circulating T-cells. Such processes are limited to autologous T cells due to the risk of graft-versus-host (GvHD) disease from allogeneic T cells through endogenous TCR expression as well as rejection through MHC incompatibility. Furthermore, prolonged ex-vivo expansion of T cells may reduce in vivo efficacy and harvesting sufficient T cells from lymphopenic patients is challenging. Direct in vitro differentiation of engineered T cells from human pluripotent stem cells (HSPCs) may overcome these problems by providing an unlimited source of cells that can be genetically edited, permitting the suppression of endogenous TCR expression through allelic exclusion, and the de novo generation of naïve antigen-specific T cells. We have developed an in vitro Artificial Thymic Organoid (ATO) system that induces highly efficient and reproducible production of mature naïve T cells from human hematopoietic stem cells and progenitor cells (HSPC). Here, we report the preclinical development of a modified ATO system that supports highly efficient in vitro differentiation and positive selection of naive human T cells from at least 5 different lines of human pluripotent stem cells (PSC), including Embryonic stem cells (ESC) and induced Pluripotent Stem Cells (iPSC). T cell differentiation from PSC was very similar phenotypically to that from HSPC. As in normal human thymopoiesis, the first evidence for T cell commitment was expression of CD7 and CD5, followed by the CD3-CD8lo "ISP8" stage, then CD4+CD8+ "DP" stage and finally production of CD3+CD8+CD4- "CD8SP" and Cd3+CD4+CD8- "CD4SP". As is typical with both monolayer cultures and ATOs (and opposite to normal thymus), CD8SP predominated over CD4SP. Surprisingly, differentiation occurred more rapidly from PSC than with HSPC. As with HSPC-ATOs, CD8SP from PSC ATOs showed a mature naïve conventional T cell phenotype i.e. CD3+TCRab+CD4- CD45RA+CD62L+CD27+ and exhibited a diverse, thymic-like TCR repertoire, and robust TCR-dependent cytokine release and proliferation. The differentiation in ATOs of an ESC line that expresses an HLA-A*02:01-restricted αβ TCR specific for NY-ESO-1 resulted in a markedly increased cell yield with an enhanced generation of naïve CD3+TCRαβ+CD8αβ+ conventional T cells, the majority of which were antigen-specific by tetramer staining. TCR-engineered T cells produced from PSC in ATOs displayed a near complete lack of endogenous TCR Vβ expression, consistent with induction of allelic exclusion by the exogenous TCR during T cell development. The TCR engineered T cells underwent polyfunctional cytokine release, and proliferation in response to artificial APCs. Moreover, the differentiation in ATOs of an ESC line that expresses a CD19-specific 2nd generation (CD28/CD3zeta) CAR construct resulted in the production of CD5+CD7+ CD45RA+ CAR T cells. As reported previously, the ESC-derived CAR T cells did not express CD4, CD8 or CD3; however, they responded to PMA/ionomycin and underwent specific cytokine release and degranulation in response to target cells expressing CD19. PSC-derivedATOs thus present a highly efficient platform for the generation of clinically relevant mature naïve and potentially non-alloreactive TCR and CAR engineered T cells for adoptive immunotherapy. Disclosures Montel-Hagen: Kite Pharma: Research Funding. Seet: Kite Pharma: Research Funding. Crooks: Kite Pharma: Research Funding.

The earliest stages of lymphoid commitment from human pluripotent hematopoietic stem cells have not been defined. A clonogenic subpopulation of CD34(+)CD38(-) cord blood cells were identified that expressed high levels of the CD7 antigen and possessed only lymphoid potential. CD34(+)CD38(-)CD7(+) (CD7(+)) cells uniformly coexpressed CD45RA and HLA-DR; c-kit and Thy-1 expression was absent to low. Clonal analysis demonstrated that single CD7(+) cells could generate B cells, natural killer cells, and dendritic cells but were devoid of myeloid or erythroid potential. In contrast, control CD34(+)CD38(-)CD7(-) (CD7(-)) cells generated both lymphoid and myelo-erythroid cells. The lymphoid potential (generation of lymphoid progeny in bulk and single cell cultures) of CD7(+) cells was equivalent to that of the pluripotent CD7(-) cells. RNA expression studies showed that CD7(+) cells expressed PU.1 and GATA-3, but did not express Pax-5, terminal deoxynucleotide transferase, or CD3epsilon. In contrast to the previously described murine common lymphoid progenitor, the alpha chain of the receptor for interleukin-7 was not detected by fluorescence-activated cell sorting analysis or RNA polymerase chain reaction in CD7(+) cells. These studies identify a clonogenic lymphoid progenitor with both B-cell and natural killer cell lineage potential with a molecular profile that suggests a developmental stage more primitive than previously identified lymphoid progenitors. The CD7(+) phenotype distinguishes primitive human lymphoid progenitors from pluripotent stem cells, thus allowing the study of regulation of early human lymphopoiesis and providing an alternative to pluripotent stem cells for genetic manipulation and transplantation. (Blood. 2001;97:3683-3690)

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, eczema, and a progressive deterioration of immune function. WAS is caused by mutations in an intracellular protein, WASP, that is involved in signal transduction and regulation of actin cytoskeleton rearrangement. Because immune dysfunction in WAS may be due to an accelerated destruction of lymphocytes, we examined the susceptibility to apoptosis of resting primary lymphocytes isolated from WAS patients in the absence of exogenous apoptogenic stimulation. We found that unstimulated WAS lymphocytes underwent spontaneous apoptosis at a greater frequency than unstimulated normal lymphocytes. Coincident with increased apoptotic susceptibility, WAS lymphocytes had markedly attenuated Bcl-2 expression, whereas Bax expression did not differ. A negative correlation between the frequency of spontaneous apoptosis and the level of Bcl-2 expression was demonstrated. These data indicate that accelerated lymphocyte destruction by spontaneous induction of apoptosis may be one pathogenic mechanism by which the progressive immunodeficiency in WAS patients develops.

The role of the homeobox gene HOXA5 in normal human hematopoiesis was studied by constitutively expressing theHOXA5 cDNA in CD34+ and CD34+CD38− cells from bone marrow and cord blood. By using retroviral vectors that contained both HOXA5and a cell surface marker gene, pure populations of progenitors that expressed the transgene were obtained for analysis of differentiation patterns. Based on both immunophenotypic and morphological analysis of cultures from transduced CD34+ cells, HOXA5expression caused a significant shift toward myeloid differentiation and away from erythroid differentiation in comparison to CD34+ cells transduced with Control vectors (P= .001, n = 15 for immunophenotypic analysis; and P < .0001, n = 19 for morphological analysis). Transduction of more primitive progenitors (CD34+CD38− cells) resulted in a significantly greater effect on differentiation than did transduction of the largely committed CD34+ population (P = .006 for difference between HOXA5 effect on CD34+v CD34+CD38−cells). Erythroid progenitors (burst-forming unit-erythroid [BFU-E]) were significantly decreased in frequency among progenitors transduced with the HOXA5 vector (P = .016, n = 7), with no reduction in total CFU numbers. Clonal analysis of single cells transduced with HOXA5 or control vectors (cultured in erythroid culture conditions) showed that HOXA5expression prevented erythroid differentiation and produced clones with a preponderance of undifferentiated blasts. These studies show that constitutive expression of HOXA5 inhibits human erythropoiesis and promotes myelopoiesis. The reciprocal inhibition of erythropoiesis and promotion of myelopoiesis in the absence of any demonstrable effect on proliferation suggests that HOXA5 diverts differentiation at a mulitpotent progenitor stage away from the erythroid toward the myeloid pathway.

Human hematopoietic stem cells are pluripotent, ie, capable of producing both lymphoid and myeloid progeny, and are therefore used for transplantation and gene therapy. An in vitro culture system was developed to study the multi-lineage developmental potential of a candidate human hematopoietic stem cell population, CD34+CD38− cells. CD34+CD38− cells cocultivated on the murine stromal line S17 generated predominantly CD19+ B-cell progenitors. Transfer of cells from S17 stroma to myeloid-specific conditions (“switch culture”) showed that a fraction of the immunophenotypically uncommitted CD19− cells generated on S17 stroma had myeloid potential (defined by expression of CD33 and generation of colony-forming unit-cells). Using the switch culture system, single CD34+CD38− cells were assessed for their lymphoid and myeloid potential. Nineteen of 50 (38%) clones generated from single CD34+CD38− cells possessed both B-lymphoid and myeloid potential. 94.7% of the CD34+CD38− cells with lympho-myeloid potential were late-proliferating (clonal appearance after 30 days), demonstrating that pluripotentiality is detected significantly more often in quiescent progenitors than in cytokine-responsive cells (P = .00002). The S17/switch culture system permits the in vitro assessment of the pluripotentiality of single human hematopoietic cells.

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

Self-renewal capacity is rapidly lost during differentiation of hematopoietic stem cells to lineage-committed progenitors. We demonstrate here that regulated intracellular signaling through the cytokine receptor Mpl induces profound expansion of not only multipotent (ie, lymphomyeloid) but also lymphoid-committed human hematopoietic progenitors. A fusion protein containing the intracellular signaling domain of Mpl and a dimerization domain was constitutively expressed in populations enriched in human lymphomyeloid progenitor/stem cells (CD34+CD38−Lin−CD7−) and multilymphoid progenitors (CD34+CD38−Lin−CD7+). Intracellular dimerization of Mpl in target cells was induced by in vitro or in vivo administration of a diffusible synthetic ligand. In vitro, Mpl dimerization produced divisions of clonogenic, multilineage CD34+ cells able to engraft immunodeficient mice. When dimerization was induced in vivo after transplantation of either lymphomyeloid or multilymphoid progenitors, donor-derived hematopoiesis was sustained for at least 12 weeks and primitive CD34+Lin− progenitors were expanded more than 1000-fold. Lineage potential of progenitors was not altered and differentiation was not prevented by synthetically induced Mpl signaling. These data demonstrate that dimerization of a single cytokine receptor can deliver a profound expansion signal in both uncommitted and lymphoid-committed human hematopoietic progenitors.

CD1d-restricted Vα24-invariant natural killer T cells (iNKTs) are important in immunoregulation. CD4+ and CD4- iNKTs develop with similar frequencies in murine thymus and depend on interleukin-15 (IL-15) in periphery. However, homeostatic requirements of iNKTs have not been analyzed in humans. We evaluated thymic production, peripheral dynamics, and functional maturation of human iNKTs. CD4+ subset comprises 90% of iNKTs in mature thymocytes and cord blood (CB) but only 40% in adult blood. Using T-cell receptor excision circle (TREC) analysis, we directly measured in vivo replicative history of CD4+ and CD4- iNKT cells. Compared to CD4+, CD4- iNKTs contain fewer TRECs, express higher levels of IL-2Rβ, and proliferate with higher rate in response to IL-15. In contrast, CD4+ cells express higher levels of IL-7Rα and better respond to IL-7. Neither thymic nor CB iNKTs are able to produce cytokines unless they are induced to proliferate. Therefore, unlike in the mouse, human CD4+ iNKTs are mainly supported by thymic output and limited peripheral expansion, whereas CD4- cells undergo extensive peripheral expansion, and both subsets develop their functions in periphery. These findings reveal important differences in homeostatic requirements and functional maturation between murine and human iNKTs that are to be considered for clinical purposes.

Mammals respond to infection by rapid production of innate immune cells. Hematopoietic stem and progenitor cells (HSPCs) have the ability to respond to pathogens directly through toll-like receptors. However, how the pathogen sensing ability of HSPCs may contribute to immune cell output remains unknown. Using a novel single-cell proteomic platform and mouse genetic models, we have shown that in response to toll-like receptor stimulation, short-term HSCs and multipotent progenitor cells produce copious amounts of diverse cytokines through NF-κB signaling. Interestingly, the cytokine production ability of HSPCs trumps mature immune cells in both magnitude and breadth. Among cytokines produced by HSPCs, IL-6 is a particularly important regulator of myeloid differentiation and HSPC proliferation. This is especially important in mediating rapid early myeloid cell recovery during neutropenia after chemotherapy or HSC transplant. This study has uncovered an important property of HSPCs that enables them to convert danger signals into versatile cytokine signals for the regulation of stress-induced hematopoiesis. To translate these findings into human hematopoietic system, we have recently developed a human cytokine chip and our preliminary analysis suggests that purified human bone marrow HSCs also have the capacity to secrete cytokines. Disclosures No relevant conflicts of interest to declare.

Successful autologous hematopoietic stem cell (HSC) transplantation in childhood acute lymphoblastic leukemia (ALL) requires the ability to either selectively kill the leukemia cells or separate normal from leukemic HSC. Based on previous studies showing that more than 95% of childhood B-lineage ALL express CD38, this study evaluated whether normal CD34(+)CD38(-) progenitors from children with B-lineage ALL could be isolated by flow cytometry. CD34(+) cells from bone marrow samples from 10 children with B-lineage ALL were isolated at day 28 of treatment, when clinical remission had been attained. The CD34(+) progenitor cells were flow cytometrically sorted into CD34(+)CD38(+) and CD34(+)CD38(-) populations. The absolute numbers of CD34(+)CD38(-) cells that could be isolated ranged from 401 to 6245. The cells were then analyzed for the presence of clonotypic rearrangements of the T-cell receptor (TCR) Vdelta2-Ddelta3 locus. Only patients whose diagnostic marrow had an informative TCR Vdelta2-Ddelta3 rearrangement were included in this study. Detection thresholds were typically 10(-4) to 10(-5) leukemic cells in normal marrow. In 6 of 10 samples analyzed, the sorted CD34(+)CD38(-) cells had no detectable Vdelta2-Ddelta3 rearrangements. In 4 cases, the clonotypic leukemic Vdelta2-Ddelta3 rearrangement was detected in the CD34(+)CD38(-) population, indicating that the putative normal HSC population also contained leukemic cells. The data indicate that although most childhood ALL cells express CD34 and CD38, leukemic cells are also frequently present in the CD34(+)CD38(-) population. Therefore, strategies to isolate and transplant normal HSC from children with ALL will require a more stringent definition of the normal HSC than the CD34(+)CD38(-) phenotype. (Blood. 2001;97:3925-3930)

Objectives Unlimited self renewal capacity and the ability to differentiate into any cell type make human pluripotent stem cells (PSC) a potential source for the ex vivo manufacture of red blood cells (RBC) for safe transfusion. Current methods of RBC differentiation from PSC suffer from low yields of RBCs, most of which contain embryonic and fetal rather than adult hemoglobins. We have previously shown that dimerization of the intracellular component of MPL (the thrombopoietin receptor), induces expansion of myelo-erythroid progenitors (MEP) from human cord blood as well as their terminal differentiation into enucleated RBC through unique, EPO-independent mechanisms (Parekh et al, 2012). Our goal was to investigate the potential of intracellular MPL dimerization to induce erythropoiesis from human PSC and to identify the signaling pathways activated by this strategy. Methods Human embryonic stem cell (hESC) lines H1 and HES3 were transduced with a lentiviral vector to express the fusion protein F36V-MPL (containing the ligand binding domain F36V and the intracytoplasmic portion of MPL). Dimerization of F36V-MPL was accomplished by addition of the synthetic ligand AP20187 (aka CID) during culture (with or without erythropoietin) on OP9 stroma in the absence of other cytokines. F36V-MPL transduced-hESC that did not receive CID and F36V-transduced hESC cultured with CID served as negative controls. Flow cytometry and Colony Forming Unit (CFU) assays were used to analyze erythroid differentiation. Phosflow and Western Blot were used to analyze cell signaling. MEP generated during hESC differentiation were defined as cells co-expressing GlyA and CD41a/CD42a. Results F36V-MPL dimerization induced significantly more Glycophorin A+ cells (P=0.0001; n=5) and 10-fold higher number of erythroid CFU (P=0.0007; n=15) as compared to negative controls. The effect was consistent across different hESC cell lines. This effect was seen in the absence of any hematopoietic cytokines, including erythropoietin (EPO), a critical cytokine for erythropoiesis and an integral component of all ex vivo PSC erythroid differentiation protocols, indicating that MPL dimerization alone is sufficient to induce erythropoiesis from hESCs. Erythroid output was further enhanced in an additive manner in the presence of EPO (P=0.006; n=5). In order to identify the point at which MPL dimerization affects erythropoiesis, CID was added during differentiation directly from hESC or to isolated MEP generated from hESC. CID and EPO increased the number of MEP compared to untreated controls, demonstrating that MPL dimerization induces the generation of early erythroid progenitors. In addition, CID drove erythroid differentiation from MEP more efficiently than EPO, demonstrated by a significantly higher frequency of total erythroid cells (P=0.02; n=3), and 4-fold increase in yield of enucleated RBC. Globin analysis by HPLC demonstrated that although no detectable beta-globin expression was observed with EPO, CID treatment induced the presence of beta-globin and increased the gamma: epsilon globin ratio, suggesting a shift toward definitive erythropoiesis. Signaling studies found that, unlike the full-length MPL receptor, which activates both STAT5/JAK2 and AKT pathways, F36V-MPL dimerization activated AKT but not STAT5 or JAK2 phosphorylation. PI3K/AKT inhibitors (LY294002 and AKT inhibitor IV) effectively inhibited erythroid differentiation of transduced hESC cultured in the presence of CID (P=0.001; n=4) indicating that MPL dimerization induced erythropoiesis is dependent on AKT signaling. Gene expression analysis by qPCR indicated that MPL dimerization upregulates a network of genes (downstream of AKT signaling) associated with the regulation of cell cycle, apoptosis, and erythroid differentiation, including GATA1, CDKN1A, RB1, VEGFA, and BCL-xL with a corresponding reduction in both apoptosis and cell cycle progression. Conclusion In summary, we have identified a novel EPO-independent approach that is not only more efficient at erythropoiesis but is also able to augment EPO induced erythropoiesis. The mechanistic insights gained from this study opens up potentially new approaches toward the generation of therapeutically relevant quantities of RBCs for transfusion. Disclosures: No relevant conflicts of interest to declare.

A system that allows manipulation of the human thymic microenvironment is needed both to elucidate the extrinsic mechanisms that control human thymopoiesis, and to develop potential cell therapies for thymic insufficiency. Implantation of intact fetal thymic fragments into immune deficient mice can be used to model human thymopoiesis in vivo, but this approach has limited capacity for cell or molecular manipulation for either experimental or translational application. We developed an implantable thymic microenvironment composed of two human thymic stroma populations critical for thymopoiesis, thymic epithelial cells (TECs) and thymic mesenchyme (TM). TECs and TM from postnatal human thymi were cultured in specific conditions, allowing cell expansion and manipulation of gene expression, prior to re-aggregation into a functional thymic unit. Human CD34+ hematopoietic stem and progenitor cells (HSPC) differentiated into T cells in the aggregates in vitro and in vivo following inguinal implantation of aggregates in immune deficient mice. Cord blood HSPC previously engrafted into murine bone marrow, were able to migrate to the implants and differentiate into functional human T cells with a broad T cell receptor repertoire. Furthermore, lentiviral mediated expression of vascular endothelial growth factor in TM enhanced TEC survival and function, resulting in significantly greater thymocyte production relative to control implants. These results demonstrate an efficient in vivo system for the generation of T cells from human HSPC, and represent the first model to allow manipulation of gene expression and cell composition in the microenvironment of the human thymus. Disclosures: No relevant conflicts of interest to declare.

Abstract 1190 The inability to derive functional hematopoietic stem cells (HSCs) in vitro from pluripotent cells prevents widespread utilization of HSCs in the clinic; however, the molecular defects compromising the in vitro generated hematopoietic stem/progenitor cells (HSPCs) are unknown. Using a two-step differentiation method in which human embryonic stem cells (hESCs) were first differentiated into embryo bodies (EBs) and then CD34+ cells from hEBs were co-cultured on OP9M2 bone marrow mesenchymal stem cell (MSC) stroma (hEB-OP9), we were able to derive HSPCs expressing the HSC immunophenotype (CD34+CD38−CD90+CD45+) (hereafter termed CD90+HSPCs). Colony forming and stroma co-culture assays demonstrated that the hEB-OP9 CD90+HSPCs were able to differentiate into myelo-erythroid lineages and T-cells. However, when comparing CD90+HSPCs from hEB-OP9 to those from fetal liver (FL)—an in vivo source of HSCs—the former remained severely functionally limited in their proliferative potential and ability to differentiate into B-cells. To identify the basis of the proliferative and differentiation defects, we performed microarray analysis to define gene expression differences between CD90+HSPCs derived from hEB-OP9, FL, early 3–5 week placenta (PL) and an earlier stage of hESC differentiation (hEB). This analysis revealed establishment of the general hematopoietic transcription factor network (e.g. SCL, RUNX1, CMYB, ETV6, HOXB4, MYB), demonstrating the successful differentiation and identification of hematopoietic cells using our two-step culturing techniques and immunophenotype criteria. Moreover, evaluation of Spearman coefficients confirmed CD90+HSPCs isolated from hEB-OP9 culture were brought into closer resemblance of the hFL CD90+HSPCs as compared to to the developmentally immature hEB and hPL CD90+HSPCs. Encouragingly, hEB-OP9 CD90+HSPCs displayed downregulation of expression of genes related to hemogenic endothelium development associated with hEB and hPL while genes critical in HSPC function, including DNA repair and chromatin modification, were upregulated to levels comparable to hFL-HSPCs. However, a subgroup of FL HSPC genes could not be induced in hEB-OP9 HSPCs, including the HOXA cluster genes and BCL11A—implicated in HSC self-renewal and B-cell formation, respectively. Interestingly, absence of HOXA genes and BCL11A and poor proliferative potential were also observed in HSPCs from early placenta, suggesting these defects are not in vitro artifacts but instead reflect an inability of hEB-OP9 HSPCs to complete developmental maturation. To validate the necessity of HOXA genes and BCL11A in proliferation potential and multipotency, we next utilized shRNAs to target MLL—the upstream regulator of the HOXA cluster—, individual HOXA genes, or BCL11A in FL-HSPCs to test whether knockdown was sufficient to recapitulate the defects observed in hESC-derived HSPCs. Knockdown of HOXA7 resulted in the loss of CD34+ cells while HOXA9 shRNA-treated cells displayed a loss of more differentiated CD38hi cells. MLL knockdown depleted both CD38+ and CD34+ populations. BCL11A silencing resulted in the loss of B-cells. These studies identify HOXA genes and BCL11A as developmentally regulated genes essential for generating self-renewing, multipotent HSCs from pluripotent cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 2291 Objectives: Unlimited self renewal capacity and the ability to differentiate into any cell type make human pluripotent stem cells (PSC) a potential source for the ex vivo manufacture of red blood cells (RBC) for safe transfusion. Current methods of RBC differentiation from PSC suffer from low yields of RBCs, most of which contain embryonic rather than adult or fetal hemoglobins. Therefore, efficient clinical translation of this strategy is critically dependent on the development of novel methods to enhance the generation of functional RBCs from PSC. We have previously shown that dimerization of the intracellular component of MPL (the thrombopoietin receptor), induces expansion of myelo-erythroid progenitors (MEP) from human cord blood as well as their terminal differentiation into enucleated RBC through unique, EPO-independent mechanisms (Parekh et al, 2012). Our goal was to investigate the potential of intracellular MPL dimerization to induce erythropoiesis from human PSC and to identify the signaling pathways activated by this strategy. Methods: Human embryonic stem cell (hESC) lines H1 and HES3 were transduced with a lentiviral vector to express the fusion protein F36V-MPL (containing the ligand binding domain F36V and the intracytoplasmic portion of MPL). Dimerization of F36V-MPL was accomplished by addition of the synthetic ligand AP20187 (aka CID) during culture (with or without erythropoietin) on OP9 stroma in the absence of other cytokines. F36V-MPL transduced-hESC that did not receive CID and F36V-transduced hESC cultured with CID served as negative controls. Flow cytometry and Colony Forming Unit (CFU) assays were used to analyze erythroid differentiation. Phosflow and Western Blot were used to analyze cell signaling. MEP generated during hESC differentiation were defined as cells co-expressing GlyA and CD41a/CD42a. Results: F36V-MPL dimerization induced significantly more Glycophorin A+ cells (P=0.0001; n=5) and 10-fold higher number of erythroid CFU (P=0.0007; n=15) as compared to negative controls. The effect was consistent across different hESC cell lines. The increased yield of erythroid cells was not due to an overall increase in cell proliferation as the total yield of cells was not statistically different between treated and untreated cultures. This effect was seen in the absence of any hematopoietic cytokines, including erythropoietin (EPO), a critical cytokine for erythropoiesis and an integral component of all ex vivo PSC erythroid differentiation protocols, indicating that MPL dimerization alone is sufficient to induce erythropoiesis from hESCs. Erythroid output was further enhanced in an additive manner in the presence of EPO (P=0.0058; n=5). In order to identify the point at which MPL dimerization affects erythropoiesis, CID was added during differentiation directly from hESC or to isolated MEP generated from hESC. CID and EPO increased the number of MEP compared to untreated controls, demonstrating that MPL dimerization induces the generation of early erythroid progenitors. In addition, CID drove erythroid differentiation from MEP more efficiently than EPO, demonstrated by a significantly higher frequency of total erythroid cells (P=0.02; n=3), and 4-fold increase in yield of enucleated RBC. This indicates that CID has a greater effect on terminal erythroid differentiation than EPO. We then investigated the signaling mechanism activated by F36V-MPL dimerization and found that, unlike the full-length MPL receptor, which activates both STAT5/JAK2 and AKT pathways, F36V-MPL dimerization activated AKT but not STAT5 or JAK2 phosphorylation. PI3K/AKT inhibitors (LY294002 and AKT inhibitor IV) effectively inhibited erythroid differentiation of transduced hESC cultured in the presence of CID (P=0.0442; n=2) indicating that MPL dimerization induced erythropoiesis is dependent on AKT signaling. Conclusion: F36V-MPL dimerization during hESC-derived hematopoiesis induces EPO-independent erythroid differentiation through AKT signaling, by both generating erythroid progenitors and promoting maturation of RBC. MPL dimerization also is more potent than EPO in inducing erythropoiesis from hESC and has an additive effect when combined with EPO, making this a potential strategy for the generation of therapeutically relevant levels of functional enucleated RBCs from PSC. Disclosures: No relevant conflicts of interest to declare.

Abstract 3119 BACKGROUND: Sickle cell disease (SCD) affects approximately 80, 000 Americans, and causes significant neurologic, pulmonary, and renal injury, as well as severe acute and chronic pain that adversely impacts quality of life. Because SCD results from abnormalities in red blood cells, which in turn are produced from adult hematopoietic stem cells, hematopoietic stem cell transplant (HSCT) from a healthy (allogeneic) donor can benefit patients with SCD, by providing a source for life-long production of normal red blood cells. However, allogeneic HSCT is limited by the availability of well-matched donors and by immunological complications of graft rejection and graft-versus-host disease. Thus, despite major improvements in clinical care, SCD continues to cause significant morbidity and early mortality. HYPOTHESIS: We hypothesize that autologous stem cell gene therapy for SCD has the potential to treat this illness without the need for immune suppression of current allogeneic HSCT approaches. Previous studies have demonstrated that addition of a β-globin gene, modified to have the anti-sickling properties of fetal (γ-) globin (βAS3), to bone marrow (BM) stem cells in murine models of SCD normalizes RBC physiology and prevents the manifestations of sickle cell disease (Levassuer Blood 102 :4312–9, 2003). The present work seeks to provide pre-clinical evidence of efficacy for SCD gene therapy using human BM CD34+ cells modified with the bAS3 lentiviral (LV) vector. RESULTS: The βAS3 globin expression cassette was inserted into the pCCL LV vector backbone to confer tat-independence for packaging. The FB (FII/BEAD-A) composite enhancer-blocking insulator was inserted into the 3' LTR (Ramezani, Stem Cells 26 :32–766, 2008). Assessments were performed transducing human BM CD34+ cells from healthy or SCD donors with βAS3 LV vectors. Efficient (1–3 vector copies/cell) and stable gene transmission were determined by qPCR and Southern Blot. CFU assays demonstrated that βAS3 gene modified SCD CD34+ cells are fully capable of maintaining their hematopoietic potential. To demonstrate the effectiveness of the erythroid-specific bAS3 gene in the context of human HSPC (Hematopoietic Stem and Progenitor Cells), we optimized an in vitro model of erythroid differentiation of huBM CD34+ cells. We successfully obtained an expansion up to 700 fold with >80% fully mature enucleated RBC derived from CD34+ cells obtained from healthy or SCD BM donors. We then assessed the expression of the βAS3 globin gene by isoelectric focusing: an average of 18% HbAS3 over the total globin present (HbS, HbA2) per Vector Copy Number (VCN) was detected in RBC derived from SCD BM CD34+. A qRT-PCR assay able to discriminate HbAS3 vs. HbA RNA, was also established, confirming the quantitative expression results obtained by isoelectric focusing. Finally, we show morphologic correction of in vitro differentiated RBC obtained from SCD BM CD34+ cells after βAS3 LV transduction; upon induction of deoxygenation, cells derived from SCD patients showed the typical sickle shape whereas significantly reduced numbers were detected in βAS3 gene modified cells. Studies to investigate risks of insertional oncogenesis from gene modification of CD34+ cells by βAS3 LV vectors are ongoing as are in vivo studies to demonstrate the efficacy of βAS3 LV vector in the NSG mouse model. CONCLUSIONS: This work provides initial evidence for the efficacy of the modification of human SCD BM CD34+ cells with βAS3 LV vector for gene therapy of sickle cell disease. This work was supported by the California Institute for Regenerative Medicine Disease Team Award (DR1-01452). Disclosures: No relevant conflicts of interest to declare.

Abstract 2394 Mesenchymal stromal (or stem-) cells (MSC) are culture-selected, heterogeneous supporting cells that can differentially regulate hematopoietic stem cell (HSC) behavior in vitro. The elusive identity of native MSC has obscured the contribution, if any, of these cells to HSC support in vivo. Having previously demonstrated that vascular pericytes (ubiquitous cells encircling endothelial cells in capillaries and microvessels) are ancestors of human MSC, we now hypothesize that pericytes are a critical component of the HSC “niche”. Consequently, pericyte isolation from total stroma would allow to develop co-culture systems for human HSC maintenance. In the present study, human cord blood CD34+ cells were cultured onto confluent human pericytes isolated from adipose tissue as CD146+CD34-CD45-CD56- cells. Co-culture of CD34+ cells on pericytes, for up to 6 weeks in the absence of any added growth factor, produced significantly i) higher numbers of CD45+ and CD34+ cells (p Disclosures: No relevant conflicts of interest to declare.

CCAAT/enhancer binding protein (C/EBP) transcription factors are involved in a variety of cellular responses including proliferation and differentiation. Although C/EBPβ and C/EBPε are believed to be most important for macrophage and granulocyte activity, respectively, experiments by others and ourselves suggest a possible overlap in their function in myelopoiesis. In order to explore further this potential redundancy, we assessed the in vivo and in vitro function of both transcription factors by generating a double knockout (KO) germline murine model (C/EBPβ/ε−/−/−/−) and compared their hematopoiesis to those of single deficient (C/EBPβ−/−, C/EBPε−/−) and wild-type (WT) mice. Gene expression analysis of bone marrow cells showed expression of C/EBPβ in C/EBPε−/− and WT mice, and vice versa. The weight of the double-KO mice was significantly less as measured at 4 weeks of age (11.5 ± 0.9 g) compared to WT (13.4 ± 0.6 g), C/EBPβ−/− (14.5 ± 1.4 g), and C/EBPε−/− mice (15.4 ± 2.3 g) (p < 0.05). The double-KO mice were prone to infections of the eyes, lungs, liver, and peritoneum. In contrast, C/EBPβ−/−, C/EBPε−/− and WT mice demonstrated no signs of infection. Microscopic imaging of peripheral blood showed metamyelocytes and myelocytes in the double-KO mice. FACS analysis found that the fraction of bone marrow cells which were Lin(−) (no expression of B220, CD3, Gr1, Ter119, and Mac1) were modestly elevated in double-KO and C/EBPβ−/− mice (8.42 % and 8.1 %, respectively) compared to C/EBPε−/− (4.24 %) and WT (3.93 %) mice. A subanalysis highlighted an elevated level of B220(−)/Gr1(−) bone marrow cells in the double-KO mice (54 %) compared to the levels in the C/EBPβ−/− (31 %), C/EBPε−/− (33 %) and WT (21.5 %) mice. Moreover, the proportion of hematopoietic stem cells in the bone marrow were significantly increased in the hematopoietic stem cell compartment [Sca1(+)/c-Kit(+)] in the double-KO mice (20.8 %) compared to the C/EBPβ−/− (6.9 %), C/EBPε−/− (5.9 %) and WT (6.9 %) mice. When given a cytotoxic stress (5-FU) to kill cycling hematopoietic progenitor cells, the mean neutrophil count at their nadir (day 4) was 0.14 × 109 cells/L in the double-KO mice compared to 0.71 × 109 cells/L in the WT mice (p < 0.001); both reached normal values again on day 10. Taken together, these results indicated a relatively higher percentage of immature hematopoietic cells in the double-KO mice compared to the WT mice. Nevertheless, clonogenic assays in methylcellulose using bone marrow cells of the double-KO showed a significant decreased number of myeloid colonies. For example, in the presence of G-CSF, GM-CSF, and SCF, a mean of 83 ± 10 hematopoietic colonies formed in the double-KO mice compared to 135 ± 6 in C/EBPβ−/−, 159 ± 12 in C/EBPε−/− and 165 ± 2 in WT mice (p < 0.001, double-KO vs. WT). Similar clonogenic results occurred when bone marrow cells were stimulated with either G-CSF, GM-CSF or SCF/G-CSF alone. Although our in vitro experiments suggested that double-KO mice had a decreased clonogenic response to G-CSF, their bone marrow cells had normal levels of phosphorylated STAT3 protein when stimulated with G-CSF. Hence, the G-CSFR and its secondary signaling pathway seemed to be intact. In further experiments, downstream targets of the C/EBP transcription factors were examined. Bone marrow macrophages activated with LPS and IFNγ from both double-KO and C/EBPβ−/− mice had decreased gene expression of IL6, IL12p35, TNFα, and G-CSF compared to the levels detected in macrophages of C/EBPε−/− and WT. Interestingly, expression levels of cathelicidin antimicrobial peptide (CAMP) were similarly robust in the macrophages from C/EBPβ−/−, C/EBPε−/−, and WT mice. In sharp contrast, CAMP expression was undetectable in the activated macrophages of the double-KO mice. In conclusion, the phenotype of the double-KO mice was often distinct from the C/EBPβ−/− and C/EBPε−/− mice suggesting a redundancy of activity of both transcription factors in myeloid hematopoiesis.