Long-term Engraftment Research Articles

The Orphan Nuclear Receptor Nr4a1 Specifies Long-Term, Myeloid-Biased HSCs and Responds To PGE2

The Nr4a family of transcription factors regulates the fate of cells from multiple tissues. The absence of Nr4a1 and Nr4a3 in the hematopoietic system results in acute myeloid leukemia, suggesting that this protein family is critically involved in the control of blood cell proliferation and development. In order to better understand the role played by Nr4a1 in particular, we have taken advantage of our Nr4a1(Nur77)GFP reporter mouse and investigated Nr4a1 expression during hematopoiesis. We find that Nr4a1 is expressed within the hematopoietic stem cell (HSC) population and most highly expressed among quiescent, long term (LT)-HSC. The association of Nr4a1 expression with LT-HSCs was directly confirmed by engraftment experiments. Sorted Lin-Sca+c-Kit+ (LSK) Nr4a1GFPhigh cell populations from CD45.2+ donors reconstituted the blood of CD45.1+ irradiated recipients for at least 20 weeks after transplant; an average of 20.3% of blood cells were CD45.2+. In contrast, Nr4a1GFPlow populations of LSK cells only transiently reconstituted the blood; well below 1% of blood cells were CD45.2+. Furthermore, blood cells arising from Nr4a1GFPhigh LSKs were dominated by myeloid cells: the lymphoid (L) to myeloid (M) ratio (ρ) was 13.7. The few Nr4a1GFPlow cells that remained after 20 weeks were almost exclusively lymphoid (L/M ratio = 99.3). Examination of the bone marrow from engrafted mice at 20 weeks showed that HSC populations derived from Nr4a1GFPhigh cells included a higher percentage of CD150+CD48- LT-HSCs than cells arising from whole bone marrow competitors. Given the association of Nr4a1 expression with long-term engraftment potential, and given that Nr4a1 has been shown to be highly responsive to environmental stimuli in a variety of tissues, we tested whether HSCs express Nr4a1 in response to signals generated by the bone marrow stroma. We evaluated the influence of a variety of factors, including GCSF, SCF, IL-6, TNFa, and PGE2. We found that PGE2 and, to a lesser extent, TNFa specifically upregulate Nr4a1 in HSCs. In summary, we show that Nr4a1 is expressed by a population of long-term, myeloid-biased HSCs, which closely resemble the potent long-term, myeloid biased HSC subset recently identified by Oguro et al. We further show that Nr4a1 expression is responsive to PGE2, which is known to enhance HSC engraftment. We hypothesize that Nr4a1 expression by HSCs reflects and integrates bone marrow environmental inductive cues critical for long-term stem cell homeostasis, including quiescence. Disclosures:No relevant conflicts of interest to declare.

A Novel Highly Conserved Hematopoietic Stem/Progenitor Cell Population for Stem Cell Transplantation and Gene Therapy

Hematopoietic stem cell (HSC)-mediated gene therapy or editing holds tremendous promise for many genetic diseases, HIV and cancer. Currently, most if not all approaches focus on the gene modification of CD34+ cells. However, CD34+enriched cell products predominantly contain lineage-committed progenitors and only very few “true” long-term engrafting HSCs which are ultimately the cells needed for sustained correction of diseases. Here we have evaluated a novel HSC-enriched phenotype for HSC transplantation and gene therapy.Most recently reported mouse xenograft transplants of purified and gene-modified human CD34+CD38- HSCs led to delayed myeloid engraftment suggesting the loss of crucial short-term engrafting progenitor cells. With the goal to identify a more refined HSC-enriched target cell population for transplantation and gene therapy, we recently performed comprehensive in vivo studies of CD34 subpopulations in our nonhuman primate (NHP) stem cell transplantation model. Comparing lentivirus (LV) gene-modified candidate cell fractions in competitive reconstitution experiments, we found an HSC-enriched phenotype (CD34+CD45RA-CD90+) demonstrating both rapid short-term recovery as well as robust long-term engraftment after myeloablative conditioning and autologous transplantation. Surprisingly and in contrast to published engraftment studies of equivalent human CD34 subpopulations in the mouse xenograft model, cell fractions enriched for multipotent progenitor cells (MPPs, CD34+CD45RA-CD90-) or lympho-myeloid progenitors (LMPs, CD34+CD45RA+CD90-) did not measurably contribute to short-term hematopoietic reconstitution in the NHP model.To determine whether the observed lack of MPP/LMP-contribution in the NHP may have been caused by ex vivo culture and/or LV gene modification conditions, engraftment studies of freshly isolated and sort-purified NHP CD34 subpopulations were performed in a novel “monkeynized” mouse model. Similar to our studies in the autologous setting, multi-lineage engraftment was exclusively driven by CD34+CD45RA-CD90+ cell fractions even freshly-isolated and transplanted without co-administration of MPPs/LMPs. Maturation of all blood lineages was found in the peripheral blood (PB), bone marrow (BM), spleen and thymus of successfully engrafted mice. Furthermore, NHP CD34+CD45RA-CD90+ cells were exclusively capable of homing into and repopulating the BM stem cell niche giving rise to phenotypical MPPs, LMPs as well as downstream progenitors.We next performed comprehensive mouse xenograft studies of freshly isolated and phenotypically identical human CD34+ subpopulations (Figure 1A) to evaluate this HSC-enrichment strategy with human stem cell sources. Similar to our studies with NHP HSPCs, multi-lineage engraftment in PB, BM, spleen and thymus was primarily restricted to CD34+CD45RA-CD90+ cell fractions (Figure 1A-E). Furthermore, engraftment and repopulation of the BM stem cell compartment was only observed for transplanted CD34+CD45RA-CD90+ HSPCs (Figure 1F). Surprisingly, we did not see engraftment of transplanted MPP- and LMP-enriched human cell fractions.In summary, these data demonstrate and confirm that both human and NHP CD34+CD45RA-CD90+ HSPCs are primarily responsible for multilineage hematopoietic reconstitution. In contrast to other HSC-enrichment strategies, the identified phenotype contains both short-term as well as long-term engraftment potential demonstrating no measurable delay in the onset of hematopoietic recovery upon transplantation in the NHP model. Most importantly, this phenotype is maintained throughout ex vivo culture and/or gene-modification, reduces the number of cells for gene-modification 20 to 30-fold, and likely increases targeting/editing efficiency as well as safety, making it a highly attractive target cell population for HSC-mediated gene and cell therapy approaches.Figure Legend - Multi-lineage engraftment potential of human CD34+ subpopulations in NSG mice: (A) Human CD34 subpopulations from GCSF-mobilized apheresis products were sort-purified and injected intravenously into sub-lethally irradiated adult NSG mice (left). Frequency of human cells in the PB over time in transplanted NSG mice (right). (B-E) Multi-lineage engraftment of human cells in the (B) PB, (C) spleen, (D) thymus and (E) BM. (F) Engraftment of human HSPCs in the murine BM. [Display omitted] DisclosuresAdair:CalImmune Inc.: Consultancy; Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Hematopoietic Stem Cell Potential within the Primary Sorted Lineage Positive Population: What Are We Losing with Hematopoietic Stem Cell Purification?

Lineage positive cells within murine whole bone marrow (WBM) are classically considered to be devoid of stem cell capacity. As such, lineage depletion prior to further selection for stem cell marker positive, lineage negative cells dominates virtually all hematopoietic stem cell (HSC) purification protocols. Here, we present data showing a significant population of long-term multi-lineage hematopoietic stem cells within the primary sorted lineage positive (Lin+) murine marrow population. Using limiting dilution competitive bone marrow transplantation assays, we found significant long-term multi-lineage engraftment capacity within the total primary sorted Lin+ population (cells positive for erythroid (TER119), myeloid (GR1, CD11b) or lymphoid (B220, CD4, CD8) markers). When 0.25 x 106 Lin+ cells were competed against 1 x 106 competitor WBM cells, average percent donor chimerism in recipient mice at 6 months post-transplant was 7.2% ± 2.9%. Donor chimerism was multi-lineage. There was still 1.9% ± 0.5% donor chimerism when only 62,500 primary sorted Lin+ cells were competed against 1 x 106 competitor WBM cells at 3 months post transplant, and further limiting dilution assays are ongoing to quantitate the stem cell compartment within the primary sorted Lin+ population. These levels of engraftment were similar to that found within un-separated donor WBM at the same dilutions, and multi-lineage engraftment capacity within the primary sorted Lin+ population persisted in secondary transplant. When further sub-fractionated into erythroid, myeloid or lymphoid sub-populations, every primary sorted individual sub-population contained long-term multi-lineage engraftment capacity at 6 months post transplant. Primary sorts were between 96-97.5% pure. When primary Lin+ sub-sets (B220+ cells, GR1+ cells, or CD11b+ cells) were double sorted, there was loss of engraftment potential within the double sorted persistently positive Lin+ subsets, but surprisingly high engraftment within those cells negative for only the single Lin+ marker on double sort (31%-59% donor chimerism at 6mo post-transplant; 1 x 105 donor cells + 3 x 105 competitor WBM cells). Immunophenotype analysis showed that these populations were not enriched in c-Kit+/Sca-1+/CD150+ cells. The high engraftment capacity of cells within the primary sorted Lin+ population, further isolated solely based on their being negative for a single Lin+ marker on the double sort prompted us to directly compare their engraftment capacity to that of purified Lineage negative (Lin-) cells. Interestingly, although isolated solely based on the presence or absence of GR1 on double sort, we found that the average percent donor chimerism at 6 months post-transplant was equal between purified Lin- cells (58.3% ± 17%) and the population of GR1 positive cells that became negative for GR1 on double sort (58.3% ± 26%; 1 x 105 donor cells + 3 x 105 competitor WBM cells). Using tritiated thymidine suicide, we found that the stem cell activity within these populations isolated from the primary sorted Lin+ fraction was due to actively cycling cells. These data further indicate that the engraftment capacity within the primary sorted Lin+ population is not simply due to conventionally purified quiescent HSCs contaminating our primary sort. In sum, there is a cycling population of stem cells within the primary sorted Lin+ fraction that appears to be distinct from quiescent highly purified HSCs. As primary sorted Lin+ cells, routinely discarded in most HSC studies, comprise up to 98% of WBM, engraftment capacity within this population represents a potentially large and understudied stem cell population within WBM worthy of further characterization. Work is currently underway to further quantitate and define this population, and delineate how cell cycle-related changes influence the phenotype and function of this cycling murine HSC population. DisclosuresNo relevant conflicts of interest to declare.

Pharmacological Control of Recipient NK Cell Function By Venetoclax Promotes Engraftment and Limits GVHD Following Reduced Intensity Conditioning without Impacting the Graft Versus AML Effect

BackgroundThe outcome of allogeneic haematopoietic stem cell transplantation (alloHSCT) is a complex interaction between conditioning regimen intensity (efficacy vs toxicity), recipient immunology (rejection vs engraftment) and donor immunology (rejection vs engraftment, and GVHD vs GVL). We must find means to lower conditioning toxicity, promote donor engraftment and limit GVHD in order to improve alloHSCT outcomes.Aims: Optimize the use of BCL2 inhibitors to modify recipient NK cell function in mouse models of alloHSCT in order to minimize GVHD severity and onset, and retain GVL responses.Hypothesis: Therapeutic targeting of recipient NK cell frequency or function pre-transplant will allow reduced intensity conditioning (RIC) and promote both donor T cell engraftment and GVL whilst reducing the risks of GVHD.MethodsMHC-mismatched mouse model of alloHSCT: Donor BM and T cells from BALB/c (H2Kd) mice were injected into irradiated C57BL/6 (H2Kb) recipients. On day 0, C57BL/6 WT, or Bcl2fl/fl (Bcl2 deleted in NKp46+NK1.1+ cells) recipients were irradiated with a RIC dose of 2 x 400 rad, before i.v. injection of 7.5e6 BALB/c BM + 1e6 T cells. C57BL/6 WT mice were also treated on day -2 and -1 by oral gavage with 100 mg/kg Venetoclax (ABT-199) or vehicle, before receiving alloHSCT. Mice were monitored for onset of GVHD and tested for early engraftment (day 7-14 post-transplant), and late engraftment (up to day 50).Graft-versus leukaemia (GVL) AML model:8e5mixed lineage leukaemia cells (MLL-AF9) were injected i.v. on day 0 into C57BL/6 WT recipients. On days 8 and 9 mice were treated by oral gavage with 100 mg/kg Venetoclax or vehicle, before receiving alloHSCT on day 10. Mouse survival and AML burden in the BM was measured on day 30.ResultsWe found that at both a myeloablative radiation dose (1200 rad) and a RIC non-myeloablative dose (800 rad), recipient NK cells were significantly more radio-resistant than either CD8+ T or myeloid cells. However, when a RIC dose of 800 rad was used during alloHSCT, the rejection threshold was not overcome and engraftment failed. We investigated both genetic and pharmacological models of recipient NK cell suppression during alloHSCT with RIC, to promote donor cell engraftment, reduce GVHD, and retain GVL.Conditional deletion of Bcl2 in NK cells results in a 90% loss of NK cells in vivo . Bcl2fl/fl alloHSCT RIC recipients showed robust donor engraftment, but absence of the pro-inflammatory cytokine storm and substantially less GVHD as determined by clinical scores and gut histology, compared to WT recipients. Pharmacological inhibition of BCL2 in WT recipients recapitulated the transplant findings in Bcl2fl/fl recipients. We found that BCL2 inhibition by Venetoclax, a BCL2 antagonist approved in the treatment of AML, resulted in NK cell apoptosis in human cells . We extended our observations in Bcl2fl/fl recipients to show that pharmacological inhibition of BCL2 in WT mice with just two doses of Venetoclax resulted in rapid depletion of NK cells. Over 35% of alloHSCT WT recipient mice pre-treated with Venetoclax developed long-term full donor engraftment even in the setting of RIC, with minimal GVHD, and retained potent GVL effects against pre-established AML.ConclusionsVenetoclax depletion of NK cells in WT alloHSCT recipients in combination with RIC was: 1) well-tolerated, 2) associated with low rates of GVHD, 3) resulted in long-term donor haematopoietic cell engraftment, and 4) maintained a GVL response. Recipient NK cell inhibition may therefore represent a means by which to deliver alloHSCT more safely. DisclosuresRitchie:Amgen Inc.: Honoraria.

Regulatory T Cells Prevent Donor Cell Rejection in a Mouse Model of Delayed Gestational Age in Utero Hematopoietic Cell Transplantation

Background: In utero hematopoietic cell transplantation (IUHCT) results in long-term, multilineage chimerism without myeloablation/immunosuppression. It has the potential to treat a number of congenital hematologic disorders, including as Sickle cell disease. Central tolerance, through which donor-reactive host T cells and host-reactive donor T cells are deleted, is instrumental to the induction of donor-specific tolerance (DST) and the prevention of graft-versus-host-disease (GVHD). The window of central tolerance induction in humans occurs at a gestational age that will pose significant technical challenges and exclude from treatment all fetuses evaluated after 14 weeks gestation. We have previously demonstrated that IUHCT results in the peripheral induction of traditional and non-traditional regulatory T cells that contribute to the suppression of GVHD (Riley et al. ASH 2016 Abstract #4540). We hypothesized that regulatory T cells also contribute to DST and, furthermore, may be used to expand the window of central tolerance induction to a later point in gestation.Methods: A mouse model of delayed gestational age IUHCT was developed in which 9.7x10^6 C57/Bl6 (B6) GFP T cell-depleted (TCD) bone marrow (BM) cells were injected intravenously into day of life 0 (P0) naive Balb/c pups via the facial vein. The ability of IUHCT-induced regulatory T cells to prevent donor cell rejection was assessed by injecting 9.7x10^6 B6 GFP TCD BM cells plus 5x10^6 CD4+ splenocytes harvested from 2-4 week old chimeric mice. Fetuses injected with 9.7x10^6 B6 GFP TCD BM cells at gestational day 14 (E14) served as positive controls for tolerance and engraftment. Peripheral blood (PB) was analyzed at 4, 8, 12, and 24 weeks of age for donor cell chimerism (%CD45+H2kb+GFP+/CD45+H2kd+) and for engraftment (defined as chimerism >1% at 4 weeks). Weight gain and histology of the skin, liver, lungs, and small bowel were analyzed to confirm the absence of GVHD. PB was analyzed at 6 months of age to confirm multilineage engraftment. An in vivo mixed lymphocyte reaction (MLR) was performed at 6 months of age to confirm long-term DST. B6-sensitized Balb/c mice served as positive controls for alloreactivity. Comparison of continuous data was performed using Student's t test. Binary data were compared using Fischer's exact test. All tests were two-sided with an alpha level set at 0.05 for significance.Results: Our model of delayed gestational age IUHCT was associated with donor cell rejection in 52 of 56 (92.9%) pups injected with TCD BM only. Co-injection of CD4+ chimeric splenocytes significantly improved engraftment (100% vs. 7.1%, p<0.001) and chimerism (7.56% vs. 0.46% at 4 weeks, p=0.003) compared to TCD BM alone. No difference in engraftment (100% vs. 90%, p=0.501) or chimerism (7.56% vs. 7.66% at 4 weeks, p=0.961) was observed compared to fetuses injected with TCD BM at E14 (Figures 1A and 1B). These trends persisted at subsequent time points. Pups injected with TCD BM plus CD4+ chimeric splenocytes displayed normal weight gain and no histologic evidence of GVHD. Survival to adulthood was 100%. Donor cells of all hematopoietic lineages were present in the PB at 6 months of age. On in vivo MLR, pups injected with TCD BM plus CD4+ chimeric splenocytes displayed significantly reduced T cell proliferation compared to B6-sensitized Balb/c mice and P0 pups injected with TCD BM only. T cell proliferation did not differ significantly from fetuses injected with TCD BM at E14 (Figure 1C).Conclusion: The induction of tolerance following IUHCT is important for maintaining successful long-term engraftment and requires injection at an early gestational age to avoid donor cell rejection by the maturing fetal immune system. This study demonstrates an important role for regulatory T cell populations in the induction of DST following IUHCT. The adoptive transfer of regulatory T cells from a chimeric mouse to a naïve P0 pup inhibits donor cell rejection in our model of delayed gestational age IUHCT, allowing central tolerance induction and long-term engraftment to occur despite the increased maturity of the recipient immune system. These findings represent proof-of-principle that augmentation of regulatory T cell-dependent peripheral tolerance is one approach by which IUHCT could be delayed to a later point in gestation, decreasing the technical difficulty of the procedure and allowing fetuses evaluated after 14 weeks to be eligible for treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Concurrent Ex Vivo Differentiation of a Vascular Niche and Hematopoietic Stem/Progenitor Cells from Human iPSCs

One of the most promising objectives of clinical hematology is to derive transplantable autologous hematopoietic stem/progenitor cells (HSPCs) ex vivo. Recent reports have described the generation of engraftable HSPCs via forced expression of key transcription factors in either endothelial cells, or hemogenic endothelium (HE) derived from induced pluripotent stem cells (iPSCs). While protocols for the generation of transgene-free engraftable HSPCs suitable for therapy remain unavailable, these studies, along with observations in the developing embryo, have shown the close association of HE populations with arterial vascular endothelium is of critical importance to HSPC production and maturation. To facilitate the development of a vascular niche supporting the emergence of transgene-free, functional HSPCs from human iPSCs, we developed a simple, monolayer-based, chemically-defined, and scalable differentiation protocol requiring no replating or embryoid body (EB) formation (commercially available as STEMdiffTM Hematopoietic Kit, Stem Cell Technologies). During the first 3 days, a mesodermal lineage specification was induced with morphogens (BMP4, bFGF and VEGF). For the subsequent 18 days, cells were further differentiated into HSPCs with the addition of hematopoietic cytokines (SCF, Flt3-L and TPO). Starting at day 5, an adherent monolayer composed in part of CD34+CD43- endothelial cells rapidly formed, followed by the initiation of endothelial-to-hematopoietic transition (EHT), as demonstrated by the emergence of round cells directly budding off the endothelial monolayer in time-lapse imaging studies (Panel A). Importantly, these cells were released within the culture supernatant and could be easily harvested by simple pipetting. Flow cytometric analysis of the supernatant cells indicated that the emerging cells initially had a predominant CD45-CD235a+ erythroid phenotype (maximum 69.9±12.4% at day 7), consistent with a primitive yolk-sac wave of hematopoiesis (PanelB). From day 7, CD45+CD34+ HSPCs increased gradually, with a maximum number (average 1x105 cells/cm2, n=10) observed at day 12, and a peak percentage at day 12 (47.4±12.4%) and 14 (48.8±9.9%) (Panel B). These cells could form colonies in clonogenic progenitor assays, albeit at reduced capacity compared to bona fide CD45+CD34+ cells. Strikingly, cells with a phenotype that enables the highest reported purity of human HSCs (CD34+ CD38- CD45RA- CD90+ CD49f+ Rholo) were highly enriched in the culture supernatant at day 10 through 14 (Panel C). However, these cells did not result in efficient, long-term engraftment in immuno-deficient (NSG) mice. To identify possible causes for the lack of durable repopulating potential of iPSC-derived HSPCs in this system, we further characterized the endothelial monolayer between day 5 and 12 of differentiation. We focused on recently described endothelial subsets within the CD34hiCD43- population, including the in vitro equivalent of definitive HE (CD34hi CD43- CD73- CD184- DLL4-), arterial vascular endothelium (CD34hi CD43- CD73mid CD184+), and venous vascular endothelium (CD34hi CD43- CD73hi CD184-) (Ditadi et al., Nat. Cell Biol 2015). At each timepoint analyzed, definitive HE comprised <1% of the total cell population (Panel D). Arterial vascular endothelium was also scarce, rising to a maximum of 6.6% at day 7 before declining to <1% by day 9 of culture (Panel D). Venous vascular endothelium accounted for approximately 2% of total cells (Panel D). The remaining cells of the monolayer expressed varying levels of CD34 and CD43, consistent with the recently demonstrated heterogeneity of cells undergoing EHT (Guibentif et al., Cell Rep 2017). Thus, our data suggest that the absence of a definitive HE population within a developing arterial niche, as occurs in the dorsal aorta (DA) during development, may explain in part the lack of engraftment potential of iPSC-derived HSPCs. The unique adaptable and scalable nature of this monolayer-based differentiation protocol will facilitate precise temporal activation or downregulation of signaling pathways essential for the generation of engraftable human HSPCs. Quantitative and temporal modulations of Wnt/Nodal pathways for mesodermal specification as well as VEGF/Notch pathways for arterial vascular endothelium are underway. [Display omitted] DisclosuresChen:Stem Cell Technologies: Patents & Royalties: StemDiff Hematopoietic Kit. Boehm:StemCell Technologies: Patents & Royalties: StemDiff Hematopoietic Kit. Larochelle:Novartis: Research Funding; StemCell Technologies: Patents & Royalties: StemDiff Hematopoietic Kit; Novartis: Research Funding.

Interferon-Gamma Pathway Regulates Emergence of Engraftable Hematopoietic Stem and Progenitor Cells from Human Pluripotent Stem Cells

Derivation of functional human hematopoietic stem cells (HSCs) from autologous human pluripotent stem cells (PSCs) has been an elusive goal in hematology research. Building upon recent evidence that HSCs are derived from definitive hemogenic endothelium (HE), we identified 7TFs (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, SPI1) that were sufficient to convert human PSC-derived HE into engraftable hematopoietic stem and progenitor cells (HSPCs) with long-term and multilineage capacity (HE-7TF cells, Sugimura et al, Nature 2017). In the current study, we attempted to define signaling pathways that further enhance engraftment of HE-7TF cells. We investigated the source of our long-term engraftable cells by sorting CD34+CD43+CD45+ triple positive cells and CD34+CD43-CD45- single positive cells from HE-7TF cells. We transplanted these two cell populations into non-irradiated c-Kit deficient immune-deficient recipients, and monitored engraftment capacity after 8 weeks of transplantation. 2 out of 5 mice transplanted with triple positive cells showed multi-lineage engraftment, whereas 5 mice transplanted with single positive cells did not, suggesting that CD34+CD43+CD45+ triple positive cells are the source of long-term engraftment in our system. We then investigated what signaling pathways regulate the emergence of CD34+CD43+CD45+ triple positive cells from HE. From high-throughput screening of 2,000 receptor ligands, cytokines/morphogens, hormone, and chemical compounds, we observed that interferon-gamma (IFNγ) increased CD34+CD43+CD45+ triple positive cells 6 to 8-fold in 5 independent experiments. The effect was abrogated by the addition of IFNα. Collectively, these data demonstrate that the IFNγ pathway promotes the emergence of engraftable HSPCs from human PSC-derived HE. DisclosuresNo relevant conflicts of interest to declare.

PI3K Inhibition with Idelalisib for Optimized Production of Chimeric Antigen Receptor T Cells

Introduction: Genetically modified T cells expressing chimeric antigen receptors (CARTs) are currently among the most encouraging treatment approaches in cancer immunotherapy. Efficacy and persistence of adoptively transferred CARTs are linked to the proportion of naïve-like T cells (TN) and stem cell memory-like (TSCM) T cells defined by stem cell-like properties such as a high proliferative and self-renewal capacity. Optimization of CART production is important to shift the balance towards these subsets. The PI3K/mTOR/AKT pathway is one of the main pathways involved in T cell differentiation. In the current study we evaluated the inhibition of PI3K by the clinically approved small molecule idelalisib during culturing of CARTs in order to increase less differentiated T cell subpopulations in the final cell product.Methods: Human peripheral blood mononuclear cells (PBMCs) of ten healthy donors were transduced with a 3rd generation retroviral CD19-specific CART vector containing CD28 and 4-1BB as costimulatory domains (kindly provided by Malcolm Brenner, Baylor College of Medicine, Houston). PBMCs were activated with anti-CD3/anti-CD28 and stimulated with IL-7/IL-15. Cultivation was performed with or without 1µM idelalisib. TN was defined as CD45RA+CCR7+, central memory-like (TCM) as CD45RA-CCR7+, effector memory-like (TEM) as CD45RA-CCR7- and effector-like (TEff) as CD45RA+CCR7- T cells. Detailed analysis of T cell subpopulations, cytokine secretion and expression of exhaustion and homing markers was performed using flow cytometry over 17 days.Results: Viability (94±3% vs 87±9%; p=0.006) and transduction efficiency (65±13% vs 60±12%; p=0.001) of CARTs were significantly higher in presence of idelalisib at day 14. Both groups showed similar evolution of CD3+/CD4+ cells (48%±13% vs 46±13%, p=0.1) and CD3+/CD8+ cells (50%±13% vs 51±13%, p=0.2) at day 14. In addition, the TN subset of CD3+ (31±11% vs 23±9%, p<0.001), CD3+/CD8+ (36±15% vs 27±12%, p<0.001) and CD3+/CD4+ (25±11% vs 19±10%, p=0.003) CARTs was significantly increased with idelalisib. Cultivation with the inhibitor reduced the expression of the exhaustion marker Tim-3 (66±10% vs 82±5%, p=0.003) and raised CD62L expression (84±9% vs 70±9%, p<0.001) of CD3+ CARTs at day 14. Both groups showed low PD-1 expression (4±3% vs 6±3%; p=0.003) after 14 days of cultivation. At day 10 of culture CARTs propagated with idelalisib showed significantly lower CD4+ T helper 1 cell (20±10% vs 23±8%; p=0.05) and higher CD4+ T helper 2 cell (72±9% vs 67±7%; p=0.004) counts. Intracellular cytokine staining at day 15 after co-culture with the CD19+ Daudi cell line showed significantly lower TNF-α (47±7% vs 58±6%; p<0.001) and IFN-γ production (33±13% vs 40±13%; p<0.001) in presence of idelalisib at CD3+ CARTs. This effect was reversible and similar cytokine production for TNF-α (64±7%) and IFN-γ (40±13%) could be achieved when CARTs were rested for one day without idelalisib.Conclusions: PI3K inhibition during CART production shifted the T cells into subpopulations that allow higher persistence, long-term engraftment and sustained antitumor activity. CARTs showed a less exhausted phenotype and a higher capacity to migrate into lymph nodes. Evaluation of the effect of idelalisib for CART cultivation in patient-derived donor cells is currently ongoing. The identification of the optimal culturing condition of CARTs will further increase the benefit of this promising treatment approach in cancer immunotherapy. DisclosuresDreger:medac: Other; medac: Other: Travel grants; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; medac: Other; Gilead: Consultancy, Speakers Bureau; medac: Other: Travel grants; medac: Other; medac: Other; medac: Other: Travel grants; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; Jansen: Consultancy; Jansen: Consultancy; Gilead: Consultancy, Speakers Bureau; Riemser: Consultancy, Research Funding; Riemser: Consultancy, Research Funding; Riemser: Consultancy, Research Funding; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; Jansen: Consultancy; Jansen: Consultancy; Jansen: Consultancy; Gilead: Consultancy, Speakers Bureau; Riemser: Consultancy, Research Funding; Riemser: Consultancy, Research Funding; Gilead: Consultancy, Speakers Bureau; Riemser: Consultancy, Research Funding; Riemser: Consultancy, Research Funding; Riemser: Consultancy, Research Funding; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; Riemser: Consultancy, Research Funding; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; Riemser: Consultancy, Research Funding; Riemser: Consultancy, Research Funding; Jansen: Consultancy; Riemser: Consultancy, Research Funding; Jansen: Consultancy; Gilead: Consultancy, Speakers Bureau; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; Jansen: Consultancy; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; Jansen: Consultancy; Gilead: Consultancy, Speakers Bureau; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Jansen: Consultancy; Gilead: Consultancy, Speakers Bureau; Jansen: Consultancy; Gilead: Consultancy, Speakers Bureau; medac: Other: Travel grants; Jansen: Consultancy; Gilead: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; medac: Other: Travel grants; medac: Other: Travel grants.

A Potential Therapy for Beta-Thalassemia (ST-400) and Sickle Cell Disease (BIVV003)

▪Background: Beta-thalassemia and sickle cell disease are genetic disorders caused by mutations in the beta-globin gene which lead to significant anemia and serious medical complications. Increases in fetal hemoglobin (HbF) have been linked to improved clinical outcomes in patients with beta-thalassemia (BT) and sickle cell disease (SCD).Methods: We have developed engineered zinc finger nucleases (ZFNs) that precisely cleave and disrupt the erythroid enhancer of the BCL11A gene, which substantially boosts HbF production in erythroid progeny of genome-edited CD34+ hematopoietic stem/progenitor cells (HSPCs). The autologous modified HSPC drug product are named ST-400 (BT) and BIVV003 (SCD).Results: We demonstrate that ST-400 / BIVV003 can be manufactured by reproducible, high-level, ZFN-driven modification in peripheral blood mobilized HSPCs at clinical production scale (>108 cells). The drug product is made in a GMP-compliant setting using a clinical-grade electroporation device to deliver the ZFN mRNAs ex vivo . Unbiased specificity studies of ST-400 / BIVV003 demonstrated an exquisite amount of specificity, with high levels of on-target modification (~80%). Erythroid colony genotyping in enhancer targeted cells, showed bi-allelic modification of the BCL11A erythroid enhancer in >50% of HSPCs, resulting in >4-fold higher levels of gamma globin mRNA and protein compared to controls. Similarly, we observed high levels of modification in research-scale preparations of HSPCs from patients with beta-thalassemia. Injection of ST-400 / BIVV003 into immune-deficient mice resulted in robust long-term (19 week) engraftment. Targeted gene modification was maintained through multi-lineage differentiation in the bone marrow and peripheral blood.Conclusions: These results support further clinical development of ST-400 / BIVV003 as a potential therapy for beta-thalassemia and sickle cell disease. DisclosuresNo relevant conflicts of interest to declare.

Poor Outcome of Domino Hematopoietic Stem Cell Transplantation in Humans - an International Case Series

BackgroundDomino hematopoietic stem cell transplantation (HSCT) refers to a situation where an HSCT recipient serves as the donor for another (HLA-identical) recipient. Several successful cases have been reported, suggesting that acquired tolerance to HLA-antigens in the first recipient allows for engraftment in the second recipient, with low risk of graft-versus-host disease (GvHD). However, no systematic analyses on the outcome of domino HSCT have been performed.MethodsA retrospective, questionnaire-based study was performed within the European Bone Marrow Transplant (EBMT) network and the Primary Immune Deficiency Treatment Consortium (PIDTC). Primary outcome measures were survival and re-transplantation; secondary outcome measures were incidence of GvHD and time to neutrophil and platelet engraftment.FindingsWe identified 11 domino-HSCTs performed in 9 different centers, between September, 1992 and March, 2016. Indications were immune deficiency (n=9), inborn errors of metabolism (n=1) and hereditary bone marrow failure (n=1). The outcome of domino recipients was poor and inferior to their donors and to published cases. Only five recipients (45%) survived with long-term donor engraftment, of whom one required repeated donor-lymphocyte infusions. Four recipients (36%) required re-transplantation and two (18%) engrafted but died from infectious complications. The time between domino-HSCT and re-transplantation ranged up to 190 months. No differences were found in CD34+ cell dose, total nucleated cell dose, nor in kinetics of engraftment between successful domino HSCTs and those resulting in re-transplantation. One domino recipient and six primary recipients developed GvHD.InterpretationThis study demonstrates the disappointing outcome of domino-HSCT. Alternative donor sources, if available, should be considered. Replicative stress on the transplanted stem cells, loss of “stem-ness”, and immune-mediated mechanisms may contribute to the limited engraftment upon serial transplantation. In addition, our results stress the importance of systematic evaluation of published and unpublished cases for unbiased clinical decision making. DisclosuresCowan:Homology Medicine: Consultancy.

Alemtuzumab/Low Dose TBI Conditioning Facilitates Stable Long-Term Donor Hematopoietic Cell Engraftment from Sibling Donors in Children with Sickle Cell Disease

BackgroundHematopoietic stem cell transplant (HCT) is the only established curative approach for sickle cell disease (SCD). Due to the high toxicity profile of myeloablative regimens, some adult patients with severe sickle cell anemia are not eligible for such therapy. However, a new adult nonmyeloablative protocol (National Institutes of Health, NIH protocol) described by Hsieh et al. (2014) was able to achieve a curative degree of mixed donor chimerisms with minimal transplant-related complications. The NIH protocol includes alemtuzumab, low dose total-body irradiation (TBI) of 300 cGy, and prolonged sirolimus for graft-versus-host disease (GVHD) prophylaxis for those recipients with a matched-sibling donor (MSD).The Alberta Children's Hospital has adopted this published regimen for MSD HCT in adolescents and younger children with SCD since the NIH data was presented in 2013.To our knowledge, there is no published literature describing the application of the NIH protocol in a pediatric population; the youngest subject in the referenced adult study was 16 years of age.MethodsThis retrospective cohort describes outcomes of nonmyeloablative allogeneic MSD HCT in children and adolescents with SCD who underwent HCT between 2013-2017. Fourteen patients with SCD under 18 years of age at the time of HCT received the NIH conditioning regimen. HLA-matched siblings had either normal hemoglobin or sickle cell trait and of the appropriate weight/height to donate peripheral blood stem cells (PBSCs) without blood priming of the apheresis circuit.The conditioning regimen consisted of alemtuzumab 1.0 mg/kg administered subcutaneously daily divided over five days (Days -7 to Day -3). Patients received a single TBI dose of 300 cGy on day -2, with testicular shielding for male recipients. GVHD prophylaxis consisted of a sirolimus loading dose of 3mg/m2/dose (PO) on day -1, followed by 1mg/m2/dose once daily starting on Day 0. PBSCs were typically collected on Day -3 to allow for cancellation of the HCT before TBI in the event of failed mobilization. Unmanipulated PBSCs were infused fresh on Day 0. Patients were eligible for early discharge post-transplant even prior to neutrophil engraftment. Weaning of sirolimus was initiated no earlier than 1-year post-HCT and if donor T-cell chimerisms were greater than 50%. Treatment failure was defined as clinical manifestation of SCD, failure to start weaning sirolimus by 2-years post-HSCT, or a failure to complete a sirolimus wean by 2.5 years post-HCT.ResultsFourteen patients (3 M,11 F) are included on this retrospective study. Median age at HCT was 11 years. Post-HCT follow-up ranges from 1 to 48 months (median 17). There were no failed donor stem cell mobilizations. Patients received a median of 11.4x106 CD34 cells per kilogram of body weight.All patients were discharged home prior to neutrophil engraftment with a median hospital stay of 7 days (range 5-19). All patients had donor neutrophil engraftment at a median of 21 days (mean= 22, SD= 4) and platelet engraftment at median of 14 days (mean= 12, SD= 14). Six patients never required platelet transfusion. Readmission after initial discharge was required in 9/14 patients with an average of 1 readmission/patient (range 1-4), typically for fever or CMV pre-emptive therapy. One patient developed septic shock with Acinetobacter ursingii related to poor central line care at home.All patients are currently alive. Post-HCT, there have been no cases of graft failure, sickling crises, veno-occlusive disease, idiopathic pneumonia syndrome, cerebral hemorrhage, PRES, or PTLD. Although 6/14 patients had CMV reactivation, only 3 patients required pre-emptive therapy. None had CMV/EBV disease.There were no treatment failures as defined by protocol. Currently, 8 patients are off sirolimus, 1 is currently weaning, with no secondary graft failure events. Only 2 patients over 1-year post-HCT have not initiated sirolimus weaning. There have been no cases of acute or chronic graft-versus-host disease.ConclusionsNonmyeloablative conditioning regimen is safe and effective as curative therapy for children with SCD. Outpatient management pre-engraftment was feasible in this population. In contrast to adult data, sirolimus weaning was possible in most patients by one-year post-HCT. Graft integrity was maintained after discontinuation of sirolimus in all eligible patients. [Display omitted] DisclosuresGuilcher:Patient Access Solutions: Consultancy; Canada Market Research: Consultancy; Jazz pharmaceuticals: Consultancy. Bruce:Bristol-Meyers-Squibb - consultant 2016-2017: Consultancy; ovartis consult and ad board 2015-2016: Consultancy; Apopharma 2015: Consultancy.

Activation of SIRT1 Deacetylase As a Therapeutic Approach for Myelodysplastic Syndromes By Restoring TET2 Function

Myelodysplastic syndromes (MDS) which arise from a primitive CD34+ hematopoitic stem/progenitor cell (HSPC), are incurable by non-transplant therapy. Better understanding of MDS HSPC regulation is critical for developing targeted therapies against the fatal disease. Tet methylcytosine dioxygenase 2 (TET2), initiating DNA demethylation through converting 5-methylcytosine to 5-hydromethylcytosine (5hmC), is the most frequently mutated gene in MDS. TET2 alterations lead to DNA hypermethylation and dysregulated gene expression in HSPCs, resulting in enhanced self-renewal and skewed myeloid specific expansion. While TET2's biological function has been well studied, the knowledge of its regulation is limited. We had demonstrated the TET2 c-terminus acetylation was reversed by SIRT1 deacetylase which maintains normal HSPCs activity under stress. We also observed that SIRT1 protein in MDS CD34+CD38- was expressed below normal levels, and that knockdown (KD) of SIRT1 enhanced in vivo growth of MDS-L cells, a patient derived MDS cell line, is associated with TET2 acetylation (Blood. 2016;128:1053). These results indicate an important role for SIRT1 in MDS HSPCs maintenance.To further assess SIRT1 function in MDS, we ectopically expressed SIRT1 in primary MDS CD34+ cells enriched for HSPCs and MDS-L cells, respectively. Overexpression (OE) of SIRT1 resulted in inhibition of colony formation of MDS CD34+ cells (p<0.01, n=3) but not normal counterpart (p>0.05, n=3). SIRT1 OE also suppressed MDSL colony formation (p<0.01) and cell growth (p<0.01). SIRT1 OE significantly reduced MDS-L cell BM engraftment in NSGs mice relative to controls (human CD45+ cells: 5.7±0.8% of BM in SIRT1 OE group vs 31.4 ± 5.8% in control, p<0.01, n=7), resulting in delayed mortality of recipient mice (median survival: 52 days vs 35 days; p<0.05, n=5). Meanwhile, treatment of SIRT1 agonist SRT1720, reduced colony formation of primary MDS CD34+ cells in a dose dependent manner (p<0.01, n=9). Similar results were also achieved in SRT1720 treated MDS-L cells. Furthermore, TET2 was hyperacetylated in primary MDS cells (n=3) compared to normal counterpart (n=3). Next, we tested whether inhibition of MDS HSPCs following SIRT1 activation was related to TET2 activation via protein deacetylation. Indeed, enhanced TET2 activity marked by 5hmC was observed in MDS CD34+ cells upon SRT1720 treatment (P<0.05, n=3). TET2 KD diminished growth inhibition induced by SIRT1 OE or SRT1720, indicating a requirement of TET2 for SIRT1 function. Through mass spectrometry analysis, three conserved acetylated lysine residuals (K1472, K1473, K1478) were identified in TET2 catalytic domain (CD). Engineered lysines to glutamines (TET2 3KQ) mutant, mimicking acetylated TET2, produced lower 5hmC, and led to increased colony growth compared to MDS-L expressing TET2 WT (p<0.01). We further evaluated the effects of SRT1720 using NUP98-HOXD13 (NHD13)MDS mouse. This murine MDS is transferable and secondary recipients receiving primary NHD13 MDS cells succumb to MDS at 4 months, allowing generation of a cohort of MDS mice for preclinical studies of therapeutics against MDS. CD45.2+ BM cells from donor NHD13+ mice showed evidence of dysplasia were harvested, and transplanted into preconditioned congenic recipients expressing CD45.1. SRT1720 treatment (100mg/kg/day for 12 weeks, p.o.) resulted in partial reversal of MDS-like disease developed in recipients receiving MDS cells, including an increase in HGB, PLT, RBC and WBC (p<0.01,n=6). Importantly, significant reduction of NHD13+ CD45.2+ chimerism in LSK and other progenitors from BM were also observed (p<0.01, n=6). As expected, 5hmC level in lineage depleted cells from SRT1720 treated mice was increased (p<0.01). Secondary transplantation was performed to evaluate the effects of SRT1720 on MDS initiating cells. We observed that the mice receiving cells from SRT1720 treated donors showed reduced NHD13+ CD45.2+ cell long-term engraftment (p<0.01, n=5).In summary, we demonstrated that genetic overexpression or pharmacological activation of SIRT1 drastically inhibited MDS maintenance, and that TET2 function was required for the inhibitory effect of SIRT1. These results support the hypothesis that SIRT1 deficiency promotes MDS cell expansion through TET2 inactivation and support the rationale for exploring SIRT1 activation as a promising approach to selectively target MDS stem cells. DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Stem Cells from Patients with Myelodysplastic Syndrome (MDS) Engraft and Migrate in-between the Humanized Niches in Immuno-Compromised Mouse Models

IntroductionMyelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized mainly by ineffective hematopoiesis. Xenograft models of MDS in immunodeficient mice have been instrumental in improving our understanding of clonal origin and evolution of this disease (Mian & Rouault-Pierre et al., Nat Commun. 2015; Rouault-Pierre & Mian et al Haematologica 2016; Leukemia, 2017); however, the low level of engraftment generally observed in these mouse models makes them impractical for testing therapeutic strategies. We used a 3D humanized scaffold in vivo system to test the MDS stem cell engraftment as well as study the cross talk between MDS-Initiating cells (MDS-ICs) and their niche.MethodologyIn this study bone marrow (BM) cells from 31 patients (27 MDS cases covering all WHO subtypes, 1 CMML, 1 MDS/MPD and 2 sAML) were studied. Initially, whole-exome sequencing was performed on all cases. Colony-forming assay was used to assess the colony forming ability of BM CD34+ cells from each patient. Mesenchymal stromal cells (MSCs) were isolated from the BM of the patient samples. We then used 3 immunodeficient mouse models (NSG, NSG-SGM3 and NBSGW) to study the engraftment kinetics and clonal evolution of the transplanted cells in gelatin-based 3D scaffolds coated with patient MSCs (RARS/RCMD, autologous; RAEB, autologous/allogenic).ResultsBM MSCs obtained from the MDS patients showed variable expansion levels, with the ability to differentiate into the adipogenic lineage whereas the osteogenic differentiation was highly aberrant.Next, 0.5x106 to 3x106 patient (n=31) mononuclear cells (MNCs T cell depleted) were seeded into 3D-scaffolds prior to implantation in NSG-SGM3 and/or NSG mice. We observed persistent long-term engraftment of hCD45+ cells ranging from 0.05% to 80% (up to 18 weeks). Importantly, 74% of the cases generated an engraftment level of >20% hCD45+ cells. Similar engraftment levels were observed in mice humanized scaffolds that were injected with BM cells from MDS/MPD, CMML and sAML (hCD45+ 10%, 22% and 86%, respectively). No difference in the hCD45+ cell engraftment between the patients with low-risk or high-risk MDS was observed.Remarkably, our 3D humanized scaffolds generated multi-lineage engraftment, with mice that received BM cells from RAEB cases yielding higher myeloid engraftment compared to RARS and RCMD (RARS hCD33+ 44%; RCMD hCD33+ 46.6%; RAEB hCD33+ 65%). Furthermore, transplantation of BM cells from 5 patients into NBSGW mice gave rise to engraftment levels of hCD45+ cells similar (hCD45+ cells, up to 40%) to the NSG and NSG-SGM3 mice. The MDS cells recovered from the mice maintained their genotypic characteristics, as demonstrated by the presence of the mutations that remained largely unchanged between the pre- and post-transplanted samples.Interestingly, Immuno-staining of the scaffolds revealed colonization by the host murine vasculature (mCD31+ endothelial cells). In line with this finding, mouse hematopoietic cells were also found to have migrated into the implanted humanized scaffolds. Surprisingly, we did not observe hCD45+ cell engraftment in the bone marrow of any of the xenotransplanted mouse strains (NSG or NSG-SGM3 mice with or without Irradiation and non-irradiated NBSGW mice). However, we demonstrated that MDS stem cells migrate by implanting a supplementary scaffold into NSG-SGM3 and NBSGW mice that was only coated with MDS-MSCs. Notably, these MDS-MSC seeded scaffolds were colonized by the circulating human MDS cells.Finally, we tested whether the cells recovered from the primary engrafted 3D humanized scaffolds would be able to generate secondary transplants. Our data shows that these cells were able to engraft in the secondary mice implanted with humanized 3D scaffolds.DiscussionOur model provides evidence that MDS BM cells are highly dependent on humanized MSC niche, confirming the importance of the cross-talk between MDS-ICs and MDS niche. Our data demonstrates that MDS stem cells do migrate out of the 3D Scaffold but require their “own niche” for colonization. Thus, our in vivo 3D model system, can be reliably used to better dissect the cross-talk between MDS stoma and MDS-ICs. This model requiring relatively low number of human MNCs, provides a robust preclinical assessment tool to study clonal evolution as well as to test therapeutic strategies on the MDS clonality prior to treatment of MDS patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Preclinical Development of HBB Gene Correction in Autologous Hematopoietic Stem and Progenitor Cells to Treat Severe Sickle Cell Disease

Sickle Cell Disease (SCD) is an autosomal recessive hemoglobinopathy affecting 70-100,000 people in the United States, and millions of people globally. The disease is caused by an adenine to thymidine single nucleotide variant in codon 6 in the human beta-globin gene (HBB gene) that leads to the substitution of glutamic acid by valine (“E6V”) in the beta-globin protein ultimatelyresulting in the formation of sickle beta-globin protein (HgbS) in red cells. While there are therapies like hydroxyurea and red blood cell transfusions that modify the course of the disease, the only curative treatment is allogeneic hematopoietic stem cell transplantation (allo-HSCT), which are available for only 15-20% of the SCD patients, severely limiting the application of allo-HSCT for patients who need it the most. Hence, there remains a tremendous unmet medical need to develop alternative treatment strategies for patients with SCD. An alternative, potentially curative, treatment strategy is to perform gene-editing by homologous recombination (HR) to directly correct the E6V mutation in the HBB gene in autologous hematopoietic stem and progenitor cells (HSPC) from sickle cell patients and then reinfuse these modified cells post-myeloablation. Here we present our IND-enabling preclinical development for CRISPR-Cas9 and rAAV6-mediated HBB genome editing of the E6V mutation in SCD-derived HSPCs as well as in plerixafor mobilized HSPCs from multiple healthy individuals. We demonstrate 30-75% E6V conversion in edited SCD-HSPCs that when differentiated into reticulocytes in vitro, corresponds in the change from HgbS to HgbA tetramers. HBB edited SCD-HSPCs displayed long-term engraftment in the bone marrow of immunodeficient NSG mice at 16 weeks post-transplant, where we identified that ~50% (range of 30-78%) of human cells were edited at the E6V position. Furthermore, by using a recombinant high fidelity Cas9 (HiFi), we were able to eliminate off-target activity as much as 20 fold in edited SCD-HSPCs while maintaining robust on-target activity. Because GCSF use is contraindicated in SCD patients in view of case reports of mortality and that there are ongoing clinical trials using plerixafor to mobilize HSPCs in SCD patients for gene therapy, we also performed IND-enabling HBB genome editing studies in plerixafor mobilized HSPCs from several healthy individuals. We achieved median gene targeting frequencies of ~25% (range of 17-27%) while maintaining high purity (CD34+ percentage >95%) and viability (>80%) across multiple independent experiments. CRISPR-Cas9 and rAAV6- HBB edited plerixafor-HSPCs displayed high potency as evidenced by multilineage methylcellulose progenitor colony formation and importantly, long-term multilineage B, myeloid, and T/NK cell engraftment in the bone marrow of NSG mice at 16-20 weeks post transplant, where a median of ~5% (range of 1-15%) of human alleles were modified at the E6V position. Moreover, In vitro erythroid differentiated human CD34+ HSPCs from NSG mouse bone marrow displayed editing frequencies of 2-15%. These results suggest long-term maintenance of HBB gene targeting in tri-lineage (myeloid/lymphoid/erythroid) plerixafor mobilized repopulating human hematopoietic stem cells. Further optimization of HSPC culture conditions resulted in 2-3 fold higher gene targeting frequencies (up to 70%) and ongoing NSG transplant studies are underway to determine whether the increases in HR frequencies also occur in long-lived repopulating hematopoietic stem cells. Altogether, these preclinical IND-enabling studies outline the framework for the manufacturing of an investigational cellular product consisting of autologous CD34+ HSPC that have been targeted for HBB gene correction of the SCD-causing E6V point mutation for the treatment of severe SCD. DisclosuresVakulskas:IDT: Employment. Behlke:IDT: Employment. Porteus:CRISPRTx: Equity Ownership.

Uncoupling key determinants of hematopoietic stem cell engraftment through cell-specific and temporally controlled recipient conditioning.

SummaryHematopoietic stem cells (HSCs) are typically characterized by transplantation into irradiated hosts in a highly perturbed microenvironment. Here, we show that selective and temporally controlled depletion of resident HSCs through genetic deletion of Gata2 constitutes efficient recipient conditioning for transplantation without irradiation. Strikingly, we achieved robust engraftment of donor HSCs even when delaying Gata2 deletion until 4 weeks after transplantation, allowing homing and early localization to occur in a completely non-perturbed environment. When HSCs from the congenic strains Ly5.1 and Ly5.2 were competitively transplanted, we found that the more proliferative state of Ly5.2 HSCs was associated with superior long-term engraftment when using conditioning by standard irradiation, while higher CXCR4 expression and a better homing ability of Ly5.1 HSCs strongly favored the outcome in our inducible HSC depletion model. Thus, the mode and timing of recipient conditioning challenges distinct functional features of transplanted HSCs.

First Multimodal, Three-Dimensional, Image-Guided Total Marrow Irradiation Model for Preclinical Bone Marrow Transplantation Studies

Total marrow irradiation(TMI) has significantly advanced radiation conditioning for hematopoietic cell transplantation inhematologicmalignancies by reducing conditioning-induced toxicities and improving survival outcomes in relapsed/refractory patients. However, the relapse rate remains high, and the lack of a preclinical TMI model has hindered scientific advancements. To accelerate TMI translation to the clinic, we developed a TMI delivery system in preclinical models. A Precision X-RAD SmART irradiator was used for TMI model development. Images acquired with whole-body contrast-enhanced computed tomography (CT) were used to reconstruct and delineate targets and vital organs for each mouse. Multiple beam and CT-guided Monte Carlo-based plans were performed to optimize doses to the targets and to vary doses to the vital organs. Long-term engraftment and reconstitution potential were evaluated by a congenic bone marrow transplantation (BMT) model and serial secondary BMT, respectively. Donor cell engraftment was measured using noninvasive bioluminescence imaging and flow cytometry. Multimodal imaging enabled identification of targets (skeleton and spleen) and vital organs (eg, lungs, gut, liver). In contrast to total body irradiation (TBI), TMI treatment allowed variation of radiation dose exposure to organs relative to the target dose. Dose reduction mirrored that in clinical TMI studies. Similar to TBI, mice treated with different TMI regimens showed full long-term donor engraftment in primary BMT and second serial BMT. The TBI-treated mice showed acute gut damage, which was minimized in mice treated with TMI. A novel multimodal image guided preclinical TMI model is reported here. TMI conditioning maintained long-term engraftment with reconstitution potential and reduced organ damage. Therefore, this TMI model provides a unique opportunity to study the therapeutic benefit of reduced organ damage and BM dose escalation to optimize treatment regimens in BMT and hematologic malignancies.

INKT cells coordinate immune pathways to enable engraftment in nonconditioned hosts.

Invariant natural killer T (iNKT) cells are a conserved population of innate T lymphocytes that interact with key antigen-presenting cells to modulate adaptive T-cell responses in ways that can either promote protective immunity, or limit pathological immune activation. Understanding the immunological networks engaged by iNKT cells to mediate these opposing functions is a key pre-requisite to effectively using iNKT cells for therapeutic applications. Using a human umbilical cord blood xenotransplantation model, we show here that co-transplanted allogeneic CD4+ iNKT cells interact with monocytes and T cells in the graft to coordinate pro-hematopoietic and immunoregulatory pathways. The nexus of iNKT cells, monocytes, and cord blood T cells led to the release of cytokines (IL-3, GM-CSF) that enhance hematopoietic stem and progenitor cell activity, and concurrently induced PGE2-mediated suppression of T-cell inflammatory responses that limit hematopoietic stem and progenitor cell engraftment. This resulted in successful long-term hematopoietic engraftment without pretransplant conditioning, including multi-lineage human chimerism and colonization of the spleen by antibody-producing human B cells. These results highlight the potential for using iNKT cellular immunotherapy to improve rates of hematopoietic engraftment independently of pretransplant conditioning.

Syngeneic hematopoietic stem cell transplantation for aplastic anemia

目的评价同基因造血干细胞移植（syn-HSCT）治疗再生障碍性贫血（AA）的疗效。方法对11例接受syn-HSCT的AA患者进行回顾性分析。结果全部11例AA患者中男4例，女7例，中位年龄为22（7～44）岁。全部11例患者移植后均获得造血重建，中位粒细胞植入时间为10（8～23）d，中位血小板植入时间为11（8～28）d。8例患者获得长期稳定植入，3例患者植入失败，其中2例行二次移植（1例获得到长期稳定植入，另1例造血重建后再次发生植入失败）。中位随访时间为53（5～135）个月，全部11例患者均存活，其中9例患者外周血象长期正常。结论syn-HSCT治疗AA可获得良好的长期生存，植入失败仍是有待解决的主要问题。

Answered and Unanswered Questions in Early-Stage Viral Vector Transduction Biology and Innate Primary Cell Toxicity for Ex-Vivo Gene Editing.

Adeno-associated virus is a highly efficient DNA delivery vehicle for genome editing strategies that employ CRISPR/Cas9 and a DNA donor for homology-directed repair. Many groups have used this strategy in development of therapies for blood and immune disorders such as sickle-cell anemia and severe-combined immunodeficiency. However, recent events have called into question the immunogenicity of AAV as a gene therapy vector and the safety profile dictated by the immune response to this vector. The target cells dictating this response and the molecular mechanisms dictating cellular response to AAV are poorly understood. Here, we will investigate the current known AAV capsid and genome interactions with cellular proteins during early stage vector transduction and how these interactions may influence innate cellular responses. We will discuss the current understanding of innate immune activation and DNA damage response to AAV, and the limitations of what is currently known. In particular, we will focus on pathway differences in cell line verses primary cells, with a focus on hematopoietic stem and progenitor cells (HSPCs) in the context of ex-vivo gene editing, and what we can learn from HSPC infection by other parvoviruses. Finally, we will discuss how innate immune and DNA damage response pathway activation in these highly sensitive stem cell populations may impact long-term engraftment and clinical outcomes as these gene-editing strategies move towards the clinic, with the aim to propose pathways relevant for improved hematopoietic stem cell survival and long-term engraftment after AAV-mediated genome editing.

Targeting protein lysine methyltransferase G9A impairs self-renewal of chronic myelogenous leukemia stem cells via upregulation of SOX6.

The application of tyrosine kinase inhibitors (TKIs) in clinic has revolutionized chronic myelogenous leukemia (CML) treatment, but fails to eliminate leukemia stem cells (LSCs), which are considered as roots of drug resistance and disease relapse. Thus, eradication of LSCs may be a promising strategy for curing CML. In this study, we found that protein lysine methyltransferase G9A was overexpressed in CML LSCs. The upregulation of G9A by BCR-ABL was independent on its tyrosine kinase activity. Knockdown of G9A by shRNAs or pharmacological inhibition of G9A by UNC0642 significantly suppressed survival and impaired self-renewal capacity of CML LSCs. Inhibition of G9a eradicated LSCs in CML mice driven by BCR-ABL gene and dramatically prolonged survival of the mice. Ex vivo treatment with G9A inhibitor inhibited long-term engraftment of CML CD34+ cells in immunodeficient mice. Mechanically, tumor suppressor SOX6 was identified as a direct target of G9A in CML LSCs by RNA-seq analysis. Silencing Sox6 at least partially rescued G9a knockdown-mediated LSCs elimination in vivo. Our findings improve the understanding of LSC regulation network and validate G9A as a therapeutic target in CML LSCs. Targeting G9A may be considered as an additional strategy for the treatment of patients with CML.